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Archive for the ‘mtDNA’ Category


Pioneers of Cancer Cell Therapy:  Turbocharging the Immune System to Battle Cancer Cells — Success in Hematological Cancers vs. Solid Tumors

Curator: Aviva Lev-Ari, PhD, RN

Chimeric Antigen Receptor T-Cell Therapy: Players in Basic & Translational Research and Biotech/Pharma

The companies are teamed with academic pioneers:

  • Novartis with University of Pennsylvania;
  • Kite Pharma with the National Cancer Institute; 
  • Juno Therapeutics with Sloan Kettering,
  • the Fred Hutchinson Cancer Research Center in Seattle and Seattle Children’s Hospital.

cancer33

IMAGE SOURCE: National Cancer Institute

 

 “CAR-T cell immunotherapy” –  genetically modified T cells that are engineered to target specific tumor antigens and/or genes that are involved in survival, proliferation, and the enhancement of effector functions have been under intense research.

 

CAR technology was originally reported by Zelig Eshhar in 1993.

https://www.weizmann.ac.il/immunology/sci/EshharPage.html

Prof. Zelig Eshhar, Ph.D., served as Chairman of the Department of Immunology at the Weizmann Institute. Prof. Eshhar has been Chair of Scientific Advisory Board at TxCell S.A. since April 2016. Prof. Eshhar has been a Member of Scientific Advisory Board at Kite Pharma, Inc. since August 8, 2013. Prof. Eshhar served as a Member of Scientific Advisory Board at Intellect Neurosciences, Inc. since April 2006.

Prof. Eshhar pioneered the CAR approach (or T-Body as he termed it) to redirect T cells to recognize, engage and kill patient’s tumor cells by engineering them with a construct that combines the anti-target specificity of an antibody with T cell activation domains. Prof. Eshhar serves on several editorial boards, including Cancer Gene Therapy, Human Gene Therapy, Gene Therapy, Expert Opinion on Therapeutics, European Journal of Immunology and the Journal of Gene Medicine. He was a Research Fellow in the Department of Pathology at Harvard Medical School and in the Department of Chemical Immunology at the Weizmann Institute in Israel. His achievements were recognized by several international awards, most recently the CAR Pioneering award by the ATTACK European Consortium. Prof. Eshhar obtained his B.Sc. in Biochemistry and Microbiology and his M.Sc. in Biochemistry from the Hebrew University, and his Ph.D. in the Department of Immunology from the Weizmann Institute of Science.

http://www.bloomberg.com/research/stocks/people/person.asp?personId=32720993&privcapId=32390485

 

Zelig Eshhar and Carl H. June honored for research on T cell engineering for cancer immunotherapy

New Rochelle, NY, November 11, 2014–Zelig Eshhar, PhD, The Weizmann Institute of Science and Sourasky Medical Center, and Carl H. June, MD, PhD, Perelman School of Medicine, University of Pennsylvania, are co-recipients of the Pioneer Award, recognized for lentiviral gene therapy clinical trials and for their leadership and contributions in engineering T-cells capable of targeting tumors with antibody-like specificity through the development of chimeric antigen receptors (CARs). Human Gene Therapy, a peer-reviewed journal from Mary Ann Liebert, Inc., publishers, is commemorating its 25th anniversary by bestowing this honor on the leading Pioneers in the field of cell and gene therapy selected by a blue ribbon panel* and publishing a Pioneer Perspective by the award recipients. The Perspectives by Dr. Eshhar and Dr. June are available free on the Human Gene Therapy website at http://www.liebertpub.com/hgt until December 11, 2014.

In his Pioneer Perspective entitled “From the Mouse Cage to Human Therapy: A Personal Perspective of the Emergence of T-bodies/Chimeric Antigen Receptor T Cells” Professor Eshhar chronicles his team’s groundbreaking contributions to the development of the CAR T-cell immunotherapeutic approach to treating cancer. He describes the method’s conceptual development including initial proof-of-concept, and the years of experimentation in mouse models of cancer. They first tested the CAR T-cells on tumors transplanted into mice then progressed to spontaneously developing cancers in immune-competent mice, which Dr. Eshhar describes as “a more suitable model that faithfully mimics cancer patients.” He recounts successful antitumor effects in mice with CAR modified T-cells injected directly into tumors, with effects seen at the injection site and at sites of metastasis, and even the potential of the CAR T-cells to prevent tumor development.

Dr. Carl H. June has led one of the clinical groups that has taken the CAR therapeutic strategy from the laboratory to the patients’ bedside, pioneering the use of CD19-specific CAR T-cells to treat patients with leukemia. In his Pioneer Perspective, “Toward Synthetic Biology with Engineered T Cells: A Long Journey Just Begun” Dr. June looks back on his long, multi-faceted career and describes how he combined his knowledge and research on immunology, cancer, and HIV to develop successful T-cell based immunotherapies. Among the lessons Dr. June has embraced throughout his career are to follow one’s passions. He also says that “accidents can be good: embrace the unexpected results and follow up on these as they are often times more scientifically interesting than predictable responses from less imaginative experiments.”

“These two extraordinary scientists made seminal contributions at key steps of the journey from bench to bedside for CAR T-cells,” says James M. Wilson, MD, PhD, Editor-in-Chief of Human Gene Therapy, and Director of the Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia.

SOURCE

http://www.eurekalert.org/pub_releases/2014-11/mali-ze111114.php

The General procedure of CAR-T cell therapy involves the follwoing steps:

1) Separate T cells from patient;

2) Engineer these T cells to express an artificial receptor, which is called “CAR” that usually targets tumor-specific antigen;

3) Expand the CAR T cells to a sufficient amount;

4) Re-introduce the CAR T cells to patient.

There are two major components that are critical to the CAR-T cell immunotherapy:

  • the design of CAR itself and
  • the choice of the targeted tumor specific antigen.

SOURCE

http://www.ochis.org/node/209

 

First publication on Adoptive transfer of genetically modified T cells is an attractive approach for generating antitumor immune responses

Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19

James N. Kochenderfer, Wyndham H. Wilson, John E. Janik, Mark E. Dudley, Maryalice Stetler-Stevenson, Steven A. Feldman, Irina Maric, Mark Raffeld, Debbie-Ann N. Nathan, Brock J. Lanier, Richard A. Morgan, Steven A. Rosenberg

Abstract

Adoptive transfer of genetically modified T cells is an attractive approach for generating antitumor immune responses. We treated a patient with advanced follicular lymphoma by administering a preparative chemotherapy regimen followed by autologous T cells genetically engineered to express a chimeric antigen receptor (CAR) that recognized the B-cell antigen CD19. The patient’s lymphoma underwent a dramatic regression, and B-cell precursors were selectively eliminated from the patient’s bone marrow after infusion of anti–CD19-CAR-transduced T cells. Blood B cells were absent for at least 39 weeks after anti–CD19-CAR-transduced T-cell infusion despite prompt recovery of other blood cell counts. Consistent with eradication of B-lineage cells, serum immunoglobulins decreased to very low levels after treatment. The prolonged and selective elimination of B-lineage cells could not be attributed to the chemotherapy that the patient received and indicated antigen-specific eradication of B-lineage cells. Adoptive transfer of anti–CD19-CAR-expressing T cells is a promising new approach for treating B-cell malignancies. This study is registered at www.clinicaltrials.gov as #NCT00924326.

SOURCE

According to Setting the Body’s ‘Serial Killers’ Loose on Cancer

After a long, intense pursuit, researchers are close to bringing to market a daring new treatment: cell therapy that turbocharges the immune system to fight cancer.

By ANDREW POLLACK  AUG. 1, 2016

http://www.nytimes.com/2016/08/02/health/cancer-cell-therapy-immune-system.html?_r=0

Dr. June’s 2011 publications did not cite Dr. Rosenberg’s paper [Blood, 2010] from the previous year, prompting Dr. Rosenberg to write a letter to The New England Journal of Medicine. Dr. June’s publications also did not acknowledge that the genetic construct he had used was the one he had obtained from Dr. Campana of St. Jude.

From the Lab to the bedside to the Out Patient Clinic

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Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle


Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Reporter: Stephen S Williams, PhD

 

Leaders in Pharmaceutical Business Intelligence would like to announce the First volume of their BioMedical E-Book Series D:

Metabolic Genomics & Pharmaceutics, Vol. I

SACHS FLYER 2014 Metabolomics SeriesDindividualred-page2

which is now available on Amazon Kindle at

http://www.amazon.com/dp/B012BB0ZF0.

This e-Book is a comprehensive review of recent Original Research on  METABOLOMICS and related opportunities for Targeted Therapy written by Experts, Authors, Writers. This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases.  It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time baccalaureate degree reader in the biological sciences.  Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career. The results of Original Research are gaining value added for the e-Reader by the Methodology of Curation. The e-Book’s articles have been published on the Open Access Online Scientific Journal, since April 2012.  All new articles on this subject, will continue to be incorporated, as published with periodical updates.

We invite e-Readers to write an Article Reviews on Amazon for this e-Book on Amazon.

All forthcoming BioMed e-Book Titles can be viewed at:

https://pharmaceuticalintelligence.com/biomed-e-books/

Leaders in Pharmaceutical Business Intelligence, launched in April 2012 an Open Access Online Scientific Journal is a scientific, medical and business multi expert authoring environment in several domains of  life sciences, pharmaceutical, healthcare & medicine industries. The venture operates as an online scientific intellectual exchange at their website http://pharmaceuticalintelligence.com and for curation and reporting on frontiers in biomedical, biological sciences, healthcare economics, pharmacology, pharmaceuticals & medicine. In addition the venture publishes a Medical E-book Series available on Amazon’s Kindle platform.

Analyzing and sharing the vast and rapidly expanding volume of scientific knowledge has never been so crucial to innovation in the medical field. WE are addressing need of overcoming this scientific information overload by:

  • delivering curation and summary interpretations of latest findings and innovations on an open-access, Web 2.0 platform with future goals of providing primarily concept-driven search in the near future
  • providing a social platform for scientists and clinicians to enter into discussion using social media
  • compiling recent discoveries and issues in yearly-updated Medical E-book Series on Amazon’s mobile Kindle platform

This curation offers better organization and visibility to the critical information useful for the next innovations in academic, clinical, and industrial research by providing these hybrid networks.

Table of Contents for Metabolic Genomics & Pharmaceutics, Vol. I

Chapter 1: Metabolic Pathways

Chapter 2: Lipid Metabolism

Chapter 3: Cell Signaling

Chapter 4: Protein Synthesis and Degradation

Chapter 5: Sub-cellular Structure

Chapter 6: Proteomics

Chapter 7: Metabolomics

Chapter 8:  Impairments in Pathological States: Endocrine Disorders; Stress

                   Hypermetabolism and Cancer

Chapter 9: Genomic Expression in Health and Disease 

 

Summary 

Epilogue

 

 

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Warburg Effect and Mitochondrial Regulation -2.1.3

Writer and Curator: Larry H Bernstein, MD, FCAP 

2.1.3 Warburg Effect and Mitochondrial Regulation

2.1.3.1 Regulation of Substrate Utilization by the Mitochondrial Pyruvate Carrier

NM Vacanti, AS Divakaruni, CR Green, SJ Parker, RR Henry, TP Ciaraldi, et a..
Molec Cell 6 Nov 2014; 56(3):425–435
http://dx.doi.org/10.1016/j.molcel.2014.09.024

Highlights

  • Oxidation of fatty acids and amino acids is increased upon MPC inhibition
    •Respiration, proliferation, and biosynthesis are maintained when MPC is inhibited
    •Glutaminolytic flux supports lipogenesis in the absence of MPC
    •MPC inhibition is distinct from hypoxia or complex I inhibition

Summary

Pyruvate lies at a central biochemical node connecting carbohydrate, amino acid, and fatty acid metabolism, and the regulation of pyruvate flux into mitochondria represents a critical step in intermediary metabolism impacting numerous diseases. To characterize changes in mitochondrial substrate utilization in the context of compromised mitochondrial pyruvate transport, we applied 13C metabolic flux analysis (MFA) to cells after transcriptional or pharmacological inhibition of the mitochondrial pyruvate carrier (MPC). Despite profound suppression of both glucose and pyruvate oxidation, cell growth, oxygen consumption, and tricarboxylic acid (TCA) metabolism were surprisingly maintained. Oxidative TCA flux was achieved through enhanced reliance on glutaminolysis through malic enzyme and pyruvate dehydrogenase (PDH) as well as fatty acid and branched-chain amino acid oxidation. Thus, in contrast to inhibition of complex I or PDH, suppression of pyruvate transport induces a form of metabolic flexibility associated with the use of lipids and amino acids as catabolic and anabolic fuels.

oxidation-of-fatty-acids-and-amino-acid

oxidation-of-fatty-acids-and-amino-acids

Graphical Abstract – Oxidation of fatty acids and amino acids is increased upon MPC inhibition

Figure 2. MPC Regulates Mitochondrial Substrate Utilization (A) Citrate mass isotopomer distribution (MID) resulting from culture with [U-13C6]glucose (UGlc). (B) Percentage of 13C-labeled metabolites from UGlc. (C) Percentage of fully labeled lactate, pyruvate, and alanine from UGlc. (D) Serine MID resulting from culture with UGlc. (E) Percentage of fully labeled metabolites derived from [U-13C5]glutamine (UGln). (F) Schematic of UGln labeling of carbon atoms in TCA cycle intermediates arising via glutaminoloysis and reductive carboxylation. Mitochondrion schematic inspired by Lewis et al. (2014). (G and H) Citrate (G) and alanine (H) MIDs resulting from culture with UGln. (I) Maximal oxygen consumption rates with or without 3 mM BPTES in medium supplemented with 1 mM pyruvate. (J) Percentage of newly synthesized palmitate as determined by ISA. (K) Contribution of UGln and UGlc to lipogenic AcCoA as determined by ISA. (L) Contribution of glutamine to lipogenic AcCoA via glutaminolysis (ISA using a [3-13C] glutamine [3Gln]) and reductive carboxylation (ISA using a [5-13C]glutamine [5Gln]) under normoxia and hypoxia. (M) Citrate MID resulting from culture with 3Gln. (N) Contribution of UGln and exogenous [3-13C] pyruvate (3Pyr) to lipogenic AcCoA. 2KD+Pyr refers to Mpc2KD cells cultured with 10 mM extracellular pyruvate. Error bars represent SD (A–E, G, H, and M), SEM(I), or 95% confidence intervals(J–L, and N).*p<0.05,**p<0.01,and ***p<0.001 by ANOVA with Dunnett’s post hoc test (A–E and G–I) or * indicates significance by non-overlapping 95% confidence intervals (J–L and N).

Figure 3. Mpc Knockdown Increases Fatty Acid Oxidation. (A) Schematic of changes in flux through metabolic pathways in Mpc2KD relative to control cells. (B) Citrate MID resulting from culture with [U-13C16] palmitate conjugated to BSA (UPalm). (C) Percentage of 13C enrichment resulting from culture with UPalm. (D) ATP-linked and maximal oxygen consumption rate, with or without 20m Metomoxir, with or without 3 mM BPTES. Culture medium supplemented with 0.5 mM carnitine. Error bars represent SD (B and C) or SEM (D). *p < 0.05, **p < 0.01, and ***p < 0.001 by two-tailed, equal variance, Student’s t test(B–D), or by ANOVA with Dunnett’s post hoc test (D).

Figure 4. Metabolic Reprogramming Resulting from Pharmacological Mpc Inhibition Is Distinct from Hypoxia or Complex I Inhibition

2.1.3.2 Oxidation of Alpha-Ketoglutarate Is Required for Reductive Carboxylation in Cancer Cells with Mitochondrial Defects

AR Mullen, Z Hu, X Shi, L Jiang, …, WM Linehan, NS Chandel, RJ DeBerardinis
Cell Reports 12 Jun 2014; 7(5):1679–1690
http://dx.doi.org/10.1016/j.celrep.2014.04.037

Highlights

  • Cells with mitochondrial defects use bidirectional metabolism of the TCA cycle
    •Glutamine supplies the succinate pool through oxidative and reductive metabolism
    •Oxidative TCA cycle metabolism is required for reductive citrate formation
    •Oxidative metabolism produces reducing equivalents for reductive carboxylation

Summary

Mammalian cells generate citrate by decarboxylating pyruvate in the mitochondria to supply the tricarboxylic acid (TCA) cycle. In contrast, hypoxia and other impairments of mitochondrial function induce an alternative pathway that produces citrate by reductively carboxylating α-ketoglutarate (AKG) via NADPH-dependent isocitrate dehydrogenase (IDH). It is unknown how cells generate reducing equivalents necessary to supply reductive carboxylation in the setting of mitochondrial impairment. Here, we identified shared metabolic features in cells using reductive carboxylation. Paradoxically, reductive carboxylation was accompanied by concomitant AKG oxidation in the TCA cycle. Inhibiting AKG oxidation decreased reducing equivalent availability and suppressed reductive carboxylation. Interrupting transfer of reducing equivalents from NADH to NADPH by nicotinamide nucleotide transhydrogenase increased NADH abundance and decreased NADPH abundance while suppressing reductive carboxylation. The data demonstrate that reductive carboxylation requires bidirectional AKG metabolism along oxidative and reductive pathways, with the oxidative pathway producing reducing equivalents used to operate IDH in reverse.

Proliferating cells support their growth by converting abundant extracellular nutrients like glucose and glutamine into precursors for macromolecular biosynthesis. A continuous supply of metabolic intermediates from the tricarboxylic acid (TCA) cycle is essential for cell growth, because many of these intermediates feed biosynthetic pathways to produce lipids, proteins and nucleic acids (Deberardinis et al., 2008). This underscores the dual roles of the TCA cycle for cell growth: it generates reducing equivalents for oxidative phosphorylation by the electron transport chain (ETC), while also serving as a hub for precursor production. During rapid growth, the TCA cycle is characterized by large influxes of carbon at positions other than acetyl-CoA, enabling the cycle to remain full even as intermediates are withdrawn for biosynthesis. Cultured cancer cells usually display persistence of TCA cycle activity despite robust aerobic glycolysis, and often require mitochondrial catabolism of glutamine to the TCA cycle intermediate AKG to maintain rapid rates of proliferation (Icard et al., 2012Hiller and Metallo, 2013).

Some cancer cells contain severe, fixed defects in oxidative metabolism caused by mutations in the TCA cycle or the ETC. These include mutations in fumarate hydratase (FH) in renal cell carcinoma and components of the succinate dehydrogenase (SDH) complex in pheochromocytoma, paraganglioma, and gastrointestinal stromal tumors (Tomlinson et al., 2002Astuti et al., 2001Baysal et al., 2000Killian et al., 2013Niemann and Muller, 2000). All of these mutations alter oxidative metabolism of glutamine in the TCA cycle. Recently, analysis of cells containing mutations in FH, ETC Complexes I or III, or exposed to the ETC inhibitors metformin and rotenone or the ATP synthase inhibitor oligomycin revealed that turnover of TCA cycle intermediates was maintained in all cases (Mullen et al., 2012). However, the cycle operated in an unusual fashion characterized by conversion of glutamine-derived AKG to isocitrate through a reductive carboxylation reaction catalyzed by NADP+/NADPH-dependent isoforms of isocitrate dehydrogenase (IDH). As a result, a large fraction of the citrate pool carried five glutamine-derived carbons. Citrate could be cleaved to produce acetyl-CoA to supply fatty acid biosynthesis, and oxaloacetate (OAA) to supply pools of other TCA cycle intermediates. Thus, reductive carboxylation enables biosynthesis by enabling cells with impaired mitochondrial metabolism to maintain pools of biosynthetic precursors that would normally be supplied by oxidative metabolism. Reductive carboxylation is also induced by hypoxia and by pseudo-hypoxic states caused by mutations in the von Hippel-Lindau (VHL) tumor suppressor gene (Metallo et al., 2012Wise et al., 2011).

Interest in reductive carboxylation stems in part from the possibility that inhibiting the pathway might induce selective growth suppression in tumor cells subjected to hypoxia or containing mutations that prevent them from engaging in maximal oxidative metabolism. Hence, several recent studies have sought to understand the mechanisms by which this pathway operates. In vitro studies of IDH1 indicate that a high ratio of NADPH/NADP+ and low citrate concentration activate the reductive carboxylation reaction (Leonardi et al., 2012). This is supported by data demonstrating that reductive carboxylation in VHL-deficient renal carcinoma cells is associated with a low concentration of citrate and a reduced ratio of citrate:AKG, suggesting that mass action can be a driving force to determine IDH directionality (Gameiro et al., 2013b). Moreover, interrupting the supply of mitochondrial NADPH by silencing the nicotinamide nucleotide transhydrogenase (NNT) suppresses reductive carboxylation (Gameiro et al., 2013a). This mitochondrial transmembrane protein catalyzes the transfer of a hydride ion from NADH to NADP+ to generate NAD+ and NADPH. Together, these observations suggest that reductive carboxylation is modulated in part through the mitochondrial redox state and the balance of substrate/products.

Here we used metabolomics and stable isotope tracing to better understand overall metabolic states associated with reductive carboxylation in cells with defective mitochondrial metabolism, and to identify sources of mitochondrial reducing equivalents necessary to induce the reaction. We identified high levels of succinate in some cells using reductive carboxylation, and determined that most of this succinate was formed through persistent oxidative metabolism of AKG. Silencing this oxidative flux by depleting the mitochondrial enzyme AKG dehydrogenase substantially altered the cellular redox state and suppressed reductive carboxylation. The data demonstrate that bidirectional/branched AKG metabolism occurs during reductive carboxylation in cells with mitochondrial defects, with oxidative metabolism producing reducing equivalents to supply reductive metabolism.

Shared metabolomic features among cell lines with cytb or FH mutations

To identify conserved metabolic features associated with reductive carboxylation in cells harboring defective mitochondrial metabolism, we analyzed metabolite abundance in isogenic pairs of cell lines in which one member displayed substantial reductive carboxylation and the other did not. We used a pair of previously described cybrids derived from 143B osteosarcoma cells, in which one cell line contained wild-type mitochondrial DNA (143Bwt) and the other contained a mutation in the cytb gene (143Bcytb), severely reducing complex III function (Rana et al., 2000Weinberg et al., 2010). The 143Bwt cells primarily use oxidative metabolism to supply the citrate pool while the 143Bcytb cells use reductive carboxylation (Mullen et al., 2012). The other pair, derived from FH-deficient UOK262 renal carcinoma cells, contained either an empty vector control (UOK262EV) or a stably re-expressed wild-type FH allele (UOK262FH). Metabolites were extracted from all four cell lines and analyzed by triple-quadrupole mass spectrometry. We first performed a quantitative analysis to determine the abundance of AKG and citrate in the four cell lines. Both 143Bcytb and UOK262EV cells had less citrate, more AKG, and lower citrate:AKG ratios than their oxidative partners (Fig. S1A-C), consistent with findings from VHL-deficient renal carcinoma cells (Gameiro et al., 2013b).

Next, to identify other perturbations, we profiled the relative abundance of more than 90 metabolites from glycolysis, the pentose phosphate pathway, one-carbon/nucleotide metabolism, the TCA cycle, amino acid degradation, and other pathways (Tables S1 and S2). Each metabolite was normalized to protein content, and relative abundance was determined between cell lines from each pair. Hierarchical clustering (Fig 1A) and principal component analysis (Fig 1B) revealed far greater metabolomic similarities between the members of each pair than between the two cell lines using reductive carboxylation. Only three metabolites displayed highly significant (p<0.005) differences in abundance between the two members of both pairs, and in all three cases the direction of the difference (i.e. higher or lower) was shared in the two cell lines using reductive carboxylation. Proline, a nonessential amino acid derived from glutamine in an NADPH-dependent biosynthetic pathway, was depleted in 143Bcytb and UOK262EV cells (Fig. 1C). 2-hydroxyglutarate (2HG), the reduced form of AKG, was elevated in 143Bcytb and UOK262EV cells (Fig. 1D), and further analysis revealed that while both the L- and D-enantiomers of this metabolite were increased, L-2HG was quantitatively the predominant enantiomer (Fig. S1D). It is likely that 2HG accumulation was related to the reduced redox ratio associated with cytb and FH mutations. Although the sources of 2HG are still under investigation, promiscuous activity of the TCA cycle enzyme malate dehydrogenase produces L-2HG in an NADH-dependent manner (Rzem et al., 2007). Both enantiomers are oxidized to AKG by dehydrogenases (L-2HG dehydrogenase and D-2HG dehydrogenase). It is therefore likely that elevated 2-HG is a consequence of a reduced NAD+/NADH ratio. Consistent with this model, inborn errors of the ETC result in 2-HG accumulation (Reinecke et al., 2011). Exposure to hypoxia (<1% O2) has also been demonstrated to reduce the cellular NAD+/NADH ratio (Santidrian et al., 2013) and to favor modest 2HG accumulation in cultured cells (Wise et al., 2011), although these levels were below those noted in gliomas expressing 2HG-producing mutant alleles of isocitrate dehydrogenase-1 or -2 (Dang et al., 2009).

Figure 1 Metabolomic features of cells using reductive carboxylation

 

Finally, the TCA cycle intermediate succinate was markedly elevated in both cell lines (Fig. 1E). We tested additional factors previously reported to stimulate reductive AKG metabolism, including a genetic defect in ETC Complex I, exposure to hypoxia, and chemical inhibitors of the ETC (Mullen et al., 2012Wise et al., 2011Metallo et al., 2012). These factors had a variable effect on succinate, with impairments of Complex III or IV strongly inducing succinate accumulation, while impairments of Complex I either had little effect or suppressed succinate (Fig. 1F).

Oxidative glutamine metabolism is the primary route of succinate formation

UOK262EV cells lack FH activity and accumulate large amounts of fumarate (Frezza et al., 2011); elevated succinate was therefore not surprising in these cells, because succinate precedes fumarate by one reaction in the TCA cycle. On the other hand, TCA cycle perturbation in 143Bcytb cells results from primary ETC dysfunction, and reductive carboxylation is postulated to be a consequence of accumulated AKG (Anastasiou and Cantley, 2012Fendt et al., 2013). Accumulation of AKG is not predicted to result in elevated succinate. We previously reported that 143Bcytb cells produce succinate through simultaneous oxidative and reductive glutamine metabolism (Mullen et al., 2012). To determine the relative contributions of these two pathways, we cultured 143Bwt and 143Bcytb with [U-13C]glutamine and monitored time-dependent 13C incorporation in succinate and other TCA cycle intermediates. Oxidative metabolism of glutamine generates succinate, fumarate and malate containing four glutamine-derived 13C nuclei on the first turn of the cycle (m+4), while reductive metabolism results in the incorporation of three 13C nuclei in these intermediates (Fig. S2). As expected, oxidative glutamine metabolism was the predominant source of succinate, fumarate and malate in 143Bwt cells (Fig. 2A-C). In 143Bcytb, fumarate and malate were produced primarily through reductive metabolism (Fig. 2E-F). Conversely, succinate was formed primarily through oxidative glutamine metabolism, with a minor contribution from the reductive carboxylation pathway (Fig. 2D). Notably, this oxidatively-derived succinate was detected prior to that formed through reductive carboxylation. This indicated that 143Bcytb cells retain the ability to oxidize AKG despite the observation that most of the citrate pool bears the labeling pattern of reductive carboxylation. Together, the labeling data in 143Bcytb cells revealed bidirectional metabolism of carbon from glutamine to produce various TCA cycle intermediates.

Figure 2  Oxidative glutamine metabolism is the primary route of succinate formation in cells using reductive carboxylation to generate citrate

Pyruvate carboxylation contributes to the TCA cycle in cells using reductive carboxylation

Because of the persistence of oxidative metabolism, we determined the extent to which other routes of metabolism besides reductive carboxylation contributed to the TCA cycle. We previously reported that silencing the glutamine-catabolizing enzyme glutaminase (GLS) depletes pools of fumarate, malate and OAA, eliciting a compensatory increase in pyruvate carboxylase (PC) to supply the TCA cycle (Cheng et al., 2011). In cells with defective oxidative phophorylation, production of OAA by PC may be preferable to glutamine oxidation because it diminishes the need to recycle reduced electron carriers generated by the TCA cycle. Citrate synthase (CS) can then condense PC-derived OAA with acetyl-CoA to form citrate. To examine the contribution of PC to the TCA cycle, cells were cultured with [3,4-13C]glucose. In this labeling scheme, glucose-derived pyruvate is labeled in carbon 1 (Fig. S3). This label is retained in OAA if pyruvate is carboxylated, but removed as CO2 during conversion of pyruvate to acetyl-CoA by pyruvate dehydrogenase (PDH).

Figure 3 Pyruvate carboxylase contributes to citrate formation in cells using reductive carboxylation

Oxidative metabolism of AKG is required for reductive carboxylation

Oxidative synthesis of succinate from AKG requires two reactions: the oxidative decarboxylation of AKG to succinyl-CoA by AKG dehydrogenase, and the conversion of succinyl-CoA to succinate by succinyl-CoA synthetase. In tumors with mutations in the succinate dehydrogenase (SDH) complex, large accumulations of succinate are associated with epigenetic modifications of DNA and histones to promote malignancy (Kaelin and McKnight, 2013Killian et al., 2013). We therefore tested whether succinate accumulation per se was required to induce reductive carboxylation in 143Bcytb cells. We used RNA interference directed against the gene encoding the alpha subunit (SUCLG1) of succinyl-CoA synthetase, the last step in the pathway of oxidative succinate formation from glutamine (Fig. 4A). Silencing this enzyme greatly reduced succinate levels (Fig. 4B), but had no effect on the labeling pattern of citrate from [U-13C]glutamine (Fig. 4C). Thus, succinate accumulation is not required for reductive carboxylation.

Figure 5 AKG dehydrogenase is required for reductive carboxylation

Figure 6 AKG dehydrogenase and NNT contribute to NAD+/NADH ratio

Finally, we tested whether these enzymes also controlled the NADP+/NADPH ratio in 143Bcytb cells. Silencing either OGDH or NNT increased the NADP+/NADPH ratio (Fig. 6F,G), whereas silencing IDH2reduced it (Fig. 6H). Together, these data are consistent with a model in which persistent metabolism of AKG by AKG dehydrogenase produces NADH that supports reductive carboxylation by serving as substrate for NNT-dependent NADPH formation, and that IDH2 is a major consumer of NADPH during reductive carboxylation (Fig. 6I).

Reductive carboxylation of AKG initiates a non-conventional form of metabolism that produces TCA cycle intermediates when oxidative metabolism is impaired by mutations, drugs or hypoxia. Because NADPH-dependent isoforms of IDH are reversible, supplying supra-physiological pools of substrates on either side of the reaction drives function of the enzyme as a reductive carboxylase or an oxidative decarboxylase. Thus, in some circumstances reductive carboxylation may operate in response to a mass effect imposed by drastic changes in the abundance of AKG and isocitrate/citrate. However, reductive carboxylation cannot occur without a source of reducing equivalents to produce NADPH. The current work demonstrates that AKG dehydrogenase, an NADH-generating enzyme complex, is required to maintain a low NAD+/NADH ratio for reductive carboxylation of AKG. Thus, reductive carboxylation not only coexists with oxidative metabolism of AKG, but depends on it. Furthermore, silencing NNT, a consumer of NADH, also perturbs the redox ratio and suppresses reductive formation of citrate. These observations suggest that the segment of the oxidative TCA cycle culminating in succinate is necessary to transmit reducing equivalents to NNT for the reductive pathway (Fig 6I).

Succinate accumulation was observed in cells with cytb or FH mutations. However, this accumulation was dispensable for reductive carboxylation, because silencing SUCLG1 expression had no bearing on the pathway as long as AKG dehydrogenase was active. Furthermore, succinate accumulation was not a universal finding of cells using reductive carboxylation. Rather, high succinate levels were observed in cells with distal defects in the ETC (complex III: antimycin, cytb mutation; complex IV: hypoxia) but not defects in complex I (rotenone, metformin, NDUFA1 mutation). These differences reflect the known suppression of SDH activity when downstream components of the ETC are impaired, and the various mechanisms by which succinate may be formed through either oxidative or reductive metabolism. Succinate has long been known as an evolutionarily conserved anaerobic end product of amino acid metabolism during prolonged hypoxia, including in diving mammals (Hochachka and Storey, 1975, Hochachka et al., 1975). The terminal step in this pathway is the conversion of fumarate to succinate using the NADH-dependent “fumarate reductase” system, essentially a reversal of succinate dehydrogenase/ETC complex II (Weinberg et al., 2000, Tomitsuka et al., 2010). However, this process requires reducing equivalents to be passed from NADH to complex I, then to Coenzyme Q, and eventually to complex II to drive the reduction of fumarate to succinate. Hence, producing succinate through reductive glutamine metabolism would require functional complex I. Interestingly, the fumarate reductase system has generally been considered as a mechanism to maintain a proton gradient under conditions of defective ETC activity. Our data suggest that the system is part of a more extensive reorganization of the TCA cycle that also enables reductive citrate formation.

In summary, we demonstrated that branched AKG metabolism is required to sustain levels of reductive carboxylation observed in cells with mitochondrial defects. The organization of this branched pathway suggests that it serves as a relay system to maintain the redox requirements for reductive carboxylation, with the oxidative arm producing reducing equivalents at the level of AKG dehydrogenase and NNT linking this activity to the production of NADPH to be used in the reductive carboxylation reaction. Hence, impairment of the oxidative arm prevents maximal engagement of reductive carboxylation. As both NNT and AKG dehydrogenase are mitochondrial enzymes, the work emphasizes the flexibility of metabolic systems in the mitochondria to fulfill requirements for redox balance and precursor production even when the canonical oxidative function of the mitochondria is impaired.

2.1.3.3 Rewiring Mitochondrial Pyruvate Metabolism. Switching Off the Light in Cancer Cells

Peter W. Szlosarek, Suk Jun Lee, Patrick J. Pollard
Molec Cell 6 Nov 2014; 56(3): 343–344
http://dx.doi.org/10.1016/j.molcel.2014.10.018

Figure 1. MPC Expression and Metabolic Targeting of Mitochondrial Pyruvate High MPC expression (green) is associated with more favorable tumor prognosis, increased pyruvate oxidation, and reduced lactate and ROS, whereas low expression or mutated MPC is linked to poor tumor prognosis and increased anaplerotic generation of OAA. Dual targeting of MPC and GDH with small molecule inhibitors may ameliorate tumorigenesis in certain cancer types.

The study by Yang et al., (2014) provides evidence for the metabolic flexibility to maintain TCA cycle function. Using isotopic labeling, the authors demonstrated that inhibition of MPCs by a specific compound (UK5099) induced glutamine-dependent acetyl-CoA formation via glutamate dehydrogenase (GDH). Consequently, and in contrast to single agent treatment, simultaneous administration of MPC and GDH inhibitors drastically abrogated the growth of cancer cells (Figure 1). These studies have also enabled a fresh perspective on metabolism in the clinic and emphasized a need for high-quality translational studies to assess the role of mitochondrial pyruvate transport in vivo. Thus, integrating the biomarker of low MPC expression with dual inhibition of

MPC and GDH as a synthetic lethal strategy (Yang et al., 2014) is testable and may offer a novel therapeutic window for patients (DeBerardinis and Thompson, 2012). Indeed, combinatorial targeting of cancer metabolism may prevent early drug resistance and lead to enhanced tumor control, as shown recently for antifolate agents combined with arginine deprivation with modulation of intracellular glutamine (Szlosarek, 2014). Moreover, it will be important to assess both intertumoral and intratumoral metabolic heterogeneity going forward, as tumor cells are highly adaptable with respect to the precursors used to fuel the TCA cycle in the presence of reduced pyruvate transport. The observation by Vacanti et al. (2014) that the flux of BCAAs increased following inhibition of MPC activity may also underlie the increase in BCAAs detected in the plasma of patients several years before a clinical diagnosis of pancreatic cancer (Mayers et al., 2014). Since measuring pyruvate transport via the MPC is technically challenging, the use of 18-FDG positron emission tomography and more recently magnetic spectroscopy with hyperpolarized 13C-labeled pyruvate will need to be incorporated into these future studies (Brindle et al., 2011).

References

Bricker, D.K., Taylor, E.B., Schell, J.C., Orsak, T., Boutron, A., Chen, Y.C., Cox, J.E., Cardon, C.M., Van Vranken, J.G., Dephoure, N., et al. (2012). Science 337, 96–100.

Brindle, K.M., Bohndiek, S.E., Gallagher, F.A., and Kettunen, M.I. (2011). Magn. Reson. Med. 66, 505–519.

DeBerardinis, R.J., and Thompson, C.B. (2012). Cell 148, 1132–1144.

Herzig, S., Raemy, E., Montessuit, S., Veuthey, J.L., Zamboni, N., Westermann, B., Kunji, E.R., and Martinou, J.C. (2012). Science 337, 93–96.

Mayers, J.R., Wu, C., Clish, C.B., Kraft, P., Torrence, M.E., Fiske, B.P., Yuan, C., Bao, Y., Townsend, M.K., Tworoger, S.S., et al. (2014). Nat. Med. 20, 1193–1198.

Metallo, C.M., and Vander Heiden, M.G. (2013). Mol. Cell 49, 388–398.

Schell, J.C., Olson, K.A., Jiang, L., Hawkins, A.J., Van Vranken, J.G., et al. (2014). Mol. Cell 56, this issue, 400–413.

Szlosarek, P.W. (2014). Proc. Natl. Acad. Sci. USA 111, 14015–14016.

Vacanti, N.M., Divakaruni, A.S., Green, C.R., Parker, S.J., Henry, R.R., et al. (2014). Mol. Cell 56, this issue, 425–435.

Yang, C., Ko, B., Hensley, C.T., Jiang, L., Wasti, A.T., et al. (2014). Mol. Cell 56, this issue, 414–424.

2.1.3.4 Betaine is a positive regulator of mitochondrial respiration

Lee I
Biochem Biophys Res Commun. 2015 Jan 9; 456(2):621-5.
http://dx.doi.org:/10.1016/j.bbrc.2014.12.005

Highlights

  • Betaine enhances cytochrome c oxidase activity and mitochondrial respiration.
    • Betaine increases mitochondrial membrane potential and cellular energy levels.
    • Betaine’s anti-tumorigenic effect might be due to a reversal of the Warburg effect.

Betaine protects cells from environmental stress and serves as a methyl donor in several biochemical pathways. It reduces cardiovascular disease risk and protects liver cells from alcoholic liver damage and nonalcoholic steatohepatitis. Its pretreatment can rescue cells exposed to toxins such as rotenone, chloroform, and LiCl. Furthermore, it has been suggested that betaine can suppress cancer cell growth in vivo and in vitro. Mitochondrial electron transport chain (ETC) complexes generate the mitochondrial membrane potential, which is essential to produce cellular energy, ATP. Reduced mitochondrial respiration and energy status have been found in many human pathological conditions including aging, cancer, and neurodegenerative disease. In this study we investigated whether betaine directly targets mitochondria. We show that betaine treatment leads to an upregulation of mitochondrial respiration and cytochrome c oxidase activity in H2.35 cells, the proposed rate limiting enzyme of ETC in vivo. Following treatment, the mitochondrial membrane potential was increased and cellular energy levels were elevated. We propose that the anti-proliferative effects of betaine on cancer cells might be due to enhanced mitochondrial function contributing to a reversal of the Warburg effect.

2.1.3.5 Mitochondrial dysfunction in human non-small-cell lung cancer cells to TRAIL-induced apoptosis by reactive oxygen species and Bcl-XL/p53-mediated amplification mechanisms

Y-L Shi, S Feng, W Chen, Z-C Hua, J-J Bian and W Yin
Cell Death and Disease (2014) 5, e1579
http://dx.doi.org:/10.1038/cddis.2014.547

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for anticancer therapy; however, non-small-cell lung carcinoma (NSCLC) cells are relatively TRAIL resistant. Identification of small molecules that can restore NSCLC susceptibility to TRAIL-induced apoptosis is meaningful. We found here that rotenone, as a mitochondrial respiration inhibitor, preferentially increased NSCLC cells sensitivity to TRAIL-mediated apoptosis at subtoxic concentrations, the mechanisms by which were accounted by the upregulation of death receptors and the downregulation of c-FLIP (cellular FLICE-like inhibitory protein). Further analysis revealed that death receptors expression by rotenone was regulated by p53, whereas c-FLIP downregulation was blocked by Bcl-XL overexpression. Rotenone triggered the mitochondria-derived reactive oxygen species (ROS) generation, which subsequently led to Bcl-XL downregulation and PUMA upregulation. As PUMA expression was regulated by p53, the PUMA, Bcl-XL and p53 in rotenone-treated cells form a positive feedback amplification loop to increase the apoptosis sensitivity. Mitochondria-derived ROS, however, promote the formation of this amplification loop. Collectively, we concluded that ROS generation, Bcl-XL and p53-mediated amplification mechanisms had an important role in the sensitization of NSCLC cells to TRAIL-mediated apoptosis by rotenone. The combined TRAIL and rotenone treatment may be appreciated as a useful approach for the therapy of NSCLC that warrants further investigation.

Abbreviations: c-FLIP, cellular FLICE-like inhibitory protein; DHE, dihydroethidium; DISC, death-inducing signaling complex; DPI, diphenylene iodonium; DR4/DR5, death receptor 4/5; EB, ethidium bromide; FADD, Fas-associated protein with death domain; MnSOD, manganese superoxide; NAC, N-acetylcysteine; NSCLC, non-small-cell lung carcinoma; PBMC, peripheral blood mononuclear cells; ROS, reactive oxygen species; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; UPR, unfolded protein response.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as a promising cancer therapeutic because it can selectively induce apoptosis in tumor cells in vitro, and most importantly, in vivo with little adverse effect on normal cells.1 However, a number of cancer cells are resistant to TRAIL, especially highly malignant tumors such as lung cancer.23 Lung cancer, especially the non-small-cell lung carcinoma (NSCLC) constitutes a heavy threat to human life. Presently, the morbidity and mortality of NSCLC has markedly increased in the past decade,4 which highlights the need for more effective treatment strategies.

TRAIL has been shown to interact with five receptors, including the death receptors 4 and 5 (DR4 and DR5), the decoy receptors DcR1 and DcR2, and osteoprotegerin.5 Ligation of TRAIL to DR4 or DR5 allows for the recruitment of Fas-associated protein with death domain (FADD), which leads to the formation of death-inducing signaling complex (DISC) and the subsequent activation of caspase-8/10.6 The effector caspase-3 is activated by caspase-8, which cleaves numerous regulatory and structural proteins resulting in cell apoptosis. Caspase-8 can also cleave the Bcl-2 inhibitory BH3-domain protein (Bid), which engages the intrinsic apoptotic pathway by binding to Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist killer (BAK). The oligomerization between Bcl-2 and Bax promotes the release of cytochrome c from mitochondria to cytosol, and facilitates the formation of apoptosome and caspase-9 activation.7 Like caspase-8, caspase-9 can also activate caspase-3 and initiate cell apoptosis. Besides apoptosis-inducing molecules, several apoptosis-inhibitory proteins also exist and have function even when apoptosis program is initiated. For example, cellular FLICE-like inhibitory protein (c-FLIP) is able to suppress DISC formation and apoptosis induction by sequestering FADD.891011

Until now, the recognized causes of TRAIL resistance include differential expression of death receptors, constitutively active AKT and NF-κB,1213overexpression of c-FLIP and IAPs, mutations in Bax and BAK gene.2 Hence, resistance can be overcome by the use of sensitizing agents that modify the deregulated death receptor expression and/or apoptosis signaling pathways in cancer cells.5 Many sensitizing agents have been developed in a variety of tumor cell models.2 Although the clinical effectiveness of these agents needs further investigation, treatment of TRAIL-resistant tumor cells with sensitizing agents, especially the compounds with low molecular weight, as well as prolonged plasma half-life represents a promising trend for cancer therapy.

Mitochondria emerge as intriguing targets for cancer therapy. Metabolic changes affecting mitochondria function inside cancer cells endow these cells with distinctive properties and survival advantage worthy of drug targeting, mitochondria-targeting drugs offer substantial promise as clinical treatment with minimal side effects.141516 Rotenone is a potent inhibitor of NADH oxidoreductase in complex I, which demonstrates anti-neoplastic activity on a variety of cancer cells.1718192021 However, the neurotoxicity of rotenone limits its potential application in cancer therapy. To avoid it, rotenone was effectively used in combination with other chemotherapeutic drugs to kill cancerous cells.22

In our previous investigation, we found that rotenone was able to suppress membrane Na+,K+-ATPase activity and enhance ouabain-induced cancer cell death.23 Given these facts, we wonder whether rotenone may also be used as a sensitizing agent that can restore the susceptibility of NSCLC cells toward TRAIL-induced apoptosis, and increase the antitumor efficacy of TRAIL on NSCLC. To test this hypothesis, we initiated this study.

Rotenone sensitizes NSCLC cell lines to TRAIL-induced apoptosis

Four NSCLC cell lines including A549, H522, H157 and Calu-1 were used in this study. As shown in Figure 1a, the apoptosis induced by TRAIL alone at 50 or 100 ng/ml on A549, H522, H157 and Calu-1 cells was non-prevalent, indicating that these NSCLC cell lines are relatively TRAIL resistant. Interestingly, when these cells were treated with TRAIL combined with rotenone, significant increase in cell apoptosis was observed. To examine whether rotenone was also able to sensitize normal cells to TRAIL-mediated apoptosis, peripheral blood mononuclear cell (PBMC) isolated from human blood were used. As a result, rotenone failed to sensitize human PBMC to TRAIL-induced apoptosis, indicating that the sensitizing effect of rotenone is tumor cell specific. Of note, the apoptosis-enhancing effect of rotenone occurred independent of its cytotoxicity, because the minimal dosage required for rotenone to cause toxic effect on NSCLC cell lines was 10 μM, however, rotenone augmented TRAIL-mediated apoptosis when it was used as little as 10 nM.

Figure 1.

Full figure and legend (310K)

http://www.nature.com/cddis/journal/v5/n12/fig_tab/cddis2014547f1.html#figure-title
To further confirm the effect of rotenone, cells were stained with Hoechst and observed under fluorescent microscope (Figure 1b). Consistently, the combined treatment of rotenone with TRAIL caused significant nuclear fragmentation in A549, H522, H157 and Calu-1 cells. Rotenone or TRAIL treatment alone, however, had no significant effect.

Caspases activation is a hallmark of cell apoptosis. In this study, the enzymatic activities of caspases including caspase-3, -8 and -9 were measured by flow cytometry by using FITC-conjugated caspases substrate (Figure 1c). As a result, rotenone used at 1 μM or TRAIL used at 100 ng/ml alone did not cause caspase-3, -8 and -9 activation. The combined treatment, however, significantly increased the enzymatic activities of them. Moreover, A549 or H522 cell apoptosis by TRAIL combined with rotenone was almost completely suppressed in the presence of z-VAD.fmk, a pan-caspase inhibitor (Figure 1d). All of these data indicate that both intrinsic and extrinsic pathways are involved in the sensitizing effect of rotenone on TRAIL-mediated apoptosis in NSCLC.

Upregulation of death receptors expression is required for rotenone-mediated sensitization to TRAIL-induced apoptosis

Sensitization to TRAIL-induced apoptosis has been explained in some studies by upregulation of death receptors,24 whereas other results show that sensitization can occur without increased TRAIL receptor expression.25 As such, we examined TRAIL receptors expression on NSCLC cells after treatment with rotenone. Rotenone increased DR4 and DR5 mRNA levels in A549 cells in a time or concentration-dependent manner (Figures 2a and b), also increased DR4 and DR5 protein expression levels (Supplementary Figure S1). Notably, rotenone failed to increase DR5 mRNA levels in H157 and Calu-1 cells (Supplementary Figure S2). To observe whether the increased DR4 and DR5 mRNA levels finally correlated with the functional molecules, we examined the surface expression levels of DR4 and DR5 by flow cytometry. The results, as shown in Figure 2c demonstrated that the cell surface expression levels of DR4 and DR5 were greatly upregulated by rotenone in either A549 cells or H522 cells.

Figure 2.

Full figure and legend (173K)

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To analyze whether the upregulation of DR4 and DR5 is a ‘side-effect’, or contrarily, necessary for rotenone-mediated sensitization to TRAIL-induced apoptosis, we blocked upregulation of the death receptors by small interfering RNAs (siRNAs) against DR4 and DR5 (Supplementary Figure S3). The results showed that blocking DR4 and DR5 expression alone significantly reduced the rate of cell apoptosis in A549 cells (Figure 2d). However, the highest inhibition of apoptosis was observed when upregulation of both receptors was blocked in parallel, thus showing an additive effect of blocking DR4 and DR5 at the same time. Similar results were also obtained in H522 cells

To analyze whether the upregulation of DR4 and DR5 is a ‘side-effect’, or contrarily, necessary for rotenone-mediated sensitization to TRAIL-induced apoptosis, we blocked upregulation of the death receptors by small interfering RNAs (siRNAs) against DR4 and DR5 (Supplementary Figure S3). The results showed that blocking DR4 and DR5 expression alone significantly reduced the rate of cell apoptosis in A549 cells (Figure 2d). However, the highest inhibition of apoptosis was observed when upregulation of both receptors was blocked in parallel, thus showing an additive effect of blocking DR4 and DR5 at the same time. Similar results were also obtained in H522 cells.

Rotenone-induced p53 activation regulates death receptors upregulation

TRAIL receptors DR4 and DR5 are regulated at multiple levels. At transcriptional level, studies suggest that several transcriptional factors including NF-κB, p53 and AP-1 are involved in DR4 or DR5 gene transcription.2 The NF-κB or AP-1 transcriptional activity was further modulated by ERK1/2, JNK and p38 MAP kinase activity. Unexpectedly, we found here that none of these MAP kinases inhibitors were able to suppress the apoptosis mediated by TRAIL plus rotenone (Figure 3a). To find out other possible mechanisms, we observed that rotenone was able to stimulate p53 phosphorylation as well as p53 protein expression in A549 and H522 cells (Figure 3b). As a p53-inducible gene, p21 mRNA expression was also upregulated by rotenone treatment in a time-dependent manner (Figure 3c). To characterize the effect of p53, A549 cells were transfected with p53 siRNA. The results, as shown in Figure 3d-1 demonstrated that rotenone-mediated surface expression levels of DR4 and DR5 in A549 cells were largely attenuated by siRNA-mediated p53 expression silencing. Control siRNA, however, failed to reveal such effect. Similar results were also obtained in H522 cells (Figure 3d-2). Silencing of p53 expression in A549 cells also partially suppressed the apoptosis induced by TRAIL plus rotenone (Figure 3e).

http://www.nature.com/cddis/journal/v5/n12/fig_tab/cddis2014547f3.html#figure-title

Rotenone suppresses c-FLIP expression and increases the sensitivity of A549 cells to TRAIL-induced apoptosis

The c-FLIP protein has been commonly appreciated as an anti-apoptotic molecule in death receptor-mediated cell apoptosis. In this study, rotenone treatment led to dose-dependent downregulation of c-FLIP expression, including c-FLIPL and c-FLIPs in A549 cells (Figure 4a-1), H522 cells (Figure 4a-2), H441 and Calu-1 cells (Supplementary Figure S4). To test whether c-FLIP is essential for the apoptosis enhancement, A549 cells were transfected with c-FLIPL-overexpressing plasmids. As shown in Figure 4b-1, the apoptosis of A549 cells after the combined treatment was significantly reduced when c-FLIPL was overexpressed. Similar results were also obtained in H522 cells (Figure 4b-2).

http://www.nature.com/cddis/journal/v5/n12/fig_tab/cddis2014547f4.html#figure-title

Bcl-XL is involved in the apoptosis enhancement by rotenone

Notably, c-FLIP downregulation by rotenone in NSCLC cells was irrelevant to p53 signaling (data not shown). To identify other mechanism involved, we found that anti-apoptotic molecule Bcl-XL was also found to be downregulated by rotenone in a dose-dependent manner (Figure 5a). Notably, both Bcl-XL and c-FLIPL mRNA levels remained unchanged in cells after rotenone treatment (Supplementary Figure S5). Bcl-2 is homolog to Bcl-XL. But surprisingly, Bcl-2 expression was almost undetectable in A549 cells. To examine whether Bcl-XL is involved, A549 cells were transfected with Bcl-XL-overexpressing plasmid. As compared with mock transfectant, cell apoptosis induced by TRAIL plus rotenone was markedly suppressed under the condition of Bcl-XL overexpression (Figure 5b). To characterize the mechanisms, surface expression levels of DR4 and DR5 were examined. As shown in Figure 5c, the increased surface expression of DR4 and DR5 in A549 cells, or in H522 cells were greatly reduced after Bcl-XLoverexpression (Figure 5c). In addition, Bcl-XL overexpression also significantly prevented the downregulation of c-FLIPL and c-FLIPs expression in A549 cells by rotenone treatment (Figure 5d).

http://www.nature.com/cddis/journal/v5/n12/fig_tab/cddis2014547f5.html#figure-title

Rotenone suppresses the interaction between BCL-XL/p53 and increases PUMA transcription

Lines of evidence suggest that Bcl-XL has a strong binding affinity with p53, and can suppress p53-mediated tumor cell apoptosis.26 In this study, FLAG-tagged Bcl-XL and HA-tagged p53 were co-transfected into cells; immunoprecipitation experiment was performed by using FLAG antibody to immunoprecipitate HA-tagged p53. As a result, we found that at the same amount of p53 protein input, rotenone treatment caused a concentration-dependent suppression of the protein interaction between Bcl-XL and p53 (Figure 6a). Rotenone also significantly suppressed the interaction between endogenous Bcl-XL and p53 when polyclonal antibody against p53 was used to immunoprecipitate cellular Bcl-XL (Figure 6b). Recent study highlighted the importance of PUMA in BCL-XL/p53 interaction and cell apoptosis.27 We found here that rotenone significantly increased PUMA gene transcription (Figure 6c) and protein expression (Figure 6d) in NSCLC cells, but not in transformed 293T cell. Meanwhile, this effect was attenuated by silencing of p53 expression (Figure 6e).

http://www.nature.com/cddis/journal/v5/n12/fig_tab/cddis2014547f6.html#figure-title

Mitochondria-derived ROS are responsible for the apoptosis-enhancing effect of rotenone

As an inhibitor of mitochondrial respiration, rotenone was found to induce reactive oxygen species (ROS) generation in a variety of transformed or non-transformed cells.2022 Consistently, by using 2′,7′-dichlorofluorescin diacetate (DCFH) for the measurement of intracellular H2O2 and dihydroethidium (DHE) for O2.−, we found that rotenone significantly triggered the .generation of H2O2(Figure 7a) and O2.− (Figure 7b) in A549 and H522 cells. To identify the origin of ROS production, we first incubated cells with diphenylene iodonium (DPI), a potent inhibitor of plasma membrane NADP/NADPH oxidase. The results showed that DPI failed to suppress rotenone-induced ROS generation (Figure 7c). Then, we generated A549 cells deficient in mitochondria DNA by culturing cells in medium supplemented with ethidium bromide (EB). These mtDNA-deficient cells were subject to rotenone treatment, and the result showed that rotenone-induced ROS production were largely attenuated in A549 ρ° cells, but not wild-type A549 cells, suggesting ROS are mainly produced from mitochondria (Figure 7d). Notably, the sensitizing effect of rotenone on TRAIL-induced apoptosis in A549 cells was largely dependent on ROS, because the antioxidant N-acetylcysteine (NAC) treatment greatly suppressed the cell apoptosis, as shown in annexin V/PI double staining experiment (Figure 7e), cell cycle analysis (Figure 7f) and caspase-3 cleavage activity assay (Figure 7g). Finally, in A549 cells stably transfected with manganese superoxide (MnSOD) and catalase, apoptosis induced by TRAIL and rotenone was partially reversed (Figure 7h). All of these data suggest that mitochondria-derived ROS, including H2O2 and O2.−, are responsible for the apoptosis-enhancing effect of rotenone.

http://www.nature.com/cddis/journal/v5/n12/fig_tab/cddis2014547f7.html#figure-title

Rotenone promotes BCl-XL degradation and PUMA transcription in ROS-dependent manner

To understand why ROS are responsible for the apoptosis-enhancing effect of rotenone, we found that rotenone-induced suppression of BCL-XL expression can be largely reversed by NAC treatment (Figure 8a). To examine whether this effect of rotenone occurs at posttranslational level, we used cycloheximide (CHX) to halt protein synthesis, and found that the rapid degradation of Bcl-XL by rotenone was largely attenuated in A549 ρ0 cells (Figure 8b). Similarly, rotenone-induced PUMA upregulation was also significantly abrogated in A549 ρ0 cells (Figure 8c). Finally, A549 cells were inoculated into nude mice to produce xenografts tumor model. In this model, the therapeutic effect of TRAIL combined with rotenone was evaluated. Notably, in order to circumvent the potential neurotoxic adverse effect of rotenone, mice were challenged with rotenone at a low concentration of 0.5 mg/kg. The results, as shown in Figure 8d revealed that while TRAIL or rotenone alone remained unaffected on A549 tumor growth, the combined therapy significantly slowed down the tumor growth. Interestingly, the tumor-suppressive effect of TRAIL plus rotenone was significantly attenuated by NAC (P<0.01). After experiment, tumors were removed and the caspase-3 activity in tumor cells was analyzed by flow cytometry. Consistently, the caspase-3 cleavage activities were significantly activated in A549 cells from animals challenged with TRAIL plus rotenone, meanwhile, this effect was attenuated by NAC (Figure 8e). The similar effect of rotenone also occurred in NCI-H441 xenografts tumor model (Supplementary Figure S6).

http://www.nature.com/cddis/journal/v5/n12/fig_tab/cddis2014547f8.html#figure-title

Restoration of cancer cells susceptibility to TRAIL-induced apoptosis is becoming a very useful strategy for cancer therapy. In this study, we provided evidence that rotenone increased the apoptosis sensitivity of NSCLC cells toward TRAIL by mechanisms involving ROS generation, p53 upregulation, Bcl-XL and c-FLIP downregulation, and death receptors upregulation. Among them, mitochondria-derived ROS had a predominant role. Although rotenone is toxic to neuron, increasing evidence also demonstrated that it was beneficial for improving inflammation, reducing reperfusion injury, decreasing virus infection or triggering cancer cell death. We identified here another important characteristic of rotenone as a tumor sensitizer in TRAIL-based cancer therapy, which widens the application potential of rotenone in disease therapy.

As Warburg proposed the cancer ‘respiration injury’ theory, increasing evidence suggest that cancer cells may have mitochondrial dysfunction, which causes cancer cells, compared with the normal cells, are under increased generation of ROS.33 The increased ROS in cancer cells have a variety of biological effects. We found here that rotenone preferentially increased the apoptosis sensitivity of cancer cells toward TRAIL, further confirming the concept that although tumor cells have a high level of intracellular ROS, they are more sensitive than normal cells to agents that can cause further accumulation of ROS.

Cancer cells stay in a stressful tumor microenvironment including hypoxia, low nutrient availability and immune infiltrates. These conditions, however, activate a range of stress response pathways to promote tumor survival and aggressiveness. In order to circumvent TRAIL-mediated apoptotic clearance, the expression levels of DR4 and DR5 in many types of cancer cells are nullified, but interestingly, they can be reactivated when cancer cells are challenged with small chemical molecules. Furthermore, those small molecules often take advantage of the stress signaling required for cancer cells survival to increase cancer cells sensitivity toward TRAIL. For example, the unfolded protein response (UPR) has an important role in cancer cells survival, SHetA2, as a small molecule, can induce UPR in NSCLC cell lines and augment TRAIL-induced apoptosis by upregulating DR5 expression in CHOP-dependent manner. Here, we found rotenone manipulated the oxidative stress signaling of NSCLC cells to increase their susceptibility to TRAIL. These facts suggest that cellular stress signaling not only offers opportunity for cancer cells to survive, but also renders cancer cells eligible for attack by small molecules. A possible explanation is that depending on the intensity of stress, cellular stress signaling can switch its role from prosurvival to death enhancement. As described in this study, although ROS generation in cancer cells is beneficial for survival, rotenone treatment further increased ROS production to a high level that surpasses the cell ability to eliminate them; as a result, ROS convert its role from survival to death.

2.1.3.6 PPARs and ERRs. molecular mediators of mitochondrial metabolism

Weiwei Fan, Ronald Evans
Current Opinion in Cell Biology Apr 2015; 33:49–54
http://dx.doi.org/10.1016/j.ceb.2014.11.002

Since the revitalization of ‘the Warburg effect’, there has been great interest in mitochondrial oxidative metabolism, not only from the cancer perspective but also from the general biomedical science field. As the center of oxidative metabolism, mitochondria and their metabolic activity are tightly controlled to meet cellular energy requirements under different physiological conditions. One such mechanism is through the inducible transcriptional co-regulators PGC1α and NCOR1, which respond to various internal or external stimuli to modulate mitochondrial function. However, the activity of such co-regulators depends on their interaction with transcriptional factors that directly bind to and control downstream target genes. The nuclear receptors PPARs and ERRs have been shown to be key transcriptional factors in regulating mitochondrial oxidative metabolism and executing the inducible effects of PGC1α and NCOR1. In this review, we summarize recent gain-of-function and loss-of-function studies of PPARs and ERRs in metabolic tissues and discuss their unique roles in regulating different aspects of mitochondrial oxidative metabolism.

Energy is vital to all living organisms. In humans and other mammals, the vast majority of energy is produced by oxidative metabolism in mitochondria [1]. As a cellular organelle, mitochondria are under tight control of the nucleus. Although the majority of mitochondrial proteins are encoded by nuclear DNA (nDNA) and their expression regulated by the nucleus, mitochondria retain their own genome, mitochondrial DNA (mtDNA), encoding 13 polypeptides of the electron transport chain (ETC) in mammals. However, all proteins required for mtDNA replication, transcription, and translation, as well as factors regulating such activities, are encoded by the nucleus [2].

The cellular demand for energy varies in different cells under different physiological conditions. Accordingly, the quantity and activity of mitochondria are differentially controlled by a transcriptional regulatory network in both the basal and induced states. A number of components of this network have been identified, including members of the nuclear receptor superfamily, the peroxisome proliferator-activated receptors (PPARs) and the estrogen-related receptors (ERRs) [34 and 5].

The Yin-Yang co-regulators

A well-known inducer of mitochondrial oxidative metabolism is the peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) [6], a nuclear cofactor which is abundantly expressed in high energy demand tissues such as heart, skeletal muscle, and brown adipose tissue (BAT) [7]. Induction by cold-exposure, fasting, and exercise allows PGC1α to regulate mitochondrial oxidative metabolism by activating genes involved in the tricarboxylic acid cycle (TCA cycle), beta-oxidation, oxidative phosphorylation (OXPHOS), as well as mitochondrial biogenesis [6 and 8] (Figure 1).

http://ars.els-cdn.com/content/image/1-s2.0-S0955067414001410-gr1.jpg

Figure 1.  PPARs and ERRs are major executors of PGC1α-induced regulation of oxidative metabolism. Physiological stress such as exercise induces both the expression and activity of PGC1α, which stimulates energy production by activating downstream genes involved in fatty acid and glucose metabolism, TCA cycle, β-oxidation, OXPHOS, and mitochondrial biogenesis. The transcriptional activity of PGC1α relies on its interactions with transcriptional factors such as PPARs (for controlling fatty acid metabolism) and ERRs (for regulating mitochondrial OXPHOS).

The effect of PGC1α on mitochondrial regulation is antagonized by transcriptional corepressors such as the nuclear receptor corepressor 1 (NCOR1) [9 and 10]. In contrast to PGC1α, the expression of NCOR1 is suppressed in conditions where PGC1α is induced such as during fasting, high-fat-diet challenge, and exercise [9 and 11]. Moreover, the knockout of NCOR1 phenotypically mimics PGC1α overexpression in regulating mitochondrial oxidative metabolism [9]. Therefore, coactivators and corepressors collectively regulate mitochondrial metabolism in a Yin-Yang fashion.

However, both PGC1α and NCOR1 lack DNA binding activity and rather act via their interaction with transcription factors that direct the regulatory program. Therefore the transcriptional factors that partner with PGC1α and NCOR1 mediate the molecular signaling cascades and execute their inducible effects on mitochondrial regulation.

PPARs: master executors controlling fatty acid oxidation

Both PGC1α and NCOR1 are co-factors for the peroxisome proliferator-activated receptors (PPARα, γ, and δ) [71112 and 13]. It is now clear that all three PPARs play essential roles in lipid and fatty acid metabolism by directly binding to and modulating genes involved in fat metabolism [1314151617,18 and 19]. While PPARγ is known as a master regulator for adipocyte differentiation and does not seem to be involved with oxidative metabolism [14 and 20], both PPARα and PPARδ are essential regulators of fatty acid oxidation (FAO) [3131519 and 21] (Figure 1).

PPARα was first cloned as the molecular target of fibrates, a class of cholesterol-lowering compounds that increase hepatic FAO [22]. The importance of PPARα in regulating FAO is indicated in its expression pattern which is restricted to tissues with high capacity of FAO such as heart, liver, BAT, and oxidative muscle [23]. On the other hand, PPARδ is ubiquitously expressed with higher levels in the digestive tract, heart, and BAT [24]. In the past 15 years, extensive studies using gain-of-function and loss-of-function models have clearly demonstrated PPARα and PPARδ as the major drivers of FAO in a wide variety of tissues.

ERRS: master executors controlling mitochondrial OXPHOS

ERRs are essential regulators of mitochondrial energy metabolism [4]. ERRα is ubiquitously expressed but particularly abundant in tissues with high energy demands such as brain, heart, muscle, and BAT. ERRβ and ERRγ have similar expression patterns, both are selectively expressed in highly oxidative tissues including brain, heart, and oxidative muscle [45]. Instead of endogenous ligands, the transcriptional activity of ERRs is primarily regulated by co-factors such as PGC1α and NCOR1 [4 and 46] (Figure 1).

Of the three ERRs, ERRβ is the least studied and its role in regulating mitochondrial function is unclear [4 and 47]. In contrast, when PGC1α is induced, ERRα is the master regulator of the mitochondrial biogenic gene network. As ERRα binds to its own promoter, PGC1α can also induce an autoregulatory loop to enhance overall ERRα activity [48]. Without ERRα, the ability of PGC1α to induce the expression of mitochondrial genes is severely impaired. However, the basal-state levels of mitochondrial target genes are not affected by ERRα deletion, suggesting induced mitochondrial biogenesis is a transient process and that other transcriptional factors such as ERRγ may be important maintaining baseline mitochondrial OXPHOS [41•42 and 43]. Consistent with this idea, ERRγ (which is active even when PGC1α is not induced) shares many target genes with ERRα [49 and 50].

Conclusion and perspectives

Taken together, recent studies have clearly demonstrated the essential roles of PPARs and ERRs in regulating mitochondrial oxidative metabolism and executing the inducible effects of PGC1α (Figure 1). Both PPARα and PPARδ are key regulators for FA oxidation. While the function of PPARα seems more restricted in FA uptake, beta-oxidation, and ketogenesis, PPARδ plays a broader role in controlling oxidative metabolism and fuel preference, with its target genes involved in FA oxidation, mitochondrial OXPHOS, and glucose utilization. However, it is still not clear how much redundancy exists between PPARα and PPARδ, a question which may require the generation of a double knockout model. In addition, more effort is needed to fully understand how PPARα and PPARδ control their target genes in response to environmental changes.

Likewise, ERRα and ERRγ have been shown to be key regulators of mitochondrial OXPHOS. Knockout studies of ERRα suggest it to be the principal executor of PGC1α induced up-regulation of mitochondrial genes, though its role in exercise-dependent changes in skeletal muscle needs further investigation. Transgenic models have demonstrated ERRγ’s powerful induction of mitochondrial biogenesis and its ability to act in a PGC1α-independent manner. However, it remains to be elucidated whether ERRγ is sufficient for basal-state mitochondrial function in general, and whether ERRα can compensate for its function.

2.1.3.7 Metabolic control via the mitochondrial protein import machinery

Opalińska M, Meisinger C.
Curr Opin Cell Biol. 2015 Apr; 33:42-48
http://dx.doi.org:/10.1016/j.ceb.2014.11.001

Mitochondria have to import most of their proteins in order to fulfill a multitude of metabolic functions. Sophisticated import machineries mediate targeting and translocation of preproteins from the cytosol and subsequent sorting into their suborganellar destination. The mode of action of these machineries has been considered for long time as a static and constitutively active process. However, recent studies revealed that the mitochondrial protein import machinery is subject to intense regulatory mechanisms that include direct control of protein flux by metabolites and metabolic signaling cascades.
2.1.3.8 The Protein Import Machinery of Mitochondria—A Regulatory Hub

AB Harbauer, RP Zahedi, A Sickmann, N Pfanner, C Meisinger
Cell Metab 4 Mar 2014; 19(3):357–372

Mitochondria are essential cell. They are best known for their role as cellular powerhouses, which convert the energy derived from food into an electrochemical proton gradient across the inner membrane. The proton gradient drives the mitochondrial ATP synthase, thus providing large amounts of ATP for the cell. In addition, mitochondria fulfill central functions in the metabolism of amino acids and lipids and the biosynthesis of iron-sulfur clusters and heme. Mitochondria form a dynamic network that is continuously remodeled by fusion and fission. They are involved in the maintenance of cellular ion homeostasis, play a crucial role in apoptosis, and have been implicated in the pathogenesis of numerous diseases, in particular neurodegenerative disorders.

Mitochondria consist of two membranes, outer membrane and inner membrane, and two aqueous compartments, intermembrane space and matrix (Figure 1). Proteomic studies revealed that mitochondria contain more than 1,000 different proteins (Prokisch et al., 2004Reinders et al., 2006Pagliarini et al., 2008 and Schmidt et al., 2010). Based on the endosymbiotic origin from a prokaryotic ancestor, mitochondria contain a complete genetic system and protein synthesis apparatus in the matrix; however, only ∼1% of mitochondrial proteins are encoded by the mitochondrial genome (13 proteins in humans and 8 proteins in yeast). Nuclear genes code for ∼99% of mitochondrial proteins. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated into mitochondria by a multicomponent import machinery. The protein import machinery is essential for the viability of eukaryotic cells. Numerous studies on the targeting signals and import components have been reported (reviewed in Dolezal et al., 2006,Neupert and Herrmann, 2007Endo and Yamano, 2010 and Schmidt et al., 2010), yet for many years little has been known on the regulation of the import machinery. This led to the general assumption that the protein import machinery is constitutively active and not subject to detailed regulation.

Figure 1. Protein Import Pathways of Mitochondria.  Most mitochondrial proteins are synthesized as precursors in the cytosol and are imported by the translocase of the outer mitochondrial membrane (TOM complex). (A) Presequence-carrying (cleavable) preproteins are transferred from TOM to the presequence translocase of the inner membrane (TIM23 complex), which is driven by the membrane potential (Δψ). The proteins either are inserted into the inner membrane (IM) or are translocated into the matrix with the help of the presequence translocase-associated motor (PAM). The presequences are typically cleaved off by the mitochondrial processing peptidase (MPP). (B) The noncleavable precursors of hydrophobic metabolite carriers are bound to molecular chaperones in the cytosol and transferred to the receptor Tom70. After translocation through the TOM channel, the precursors bind to small TIM chaperones in the intermembrane space and are membrane inserted by the Δψ-dependent carrier translocase of the inner membrane (TIM22 complex).
(C) Cysteine-rich proteins destined for the intermembrane space (IMS) are translocated through the TOM channel in a reduced conformation and imported by the mitochondrial IMS import and assembly (MIA) machinery. Mia40 functions as precursor receptor and oxidoreductase in the IMS, promoting the insertion of disulfide bonds into the imported proteins. The sulfhydryl oxidase Erv1 reoxidizes Mia40 for further rounds of oxidative protein import and folding. (D) The precursors of outer membrane β-barrel proteins are imported by the TOM complex and small TIM chaperones and are inserted into the outer membrane by the sorting and assembly machinery (SAM complex). (E) Outer membrane (OM) proteins with α-helical transmembrane segments are inserted into the membrane by import pathways that have only been partially characterized. Shown is an import pathway via the mitochondrial import (MIM) complex

Studies in recent years, however, indicated that different steps of mitochondrial protein import are regulated, suggesting a remarkable diversity of potential mechanisms. After an overview on the mitochondrial protein import machinery, we will discuss the regulatory processes at different stages of protein translocation into mitochondria. We propose that the mitochondrial protein import machinery plays a crucial role as regulatory hub under physiological and pathophysiological conditions. Whereas the basic mechanisms of mitochondrial protein import have been conserved from lower to higher eukaryotes (yeast to humans), regulatory processes may differ between different organisms and cell types. So far, many studies on the regulation of mitochondrial protein import have only been performed in a limited set of organisms. Here we discuss regulatory principles, yet it is important to emphasize that future studies will have to address which regulatory processes have been conserved in evolution and which processes are organism specific.

Protein Import Pathways into Mitochondria

The classical route of protein import into mitochondria is the presequence pathway (Neupert and Herrmann, 2007 and Chacinska et al., 2009). This pathway is used by more than half of all mitochondrial proteins (Vögtle et al., 2009). The proteins are synthesized as precursors with cleavable amino-terminal extensions, termed presequences. The presequences form positively charged amphipathic α helices and are recognized by receptors of the translocase of the outer mitochondrial membrane (TOM complex) (Figure 1A) (Mayer et al., 1995Brix et al., 1997van Wilpe et al., 1999Abe et al., 2000Meisinger et al., 2001 and Saitoh et al., 2007). Upon translocation through the TOM channel, the cleavable preproteins are transferred to the presequence translocase of the inner membrane (TIM23 complex). The membrane potential across the inner membrane (Δψ, negative on the matrix side) exerts an electrophoretic effect on the positively charged presequences (Martin et al., 1991). The presequence translocase-associated motor (PAM) with the ATP-dependent heat-shock protein 70 (mtHsp70) drives preprotein translocation into the matrix (Chacinska et al., 2005 and Mapa et al., 2010). Here the presequences are typically cleaved off by the mitochondrial processing peptidase (MPP). Some cleavable preproteins contain a hydrophobic segment behind the presequence, leading to arrest of translocation in the TIM23 complex and lateral release of the protein into the inner membrane (Glick et al., 1992Chacinska et al., 2005 and Meier et al., 2005). In an alternative sorting route, some cleavable preproteins destined for the inner membrane are fully or partially translocated into the matrix, followed by insertion into the inner membrane by the OXA export machinery, which has been conserved from bacteria to mitochondria (“conservative sorting”) (He and Fox, 1997Hell et al., 1998Meier et al., 2005 and Bohnert et al., 2010).  …

Regulatory Processes Acting at Cytosolic Precursors of Mitochondrial Proteins

Two properties of cytosolic precursor proteins are crucial for import into mitochondria. (1) The targeting signals of the precursors have to be accessible to organellar receptors. Modification of a targeting signal by posttranslational modification or masking of a signal by binding partners can promote or inhibit import into an organelle. (2) The protein import channels of mitochondria are so narrow that folded preproteins cannot be imported. Thus preproteins should be in a loosely folded state or have to be unfolded during the import process. Stable folding of preprotein domains in the cytosol impairs protein import.  …

Import Regulation by Binding of Metabolites or Partner Proteins to Preproteins

Binding of a metabolite to a precursor protein can represent a direct means of import regulation (Figure 2A, condition 1). A characteristic example is the import of 5-aminolevulinate synthase, a mitochondrial matrix protein that catalyzes the first step of heme biosynthesis (Hamza and Dailey, 2012). The precursor contains heme binding motifs in its amino-terminal region, including the presequence (Dailey et al., 2005). Binding of heme to the precursor inhibits its import into mitochondria, likely by impairing recognition of the precursor protein by TOM receptors (Lathrop and Timko, 1993González-Domínguez et al., 2001,Munakata et al., 2004 and Dailey et al., 2005). Thus the biosynthetic pathway is regulated by a feedback inhibition of mitochondrial import of a crucial enzyme, providing an efficient and precursor-specific means of import regulation dependent on the metabolic situation.

Figure 2. Regulation of Cytosolic Precursors of Mitochondrial Proteins

(A) The import of a subset of mitochondrial precursor proteins can be positively or negatively regulated by precursor-specific reactions in the cytosol. (1) Binding of ligands/metabolites can inhibit mitochondrial import. (2) Binding of precursors to partner proteins can stimulate or inhibit import into mitochondria. (3) Phosphorylation of precursors in the vicinity of targeting signals can modulate dual targeting to the endoplasmic reticulum (ER) and mitochondria. (4) Precursor folding can mask the targeting signal. (B) Cytosolic and mitochondrial fumarases are derived from the same presequence-carrying preprotein. The precursor is partially imported by the TOM and TIM23 complexes of the mitochondrial membranes and the presequence is removed by the mitochondrial processing peptidase (MPP). Folding of the preprotein promotes retrograde translocation of more than half of the molecules into the cytosol, whereas the other molecules are completely imported into mitochondria.

Regulation of Mitochondrial Protein Entry Gate by Cytosolic Kinases

Figure 3. Regulation of TOM Complex by Cytosolic Kinases

(A) All subunits of the translocase of the outer mitochondrial membrane (TOM complex) are phosphorylated by cytosolic kinases (phosphorylated amino acid residues are indicated by stars with P). Casein kinase 1 (CK1) stimulates the assembly of Tom22 into the TOM complex. Casein kinase 2 (CK2) stimulates the biogenesis of Tom22 as well as the mitochondrial import protein 1 (Mim1). Protein kinase A (PKA) inhibits the biogenesis of Tom22 and Tom40, and inhibits the activity of Tom70 (see B). Cyclin-dependent kinases (CDK) are possibly involved in regulation of TOM. (B) Metabolic shift-induced regulation of the receptor Tom70 by PKA. Carrier precursors bind to cytosolic chaperones (Hsp70 and/or Hsp90). Tom70 has two binding pockets, one for the precursor and one for the accompanying chaperone (shown on the left). When glucose is added to yeast cells (fermentable conditions), the levels of intracellular cAMP are increased and PKA is activated (shown on the right). PKA phosphorylates a serine of Tom70 in vicinity of the chaperone binding pocket, thus impairing chaperone binding to Tom70 and carrier import into mitochondria.

Casein Kinase 2 Stimulates TOM Biogenesis and Protein Import

Metabolic Switch from Respiratory to Fermentable Conditions Involves Protein Kinase A-Mediated Inhibition of TOM

Network of Stimulatory and Inhibitory Kinases Acts on TOM Receptors, Channel, and Assembly Factors

Protein Import Activity as Sensor of Mitochondrial Stress and Dysfunction

Figure 4. Mitochondrial Quality Control and Stress Response

(A) Import and quality control of cleavable preproteins. The TIM23 complex cooperates with several machineries: the TOM complex, a supercomplex consisting of the respiratory chain complexes III and IV, and the presequence translocase-associated motor (PAM) with the central chaperone mtHsp70. Several proteases/peptidases involved in processing, quality control, and/or degradation of imported proteins are shown, including mitochondrial processing peptidase (MPP), intermediate cleaving peptidase (XPNPEP3/Icp55), mitochondrial intermediate peptidase (MIP/Oct1), mitochondrial rhomboid protease (PARL/Pcp1), and LON/Pim1 protease. (B) The transcription factor ATFS-1 contains dual targeting information, a mitochondrial targeting signal at the amino terminus, and a nuclear localization signal (NLS). In normal cells, ATFS-1 is efficiently imported into mitochondria and degraded by the Lon protease in the matrix. When under stress conditions the protein import activity of mitochondria is reduced (due to lower Δψ, impaired mtHsp70 activity, or peptides exported by the peptide transporter HAF-1), some ATFS-1 molecules accumulate in the cytosol and can be imported into the nucleus, leading to induction of an unfolded protein response (UPRmt).

Regulation of PINK1/Parkin-Induced Mitophagy by the Activity of the Mitochondrial Protein Import Machinery

Figure 5.  Mitochondrial Dynamics and Disease

(A) In healthy cells, the kinase PINK1 is partially imported into mitochondria in a membrane potential (Δψ)-dependent manner and processed by the inner membrane rhomboid protease PARL, which cleaves within the transmembrane segment and generates a destabilizing N terminus, followed by retro-translocation of cleaved PINK1 into the cytosol and degradation by the ubiquitin-proteasome system (different views have been reported if PINK1 is first processed by MPP or not; Greene et al., 2012, Kato et al., 2013 and Yamano and Youle, 2013). Dissipation of Δψ in damaged mitochondria leads to an accumulation of unprocessed PINK1 at the TOM complex and the recruitment of the ubiquitin ligase Parkin to mitochondria. Mitofusin 2 is phosphorylated by PINK1 and likely functions as receptor for Parkin. Parkin mediates ubiquitination of mitochondrial outer membrane proteins (including mitofusins), leading to a degradation of damaged mitochondria by mitophagy. Mutations of PINK1 or Parkin have been observed in monogenic cases of Parkinson’s disease. (B) The inner membrane fusion protein OPA1/Mgm1 is present in long and short isoforms. A balanced formation of the isoforms is a prerequisite for the proper function of OPA1/Mgm1. The precursor of OPA1/Mgm1 is imported by the TOM and TIM23 complexes. A hydrophobic segment of the precursor arrests translocation in the inner membrane, and the amino-terminal targeting signal is cleaved by MPP, generating the long isoforms. In yeast mitochondria, the import motor PAM drives the Mgm1 precursor further toward the matrix such that a second hydrophobic segment is cleaved by the inner membrane rhomboid protease Pcp1, generating the short isoform (s-Mgm1). In mammals, the m-AAA protease is likely responsible for the balanced formation of long (L) and short (S) isoforms of OPA1. A further protease, OMA1, can convert long isoforms into short isoforms in particular under stress conditions, leading to an impairment of mitochondrial fusion and thus to fragmentation of mitochondria.

….

Mitochondrial research is of increasing importance for the molecular understanding of numerous diseases, in particular of neurodegenerative disorders. The well-established connection between the pathogenesis of Parkinson’s disease and mitochondrial protein import has been discussed above. Several observations point to a possible connection of mitochondrial protein import with the pathogenesis of Alzheimer’s disease, though a direct role of mitochondria has not been demonstrated so far. The amyloid-β peptide (Aβ), which is generated from the amyloid precursor protein (APP), was found to be imported into mitochondria by the TOM complex, to impair respiratory activity, and to enhance ROS generation and fragmentation of mitochondria (Hansson Petersen et al., 2008, Ittner and Götz, 2011 and Itoh et al., 2013). An accumulation of APP in the TOM and TIM23 import channels has also been reported (Devi et al., 2006). The molecular mechanisms of how mitochondrial activity and dynamics may be altered by Aβ (and possibly APP) and how mitochondrial alterations may impact on the pathogenesis of Alzheimer’s disease await further analysis.

It is tempting to speculate that regulatory changes in mitochondrial protein import may be involved in tumor development. Cancer cells can shift their metabolism from respiration toward glycolysis (Warburg effect) (Warburg, 1956, Frezza and Gottlieb, 2009, Diaz-Ruiz et al., 2011 and Nunnari and Suomalainen, 2012). A glucose-induced downregulation of import of metabolite carriers into mitochondria may represent one of the possible mechanisms during metabolic shift to glycolysis. Such a mechanism has been shown for the carrier receptor Tom70 in yeast mitochondria (Schmidt et al., 2011). A detailed analysis of regulation of mitochondrial preprotein translocases in healthy mammalian cells as well as in cancer cells will represent an important task for the future.

Conclusion

In summary, the concept of the “mitochondrial protein import machinery as regulatory hub” will promote a rapidly developing field of interdisciplinary research, ranging from studies on molecular mechanisms to the analysis of mitochondrial diseases. In addition to identifying distinct regulatory mechanisms, a major challenge will be to define the interactions between different machineries and regulatory processes, including signaling networks, preprotein translocases, bioenergetic complexes, and machineries regulating mitochondrial membrane dynamics and contact sites, in order to understand the integrative system controlling mitochondrial biogenesis and fitness.

2.1.3.9 Exosome Transfer from Stromal to Breast Cancer Cells Regulates Therapy Resistance Pathways

MC Boelens, Tony J. Wu, Barzin Y. Nabet, et al.
Cell 23 Oct 2014; 159(3): 499–513
http://www.sciencedirect.com/science/article/pii/S0092867414012392

Highlights

  • Exosome transfer from stromal to breast cancer cells instigates antiviral signaling
    • RNA in exosomes activates antiviral STAT1 pathway through RIG-I
    • STAT1 cooperates with NOTCH3 to expand therapy-resistant cells
    • Antiviral/NOTCH3 pathways predict NOTCH activity and resistance in primary tumors

Summary

Stromal communication with cancer cells can influence treatment response. We show that stromal and breast cancer (BrCa) cells utilize paracrine and juxtacrine signaling to drive chemotherapy and radiation resistance. Upon heterotypic interaction, exosomes are transferred from stromal to BrCa cells. RNA within exosomes, which are largely noncoding transcripts and transposable elements, stimulates the pattern recognition receptor RIG-I to activate STAT1-dependent antiviral signaling. In parallel, stromal cells also activate NOTCH3 on BrCa cells. The paracrine antiviral and juxtacrine NOTCH3 pathways converge as STAT1 facilitates transcriptional responses to NOTCH3 and expands therapy-resistant tumor-initiating cells. Primary human and/or mouse BrCa analysis support the role of antiviral/NOTCH3 pathways in NOTCH signaling and stroma-mediated resistance, which is abrogated by combination therapy with gamma secretase inhibitors. Thus, stromal cells orchestrate an intricate crosstalk with BrCa cells by utilizing exosomes to instigate antiviral signaling. This expands BrCa subpopulations adept at resisting therapy and reinitiating tumor growth.

stromal-communication-with-cancer-cells

stromal-communication-with-cancer-cells

Graphical Abstract

2.1.3.10 Emerging concepts in bioenergetics and cancer research

Obre E, Rossignol R
Int J Biochem Cell Biol. 2015 Feb; 59:167-81
http://dx.doi.org:/10.1016/j.biocel.2014.12.008

The field of energy metabolism dramatically progressed in the last decade, owing to a large number of cancer studies, as well as fundamental investigations on related transcriptional networks and cellular interactions with the microenvironment. The concept of metabolic flexibility was clarified in studies showing the ability of cancer cells to remodel the biochemical pathways of energy transduction and linked anabolism in response to glucose, glutamine or oxygen deprivation. A clearer understanding of the large-scale bioenergetic impact of C-MYC, MYCN, KRAS and P53 was obtained, along with its modification during the course of tumor development. The metabolic dialog between different types of cancer cells, but also with the stroma, also complexified the understanding of bioenergetics and raised the concepts of metabolic symbiosis and reverse Warburg effect. Signaling studies revealed the role of respiratory chain-derived reactive oxygen species for metabolic remodeling and metastasis development. The discovery of oxidative tumors in human and mice models related to chemoresistance also changed the prevalent view of dysfunctional mitochondria in cancer cells. Likewise, the influence of energy metabolism-derived oncometabolites emerged as a new means of tumor genetic regulation. The knowledge obtained on the multi-site regulation of energy metabolism in tumors was translated to cancer preclinical studies, supported by genetic proof of concept studies targeting LDHA, HK2, PGAM1, or ACLY. Here, we review those different facets of metabolic remodeling in cancer, from its diversity in physiology and pathology, to the search of the genetic determinants, the microenvironmental regulators and pharmacological modulators.

2.1.3.11 Protecting the mitochondrial powerhouse

M Scheibye-Knudsen, EF Fang, DL Croteau, DM Wilson III, VA Bohr
Trends in Cell Biol, Mar 2015; 25(3):158–170

Highlights

  • Mitochondrial maintenance is essential for cellular and organismal function.
    • Maintenance includes reactive oxygen species (ROS) regulation, DNA repair, fusion–fission, and mitophagy.
    • Loss of function of these pathways leads to disease.

Mitochondria are the oxygen-consuming power plants of cells. They provide a critical milieu for the synthesis of many essential molecules and allow for highly efficient energy production through oxidative phosphorylation. The use of oxygen is, however, a double-edged sword that on the one hand supplies ATP for cellular survival, and on the other leads to the formation of damaging reactive oxygen species (ROS). Different quality control pathways maintain mitochondria function including mitochondrial DNA (mtDNA) replication and repair, fusion–fission dynamics, free radical scavenging, and mitophagy. Further, failure of these pathways may lead to human disease. We review these pathways and propose a strategy towards a treatment for these often untreatable disorders.

Discussion

Radoslav Bozov –

Larry, pyruvate is a direct substrate for synthesizing pyrimidine rings, as well as C-13 NMR study proven source of methyl groups on SAM! Think about what cancer cells care for – dis-regulated growth through ‘escaped’ mutability of proteins, ‘twisting’ pathways of ordered metabolism space-time wise! mtDNA is a back up, evolutionary primitive, however, primary system for pulling strings onto cell cycle events. Oxygen (never observed single molecule) pulls up electron negative light from emerging super rich energy carbon systems. Therefore, ATP is more acting like a neutralizer – resonator of space-energy systems interoperability! You cannot look at a compartment / space independently , as dimension always add 1 towards 3+1.

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Summary of Cell Structure, Anatomic Correlates of Metabolic Function

Author and Curator: Larry H. Bernstein, MD, FCAP  

 

This chapter has been concerned with the subcellular ultrastructure of organelles, and importantly, their function.  There is no waste in the cell structure. The nucleus has the instructions necessary to carry out the cell’s functions.  In the Eukaryotic cell there is significant differentiation so that the cells are regulated for the needs that they uniquely carry out.  When there is disregulation, it leads to remodeling or to cell death.

Here I shall note some highlights of this chapter.

  1. In every aspect of cell function, proteins are involved embedded in the structure, for most efficient functioning.
  2. Metabolic regulation is dependent on pathways that are also linkages of proteins.
  3. Energy utilization is dependent on enzymatic reactions, often involving essential metal ions of high valence numbers, which facilitates covalent and anion binding, and has an essential role in allostericity.

Mitochondria

Mitochondria,_mammalian_lung

Mitochondria,_mammalian_lung

http://en.wikipedia.org/wiki/File:Mitochondria,_mammalian_lung_-_TEM.jpg

Mitochondria range from 0.5 to 1.0 micrometer (μm) in diameter. These structures are sometimes described as “cellular power plants” because they generate most of the cell’s supply of adenosine triphosphate (ATP), used as a source of chemical energy. In addition to supplying cellular energy, mitochondria are involved in other tasks such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. Mitochondria have been implicated in several human diseases, including mitochondrial disorders and cardiac dysfunction.

The number of mitochondria in a cell can vary widely by organism, tissue, and cell type. For instance, red blood cells have no mitochondria, whereas liver cells can have more than 2000. The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. Mitochondrial proteins vary depending on the tissue and the species. The mitochondrial proteome is thought to be dynamically regulated. Although most of a cell’s DNA is contained in the cell nucleus, the mitochondrion has its own independent genome. Further, its DNA shows substantial similarity to bacterial genomes.

In 1913 particles from extracts of guinea-pig liver were linked to respiration by Otto Heinrich Warburg, which he called “grana”. Warburg and Heinrich Otto Wieland, who had also postulated a similar particle mechanism, disagreed on the chemical nature of the respiration. It was not until 1925 when David Keilin discovered cytochromes that the respiratory chain was described.  In 1939, experiments using minced muscle cells demonstrated that one oxygen atom can form two adenosine triphosphate molecules, and, in 1941, the concept of phosphate bonds being a form of energy in cellular metabolism was developed by Fritz Albert Lipmann. In the following years, the mechanism behind cellular respiration was further elaborated, although its link to the mitochondria was not known. The introduction of tissue fractionation by Albert Claude allowed mitochondria to be isolated from other cell fractions and biochemical analysis to be conducted on them alone. In 1946, he concluded that cytochrome oxidase and other enzymes responsible for the respiratory chain were isolated to the mitchondria.

The first high-resolution micrographs appeared in 1952, replacing the Janus Green stains as the preferred way of visualising the mitochondria. This led to a more detailed analysis of the structure of the mitochondria, including confirmation that they were surrounded by a membrane. It also showed a second membrane inside the mitochondria that folded up in ridges dividing up the inner chamber and that the size and shape of the mitochondria varied from cell to cell.  In 1967, it was discovered that mitochondria contained ribosomes. In 1968, methods were developed for mapping the mitochondrial genes, with the genetic and physical map of yeast mitochondria being completed in 1976.

A mitochondrion contains outer and inner membranes composed of phospholipid bilayers and proteins. The two membranes have different properties. Because of this double-membraned organization, there are five distinct parts to a mitochondrion. They are:

  1. the outer mitochondrial membrane,
  2. the intermembrane space (the space between the outer and inner membranes),
  3. the inner mitochondrial membrane,
  4. the cristae space (formed by infoldings of the inner membrane), and
  5. the matrix (space within the inner membrane).

Mitochondria stripped of their outer membrane are called mitoplasts.

Mitochondrion_structure_drawing

Mitochondrion_structure_drawing

http://upload.wikimedia.org/wikipedia/commons/thumb/9/9e/Mitochondrion_structure_drawing.svg/500px-Mitochondrion_structure_drawing.svg.png

Mitochondrion ultrastructure (interactive diagram) A mitochondrion has a double membrane; the inner one contains its chemiosmotic apparatus and has deep grooves which increase its surface area. While commonly depicted as an “orange sausage with a blob inside of it” (like it is here), mitochondria can take many shapes and their intermembrane space is quite thin.

The intermembrane space is the space between the outer membrane and the inner membrane. It is also known as perimitochondrial space. Because the outer membrane is freely permeable to small molecules, the concentrations of small molecules such as ions and sugars in the intermembrane space is the same as the cytosol. However, large proteins must have a specific signaling sequence to be transported across the outer membrane, so the protein composition of this space is different from the protein composition of the cytosol. One protein that is localized to the intermembrane space in this way is cytochrome c.

The inner mitochondrial membrane contains proteins with five types of functions:

  1. Those that perform the redox reactions of oxidative phosphorylation
  2. ATP synthase, which generates ATP in the matrix
  3. Specific transport proteins that regulate metabolite passage into and out of the matrix
  4. Protein import machinery.
  5. Mitochondria fusion and fission protein.

It contains more than 151 different polypeptides, and has a very high protein-to-phospholipid ratio (more than 3:1 by weight, which is about 1 protein for 15 phospholipids). The inner membrane is home to around 1/5 of the total protein in a mitochondrion. In addition, the inner membrane is rich in an unusual phospholipid, cardiolipin. This phospholipid was originally discovered in cow hearts in 1942, and is usually characteristic of mitochondrial and bacterial plasma membranes. Cardiolipin contains four fatty acids rather than two, and may help to make the inner membrane impermeable. Unlike the outer membrane, the inner membrane doesn’t contain porins, and is highly impermeable to all molecules. Almost all ions and molecules require special membrane transporters to enter or exit the matrix. Proteins are ferried into the matrix via the translocase of the inner membrane (TIM) complex or via Oxa1. In addition, there is a membrane potential across the inner membrane, formed by the action of the enzymes of the electron transport chain.

The inner mitochondrial membrane is compartmentalized into numerous cristae, which expand the surface area of the inner mitochondrial membrane, enhancing its ability to produce ATP. For typical liver mitochondria, the area of the inner membrane is about five times as large as the outer membrane. This ratio is variable and mitochondria from cells that have a greater demand for ATP, such as muscle cells, contain even more cristae. These folds are studded with small round bodies known as F1 particles or oxysomes. These are not simple random folds but rather invaginations of the inner membrane, which can affect overall chemiosmotic function. One recent mathematical modeling study has suggested that the optical properties of the cristae in filamentous mitochondria may affect the generation and propagation of light within the tissue.

Mitochondrion

Mitochondrion

http://upload.wikimedia.org/wikipedia/commons/thumb/d/d8/MitochondrionCAM.jpg/250px-MitochondrionCAM.jpg

The matrix is the space enclosed by the inner membrane. It contains about 2/3 of the total protein in a mitochondrion. The matrix is important in thThe MAM is enriched in enzymes involved in lipid biosynthesis, such as phosphatidylserine synthase on the ER face and phosphatidylserine decarboxylase on the mitochondrial face.[28][29] Because mitochondria are dynamic organelles constantly undergoing fission and fusion events, they require a constant and well-regulated supply of phospholipids for membrane integrity.[30][31] But mitochondria are not only a destination for the phospholipids they finish synthesis of; rather, this organelle also plays a role in inter-organelle trafficking of the intermediates and products of phospholipid biosynthetic pathways, ceramide and cholesterol metabolism, and glycosphingolipid anabolisme production of ATP with the aid of the ATP synthase contained in the inner membrane. The matrix contains a highly concentrated mixture of hundreds of enzymes, special mitochondrial ribosomes, tRNA, and several copies of the mitochondrial DNA genome. Of the enzymes, the major functions include oxidation of pyruvate and fatty acids, and the citric acid cycle.

Purified MAM from subcellular fractionation has shown to be enriched in enzymes involved in phospholipid exchange, in addition to channels associated with Ca2+ signaling. The mitochondria-associated ER membrane (MAM) is another structural element that is increasingly recognized for its critical role in cellular physiology and homeostasis. Once considered a technical snag in cell fractionation techniques, the alleged ER vesicle contaminants that invariably appeared in the mitochondrial fraction have been re-identified as membranous structures derived from the MAM—the interface between mitochondria and the ER. Physical coupling between these two organelles had previously been observed in electron micrographs and has more recently been probed with fluorescence microscopy. Such studies estimate that at the MAM, which may comprise up to 20% of the mitochondrial outer membrane, the ER and mitochondria are separated by a mere 10–25 nm and held together by protein tethering complexes.

Such trafficking capacity depends on the MAM, which has been shown to facilitate transfer of lipid intermediates between organelles. In contrast to the standard vesicular mechanism of lipid transfer, evidence indicates that the physical proximity of the ER and mitochondrial membranes at the MAM allows for lipid flipping between opposed bilayers. Despite this unusual and seemingly energetically unfavorable mechanism, such transport does not require ATP. Instead, in yeast, it has been shown to be dependent on a multiprotein tethering structure termed the ER-mitochondria encounter structure, or ERMES, although it remains unclear whether this structure directly mediates lipid transfer or is required to keep the membranes in sufficiently close proximity to lower the energy barrier for lipid flipping.

A critical role for the ER in calcium signaling was acknowledged before such a role for the mitochondria was widely accepted, in part because the low affinity of Ca2+ channels localized to the outer mitochondrial membrane seemed to fly in the face of this organelle’s purported responsiveness to changes in intracellular Ca2+ flux. But the presence of the MAM resolves this apparent contradiction: the close physical association between the two organelles results in Ca2+ microdomains at contact points that facilitate efficient Ca2+ transmission from the ER to the mitochondria. Transmission occurs in response to so-called “Ca2+ puffs” generated by spontaneous clustering and activation of IP3R, a canonical ER membrane Ca2+ channel.

The properties of the Ca2+ pump SERCA and the channel IP3R present on the ER membrane facilitate feedback regulation coordinated by MAM function. In particular, clearance of Ca2+ by the MAM allows for spatio-temporal patterning of Ca2+ signaling because Ca2+ alters IP3R activity in a biphasic manner. SERCA is likewise affected by mitochondrial feedback: uptake of Ca2+ by the MAM stimulates ATP production, thus providing energy that enables SERCA to reload the ER with Ca2+ for continued Ca2+ efflux at the MAM. Thus, the MAM is not a passive buffer for Ca2+ puffs; rather it helps modulate further Ca2+ signaling through feedback loops that affect ER dynamics.

Regulating ER release of Ca2+ at the MAM is especially critical because only a certain window of Ca2+ uptake sustains the mitochondria, and consequently the cell, at homeostasis. Sufficient intraorganelle Ca2+ signaling is required to stimulate metabolism by activating dehydrogenase enzymes critical to flux through the citric acid cycle. However, once Ca2+ signaling in the mitochondria passes a certain threshold, it stimulates the intrinsic pathway of apoptosis in part by collapsing the mitochondrial membrane potential required for metabolism.  Studies examining the role of pro- and anti-apoptotic factors support this model; for example, the anti-apoptotic factor Bcl-2 has been shown to interact with IP3Rs to reduce Ca2+ filling of the ER, leading to reduced efflux at the MAM and preventing collapse of the mitochondrial membrane potential post-apoptotic stimuli. Given the need for such fine regulation of Ca2+ signaling, it is perhaps unsurprising that dysregulated mitochondrial Ca2+ has been implicated in several neurodegenerative diseases, while the catalogue of tumor suppressors includes a few that are enriched at the MAM.

…more

http://en.wikipedia.org/wiki/Mitochondrion

Lysosome and Apoptosis

Role of autophagy in cancer

R Mathew, V Karantza-Wadsworth & E White

Nature Reviews Cancer 7, 961-967 (Dec 2007) |  http://dx.doi.org:/10.1038/nrc2254

Autophagy is a cellular degradation pathway for the clearance of damaged or superfluous proteins and organelles. The recycling of these intracellular constituents also serves as an alternative energy source during periods of metabolic stress to maintain homeostasis and viability. In tumour cells with defects in apoptosis, autophagy allows prolonged survival. Paradoxically, autophagy defects are associated with increased tumorigenesis, but the mechanism behind this has not been determined. Recent evidence suggests that autophagy provides a protective function to limit tumour necrosis and inflammation, and to mitigate genome damage in tumour cells in response to metabolic stress.

Sustained Activation of mTORC1 in Skeletal Muscle Inhibits Constitutive and Starvation-Induced Autophagy and Causes a Severe, Late-Onset Myopathy

P Castets, S Lin, N Rion, S Di Fulvio, et al.
cell-metabolism 7 May, 2013; 17(5): p731–744   http://dx.doi.org/10.1016/j.cmet.2013.03.015

  • mTORC1 inhibition is required for constitutive and starvation-induced autophagy
  • Sustained activation of mTORC1 causes a severe myopathy due to autophagy impairment
  • TSC1 depletion is sufficient to activate mTORC1 irrespective of other stimuli
  • mTORC1 inactivation is sufficient to trigger LC3 lipidation

Autophagy is a catabolic process that ensures homeostatic cell clearance and is deregulated in a growing number of myopathological conditions. Although FoxO3 was shown to promote the expression of autophagy-related genes in skeletal muscle, the mechanisms triggering autophagy are unclear. We show that TSC1-deficient mice (TSCmKO), characterized by sustained activation of mTORC1, develop a late-onset myopathy related to impaired autophagy. In young TSCmKO mice,

  • constitutive and starvation-induced autophagy is blocked at the induction steps via
  • mTORC1-mediated inhibition of Ulk1, despite FoxO3 activation.

Rapamycin is sufficient to restore autophagy in TSCmKO mice and

  • improves the muscle phenotype of old mutant mice.

Inversely, abrogation of mTORC1 signaling by

  • depletion of raptor induces autophagy regardless of FoxO inhibition.

Thus, mTORC1 is the dominant regulator of autophagy induction in skeletal muscle and

  • ensures a tight coordination of metabolic pathways.

These findings may open interesting avenues for therapeutic strategies directed toward autophagy-related muscle diseases.

Histone deacetylases 1 and 2 regulate autophagy flux and skeletal muscle homeostasis in mice

Viviana Moresi, et al.   PNAS Jan 31, 2012; 109(5): 1649-1654
http://dx.doi.org:/10.1073/pnas.1121159109
http://www.pnas.org/content/109/5/1649/F6.medium.gif

HDAC1 activates FoxO and is both sufficient and required for skeletal muscle atrophy

Beharry, PB. Sandesara, BM. Roberts, et al.
J. Cell Sci. Apr 2014 127 (7) 1441-1453   http://dx.doi.org:/10.1242/​jcs.136390

The Forkhead box O (FoxO) transcription factors are activated, and necessary for the muscle atrophy, in several pathophysiological conditions, including muscle disuse and cancer cachexia. However, the mechanisms that lead to FoxO activation are not well defined. Recent data from our laboratory and others indicate that

  • the activity of FoxO is repressed under basal conditions via reversible lysine acetylation,
  • which becomes compromised during catabolic conditions.

Therefore, we aimed to determine how histone deacetylase (HDAC) proteins contribute to

  • activation of FoxO and induction of the muscle atrophy program.

Through the use of various pharmacological inhibitors to block HDAC activity, we demonstrate that

  • class I HDACs are key regulators of FoxO and the muscle-atrophy program
  • during both nutrient deprivation and skeletal muscle disuse.

Furthermore, we demonstrate, through the use of wild-type and dominant-negative HDAC1 expression plasmids,

  • that HDAC1 is sufficient to activate FoxO and induce muscle fiber atrophy in vivo and
  • is necessary for the atrophy of muscle fibers that is associated with muscle disuse.

The ability of HDAC1 to cause muscle atrophy required its deacetylase activity and

  • was linked to the induction of several atrophy genes by HDAC1,
  • including atrogin-1, which required deacetylation of FoxO3a.

Moreover, pharmacological inhibition of class I HDACs during muscle disuse, using MS-275,

  • significantly attenuated both disuse muscle fiber atrophy and contractile dysfunction.

Together, these data solidify the importance of class I HDACs in the muscle atrophy program and

  • indicate that class I HDAC inhibitors are feasible countermeasures to impede muscle atrophy and weakness.

Autophagy and thyroid carcinogenesis: genetic and epigenetic links
F Morani, R Titone, L Pagano, et al.  Endocr Relat Cancer Feb 1, 2014 21 R13-R29
http://dx.doi.org:/10.1530/ERC-13-0271

Autophagy is a vesicular process for the lysosomal degradation of protein aggregates and

  • of damaged or redundant organelles.

Autophagy plays an important role in cell homeostasis, and there is evidence that

  • this process is dysregulated in cancer cells.

Recent in vitro preclinical studies have indicated that autophagy is

  • involved in the cytotoxic response to chemotherapeutics in thyroid cancer cells.

Indeed, several oncogenes and oncosuppressor genes implicated in thyroid carcinogenesis

  • also play a role in the regulation of autophagy.

In addition, some epigenetic modulators involved in thyroid carcinogenesis also influence autophagy. In this review, we highlight the genetic and epigenetic factors that

  • mechanistically link thyroid carcinogenesis and autophagy, thus substantiating the rationale for
  • an autophagy-targeted therapy of aggressive and radio-chemo-resistant thyroid cancers.

Read Full Post »


Somatic, germ-cell, and whole sequence DNA in cell lineage and disease profiling

Curator: Larry H Bernstein, MD, FCAP

In humans, mitochondrial DNA spans about 16,500 DNA building blocks (base pairs), representing a small fraction of the total DNA in cells. Mitochondrial DNA contains 37 genes, essential for normal mitochondrial function and thirteen of them provide instructions for making enzymes involved in inner membrane function. The remaining 24 genes are transcribed into transfer RNA (tRNA) and ribosomal RNA (rRNA), which are needed to transfer amino acids into proteins.

Somatic mutations occur in the DNA of certain cells during a person’s lifetime and typically are not passed to future generations.  They differ from germ-line mutations that have a lineal descent from the maternal parent, and they occur later in life.  Mutations in the sperm DNA are not carried on to future generations, as the sperm mitochondria are destroyed after the egg is fertilized.

There is limited evidence linking somatic mutations in mitochondrial DNA with certain cancers, including breast, colon, stomach, liver, and kidney tumors. These mutations might also be associated with cancer of blood-forming tissue (leukemia) and cancer of immune system cells (lymphoma).  There are many heritable diseases that are related to germ-line mutations, and germ-line mutations have a role in many common diseases.  Mitochondrial DNA is particularly vulnerable to the effects of reactive oxygen species (ROS), and with a limited ability of the mitochondrion to repair itself, ROS easily damage mitochondrial DNA.  The repair mechanism is tied to ubiquitinylation system.  A  list of disorders associated with mitochondrial genes  is provided from Wikipedia.

Inherited changes in mitochondrial DNA may be associated with pathologies in growth and development, and multiorgan system disorders, as mutations disrupt the mitochondria’s ability to generate the cell’s energy. The effects of these conditions are most pronounced in organs and tissues with high energy requirements (such as the heart, brain, and muscles). Although the health consequences of inherited mitochondrial DNA mutations vary widely, some frequently observed features include muscle weakness and wasting, problems with movement, diabetes, kidney failure, heart disease, loss of intellectual functions (dementia), hearing loss, and abnormalities involving the eyes and vision.

A buildup of somatic mutations in mitochondrial DNA has been considered to have a role in or associated with increased risk of certain age-related disorders such as heart disease, Alzheimer disease, and Parkinson disease, and the severity of many mitochondrial disorders is thought to be associated with the percentage of mitochondria affected by a particular genetic change. Consequently, the progressive accumulation of these mutations over a person’s lifetime may play a role in aging.

Mitochondrial DNA is typically diagrammed as a circular structure with genes and regulatory regions labeled.

Mitochondrial DNA

Mitochondrial DNA

http://ghr.nlm.nih.gov/html/images/chromosomeIdeograms/mitochondria/wholeMitochondria.jpg

Additional Resources:

  • Additional NIH Resources – National Institutes of Health

NHGRI Talking Glossary: Mitochondrial DNA

mtDNA : The Eve Gene –  by Stephen Oppenheimer

Mutations are a cumulative dossier of our own maternal prehistory. The main task of DNA is to copy itself to each new generation. We can use these mutations to reconstruct a genetic tree of mtDNA, because each new mtDNA mutation in a prospective mother’s ovum will be transferred in perpetuity to all her descendants down the female line. Each new female line is thus defined by the old mutations as well as the new ones.

By looking at the DNA code in a sample of people alive today, and piecing together the changes in the code that have arisen down the generations, biologists can trace the line of descent back in time to a distant shared ancestor. Because we inherit mtDNA only from our mother, this line of descent is a picture of the female genealogy of the human species.

formation of gene trees

formation of gene trees

The diagram above shows the drawing of gene trees using single mutations

http://www.bradshawfoundation.com/journey/images/gene-diagram3.gif

Not only can we retrace the tree, but by taking into account here the sampled people came from, we can see where certain mutations occurred – for example, whether in Europe, or Asia, or Africa. What’s more, because the changes happen at a statistically consistent (though random) rate, we can approximate the time when they happened.  This has made it possible, during the late 1990s and in the new century, for us to do something that anthropologists of the past could only have dreamt of: we can now trace the migrations of modern humans around our planet.

It turns out that the oldest changes in our mtDNA took place in Africa 150,000 – 190,000 years ago. Then new mutations start to appear in Asia, about 60,000 – 80,000 years ago. This tells us that modern humans evolved in Africa, and that some of us migrated out of Africa into Asia after 80,000 years ago.  A method established in 1996, which dates each branch of the gene tree by averaging the number of new mutations in daughter types of that branch, has stood the test of time.

A final point on the methods of genetic tracking of migrations: it is important to distinguish this new approach to tracing the history of molecules on a DNA tree, known as phylogeography (literally ‘tree-geography’), from the mathematical study of the history of whole human populations, which has been used for decades and is known as classical population genetics.

The two disciplines are based on the same Mendelian biological principles, but have quite different aims and assumptions, and the difference is the source of much misunderstanding and controversy. The simplest way of explaining it is that phylogeography studies the prehistory of individual DNA molecules, while population genetics studies the prehistory of populations. Put another way, each human population contains multiple versions of any particular DNA molecule, each with its own history and different origin.

gene-diagram

gene-diagram

The diagram above shows the tracing of gene spread geographically.
Green disks represent migrant new growth on the tree
http://www.bradshawfoundation.com/journey/images/gene-diagram4.gif

http://www.bradshawfoundation.com/journey/eve.html

David Moskowitz, MD, PhD
Founder and President, GenoMed

 

Germline genes make the best drug targets

  • They operate earliest in the disease pathway
  • Unlike tissue-expressed genes, which operate years after the disease began
  • But which everybody else is using as drug targets

Variation in germline DNA is where all disease starts

  • Cancer patients overexpress oncogenes and underexpress tumor suppressors

beginning in their germline DNA

  • Mutations in tumor DNA are “private”
  • Each tumor is a “snowflake”

Tumor-expressed genes can be compensatory, not causative

  • “Passengers, not drivers”
  • We have the drivers

Tumorigenesis SNPs

Using a SNPnet™ covering only 1/3 of the genome, we found about

2,500 genes associated with each of 6 different cancers in whites

  • Nobody else has found any yet
  • This will change in 2-3 years

We estimate 10,000 genes per cancer

What cellular program takes up 1/3-1/2 of the genome?

What program takes up >1/3 of the genome?

  • Differentiation…

Does sporadic cancer arise when a tissue stem cell fails to differentiate?

  • In the embryo, the surrounding tissue expresses “fields”

Lent C. Johnson published a “field” based hypothesis of bone tumors that coincides with differentiation at the

  1. METAPHYSIS
  2. HYPOPHYSIS

and the type CELL – chondroblast, osteoblast, giant cell (osteoclast), fibroblast

Orthopedic surgeons use magnetic fields for healing

  • of powerful transcription factors.
  • Not so in adult life: a proliferating tissue stem cell is literally on its own.

Germlines hold the key to effective “differentiation therapy”

  • Ideal for patients with stage 3-4 cancer
  • Examples of differentiation therapy:
  1. 1,25-vitamin D and
  2. retinoic acid

Non-toxic but more effective treatment for late stage disease,

GenoMed’s 2,500 cancer-causing genes:

  • ½ are oncogenes,
  • ½ are tumor suppressors

Design inhibitors to oncogenes

  • Screen 1st for toxicity;
  • genomic epidemiology guarantees clinical efficacy

 

Jewish Heritage Written in DNA

By Kate Yandell | Sept 9, 2014

Fully sequenced genomes of more than 100 Ashkenazi people clarify the group’s history and provide a reference for researchers and physicians trying to pinpoint disease-associated genes.

A whole-genome sequence study from 128 healthy Jewish people is aimed at identifying disease-associated variants in the jewish population of Ashkenazi ancestry, according to a study published Sept 9 in Nature Communications. The library of sequences confirms earlier conclusions about Ashkenazi history hinted at by more limited DNA sequencing studies. The sequences point to an approximate 350-person bottleneck in the Ashkenazi population as recently as 700 years ago (1400 A.D.), and suggest that the population has a mixture of European and Middle Eastern ancestry.

The study “provides a very nice reference panel for the very unique population of Ashkenazi Jews,” said Alon Keinan, who studies human population genomics at Cornell University in New York. Keinan
is acknowledged in the study but was not involved in the research.

“One might have thought that, after many years of genetic studies relating to Ashkenazi Jews . . . there would be little room for additional insights,” Karl Skorecki of the Rambam Healthcare Campus
in Israel who also was not involved in the study wrote in an e-mail to The Scientist. The study, he added, provides “a powerful further validation and further resolution of the demographic history of
the Ashkenazi Jews in relation to non-Jewish Europeans that is reassuringly consistent with inferences drawn from two decades of studies using uniparental regions . . . and from array-based data.”

Itsik Pe’er, coauthor of the new study and an associate professor of computer science at Columbia University in New York City, recalled that several years ago, he and his colleagues kept running into the same problem as they tried to understand the genetics of disease in Ashkenazi populations. They were comparing their Ashkenazi samples to the only control genomes that were available, which were of largely non-Jewish European origin. The Ashkenazi genomes had variation that was absent in these general European genomes, making it hard to distinguish rare variants in Ashkenazi people.

“Technology is there to tell us everything in that [Ashkenazi] patient’s genome, but the genome was not there to distinguish the variants that are there and to tell us whether they are normal or whether we should get worried,” said Pe’er. Pe’er’s group teamed up with researchers from additional universities and hospitals in the U.S., Belgium, and Israel to sequence a collection of healthy Ashkenazi people’s genomes. The panel of reference sequences performs better than a group of European genomes at filtering out harmless variants from Ashkenazi Jewish genomes, thereby making it easier to identify potentially harmful ones. According to Pe’er, researchers will also be able to use the panel to infer
more complete sequences from partially sequenced genomes by looking for familiar sequences from the reference genomes.

The team also used its data to better understand the history of the Ashkenazi Jewish people through analyzing both level of similarity within Ashkenazi genomes and between Ashkenazi and non-Jewish
European genomes. By analyzing the length of identical DNA sequences that Ashkenazi individuals share, the researchers were able to estimate that 25 to 32 generations ago, the Ashkenazi Jewish population shrunk to just several hundred people, before expanding rapidly to eventually include the millions of Ashkenazi Jews alive today. Further, the researchers concluded that modern Ashkenazi Jews likely have an approximately even mixture of European and Middle Eastern ancestry. This suggests that after the Jewish people migrated from the Middle East to Europe, they recruited people from local European populations.

These results are compatible with those of prior work on mitochondrial DNA (mtDNA), which is passed on maternally. This prior work suggested that Ashkenazi men from the Middle East intermarried with local European women. The Ashkenazi population “hasn’t been likely as isolated as at least some researchers considered,” said Keinan.

Finally, the newly sequenced genomes shed light on the deeper history of Europe, showing that the European and Middle Eastern portions of Ashkenazi ancestry diverged just around 20,000 years ago.

“This is, I think, the first evidence from whole human genomes that the most important wave of settlement from the Near East was most likely shortly after the Last Glacial Maximum  . . . and, notably, before the Neolithic transitionwhich is what researchers working on mitochondrial DNA have been arguing for some years,” Martin Richards, an archeogeneticist at the University of Huddersfield in the U.K., told The Scientist in an e-mail.

Skorecki noted that the new study “demonstrates the utility of sequencing whole genomes in a diverse population… with sufficient numbers of samples, parent population information, and
computational analytic power, we can expect important and surprising utilities for personal genomic and insights in terms of human demographic history from whole genomes.”

  1. Carmi et al., “Sequencing an Ashkenazi reference panel supports population-targeted personal genomics and illuminates Jewish and European origins,” Nature
    Communications,
    http://dx.doi.org:/10.1038/ncomms5835, 2014.

Added Layers of Proteome Complexity

By Anna Azvolinsky | July 17, 2014

Scientists discover a broad spectrum of alternatively spliced human protein variants within a well-studied family of genes.

There may be more to the human proteome than previously thought. Some genes are known to have several different alternatively spliced protein variants, but the Scripps Research Institute’s Paul Schimmel and his colleagues have now uncovered almost 250 protein splice variants of an essential, evolutionarily conserved family of human genes. The results were published today (July 17) in Science.

Focusing on the 20-gene family of aminoacyl tRNA synthetases (AARSs), the team captured AARS transcripts from human tissues—some fetal, some adult—and showed that many of these messenger RNAs (mRNAs) were translated into proteins. Previous studies have identified
several splice variants of these enzymes that have novel functions, but uncovering so many more variants was unexpected, Schimmel said. Most of these new protein products lack the catalytic domain but retain other AARS non-catalytic functional domains. “The main point is that a vast new area of biology, previously missed, has been uncovered,”
said Schimmel.

“This is an incredible study that fundamentally changes how we look at the protein-synthesis machinery,” Michael Ibba, a protein translation researcher at Ohio State University who was not involved in the work, told The Scientist in an e-mail. “The unexpected and potentially vast
expanded functional networks that emerge from this study have the potential to influence virtually any aspect of cell growth.”

The team—including researchers at the Hong Kong University of Science and Technology, Stanford University, and aTyr Pharma, a San Diego-based biotech company that Schimmel co-founded—comprehensively captured and sequenced the AARS mRNAs from six human tissue types using high-throughput deep sequencing. While many of the transcripts were expressed in each of the tissues, there was also some tissue specificity.

Next, the team showed that a proportion of these transcripts, including those missing the catalytic domain, indeed resulted in stable protein products: 48 of these splice variants associated with polysomes. In vitro translation assays and the expression of more than 100 of these variants in cells confirmed that many of these variants could be made into
stable protein products.

The AARS enzymes—of which there’s one for each of the 20 amino acids—bring together an amino acid with its appropriate transfer RNA (tRNA) molecule. This reaction allows a ribosome to add the amino acid to a growing peptide chain during protein translation. AARS
enzymes can be found in all living organisms and are thought to be among the first proteins to have originated on Earth.

To understand whether these non-catalytic proteins had unique biological activities, the researchers expressed and purified recombinant AARS fragments, testing them in cell-based assays for proliferation, cell differentiation, and transcriptional regulation, among other
phenotypes. “We screened through dozens of biological assays and found that these variants operate in many signaling pathways,” said Schimmel.

“This is an interesting finding and fits into the existing paradigm that, in many cases, a single gene is processed in various ways [in the cell] to have alternative functions,” said Steven Brenner, a computational genomics researcher at the University of California, Berkeley.

The team is now investigating the potentially unique roles of these protein splice variants in greater detail—in both human tissue as well as in model organisms. For example, it is not yet clear whether any of these variants directly bind tRNAs.

“I do think [these proteins] will play some biological roles,” said Tao Pan, who studies the functional roles of tRNAs at the University of Chicago. “I am very optimistic that interesting biological functions will come out of future studies on these variants.”

Brenner agreed. “There could be very different biological roles [for some of these proteins]. Biology is very creative that way, [it’s] able to generate highly diverse new functions using combinations of existing protein domains.” However, the low abundance of these variants
is likely to constrain their potential cellular functions, he noted.

Because AARSs are among the oldest proteins, these ancient enzymes were likely subject to plenty of change over time, said Karin Musier-Forsyth, who studies protein translational
at the Ohio State University. According to Musier-Forsyth, synthetases are already known to have non-translational functions and differential localizations. “Like the addition of post-translational modifications, splicing variation has evolved as another way to repurpose protein function,” she said.

One of the protein variants was able to stimulate skeletal muscle fiber formation ex vivo and upregulate genes involved in muscle cell differentiation and metabolism in primary human skeletal myoblasts. “This was really striking,” said Musier-Forsyth. “This suggests
that, perhaps, peptides derived from these splice variants could be used as protein-based therapeutics for a variety of diseases.”

W.S. Lo et al., “Human tRNA synthetase catalytic nulls with diverse functions,” Science, http://dx.doi.org:/10.1126/science.1252943, 2014.

It’s Not Only in DNA’s Hands

By Ilene Schneider  LabRoots   Aug 22, 2014

Blood stem cells have the potential to turn into any type of blood cell, whether it is the oxygen-carrying red blood cells or the immune system’s many types of white blood cells that help fight infection. How exactly is the fate of these stem cells regulated? Preliminary findings from research conducted by scientists from the Weizmann Institute of Science and the Hebrew University are starting to reshape the conventional understanding of the way blood stem cell fate decisions are controlled, thanks to a new technique for epigenetic analysis developed at these institutions. Understanding epigenetic mechanisms (environmental influences other than genetics) of cell fate could lead to the deciphering of the molecular mechanisms of many diseases,
including immunological disorders, anemia, leukemia, and many more. The study of epigenetics also lends strong support to findings that environmental factors and lifestyle play a more prominent
role in shaping our destiny than previously realized.

 

The process of differentiation – in which a stem cell becomes a specialized mature cell – is controlled by a cascade of events in which specific genes are turned “on” and “off” in a highly regulated and accurate order. The instructions for this process are contained within the DNA itself in short regulatory sequences.

  • These regulatory regions are normally in a “closed” state, masked by special proteins called histones to ensure against unwarranted activation. Therefore, to access and “activate”
    the instructions,
  • this DNA mask needs to be “opened” by epigenetic modifications of the histones so it can be read by the necessary machinery.

In a paper published in Science, Dr. Ido Amit and David Lara-Astiaso of the Weizmann Institute’s Department of Immunology, along with Prof. Nir Friedman and Assaf Weiner of the Hebrew University of Jerusalem, charted – for the first time – histone dynamics during blood development. Thanks to the new technique for epigenetic profiling they developed, in which just a handful of cells – as few as 500 – can be sampled and analyzed accurately, they have identified the exact
DNA sequences, as well as the various regulatory proteins, that are involved in regulating the process of blood stem cell fate.

This research has also yielded unexpected results: As many as

  • 50% of these regulatory sequences are established and opened during intermediate stages of cell development.

The meaning of the research is that epigenetics can be active at stages in which it had been thought that cell destiny was already set. “This changes our whole understanding of the process of blood stem cell fate decisions,” says Lara-Astiaso, “suggesting that the process is more
dynamic and flexible than previously thought.”

Although this research was conducted on mouse blood stem cells, the scientists believe that the mechanism may hold true for other types of cells. “This research creates a lot of excitement in the field, as it sets the groundwork to study these regulatory elements in humans,” says Weiner.

Largest Cancer Genetic Analysis Reveals New Way of Classifying Cancer

http://www.biosciencetechnology.com/news/2014/08/largest-cancer-genetic-analysis-reveals-new-way-classifying-cancer

Thu, 08/07/2014 – 2:24pm

Researchers with The Cancer Genome Atlas (TCGA) Research Network have completed the largest, most diverse tumor genetic analysis ever conducted, revealing a new approach to classifying cancers. The work, led by researchers at the UNC Lineberger Comprehensive
Cancer Center at the University of North Carolina at Chapel Hill and other TCGA sites, not only

  • revamps traditional ideas of how cancers are diagnosed and treated, but could also have
  • a profound impact on the future landscape of drug development.

“We found that one in 10 cancers analyzed in this study would be classified differently using this new approach,” said Chuck Perou, PhD, professor of genetics and pathology, UNC Lineberger member and senior author of the paper, which appears online Aug. 7 in Cell.
“That means that

  • 10 percent of the patients might be better off getting a different therapy—that’s huge.”

Since 2006, much of the research has identified cancer as not a single disease, but many types and subtypes and has defined these disease types based on the tissue—breast, lung, colon, etc.—in which it originated. In this scenario, treatments were tailored to which
tissue was affected, but questions have always existed because some treatments work, and fail for others, even when a single tissue type is tested.

In their work, TCGA researchers analyzed more than 3,500 tumors across 12 different tissue types to see how they compared to one another — the largest data set of tumor genomics ever assembled, explained Katherine Hoadley, PhD, research assistant professor
in genetics and lead author. They found that

  • cancers are more likely to be genetically similar based on the type of cell in which the cancer originated, compared to the type of tissue in which it originated. 

This is fundamental premise of pathology! (Larry Bernstein)  It goes back to Rudolph Virchow. 

“In some cases, the cells in the tissue from which the tumor originates are the same,” said Hoadley. “But in other cases, the tissue in which the cancer originates is made up of multiple types of cells that can each give rise to tumors. Understanding the cell in which the cancer originates appears to be very important in determining the subtype of a tumor
and, in turn, how that tumor behaves and how it should be treated.”

Perou and Hoadley explain that the new approach may also shift how cancer drugs are developed, focusing more on the development of drugs targeting larger groups of cancers with genomic similarities, as opposed to a single tumor type as they are currently developed.

One striking example of the genetic differences within a single tissue type is breast cancer.
The breast, a highly complex organ with multiple types of cells, gives rise to multiple types of breast cancer; luminal A, luminal B, HER2-enriched and basal-like, which was previously known. In this analysis, the basal-like breast cancers looked more like ovarian cancer
and cancers of a squamous-cell type origin, a type of cell that composes the lower-layer of a tissue, rather than other cancers that arise in the breast.

“This latest research further solidifies that basal-like breast cancer is an entirely unique disease and is completely distinct from other types of breast cancer,” said Perou. In addition, bladder cancers were also quite diverse and might represent at least three different disease types that also showed differences in patient survival.

As part of the Alliance for Clinical Trials in Oncology, a national network of researchers conducting clinical trials, UNC researchers are already testing the effectiveness of carboplatin—a common treatment for ovarian cancer—on top of standard of care chemotherapy for triple-negative breast cancer (TNBC) patients, of which 80 percent are the basal-like subtype. The results of this study (called CALGB40603)
were just published on Aug. 6 in the Journal of Clinical Oncology and showed a benefit of carboplatin in TNBC patients. This new clinical trial result suggests that there may be great value in comparing clinical results across tumor types for which this study highlights as having common genomic similarities.

As participants in TCGA, UNC Lineberger scientists have been involved in multiple individual tissue type studies including most recently an analysis of a comprehensive genomic profile of lung adenocarcinoma. Perou’s seminal work in 2000 led to the first discovery of breast
cancer as not one, but in fact, four distinct subtypes of disease.  These most recent findings should continue to lay the groundwork for what could be the next generation of cancer diagnostics.

Source: University of North Carolina at Chapel Hill School of Medicine

New Gene Tied to Breast Cancer Risk

Wed, 08/06/2014

Marilynn Marchione – AP Chief Medical Writer – Associated Press

It’s long been known that faulty BRCA genes greatly raise the risk for breast cancer. Now, scientists say a more recently identified, less common gene can do the same.

Mutations in the gene can make breast cancer up to nine times more likely to develop, an international team of researchers reports in this week’s New England Journal of Medicine.

About 5 to 10 percent of breast cancers are thought to be due to bad BRCA1 or BRCA2 genes. Beyond those, many other genes are thought to play a role but how much each one raises risk has not been known, said Dr. Jeffrey Weitzel, a genetics expert at City of Hope Cancer Center
in Duarte, Calif.

The new study on the gene- called PALB2 – shows “this one is serious,” and probably is the most dangerous in terms of breast cancer after the BRCA genes, said Weitzel, one of leaders of the study.

It involved 362 members of 154 families with PALB2 mutations – the largest study of its kind. The faulty gene seems to give a woman a 14 percent chance of breast cancer by age 50 and 35 percent by age 70 and an even greater risk if she has two or more close relatives with the disease.

That’s nearly as high as the risk from a faulty BRCA2 gene, Dr. Michele Evans of the National Institute on Aging and Dr. Dan Longo of the medical journal staff write in a commentary in the journal.

The PALB2 gene works with BRCA2 as a tumor suppressor, so when it is mutated, cancer can flourish.

How common the mutations are isn’t well known, but it’s “probably more than we thought because people just weren’t testing for it,” Weitzel said. He found three cases among his own breast cancer
patients in the last month alone.

Among breast cancer patients, BRCA mutations are carried by 5 percent of whites and 12 percent of Eastern European (Ashkenazi) Jews. PALB2 mutations have been seen in up to 4 percent of families with a history of breast cancer.

 Men with a faulty PALB2 gene also have a risk for breast cancer that is eight times greater than men in the general population.

Testing for PALB2 often is included in more comprehensive genetic testing, and the new study should give people with the mutation better information on their risk, Weitzel said. Doctors say that people with faulty cancer genes should be offered genetic counseling and may want to consider more frequent screening and prevention options, which can range from hormone-blocking pills to breast removal.

The actress Angelina Jolie had her healthy breasts removed last year after learning she had a defective BRCA1 gene.

The study was funded by many government and cancer groups around the world and was led by Dr. Marc Tischkowitz of the University of Cambridge in England. The authors include Mary-Clare King, the University of Washington scientist who discovered the first breast
cancer predisposition gene, BRCA1.

Study: http://www.nejm.org/doi/full/10.1056/NEJMoa1400382

Gene info: http://ghr.nlm.nih.gov/gene/PALB2

Structure of the DDB1–CRBN E3 ubiquitin ligase in complex with thalidomide

Eric S. Fischer, Kerstin Böhm, John R. Lydeard, Haidi Yang, …, J. Wade Harper, Jeremy L. Jenkins & Nicolas H. Thomä

Nature (07 Aug 2014); 512, 49–53  http://dx.doi.org:/10.1038/nature13527

Published online 16 July 2014

In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide,

  • these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for
    multiple myeloma and 5q-deletion-associated dysplasia.
  • IMiDs target the E3 ubiquitin ligase CUL4–RBX1–DDB1–CRBN (known as CRL4CRBN) and
  • promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4CRBN.

Here we present crystal structures of the DDB1–CRBN complex bound to thalidomide,
lenalidomide and pomalidomide. The structure establishes that

  • CRBN is a substrate receptor within CRL4CRBN and enantioselectively binds IMiDs.

Using an unbiased screen, we identified the

  • homeobox transcription factor MEIS2 as an endogenous substrate of CRL4CRBN.

Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4CRBN while the ligase complex is recruiting IKZF1 or IKZF3 for degradation.

This dual activity implies that

  • small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins.

Curator’s Viewpoint:

The short pieces may not appear to be so closely connected, except for the last subject on the pharmaceutical targeting of an E3 ubiquitin ligase ubiquitination of proteins, but even in that case, we have to keep in mind that protein formation by amino acid transcription, remodeling, and recapture of amino acids are in equilibrium through ubiquitylation. So I put it there.  The DNA in populations ties some mutations to disease that is tied specifically to populations, not only the sephardic population, but in Asia as well.

The next article for consideration is methodological considerations.  The BRCA2 in the sephardic population is one of a number of mutations we can identify, extending to Tay Sachs disease, for instance.  How this might have occurred in the history of the jewish people is not so obvious, except perhaps in the segregation of the jewish population for centuries.  The mutation would be confined within the population with limited marriage outside of the jewish community.  It has been known for some time that there is a Cohen gene that traces back to the priests (Kohanim) of the Holy Temple, the descendents of Aaron (Aharon), the brother of Moses.  The priests would stand at the Ark and bless the congregation in the most holy convocation of Yom Kippur, according to tradition.  Marriages were arranged between the bride and the groom.  Of course, arranged marriages were also the case in other ethnic communities, and between the privileged.

That was dramatically the case during the reign of Queen Victoria of England, with Royal arrangements across Europe.
That would be a factor in the transmission of hemophilia, and in mental disorders in the Royal families. Haemophilia figured prominently in the history of European royalty in the 19th and 20th centuries. Britain’s Queen Victoria, through two of her five daughters (Princess Alice and Princess Beatrice), passed the mutation to various royal houses across the continent, including the royal families of Spain, Germany and Russia. Victoria’s son Prince Leopold, Duke of Albany suffered from the disease.  The Prince Leopold, Duke of Albany KG KT GCSI GCMG GCStJ (Leopold George Duncan Albert; 7 April 1853 – 28 March 1884) was the eighth child and fourth son of Queen Victoria and Prince Albert of Saxe-Coburg and Gotha. Leopold was later created Duke of Albany, Earl of Clarence, and Baron Arklow. He had haemophilia, which led to his death at the age of 30.  The sex-linked X chromosome disorder manifests almost entirely in males, although the gene for the disorder is located on the X chromosome and may be inherited from either mother or father. Expression of the disorder is much more common in males than in females. This is because, although the trait is recessive, males only inherit one X chromosome, from their mothers. Of course, this is classical Mendelian genetics. Victoria appears to have been a spontaneous or de novo mutation and is usually considered the source of the disease in modern cases of haemophilia among royalty. The mutation would probably be assumed today to have occurred at the conception of Princess Alice, as she was the only known carrier among Victoria and Albert’s first seven children. Leopold was a sufferer of haemophilia and her daughters Alice and Beatrice were confirmed carriers of the gene.

Cousin marriage is marriage between people with a common grandparent or other more distant ancestor. In various cultures and legal jurisdictions,  Marriages between first and second cousins account for over 10% of marriages worldwide, and they are common in the Middle East, where in some nations they account for over half of all marriages. Proportions of first-cousin marriage in the United States, Europe and other Western countries like Brazil have declined since the 19th century, though even during that period they were not more than 3.63 percent of all unions in Europe. Cousin marriage is allowed throughout the Middle East for all recorded history, and is used mostly in Syria. It has often been chosen to keep cultural values intact through many generations and preserve familial wealth. In Iraq the right of the cousin has also traditionally been followed and a girl breaking the rule without the consent of the ibn ‘amm could have ended up murdered by him. The Syrian city of Aleppo during the 19th century featured a rate of cousin marriage among the elite of 24% according to one estimate, a figure that masked widespread variation: some leading families had none or only one cousin marriage, while others had rates approaching 70%. Cousin marriage rates were highest among women, merchant families, and older well-established families.  The percentage of Iranian cousin marriages increased from 34 to 44% between the 1940s and 1970s. Cousin marriage among native Middle Eastern Jews is generally far higher than among the European Ashkenazim, who assimilated European marital practices after the diaspora.

The essential elements of the marriage contract were now an offer by the man, an acceptance by the woman, and the performance of such conditions as the payment of dowry. According to anthropologist Ladislav Holý, cousin marriage is not an independent phenomenon but rather one expression of a wider Middle Eastern preference for agnatic solidarity, or solidarity with one’s father’s lineage.

A 2009 study found that many Arab countries display some of the highest rates of consanguineous marriages in the world, and that first cousin marriages which may reach 25-30% of all marriages. Research among Arabs and worldwide has indicated that consanguinity could have an effect on some reproductive health parameters such as postnatal mortality and rates of congenital malformations.

In the terraced streets of Bradford, Yorkshire, a child’s death is anything but rare. At the boy’s inquest, coroner Mark Hinchliffe said Hamza Rehman had died because his Pakistan-born parents (shopkeeper Abdul and housewife Rozina) are first cousins. Muslims have practiced marriages between first cousins in non-prohibited countries since the time of the Quran.

Four years before, Hamza’s older sister, three-month-old Khadeja, had died of the same brain disorder which causes fits, sickness and chest infections. The couple had another baby born with equally devastating neurological problems.

A heartbroken Mr Rehman told the inquest that he and his wife were unsure whether to have any more children. The coroner expressed deep sympathy before saying that Hamza’s death should serve as a warning to others.

I have diverged somewhat onto the genetic risks of consanguinous marriages, which George Darwin, son of Charles Darwin, argues were had a small effect in then English society.  But most importantly, we see the larger factor here of social and familial inheritance, and also the concept of cultural identity.

Insofar as the somatic and mitochondrial mutations are concerned, I call attention to the finding in the GWAS study above discussed that the results were supportive of the conclusions from mtDNA.  This gives some reason to consider whether sufficient information is obtained from the mtDNA, without the more robust GWAS.  One cannot fully consider this without some knowledge of the methodology of specimen preparation.

It is not difficult to prepare mitochondria from cells and obtain a very good preparation before further analysis, whether of the membrane structures, the enzymatic activity, or of the DNA and RNA polynucleotides.  The separation is easily achieved with differential centrifugation.  On the other hand, the finding of the basal layer of epithelium having a different signature than the superficial layer, established by the genomic studies, but known histologically for non-neoplastic tissue, is a matter for cell separation methods that are not easy.  It is from the lower layer of cells that we derive carcinoma in-situ.  These cells were identified in breast, are expected to be found in uterus, and were like the cells in ovarian-cancer, which suggested the use of a common treatment regimen as adjunct in triple negative breast cancer and ovarian cancer.  The importance of a suuficiently prepared cellular specimen as opposed to tissue specimen can’t be taken for granted.

 

 

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RNA and the Transcription the Genetic Code

Curator: Larry H. Bernstein, MD, FCAP

 

 

This portion of the series is a followup on the series on the replication of the genetic code (DNA).  It may be considered alone, or as a tenth article.  Just as DNA has become far more than it was envisioned 60 years ago, the RNA, which was opened to further investigation by Roger Kornberg, Nobel Laureate, and son of the Nobel Laureate, Arthur Kornberg, who studied DNA polymerase, and with his Nobel Associate, attracted the finest minds in biochemistry and built the best academic department of Biochemistry at Stanford University.  RNA is associated with RNA polymerase as DNA is associated with DNA polymerase.  We have already highlighted the several critical reactions involved in the step-by-step replication of DNA.  The classic model has dictated DNA-RNA-protein.  We shall here look at the amazing view that RNA is heterogeneous, and is involved in living processes in health and disease.

 

 

Transcription (Wikipedia)

Transcription is the first step of gene expression, in which a particular segment of DNA is copied into RNA

Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language

  • that can be converted back and forth from DNA to RNA by the action of the correct enzymes.

During transcription, a DNA sequence is read by an RNA polymerase,

As opposed to DNA replication, transcription results in

  1. an RNA complement that includes the nucleotide uracil (U) in all instances
  • where thymine (T) would have occurred in a DNA complement.

Also unlike DNA replication where DNA is synthesized, transcription does not involve an RNA primer to initiate RNA synthesis.

Eukaryotic transcription is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of RNA replica. Gene transcription occurs in both eukaryotic and prokaryotic cells.
A eukaryotic cell has a nucleus that separates the processes of transcription and translation. Eukaryotic transcription occurs

The complexity of the eukaryotic genome necessitates a great variety and complexity of gene expression control.

Transcription can be reduced to the following steps, each moving like a wave along the DNA.

  1. One or more sigma factors initiate transcription of a gene by enabling binding of RNA polymerase to promoter DNA.
  2. RNA polymerase moves a transcription bubble, like the slider of a zipper, which splits the double helix DNA molecule into two strands of unpaired DNA nucleotides, by breaking the hydrogen bonds between complementary DNA nucleotides.
  3. RNA polymerase adds matching RNA nucleotides that are paired with complementary DNA nucleotides of one DNA strand.
  4. RNA sugar-phosphate backbone forms with assistance from RNA polymerase to form an RNA strand.
  5. Hydrogen bonds of the untwisted RNA + DNA helix break, freeing the newly synthesized RNA strand.
  6. If the cell has a nucleus, the RNA may be further processed (with the addition of a 3’UTR poly-A tail and a 5’UTR cap) and exits to the cytoplasm through the nuclear pore complex.

The stretch of DNA transcribed into an RNA molecule is called a transcription unit and encodes at least one gene. If the gene transcribed encodes a protein, the result of transcription is messenger RNA (mRNA), which will then be used to create that protein via the process of translation. Alternatively, the transcribed gene may encode for either non-coding RNA genes (such as microRNA, lincRNA, etc.) or ribosomal RNA (rRNA) or transfer RNA (tRNA), other components of the protein-assembly process, or other ribozymes.[1]

Making RNA replication of gene in eukaryotic cells

Transcription is the process of copying genetic information stored in a DNA strand into a transportable complementary strand of RNA.[1] Eukaryotic transcription takes place in the nucleus of the cell and proceeds in three sequential stages: initiation, elongation, and termination.[1] The transcriptional machinery that catalyzes this complex reaction has at its core three multi-subunit RNA polymerases.[1]

Protein coding genes are transcribed into messenger RNAs (mRNAs) that carry the information from DNA to the site of protein synthesis.[1] Although mRNAs possess great diversity, they are not the most abundant RNA species made in the cell. The so-called non-coding RNAs account for the large majority of the transcriptional output of a cell.[2] These non-coding RNAs perform a variety of important cellular functions.[2]

RNA Polymerase

Eukaryotes have three nuclear RNA polymerases, each with distinct roles and properties

Name Location Product
RNA Polymerase I (Pol I, Pol A) nucleolus larger ribosomal RNA (rRNA) (28S, 18S, 5.8S)
RNA Polymerase II (Pol II, Pol B) nucleus messenger RNA (mRNA), most small nuclear RNAs (snRNAs), small interfering RNA (siRNAs) and micro RNA (miRNA).
RNA Polymerase III (Pol III, Pol C) nucleus (and possibly the nucleolus-nucleoplasm interface) transfer RNA (tRNA), other small RNAs (including the small 5S ribosomal RNA (5s rRNA), snRNA U6, signal recognition particle RNA (SRP RNA) and other stable short RNAs

RNA polymerase I (Pol I)

  • catalyzes the transcription of all rRNA genes except 5S.[3][4]

These rRNA genes are organized into a single transcriptional unit and are transcribed into a continuous transcript. This precursor is then processed into

  • three rRNAs: 18S, 5.8S, and 28S.

The transcription of rRNA genes

  1. takes place in a specialized structure of the nucleus called the nucleolus,[5] where
  2. the transcribed rRNAs are combined with proteins to form ribosomes.[6]

RNA polymerase II (Pol II)

  • is responsible for the transcription of all mRNAs, some snRNAs, siRNAs, and all miRNAs.[3][4]

Many Pol II transcripts exist transiently as single strand precursor RNAs (pre-RNAs) that

  • are further processed to generate mature RNAs.[1]
  1.  precursor mRNAs (pre-mRNAs)are extensively processed
  2. before exiting into the cytoplasm through the nuclear pore for protein translation.

RNA polymerase III (Pol III) transcribes small non-coding RNAs, including tRNAs, 5S rRNA, U6 snRNA, SRP RNA, and other stable short RNAs such as ribonuclease P RNA.[7]

Structure of eukaryotic RNA polymerase II (light blue) in complex with α-amanitin (red), a strong poison found in death cap mushrooms that targets this vital enzyme

RNA Polymerases I, II, and III contain 14, 12, and 17 subunits, respectively.[8] All three eukaryotic polymerases have five core subunits that exhibit

  • homology with the β, β’, αI, αII, and ω subunits of E. coli RNA polymerase.

An identical ω-like subunit (RBP6) is used by all three eukaryotic polymerases,

  • while the same α-like subunits are used by Pol I and III.

The three eukaryotic polymerases share four other common subunits among themselves. The remaining subunits are unique to each RNA polymerase.

The additional subunits found in Pol I and Pol III relative to Pol II, are

  • homologous to Pol II transcription factors.[8]

Crystal structures of RNA polymerases I[9] and II [10] provide an opportunity to understand the interactions among the subunits and the molecular mechanism of eukaryotic transcription in atomic detail.

The carboxyl terminal domain (CTD) of RPB1, the largest subunit of RNA polymerase II,

  • plays an important role in bringing together the machinery necessary for the synthesis and processing of Pol II transcripts.[11]

Long and structurally disordered, the CTD

  • contains multiple repeats of heptapeptide sequence YSPTSPS
  1. that are subject to phosphorylation and
  2. other posttranslational modifications during the transcription cycle.

These modifications and their regulation constitute

  • the operational code for the CTD to control
  1. transcription initiation,
  2. elongation and
  3. termination and
  • to couple transcription and RNA processing.[11]

A DNA transcription unit encoding for a protein contains

  • not only the sequence that will eventually be directly translated into the protein (the coding sequence)
  • but also regulatory sequences that direct and regulate the synthesis of that protein.

The regulatory sequence before (i.e., upstream from) the coding sequence is called

the sequence following (downstream from) the coding sequence is called

Initiation

The initiation of gene transcription in eukaryotes occurs in specific steps.[1]

First, an RNA polymerase along with general transcription factors binds to the promoter region of the gene

The subsequent transition of the complex from the closed state to the open state results in

  • the melting or separation of the two DNA strands and
  • the positioning of the template strand to the active site of the RNA polymerase.

Without the need of a primer

  1. RNA polymerase can initiate the synthesis of a new RNA chain using the template DNA strand
  2. to guide ribonucleotide selection and polymerization chemistry.[1]

However, many of the initiated syntheses are aborted

  • before the transcripts reach a significant length (~10 nucleotides).

During these abortive cycles, the polymerase keeps making and releasing short transcripts

  • until it is able to produce a transcript that surpasses ten nucleotides in length.

Once this threshold is attained, RNA polymerase escapes the promoter and

  • transcription proceeds to the elongation phase.[1]

Eukaryotic promoters and general transcription factors

Pol II-transcribed genes contain a region

  • in the immediate vicinity of the transcription start site (TSS) that binds and positions the preinitiation complex.

This region is called the core promoter because of its essential role in transcription initiation.[12][13] Different classes

  • of sequence elements are found in the promoters. For example,
  • the TATA box is the highly conserved DNA recognition sequence for the TATA box binding protein,
  • TBP, whose binding initiates transcription complex assembly at many genes.

Eukaryotic genes

  • contain regulatory sequences beyond the core promoter.

These cis-acting control elements

  • bind transcriptional activators or repressors to increase or decrease transcription from the core promoter.

Well-characterized regulatory elements include

These regulatory sequences

  • can be spread over a large genomic distance, sometimes located
  • hundreds of kilobases from the core promoters.[1]

General transcription factors are

  • a group of proteins involved in transcription initiation and regulation.[1]

These factors typically have DNA-binding domains that bind

  1. specific sequence elements of the core promoter and
  2. help recruit RNA polymerase to the transcriptional start site.

General transcription factors for RNA polymerase II include TFIID, TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH.[1][14][15]

Transcription has some proofreading mechanisms

  • but they are fewer and less effective than the controls for copying DNA; therefore, transcription has a lower copying fidelity than DNA replication.[2]

As in DNA replication, DNA is read from 3′ end → 5′ end during transcription. Meanwhile,

  • the complementary RNA is created from the 5′ end → 3′ end direction.

This means its 5′ end is created first in base pairing. Although DNA is arranged as two antiparallel strands in a double helix, only

one of the two DNA strands, called the template strand, is used for transcription.

This is because RNA is only single-stranded, as opposed to double-stranded DNA. The other DNA strand (the non-template strand) is called the coding strand,

  • because its sequence is the same as the newly created RNA transcript (except for the substitution of uracil for thymine).

The use of only the 3′ end → 5′ end strand eliminates the need for the Okazaki fragments seen in DNA replication.[1]

In virology, the term may also be used when referring to mRNA synthesis from a RNA molecule (i.e. RNA replication). For instance,

  • the genome of an negative-sense single-stranded RNA (ssRNA -) virus
  1. may serve as a template to transcribe a positive-sense single-stranded RNA (ssRNA +) molecule,
  • since the positive-sense strand contains the information needed
  • to translate the viral proteins for viral replication afterwards.

This process is catalysed by a viral RNA replicase.

Transcription is divided into pre-initiation, initiation, promoter clearance, elongation and termination.

Pre-initiation

In eukaryotes, RNA polymerase, and therefore the initiation of transcription, requires

  • the presence of a core promoter sequence in the DNA.

Promoters are regions of DNA that promote transcription and, in eukaryotes, are found at -30, -75, and -90 base pairs

  • upstream from the transcription start site (abbreviated to TSS).

Core promoters are sequences within the promoter that are essential for transcription initiation. RNA polymerase is able to

The most characterized type of core promoter in eukaryotes is

  • a short DNA sequence known as a TATA box, found 25-30 base pairs upstream from the TSS.

The TATA box, as a core promoter, is the binding site for

  1. a transcription factor known as TATA-binding protein (TBP), which
  2. is itself a subunit of another transcription factor, called Transcription Factor II D (TFIID).

After TFIID binds to the TATA box via the TBP,

  • five more transcription factors and RNA polymerase combine around the TATA box
  • in a series of stages to form a preinitiation complex.

One transcription factor, Transcription factor II H, has two components

  • with helicase activity and so
  • is involved in the separating of opposing strands of double-stranded DNA
  • to form the initial transcription bubble.

However, only a low, or basal, rate of transcription is driven by the preinitiation complex alone. Other proteins known as

  1. activators and repressors,
  2. along with any associated coactivators or corepressors,
  3. are responsible for modulating transcription rate.

Thus, preinitiation complex contains:

  1. Core Promoter Sequence
  2. Transcription Factors
  3. RNA Polymerase
  4. Activators and Repressors.

The transcription preinitiation in archaea is, in essence, homologous to that of eukaryotes, but is much less complex.[3]

The archaeal preinitiation complex assembles at a TATA-box binding site; however,

  • in archaea, this complex is composed of only RNA polymerase II, TBP, and TFB (the archaeal homologue of eukaryotic transcription factor II B (TFIIB)).[4][5]

Initiation

Simple diagram of transcription initiation. RNAP = RNA polymerase

In bacteria, transcription begins with the binding of RNA polymerase to the promoter in DNA. RNA polymerase is a core enzyme consisting of five subunits: 2 α subunits, 1 β subunit, 1 β’ subunit, and 1 ω subunit. At the start of initiation,

  • the core enzyme is associated with a sigma factor that
  • aids in finding the appropriate -35 and -10 base pairs downstream of promoter sequences.[6]

When the sigma factor and RNA polymerase combine, they form a holoenzyme.

Transcription initiation is more complex in eukaryotes. Eukaryotic RNA polymerase

  • does not directly recognize the core promoter sequences. Instead,
  • a collection of proteins called transcription factors mediate
  • the binding of RNA polymerase and the initiation of transcription.

Only after certain transcription factors are attached to the promoter does the RNA polymerase bind to it. The completed assembly of

  • transcription factors and RNA polymerase bind to the promoter,
  • forming a transcription initiation complex.

Transcription in the archaea domain is similar to transcription in eukaryotes.[7]

Promoter clearance

After the first bond is synthesized, the RNA polymerase must clear the promoter. During this time

  • there is a tendency to release the RNA transcript and produce truncated transcripts. This is called
  • abortive initiation and is common for both eukaryotes and prokaryotes.[8]

In prokaryotes, abortive initiation continues to occur

  • until an RNA product of a threshold length of approximately 10 nucleotides is synthesized,
  • at which point promoter escape occurs and a transcription elongation complex is formed.

The σ factor is released according to a stochastic model.[9] Mechanistically, promoter escape occurs through a scrunching mechanism, where

  • the energy built up by DNA scrunching provides the energy needed to break interactions between RNA polymerase holoenzyme and the promoter.[10]

In eukaryotes, after several rounds of 10nt abortive initiation,

  • promoter clearance coincides with the TFIIH’s phosphorylation of serine 5 on the carboxy terminal domain of RNAP II,
  • leading to the recruitment of capping enzyme (CE).[11][12]

The exact mechanism of how CE induces promoter clearance in eukaryotes is not yet known.

Elongation

Simple diagram of transcription elongation

One strand of the DNA, the template strand (or noncoding strand), is used as a template for RNA synthesis. As transcription proceeds,

  • RNA polymerase traverses the template strand and uses base pairing complementarity with the DNA template to create an RNA copy.

Although RNA polymerase traverses the template strand from 3′ → 5′, the coding (non-template) strand and newly formed RNA can also be used as reference points,

  • so transcription can be described as occurring 5′ → 3′.

This produces an RNA molecule from 5′ → 3′, an exact copy of the coding strand (except that thymines are replaced with uracils, and the nucleotides are composed of a ribose (5-carbon) sugar where DNA has deoxyribose (one fewer oxygen atom) in its sugar-phosphate backbone).

mRNA transcription can involve multiple RNA polymerases on a single DNA template and multiple rounds of transcription (amplification of particular mRNA),

  • so many mRNA molecules can be rapidly produced from a single copy of a gene.

Elongation also involves a proofreading mechanism

  • that can replace incorrectly incorporated bases.

In eukaryotes,

  • short pauses during transcription allow appropriate RNA editing factors to bind.

These pauses may be intrinsic to the RNA polymerase or due to chromatin structure.

Termination

Main article: Terminator (genetics)

Bacteria use two different strategies for transcription termination –

  1. Rho-independent termination and
  2. Rho-dependent termination.

In Rho-independent transcription termination, also called intrinsic termination,

RNA transcription stops when the newly synthesized RNA molecule forms

  1. a G-C-rich hairpin loop followed by a run of Us. When the hairpin forms,
  2. the mechanical stress breaks the weak rU-dA bonds,
  3. now filling the DNA-RNA hybrid. This pulls the poly-U transcript out of the active site of the RNA polymerase,
  4. in effect, terminating transcription.

In the “Rho-dependent” type of termination, a protein factor called “Rho

  • destabilizes the interaction between the template and the mRNA, thus
  • releasing the newly synthesized mRNA from the elongation complex.[13]

Transcription termination in eukaryotes is less understood but involves cleavage of the new transcript followed by template-independent addition of As at its new 3′ end, in a process called polyadenylation.[14]

Inhibitors

Transcription inhibitors can be used as antibiotics against, for example, pathogenic bacteria (antibacterials) and fungi (antifungals). An example of such an antibacterial is

8-Hydroxyquinoline is an antifungal transcription inhibitor.[15] The effects of histone methylation may also work to inhibit the action of transcription.

Transcription factories

Active transcription units are clustered in the nucleus, in discrete sites called transcription factories or euchromatin. Such sites can be visualized by allowing engaged polymerases to extend their transcripts in tagged precursors (Br-UTP or Br-U) and immuno-labeling the tagged nascent RNA. Transcription factories can also be localized using fluorescence in situ hybridization or marked by antibodies directed against polymerases. There are ~10,000 factories in the nucleoplasm of a HeLa cell, among which are ~8,000 polymerase II factories and ~2,000 polymerase III factories. Each polymerase II factory contains ~8 polymerases. As most active transcription units are associated with only one polymerase, each factory usually contains ~8 different transcription units. These units might be associated through promoters and/or enhancers, with loops forming a ‘cloud’ around the factor.[16]

History

A molecule that allows the genetic material to be realized as a protein was first hypothesized by François Jacob and Jacques Monod. Severo Ochoa won a Nobel Prize in Physiology or Medicine in 1959 for developing a process for synthesizing RNA in vitro with polynucleotide phosphorylase, which was useful for cracking the genetic code. RNA synthesis by RNA polymerase was established in vitro by several laboratories by 1965; however, the RNA synthesized by these enzymes had properties that suggested the existence of an additional factor needed to terminate transcription correctly.

In 1972, Walter Fiers became the first person to actually prove the existence of the terminating enzyme.

Roger D. Kornberg won the 2006 Nobel Prize in Chemistry “for his studies of the molecular basis of eukaryotic transcription”.

Reverse transcription

Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA. HIV has an RNA genome that is reverse transcribed into DNA. The resulting DNA can be merged with the DNA genome of the host cell. The main enzyme responsible for synthesis of DNA from an RNA template is called reverse transcriptase.

Some eukaryotic cells contain an enzyme with reverse transcription activity called telomerase. Telomerase is a reverse transcriptase that lengthens the ends of linear chromosomes. Telomerase carries an RNA template from which it synthesizes a repeating sequence of DNA, or “junk” DNA. This repeated sequence of DNA is called a telomere and can be thought of as a “cap” for a chromosome. It is important because every time a linear chromosome is duplicated, it is shortened. With this “junk” DNA or “cap” at the ends of chromosomes, the shortening eliminates some of the non-essential, repeated sequence rather than the protein-encoding DNA sequence, that is farther away from the chromosome end.

Telomerase is often activated in cancer cells to enable cancer cells to duplicate their genomes indefinitely without losing important protein-coding DNA sequence. Activation of telomerase could be part of the process that allows cancer cells to become immortal. The immortalizing factor of cancer via telomere lengthening due to telomerase has been proven to occur in 90% of all carcinogenic tumors in vivo with the remaining 10% using an alternative telomere maintenance route called ALT or Alternative Lengthening of Telomeres.[20]

RNA-Seq Dissects the Transcriptome

Transcript Targeting  Kathy Liszewski
GEN    Jul 1, 2014 (Vol. 34, No. 13)

With the rapid rise of next-generation sequencing (NGS), one of its technologies, RNA sequencing (RNA-Seq), has taken center stage for analyzing whole transcriptomes.

Although RNA-Seq is still the new kid on the block,

  • this technology has the potential to revolutionize transcriptomics,
  • revealing the architecture of gene expression in unprecedented detail.

RNA-Seq applications are proliferating and include

  • the elucidation of disease processes,
  • targeted drug development, and
  • personalized medicine.

To orient researchers who are unfamiliar with the differences between  RNA-Seq platforms, Kelli Bramlett, R&D scientist, Life Technologies, poses two key questions:

1. Are you interested in pure discovery, in a nonguided fashion, of every RNA species present in your test samples?

2. Are you mainly focused on measuring expression levels of well-annotated coding RNA transcripts?

You might have a set of genes crucial to

 

  • identifying a disease state, or
  • profiling the stage of a specific type of cancer, or
  • monitoring development in your experimental system,

You then would want to employ a system that

  • “allows you to quickly and efficiently focus on just your genes of interest and screen through many different samples in a short amount of time.”

RNA-Seq allows for true discovery but

  • “requires sequencing depth and
  • requires significant additional time for analysis
  • If a focused panel targeting specific RNAs will better meet your needs, this can be accomplished on systems with
  • much faster turnaround time and less sequencing depth.”( according to Dr. Bramlett)

Enhancing Sensitivity

RNA-Seq has advanced our ability to characterize transcriptomes at high resolution, and the laboratory and data analysis techniques used for this NGS application continue to mature, notes John Tan, Ph.D., senior scientist, Roche NimbleGen. “High sequencing costs combined with the omnipresence of pervasive, abundant transcripts decrease our power to study rare transcripts, decrease throughput, and limit the routine use of this technology.”

For example, notes Dr. Tan, a small number of

  • highly expressed housekeeping genes can be responsible for a large fraction of total sequence reads in an experiment, thus
  • increasing the amount of sequencing required to characterize less abundant transcripts of interest.

To improve the cost-effectiveness, throughput, and sensitivity of RNA-Seq, Dr. Tan and colleagues are developing methods to perform targeted RNA-Seq.
“Targeted enrichment of transcripts of interest

  • circumvents the need to perform separate rRNA depletion or polyA enrichment steps on input RNA,” explains Dr. Tan.

“By targeting their sequencing, researchers can avoid wasting resources on

  • housekeeping transcripts and focus instead on genes or genomic regions of interest.”

Targeted RNA-Seq can allow deeper sequence coverage, increased sensitivity for low-abundance transcripts, less total sequencing per sample, and more samples processed per sequencing instrument run. “Significantly, we observe that the enrichment step also preserves quantitative information very well,” adds Dr. Tan. “These advances will facilitate a more routine use of RNA-Seq technology.”

  • Sample Integrity Issues

“Formalin-fixed, paraffin-embedded (FFPE) patient tissue archives and the clinical data associated with them may provide only limited amounts of sample that may also be degraded,” comments Gary Schroth, Ph.D., distinguished scientist, Illumina. Dr. Schroth says that most labs currently gauge RNA integrity via the RIN (RNA integrity number). but the RIN number from FFPE samples is not a sensitive measure of RNA quality or a good predictor for library preparation. A better predictor is RNA fragment size. We developed the DV200 metric, the percentage of RNA fragments greater than 200 nucleotides, a size needed for accurate construction of libraries.”

Illumina offers its TruSeq® RNA Access Library Preparation Kit especially for FFPE samples. This kit, when used with the DV200 metric, provides cleaner and more accurate library preparation. This new approach allows researchers to start with five-to tenfold less material when making libraries from FFPE samples.

  • Strand Specificity

Most NGS requires initial construction of libraries that may not provide the specificity desired even when prepared from mRNA. “Traditional RNA-Seq library preparation loses the strandedness of transcripts—information that is critical in understanding cellular transcription,” says Jungsoo Park, senior marketing and sales manager, Lexogen.

According to Park, Lexogen tackled this problem

  • by developing a method to generate libraries with greater than 99.9% strand specificity with a simplified process that takes 4.5 hours to complete.

Lexogen’s SENSE mRNA-Seq library kit initially isolates mRNA via

  • the poly A tail and utilizes random hybridization of the transcripts that
  • are bound to the magnetic beads without transcript fragmentation.

“This is a revolutionary method, which keeps high strandedness of the transcripts,” asserts Park.

One of the novel aspects of this approach is the use of starter/stopper heterodimers containing platform-specific linkers that hybridize to the mRNA.
“The starters serve as primers for reverse transcription, which then

  • terminates once the stopper from the next heterodimer is reached,

“At this point, the newly synthesized cDNA and the stopper are ligated while still bound to the RNA template.” According to Park,

  • there is no need for a time-consuming fragmentation step, and library size is determined simply by the protocol itself.

For researchers only intending to see the expression levels, sequencing of the entire mRNA transcript will require subsequent bioinformatics processes such as RPKM, a measure of relative molar RNA concentration.

  RNA-Seq Libraries

NuGEN Technologies offers its Ovation Human Blood RNA-Seq Library System as an end-to-end solution for strand-specific RNA-Seq library construction. NuGEN’s Insert Dependent Adaptor Cleavage (InDA-C) technology can provide targeted depletion of unwanted high-abundance transcripts.
  • Cells possess many thousands of transcripts.
  • uninformative transcript species that can compromise data quality and the cost-effectiveness of sequencing
  • NuGEN Technologies has developed a method for targeted depletion of unwanted transcripts following construction of RNA-Seq libraries. (Insert Dependent Adaptor Cleavage (InDA-C),

employs customized primers that target specific transcripts, such as ribosomal and globin RNAs, to exclude from final RNA-Seq libraries. (hemoglobin RNA derived from blood accounts for at least 60% of transcripts)  “By depleting these two transcript classes, InDA-C quadruples informative reads. and it avoids off-target mRNA cross-hybridization events that can potentially introduce bias. The species and transcript specificity of the workflow relies on the design of InDA-C primers, which can be constructed

  • to target virtually any class of unwanted transcripts for targeted depletion,”  according to Dr. Kain.

NuGEN has developed Single Primer Enrichment Technology, which can be used to prepare targeted NGS libraries from both gDNA or cDNA,

  •  used to identify gene fusion products and alternative splicing patterns from enriched cDNA libraries.

platforms automate the RNA sequencing sample preparation process [Beckman Coulter]

Preparation of libraries for RNA-Seq entails an intensive workflow.  according to Alisa Jackson, senior marketing manager, Genomic Solutions, Beckman Coulter, automation provides four key advantages:

  • Creation of high-quality mRNA libraries. Initial steps in this process include depleting samples of ribosomal RNA. Although it has the greatest abundance, rRNA gives the least amount of information.
  • “We’ve automated this process on our Biomek instruments using popular sample preparation kits from Illumina and New England Biolabs,” notes Jackson. “Accurate pipetting and thorough mixing are critical for this process. The Biomek liquid handler’s 96-channel pipetting head is used in combination with an on-deck orbital shaker to vigorously mix samples. Results show this ‘mix and shake’ approach works well.”
  • Limited exposure to RNAses from human contact. Every scientist’s nemesis when working with RNA is the universal presence of RNA-degrading RNAses. To help overcome this problem, says Jackson, “Biomek consumables such as pipette tips are DNase and RNase-free.”
  • Reduced exposure to toxic chemicals. “An instrument dispenses all reagents involved in the various steps of process.”
  • Enhanced reproducibility. “This is still a very expensive process,” asserts Jackson. “Obtaining accurate results the first time prevents costly repetitions. For this reason, we provide Biomek methods for many NGS library preparation kits. By fully testing these methods with real-life samples, we ensure reliable and repeatable creation of sequence-ready RNA libraries, whether stranded or nonstranded, mRNA or total RNA.”
  • What’s Next?

RNA-seq data analysis

RNA-seq data analysis for target identification. [Boehringer Ingelheim]

  •  “With RNA-Seq, we are closing in on personalized medicine,” suggests Qichao Zhu, Ph.D., principal scientist, Boehringer Ingelheim. “This technology allows more exact identification of patient subgroups. Instead of ‘one drug fits all,’ we can now begin to more appropriately define which drugs will work in which patients. Diseases such as cancer and cystic fibrosis as well as neurodegenerative illnesses have many patient subcategories. Future pharmaceutical drug discovery will be better able to develop targeted therapeutics with the help of RNA-Seq.
  • ”There are still many challenges in the field, however. “A critical aspect is accuracy. Given the large scale set of RNA-Seq, even 99.99% accuracy is not good enough for diagnostics,” insists Dr. Zhu. “Further, as we move forward, we will need to improve many aspects of the technology including
  • disease tissue sample isolation,
  • library construction methodologies, as well as
  • analysis of massive datasets.

“In the future, a patient will go into the doctor’s office and have a whole transcriptome profile test performed.“When PCR technology was discovered, no one knew just how powerful it would become or how many applications it would generate. Now, it is used everywhere. NGS technology and RNA-Seq have a similar potential. ”

 

Gene Paces microRNAs to Set Developmental Rhythms

Kevin Mayer   Jul 18, 2014   GEN News Highlights

http:/www.genengnews.com/gen-news-highlights/gene-paces-micrornas-to-set-developmental-rhythms/81250124/

Using C. elegans as a model researchers identified LIN-42, a gene that is found in animals across the evolutionary tree, as a potent regulator of numerous developmental processes. [C. Hammell, Cold Spring Harbor Laboratory]

  • Although the how of a gene’s function is important, the when, too, is crucial. The ebb and flow ofgene expression can influence a cell’s fate during development, the maturation of entire organisms, and even the evolution of species—helping to explain how species with very similar gene content can differ so dramatically.

Nature’s developmental clockwork

  • depends on the activation or repression of a specific and unique complement of genes. And these genes, in turn,
  • are regulated by microRNA molecules. And, finally,
  • the microRNAs are also subject to regulation.
  •  one must then study the regulators of the regulators of the regulators.

Little is known of the ultimate regulators—the elements that determine the activities of microRNAs. These elements, however, are presumably as subtle as they are powerful—

  1. subtle because microRNAs defined temporal gene expression and cell lineage patterns in a dosage-dependent manner;
  2. powerful because a single microRNA gene can control hundreds of other genes at once.
  3. as always, timing is everything: If a microRNA turns off genes too early or too late, the organism that depends on them will likely suffer severe developmental defects.

To undertake a search for genes that control developmental timing through microRNAs, a team of researchers at Cold Spring Harbor Laboratory relied on a tried-and-true model of animal development, Caenorhabditis elegans. These worms have a fixed number of cells, and each cell division is precisely timed.  “It enables us to understand

  • exactly how a mutation affects development,
  • whether maturation is precocious or delayed,
  • by directly observing defects in the timing of gene expression.” (said team leader Christopher Hammell, Ph.D.)

The researchers described their work in an article entitled, “LIN-42, the Caenorhabditis elegans PERIOD  homolog, Negatively Regulates MicroRNA Transcription,” which appeared July 17 in PLoS Genetics.

the goal to unveil factors that regulate the expression of microRNAs that control developmental timing –

  • they  identified LIN-42, the C. elegans homolog of the human and Drosophila period gene implicated in circadian gene regulation, as a negative regulator of microRNA expression

“By analyzing the transcriptional expression patterns of representative microRNAs, we found that the transcription of many microRNAs is normally highly dynamic and coupled aspects of post-embryonic growth and behavior.”

“LIN-42 shares a significant amount of similarity to the genes that control circadian rhythms in organisms such as mice and humans,” explained Roberto Perales, Ph.D., one of the lead authors of the study. “These are genes that control the timing of cellular processes on a daily basis for you and me. In the worm, these same genes and mechanisms control development, growth, and behavior. This system will provide us with leverage to understand how all of these things are coordinated.”

  1.  LIN-42 controls the repression of numerous genes in addition to microRNAs.
  2.  levels of the protein encoded by LIN-42 tend to
  • oscillate over the course of development and form a part of a developmental clock.

“LIN-42 provides the organism with a kind of cadence or temporal memory, so that

  1. it can remember that it has completed one developmental step before it moves on to the next,” emphasized Dr. Hammell. “This way, LIN-42 coordinates optimal levels of the genes required throughout development.”

 

Intracellular RNA-Seq

This literature review highlights a study led by George Church describing FISSEQ, or fluorescent in situ RNA sequencing.

Anton Simeonov, Ph.D.   Jul 25, 2014

http://www.genengnews.com/insight-and-intelligence/intracellular-rna-seq/77900207/

 

 FISSEQ appears to be sensitive to genes associated with cell type and function, and this in turn could be used for cell typing. [© Alila Medicinal Media – Fotolia.com]

  • Methods such as fluorescence in situ hybridization (FISH) allow gene expression to be observed at the tissue and cellular level; however, only a limited number of genes can be monitored in this manner, making transcriptome-wide studies impractical. George Church’s group* is presenting the further development of their original approach called
  • fluorescent in situ sequencing (FISSEQ) to incorporate a spatially structured sequencing library and an imaging method capable of resolving the amplicons (see Figure 1).

In fixed cells, RNA was reverse transcribed with tagged random hexamers to produce cDNA amplicons.

  1. Aminoallyl deoxyuridine 5-triphosphate (dUTP) was incorporated during reverse transcription and
  2. after the cDNA fragments were circularized before rolling circle amplification (RCA),
  3. an amine-reactive linker was used to cross-link the RCA amplicons containing aminoallyl dUTP.

The team generated RNA sequencing libraries in different cell types, tissue sections, and whole-mount embryos for three-dimensional (3D) visualization that spanned multiple resolution scales (see Figure 1).

Click Image To Enlarge +
Figure 1
  • Figure 1. Construction of 3D RNA-seq libraries in situ. After RT using random hexamers with an adapter sequence in fixed cells, the cDNA is amplified and cross-linked in situ. (A) A fluorescent probe is hybridized to the adapter sequence and imaged by confocal microscopy in human iPS cells (hiPSCs; scale bar: 10 μm) and fibroblasts (scale bar: 25 μm). (B) FISSEQ can localize the total RNA transcriptome in mouse embryo and adult brain sections (scale bar: 1 mm) and whole-mount Drosophila embryos (scale bar: 5 μm), although we have not sequenced these samples. (C) 3D rendering of gene-specific or adapter-specific probes hybridized to cDNA amplicons. 3D, three-dimensional; RT, reverse transcription; FISSEQ, fluorescent in situ sequencing; FISH, fluorescence in situ hybridization.
  • In a proof-of-concept experiment (see Figure 2) the authors sequenced primary fibroblasts in situ after simulating a response to injury, which yielded 156,762 reads, mapped to 8,102 annotated genes. When the 100 highest ranked genes were clustered, cells kept in fetal bovine serum medium were enriched for fibroblast-associated gene hits, while the rapidly dividing cells in epidermal growth factor medium were less fibroblast-like, reaffirming that the FISSEQ platform output reflects the change in transcription status as a function of the cellular environment and stress factors.

 

  • Figure 2. Overcoming resolution limitations and enhancing the signal-to-noise ratio. Ligation of fluorescent oligonucleotides occurs when the sequencing primer ends are perfectly complementary to the template. Extending sequencing primers by one or more bases, one can randomly sample amplicons at 1/4th, 1/16th, and 1/256th of the original density in fibroblasts (scale bar: 5 μm). N, nucleus; C, cytoplasm.
  • The authors further noted that FISSEQ appears to be sensitive to genes associated with cell type and function, and this in turn could be used for cell typing. It was also speculated that FISSEQ might allow for a combined transcriptome profiling and mutation detection in situ.
  • *Abstract from Science 2014, Vol. 343:1360–1363

Understanding the spatial organization of gene expression with single-nucleotide resolution requires

  • localizing the sequences of expressed RNA transcripts within a cell in situ.

Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked complementary DNA (cDNA) amplicons are sequenced within a biological sample.

  1. Using 30-base reads from 8102 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay.
  2. FISSEQ is compatible with tissue sections and whole-mount embryos and
  3. reduces the limitations of optical resolution and noisy signals on single-molecule detection.

Our platform enables massively parallel detection of genetic elements, including

  • gene transcripts and molecular barcodes, and can be used
  • to investigate cellular phenotype, gene regulation, and environment in situ.

Anton Simeonov, Ph.D., works at the NIH.

ASSAY & Drug Development Technologies, is published by Mary Ann Liebert, Inc.
GEN presents here one article that was analyzed in the “Literature Search and Review” column, a paper published in Science titled “Highly multiplexed subcellular RNA sequencing in situ.” Authors of the paper are Lee JH, Daugharthy ER, Scheiman J, Kalhor R, Yang JL, Ferrante TC, Terry R, … and Church GM.

 

Completely ablate microRNA genes on the genomic level

  • miR-KOs are transcription activator-like effector (TALE) nucleases that
  • precisely edit specific miRNAs in mammalian cells.
  • SBI designed miR-TALE-nucleases to cleave within the miRNA seed region.

In the absence of HR donor vectors, the cellular machinery repairs such breaks via

  • non-homologous end joining (NHEJ).

This is an error-prone system that typically generates small deletions or insertions (indels) at or near the site of cleavage. Since the seed region (defined as bases 2-8 of the microRNA) directs miRNA binding to its target DNA, indels within the seed region completely abolish miRNA function.

 

Design of miR-KO TALE Nucleases

The miR-KOs are designed to disrupt the miRNA seed region. Pairing miR-KOs with an HR donor

  • replaces the entire miRNA hairpin structure with an insulated selectable marker cassette.

Sample data for miR-KO 21 Knockout

Selection for HR events by puromycin or by FACS-based sorting for RFP can enrich for properly knocked-out alleles. The enriched cell populations are then

  • genotyped to determine whether the knockout is at a single allele or bi-allelic (as in the case of hsa-miR-21).

Genotyping for HR events is performed via junction PCR of genomic DNA-insert junctions at 5′ and/or 3′ ends of an HR site. PCR primer pairs are designed with one of the primer sequences corresponding to the targeted genomic DNA region and the other corresponding to the HR vector.

Primer design strategy for HR-directed genotyping

Genomic DNA PCR was used to to detect HR integration in one or both alleles of hsa-miR-21. Individual cellular clones that display one HR event typically display mutated seed regions in the other allele. miR-KOs, when combined with HR donor vectors have been shown to be highly efficient in generating double miRNA knockouts. For example, a miR-KO strategy against human miR-21 in HEK293T cells resulted in 30 puromycin-resistant lines out of 96 single cell-derived clones. Subsequent PCR-based genotyping of 23 successful PCR amplifications revealed that ~96% (22/23) were mono-allelic (i.e. one allele with HR and other with NHEJ or WT) and ~4% (1/23) were bi-allelic (e.g. both alleles undergone HR) for HR-induced miR-21 deletion. Furthermore, sequencing of PCR products spanning the targeted seed region of miR-21 revealed that 91% (10/11) were NHEJ-modified.

Taken together, these results show a 87% bi-allelic modification rate (20 out of 23 clones)

  • when the miR-KOs are combined with an HR donor vector.

Validation and phenotypic analysis of miR-KO of hsa-miR-21

To confirm complete loss of miRNA-21 expression, we quantified miR-21 expression in three independent miR-21 double knockouts by qPCR.

  1. Clone #1 and #7 carry one deletion of the miR-21 hairpin structure (via HR) and
  2. one indel within the seed region (via NHEJ);
  3. clone #5 carries bi-allelic deletions of the hairpin structure (bi-allelic HR).

We found complete abolishment of miR-21 expression in all three cell lines.

Growth phenotype uncovered in miR-21 KO cell lines

MicroRNA-21 has been characterized as a cell-promoting OncomiR. The abalation of the genomic hsa-miR-21 in human cells resulted in reduced proliferation in all three miR-21 knockout lines tested. Growth curves were plotted for the parental HEK293 cells as well as the three independent knockout lines.

Increase the ease and efficiency of obtaining KOs with matched HR vectors

While the use of miR-KOs alone can successfully abolish miRNA function,

  • screening for bi-allelic indels can be laborious.

Due to the small changes seen with indels, many clonal lines have to be established through limited dilution or single-cell sorting techniques, and

  • subsequently genomic DNA is PCR-amplified,
  • cloned into vectors and
  • subjected to genotyping by Sanger sequencing.

Since many cells will only have either zero or one alleles modified, tremendous work is often required to obtain bi-allelic indels.

To facilitate the screening process,

  • one may combine miRNA-specific TALE-nucleases with HR donor vectors, which enables positive selection and convenient screening of targeted cells.

Because NHEJ occurs more frequently than HR donor integration,

  • the majority of cells that undergo HR integration on one allele carry an indel in the miRNA seed region of the second allele.

This strategy has been shown to be highly efficient in generating bi-allelic miRNA knockouts. A positive selection strategy reveals puromycin-resistant and RFP-positive single-cell derived colonies, majority of which are double knockouts (i.e. HR event on one allele and indel in seed region of second allele).

Shown above is an overview of miR-KO strategies with miR-KOs alone and in combination with an HR donor vector. The HR donor vector enables positive selection, which allows for simple and efficient generation of cells harboring double knockouts.
Gene Described as Critical to Stem Cell Development

GEN News Highlights  Jul 18, 2014
http://www.genengnews.com/gen-news-highlights/gene-described-as-critical-to-stem-cell-development/81250121/

  • Scientists at Michigan State University say they have found that a gene known as ASF1A could be critical to the development of stem cells. ASF1A is at least one of the genes responsible for the mechanism of cellular reprogramming, a phenomenon that can turn one cell type into another, which is key to the making of stem cells, according to the researchers.

In a paper (“Histone chaperone ASF1A is required for maintenance of pluripotency and cellular reprogramming”) published in Science, the MSU team describes

  • how they analyzed more than 5,000 genes from a human oocyte before determining that
  • the ASF1A, along with another gene known as OCT4 and a helper soluble molecule, were the ones responsible for the reprogramming.

In 2006, an MSU team identified the thousands of genes that reside in the oocyte. In 2007, a team of Japanese researchers found that

  • by introducing four other genes into cells, induced pluripotent stem cells (iPSCs) could be created without the use of a human egg.

The researchers say that the genes ASF1A and OCT4 work in tandem with a ligand,

  • a hormone-like substance that also is produced in the oocyte called GDF9, to facilitate the reprogramming process.
  • overexpression of just ASF1A and OCT4 in hADFs exposed to the oocyte-specific paracrine growth factor GDF9 can reprogram hADFs into pluripotent cells

The report underscores the importance of studying the unfertilized MII [metaphase II human] as a means

  • to understand the molecular pathways governing somatic cell reprogramming.

“We believe that ASF1A and GDF9 are two players among many others that remain to be discovered, which are part of the cellular-reprogramming process,” noted Dr. Cibelli. “We hope that in the near future, with what we have learned here, we will be able to test new hypotheses that will reveal more secrets the oocyte is hiding from us. In turn, we will be able to develop new and safer cell therapy strategies.”

  • Although the how of a gene’s function is important, the when, too, is crucial. The ebb and flow of gene expression can influence a cell’s fate during development, the maturation of entire organisms, and even the evolution of species—helping to explain how species with very similar gene content can differ so dramatically.

 

Identification and Insilico Analysis of Retinoblastoma Serum microRNA Profile and Gene Targets Towards Prediction of Novel Serum Biomarkers

M Beta, A Venkatesan, M Vasudevan, U Vetrivel, et al. Identification and Insilico Analysis of Retinoblastoma Serum microRNA Profile and Gene Targets Towards Prediction of Novel Serum Biomarkers.

Bioinformatics and Biology Insights 2013:7 21–34.   http://dx.doi.org:/10.4137/BBI.S10501

This study was undertaken

  • to identify the differentially expressed miRNAs in the serum of children with RB in comparison with the normal age matched serum,
  • to analyze its concurrence with the existing RB tumor miRNA profile,
  • to identify its novel gene targets specific to RB, and
  • to study the expression of a few of the identified oncogenic miRNAs in the advanced stage primary RB patient’s serum sample.

MiRNA profiling performed on 14 pooled serum from chil­dren with advanced RB and 14 normal age matched serum samples

  • 21 miRNAs found to be upregulated (fold change > 2.0, P < 0.05) and
  • 24 downregulated (fold change > 2.0, P < 0.05).

Intersection of 59 significantly deregulated miRNAs identified from RB tumor profiles with that of miRNAs detected in serum profile revealed that

  • 33 miRNAs had followed a similar deregulation pattern in RB serum.

Later we validated a few of the miRNAs (miRNA 17-92) identified by microarray in the RB patient serum samples (n = 20) by using qRT-PCR.

Expression of the oncogenic miRNAs, miR-17, miR-18a, and miR-20a by qRT-PCR was significant in the serum samples

  • exploring the potential of serum miRNAs identification as noninvasive diagnosis.

Moreover, from miRNA gene target prediction, key regulatory genes of

  • cell proliferation,
  • apoptosis, and
  • positive and negative regulatory networks

involved in RB progression were identified in the gene expression profile of RB tumors.
Therefore, these identified miRNAs and their corresponding target genes could give insights on

  • potential biomarkers and key events involved in the RB pathway.

 

Prediction of Breast Cancer Metastasis by Gene Expression Profiles: A Comparison of Metagenes and Single Genes

(M Burton, M Thomassen, Q Tan, and TA Kruse.) Cancer Informatics 2012:11 193–217

http://dx.doi.org:/10.4137/CIN.S10375

The popularity of a large number of microarray applications has in cancer research led to the development of predictive or prognostic gene expression profiles. However, the diversity of microarray platforms has made the full validation of such profiles and their related gene lists across studies difficult and, at the level of classification accuracies, rarely validated in multiple independent datasets. Frequently, while the individual genes between such lists may not match, genes with same function are included across such gene lists. Development of such lists does not take into account the fact that

  • genes can be grouped together as metagenes (MGs) based on common characteristics such as pathways, regulation, or genomic location.

In this study we compared the performance of either metagene- or single gene-based feature sets and classifiers using random forest and two support vector machines for classifier building. The performance

  • within the same dataset,
  • feature set validation perfor­mance, and
  • validation performance of entire classifiers in strictly independent datasets

were assessed by

  • 10 times repeated 10-fold cross validation,
  • leave-one-out cross validation, and
  • one-fold validation, respectively.

To test the significance of the performance difference between MG- and SG-features/classifiers, we used a repeated down-sampled binomial test approach.

MG- and SG-feature sets are transferable and perform well for training and testing prediction of metastasis outcome

  • in strictly independent data sets, both
  • between different and
  • within similar microarray platforms, while
  • classifiers had a poorer performance when validated in strictly independent datasets.

The study showed that MG- and SG-feature sets perform equally well in classifying indepen­dent data. Furthermore, SG-classifiers significantly outperformed MG-classifier

  • when validation is conducted between datasets using similar platforms, while
  • no significant performance difference was found when validation was performed between different platforms.

Prediction of metastasis outcome in lymph node–negative patients by MG- and SG-classifiers showed that SG-classifiers performed significantly better than MG-classifiers when validated in independent data based on the same microarray platform as used for developing the classifier. However, the MG- and SG-classifiers had similar performance when conducting classifier validation in independent data based on a different microarray platform. The latter was also true when only validating sets of MG- and SG-features in independent datasets, both between and within similar and different platforms.

 

Molecular basis of transcription pausing

Jeffrey W. Roberts

Science 13 June 2014;  344(6189), pp. 1226-1227   http://dx.doi.org:/10.1126/science.1255712

+Author Affiliations

  1. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
  2. E-mail: jwr7@cornell.edu

During RNA synthesis, RNA polymerase moves erratically along DNA,

  1. frequently resting as it produces an RNA copy of the DNA sequence.

Such pausing helps coordinate the appearance of a transcript with its utilization by cellular processes; to this end,

  • the movement of RNA polymerase is modulated by mechanisms that determine its rate. For example,
  1. pausing is critical to regulatory activities of the enzyme such as the termination of transcription. It is also essential
  2. during early modifications of eukaryotic RNA polymerase II that activate the enzyme for elongation.

Two reports analyzing transcription pausing on a global scale in Escherichia coli, by Larson et al. (1) and by Vvedenskaya et al. (2) on page 1285 of this issue, suggest new functions of pausing and reveal important aspects of its molecular basis.

The studies of Larson et al. and Vvedenskaya et al. follow decades of analysis of bacterial transcription that has illuminated

  • the molecular basis of polymerase pausing events that serve critical regulatory functions.

A transcription pause specified by the DNA sequence

  • synchronizes the translation of RNA into protein with
  • the transcription of leader regions of operons (groups of genes transcribed together) for amino acid biosynthesis;
  • this coordination controls amino acid synthesis in response to amino acid availability (3).

A protein-induced pause occurs when the E. coli initiation factor σ70 restrains RNA polymerase

  • by binding a second occurrence of the “−10” promoter element.

This paused polymerase provides a structure for

  1. engaging a transcription antiterminator (the bacteriophage λ Q protein) (4) that,
  2. inhibits transcription pauses, including those essential for transcription termination.

Knowledge about the interactions between nucleic acids and RNA polymerase that induce pausing

  • comes partly from studies on the E. coli histidine biosynthesis operon.

RNA polymerase pauses at the leader region of this cluster of genes (the “his pause”),

  • allowing an essential RNA hairpin structure to form just upstream of the RNA-DNA hybrid
  • where RNA synthesis is templated in the polymerase’s catalytic cleft.

Importantly, however, other sequence elements are required to induce and stabilize the his pause—particularly

  • the nucleotide at the newly formed, growing end of the RNA (pausing is favored by pyrimidines rather than purines) (5), and
  • at the incoming nucleotide position [pausing is favored particularly by guanine (G)] (6), as well as surrounding elements.

Biochemical and structural analyses have identified an endpoint of the pausing process called the “elemental pause” in which

  • the catalytic structure in the active site is distorted, preventing further nucleotide addition (7).

The elemental paused state also involves distinct conformational changes in the polymerase

  1. that may favor transcription termination and
  2. allow the his and related pauses to be stabilized by RNA hairpins (8).

ILLUSTRATION: V. ALTOUNIAN/SCIENCE

Single-molecule analysis of transcribing RNA polymerase, at nearly single-nucleotide resolution, identified many specific pause sites in the E. coli genome (9). Pausing occurs on essentially any DNA, and very frequently—every 100 nucleotides or so. These “ubiquitous” pauses are only partly efficient (i.e., not always recognized as the enzyme transits), and mostly have not been associated with specific functions. However, their existence is consistent with biochemical experiments showing that the progress of RNA polymerase is generally erratic. A consensus sequence for ubiquitous pauses was identified, with two important elements:

  • a preference for pyrimidine [mostly cytosine (C)] at the newly formed RNA end,
  • followed by G to be incorporated next—just as found for the his pause; and
  • a preference for G at position −10 of the RNA (10 nucleotides before the 3′ end), which is
  • at the upstream boundary of the RNA-DNA templating hybrid.

Remarkably, the tendency of a G in this position to induce pausing was recognized earlier, when DNA could be sequenced only through its transcript (10); it was thought that inhibited unwinding of the RNA-DNA hybrid underlies the pause.

 

Polyymerase, paused.

During transcription, RNA exists in two states as RNA polymerase progresses:

  1. pretranslocated, just after the addition of the last nucleotide [here, cytosine (C)]; and
  2. posttranslocated, after all nucleic acids have shifted in register by one nucleotide relative to the enzyme,
  • exposing the active site for binding of the next substrate molecule [here, guanine (G)].

The pretranslocated state is dominant in the pause. The critical G-C base (RNA-DNA) pair at position −10 in pretranslocated state and

  • the nontemplate DNA strand G bound in the polymerase in the posttranslocated state are marked with an asterisk.

ILLUSTRATION: V. ALTOUNIAN/SCIENCE

This ubiquitous pausing consensus sequence now has been refined and mapped exhaustively in the E. coligenome by Larson et al. and Vvedenskaya et al. (see the figure). In an analysis called native elongating transcript sequencing (NET-Seq) (11), transcripts associated with the whole cellular population of RNA polymerase are isolated from abruptly frozen cells and their growing ends are sequenced, giving a snapshot at nucleotide resolution of global transcription activity; DNA sites that are highly populated by RNA polymerase represent pauses. Larson et al. identified ∼20,000 transcription pause sites in the E. coli genome, including those expected from previous analysis of known sites like the his pause. Their analysis raises interesting questions about the role of such abundant pausing sequences.

Primarily, Larson et al. note that pauses frequently occur

  • exactly at the site of translation initiation, suggesting an important role in gene expression.

This coincidence of events is understandable when you examine the sequences. The consensus sequence in RNA for RNA polymerase pausing is G−10Y−1G+1 [G at position −10 and at the site after the pause; Y denotes either C or uracil (U) at the RNA end] according to Larson et al. and Vvedenskaya et al. The Shine-Dalgarno consensus sequence in RNA that the small-subunit ribosome recognizes is AGGAGG [adenine (A)] providing the G at the −10 position;

  • the downstream initiation codon for RNA translation is AUG, providing (for E. coli) the U at the pause end at position −1, with a following G at position +1.

A slightly modified pausing consensus sequence in the bacterium Bacillus subtilis accommodates the difference in spacing between the Shine-Dalgarno sequence and the initiation codon. What might be the role of a pause exactly at the translation initiation site? Because the ribosome binding site is physically concealed by RNA at the pause,

  • pausing may enable some process that prepares the RNA for translation once RNA polymerase transits the pause site.

Larson et al. suggest that the pause allows upstream RNA secondary structure to resolve in order to present the initiation region properly to the ribosome.

A particularly informative application of NET-Seq that provides new mechanistic information about pausing is based on the discovery of a specific binding site in RNA polymerase [the core recognition element (CRE)] for G in the non-template DNA strand (the strand not transcribed), at position +1 in the “posttranslocated” structure (12).

  • It could be that specific binding of a nucleotide to the enzyme in this position enhances pausing by slowing translocation;

surprisingly, however, Vvedenskaya et al. find the opposite. Cells altered to destroy the G binding site have up to twice as many sites of pausing as in wild-type cells, with

  • a greater preference for G as the incoming nucleotide.

However, this result is understandable in terms of the translocation cycle of RNA polymerase and the ubiquitous pausing sequence that has G at position +1. Binding of G at position +1 to CRE only occurs in the posttranslocated state, which would thus be favored over the pretranslocated state. Hence,

  • if G binding inhibits pausing, then the rate-limiting paused structure must be in the pretranslocated state (a conclusion also made by Larson et al. from biochemical experiments).

This is an important insight into the sequence of protein–nucleic acid interactions that occur in pausing. Vvedenskaya et al. suggest that the actual role of the G binding site is to promote translocation and thus inhibit pausing, to smooth out adventitious pauses in genomic DNA.

The studies by Larson et al. and Vvedenskaya et al. provide a refined and detailed analysis of DNA sequence–induced transcription pausing. As a core process in gene expression, this understanding is relevant not only for the basic biology of transcription, but also has applications in synthetic biology and the design of genetic circuits.

References

    1. H. Larson
    2. et al

., Science 344, 1042 (2014).

 

Abstract/FREE Full Text

O. Vvedenskaya et al.

,Science 344, 1285 (2014).

Abstract/FREE Full Text

  1. Landick, Turnbough, , C. Yanofsky

, in Escherichia coli and Salmonella, F. Neidhardt , Ed. (American Society for Microbiology, Washington, DC, 1996), vol. 1, pp. 1263–1286.

Google Scholar

  1. A. Perdue, W. Roberts

, J. Mol. Biol. 412, 782 (2011).

CrossRefMedlineGoogle Scholar

  1. L. Chan Landick

, J. Mol. Biol. 233, 25 (1993)

CrossRefMedlineWeb of ScienceGoogle Scholar

  1. N. Lee, Phung,Stewart, Landick

, J. Biol. Chem. 265, 15145 (1990).

Abstract/FREE Full Text

  1. Toulokhonov, Zhang,Palangat, Landick

, Mol. Cell 27, 406 (2007).

CrossRefMedlineWeb of ScienceGoogle Scholar

  1. Weixlbaumer, Leon, Landick, A. Darst

, Cell 152, 431 (2013).

CrossRefMedlineWeb of ScienceGoogle Scholar

9. M. Herbert, et al

., Cell 125, 1083 (2006).

CrossRefMedlineWeb of ScienceGoogle Scholar

10. Gilbert

, in RNA Polymerase, R. L. a. M. J. Chamberl1n , Ed. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1976), pp. 193–205.

Google Scholar

10. Churchman,, S. Weissman

, Nature 469, 368 (2011).

 

CrossRefMedlineWeb of ScienceGoogle Scholar

12.Zhan et al

., Science 338, 1076 (2012).

 

The editors suggest the following Related Resources on Science sites

In Science Magazine

REPORT Interactions between RNA polymerase and the “core recognition element” counteract pausing

Irina O. Vvedenskaya,  Hanif Vahedian-Movahed, Jeremy G. Bird, Jared G. Knoblauch, Seth R. Goldman,

Yu Zhang, Richard H. Ebright, and Bryce E. Nickels

Science 13 June 2014: 1285-1289.

 

“miR”roring Lupus Control

Angela Colmone

Sci.Signal., 29 July 2014;; 7(336),, p. ec202   http://dx.doi.org:/10.1126/scisignal.2005732

Decreased expression of the B cell signaling inhibitor PTEN may contribute to lupus pathology. Wu et al. found that microRNA (miR)–mediated regulation of PTEN is altered in patients with the autoimmune disease systemic lupus erythematosus (SLE). Patients with SLE have hyperactivated B cells, which results in the production of autoantibodies. The authors found that decreased expression of PTEN in B cells from SLE patients contributes to this B cell hyperactivation. What’s more, they found that PTEN expression in these cells was regulated by miRs and that blocking miR-7 could restore PTEN expression and function to that of healthy controls. These data support exploring miR-7 and PTEN as therapeutic targets for SLE.

X-n. Wu, Y-x. Ye, J-w. Niu, Y. Li, X. Li, X. You, H. Chen, L-d. Zhao, X-f. Zeng, F-c. Zhang, F-l. Tang, W. He, X-t. Cao, X. Zhang, P. E. Lipsky, Defective PTEN regulation contributes to B cell hyperresponsiveness in systemic lupus erythematosus. Sci. Transl. Med. 6, 246ra99 (2014). [Full Text]

Citation:

  1. Colmone, “miR”roring Lupus Control. Sci. Signal.7, ec202 (2014).

 

Long Noncoding RNA Regulating Apoptosis Discovered

Source: © Dmitry Sunagatov – Fotolia.com

  • Scientists from the University of São Paulo (USP) have identified an RNA molecule known as INXS that, although containing no instructions for the production of a protein, modulates the action of an important gene that impactsapoptosis.

According to Sergio Verjovski-Almeida, Ph.D., professor at the USP Chemistry Institute, INXS expression is generally diminished in cancer cells, and methods that are capable of stimulating the production of this noncoding RNA can be used to treat tumors. In experiments on mice, the USP scientists were able to effect a 10-fold reduction in the volume of subcutaneous malignant tumors by administering local injections of a plasmid containing INXS.

The team’s findings (“Long noncoding RNA INXS is a critical mediator of BCL-XS induced apoptosis”) were published in Nucleic Acids Research.

The group headed by Dr. Verjovski-Almeida at USP has been investigating the regulatory role of so-called intronic nonprotein-coding genes—those found in the same region of the genome as a coding gene but on the opposite DNA strand. INXS, for example, is an RNA expressed on the opposite strand of a gene coding for  the BCL-X protein.

“We were studying several protein-coding genes involved in cell death in search of evidence that one of them was regulated by intronic noncoding RNA. That was when we found the gene for BCL-X, which is located on chromosome 20,” he explained.

BCL-X is present in cells in two different forms: one that inhibits apoptosis (BCL-XL) and one that induces the process of cell death (BCL-XS). The two isoforms act on the mitochondria but in opposite ways. The BCL-XS isoform is considered a tumor suppressor because it activates caspases, which are required for the activation of other genes that cause cell death.

“In a healthy cell, there is a balance between the two BCL-X isoforms. Normally, there is already a smaller number of the pro-apoptotic form (BCL-XS). However, in comparing tumor cells to nontumor cells, we observed that tumor cells contain even fewer of the pro-apoptotic form, as well as reduced levels of INXS. We suspect that one thing affects the other,” continued Dr. Verjovski-Almeida.

To confirm the hypothesis, the group silenced INXS expression in a normal cell lineage and the result, as expected, was an increase in the BCL-XL (anti-apoptotic) isoform. “The rate between the two—which was 0.25—decreased to 0.15; in other words, the pro-apoptotic form that previously represented one fourth of the total began to represent only one sixth,” noted Dr. Verjovski-Almeida.

The opposite occurred when the researchers artificially increased the amount of INXS using plasmid expression in a kidney cancer cell line, with the noncoding RNA being reduced. “The pro-apoptotic form increased, and the anti-apoptotic form decreased,” he added.

“In a mouse xenograft model, intra-tumor injections of an INXS-expressing plasmid caused a marked reduction in tumor weight, and an increase in BCL-XS isoform, as determined in the excised tumors,” wrote the investigators. “We revealed an endogenous lncRNA that induces apoptosis, suggesting that INXS is a possible target to be explored in cancer therapies.

 

Scientists map one of the most important proteins in life—and cancer

Mon, 07/21/2014

Scientists have revealed the structure of one of the most important and complicated proteins in cell division—a fundamental process in life and the development of cancer—in research published in Nature.

Images of the gigantic protein in unprecedented detail will transform scientists’ understanding of exactly how cells copy their chromosomes and divide, and could reveal binding sites for future cancer drugs.

A team from The Institute of Cancer Research, London, and the Medical Research Council Laboratory of Molecular Biology in Cambridge produced the first detailed images of the anaphase-promoting complex (APC/C).

The APC/C performs a wide range of vital tasks associated with mitosis,

  1. the process during which a cell copies its chromosomes and
  2. pulls them apart into two separate cells.
  3. Mitosis is used in cell division by all animals and plants.

Discovering its structure could ultimately lead to new treatments for cancer, which

  • hijacks the normal process of cell division to make thousands of copies of harmful cancer cells.

In the study, which was funded by Cancer Research UK,

the researchers reconstituted human APC/C and used a combination of electron microscopy and imaging software to visualize it at a resolution of less than a billionth of a meter.

The resolution was so fine that it allowed the researchers to see the secondary structure—

  • the set of basic building blocks which combine to form every protein.

Alpha-helix rods and folded beta-sheet constructions were clearly visible within the 20 subunits of the APC/C, defining the overall architecture of the complex.

Previous studies led by the same research team had shown

  • a globular structure for APC/C in much lower resolution, but
  • the secondary structure had not previously been mapped.

The new study could identify binding sites for potential cancer drugs.

Each of the APC/C’s subunits bond and mesh with other units at different points in the cell cycle,

  1. allowing it to control a range of mitotic processes including the initiation of DNA replication,
  2. the segregation of chromosomes along protein ‘rails’ called spindles, and
  3. the ultimate splitting of one cell into two, called cytokinesis.

Disrupting each of these processes could

  • selectively kill cancer cells or prevent them from dividing.

Dr David Barford, who led the study as Professor of Molecular Biology at The Institute of Cancer Research, London, before taking up a new position at the Medical Research Council Laboratory of Molecular Biology in Cambridge, said:

“It’s very rewarding to finally tie down the detailed structure of this important protein, which is both

  • one of the most important and most complicated found in all of nature.

We hope our discovery will open up whole new avenues of research that increase our understanding of the process of mitosis, and ultimately lead to the discovery of new cancer drugs.”

Professor Paul Workman, Interim Chief Executive of The Institute of Cancer Research, London, said: “The fantastic insights into molecular structure

  • provided by this study are a vivid illustration of the critical role played by fundamental cell biology in cancer research.

“The new study is a major step forward in our understanding of cell division. When this process goes awry

  • it is a critical difference that separates cancer cells from their healthy counterparts.

Understanding exactly how cancer cells divide inappropriately is crucial to

  • the discovery of innovative cancer treatments to improve outcomes for cancer patients.”

Dr Kat Arney, Science Information Manager at Cancer Research UK, said “Figuring out how the fundamental molecular ‘nuts and bolts’ of cells work is vital

  • if we’re to make progress understanding what goes wrong in cancer cells and how to tackle them more effectively.

Revealing the intricate details of biological shapes is a hugely important step towards identifying targets for future cancer drugs.”

Source: The Institute of Cancer Research, London

 

A cell death avenue evolved from a life-saving path

  1. Harm H. Kampinga

+Author Affiliations

  1. Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, Netherlands.
  2. E-mail: h.kampinga@umcg.nl

Related Resources

In Science Magazine

Science 20 June 2014: 1389-1392.Published online 22 May 2014

In Science Signaling

Sci. Signal. 24 June 2014: ec175.

Yeast metacaspases are the ancestral enzymes of caspases that execute cellular suicide (“programmed cell death”) in multicellular organisms. Studies on metacaspase 1 (Mca1)

  • have suggested that single-cell eukaryotes can also commit programmed cell death (12). However,

on page 1389 of this issue, Malmgren Hill et al. (3) show that

  • Mca1 has positive rather than negative effects on the life span of the budding yeast Saccharomyces cerevisiae,
  • especially when protein homeostasis is impaired.

Mca1 helps to degrade misfolded proteins that accumulate during aging or that are generated by acute stress, and

  • thereby ensures the continuous and healthy generation of daughter cells
  • that are free of insoluble aggregates that otherwise would limit life span.

View larger version:

 

ILLUSTRATION: V. ALTOUNIAN/SCIENCE

Loss of Mca1 activity has been associated with a reduced appearance of programmed cell death markers (14),

  • implying that its overexpression should decrease the replicative life span of yeast (the number of daughter cells a mother cell can produce throughout its life). Cells lacking Mca1
  • have increased amounts of protein aggregates and oxidized proteins (45).

Malmgren Hill et al. not only show that this is related to decreased survival,

  • but also provide mechanistic insights into the mode of action of Mca1.

Its pro-life action depends on the chaperone heat shock protein 104 (Hsp104), a protein that

  1. can disentangle protein aggregates and
  2. is crucial for the asymmetric segregation of protein aggregates in dividing cells.

Mca1 deficiency does not affect life span of wild-type strains, but

  1. further decreases life span in strains already compromised in protein quality control. In particular,
  2. replicative aging is accelerated in strains lacking the Hsp70 co-chaperone Ydj1.

Mca1 does not improve protein folding but supports

  • degradation of terminally misfolded proteins.

Malmgren Hill et al. show that Mca1 requires proteasomes (protein structures that break down proteins) for all its effects.

The study by Malmgren Hill et al. challenges the idea that

  1. caspases are activated as an altruistic suicide mechanism in single-cell eukaryotes
  2. as a means to provide nutrients for younger and fitter cells in the population (2). Rather,
  3. the data suggest that from an evolutionary perspective, caspase activation is an integrated part of a protective response
  4. to help cells survive toxic stress caused by the accumulation of misfolded proteins.

When, however, activated incorrectly (e.g., in the absence of proteotoxic stress) or too strongly (e.g., in the case of excessive damage to the cell),

  1. the caspase activity may become nonselective and thus
  2. lead to the typical Mca1-dependent hallmarks of programmed cell death (124). Also,
  3. caspase activation in metazoa may function primarily in cell-autonomous protection and cellular remodeling or
  4. pruning. Its role in programmed cell death may also simply reflect overactivation upon severe cellular damage or
  5. hijacking of the caspases in the absence of stress to serve in non–cell-autonomous regulated tissue homeostasis.

View larger version:

Defense against protein damage.

Stress-damaged proteins that form aggregates in cells can be reactivated with the Hsp104-Ssa-Ydj1 chaperone machinery. Mca1 may act

  • in parallel by binding to misfolded proteins during early stages of aggregation for proteasomal degradation (this is independent of Mca1’s enzymatic activity). Alternatively,
  • Mca1 may associate with misfolded proteins formed at late stages of aggregation (together with Hsp104 and Ssa), helping to disentangle
  • the aggregates by its protease cleavage activity before shunting them to the proteasome for degradation.

ILLUSTRATION: V. ALTOUNIAN/SCIENCE

The results of Malmgren Hill et al. also highlight the importance of protein quality control for cellular aging. A collapse of protein homeostasis

  • has been implicated mostly in chronological aging of differentiated cells and, for example,
  • as a cause of neurodegenerative diseases (6).

The authors show that it also plays a prominent role in replicative aging.

  • This supports early findings in yeast (7) and may also be relevant to metazoa,
  • in which stem cells have extremely efficient protein degradation mechanisms (8) and
  • also use asymmetric segregation of protein damage for rejuvenation (9).

The data of Malmgren Hill et al. also suggest the existence of an additional layer of control of protein homeostasis. Beyond the

  • activation and induction of chaperones that assist in protein sorting, refolding, and protein degradation via proteasomes and
  • autophagosomes (membrane structures that deliver proteins to lysosomes for enzymatic destruction) (10),
  • Malmgren Hill et al. show that activation of caspases also belongs to the cell’s repertoire of defense mechanisms against protein damage.
  • Mca1 might act in parallel to the Ssa-Ydj1 machinery. Although
  • Ssa-Ydj1 collaborates with Hsp104 to refold proteins after their aggregation (11),
  • Mca1 primarily supports protein degradation, as its actions require not only Hsp104 but also proteasomal activity (3).

Precisely how Mca1 exerts its effect is yet unclear. It can associate with aggregates independent of other chaperones (35) and

  • independent of its catalytic activity (5), suggesting that
  • it binds directly to misfolded proteins [likely through its amino-terminal “pro-domain”
  • that is rich in glutamine and asparagine repeats].

This interaction may exert chaperone-like activity by keeping unfolded proteins

  • in a proteasome-competent form, which explains why part of Mca1’s protective actions in wild-type strains is independent of its protease activity.

However, the caspase activity of Mca1 is required for protein homeostasis and control of life span in Ydj1-deficient strains. It could be that

  • for more terminally misfolded proteins that accumulate in the absence of Ydj1,
  • protease cleavage may help to dismantle such aggregates in concert with Ssa and Hsp104 (see the figure).

This would also explain why the strongest phenotypes of Mca1 are seen under conditions in which Ydj1 is absent. More biochemical data with purified proteins will be needed to test these ideas.

The study of Malmgren Hill et al. suggests that altruism may not exist among cells. However, life and death seem to be close neighbors, and the things that are life saving may also become lethal. It will therefore be a challenge

  • to make use of these insights into caspase function in order to treat diseases by selectively tipping the balance toward life (e.g., in neurodegenerative diseases) or death (e.g., in cancer).

References

  1. Madeo et al

., Mol. Cell 9, 911 (2002).

 

CrossRefMedlineWeb of ScienceGoogle Scholar

  1. Herker et al

., J. Cell Biol. 164, 501 (2004).

 

Abstract/FREE Full Text

  1. Malmgren Hill et al

., Science 344, 1389 (2014).

 

Abstract/FREE Full Text

  1. A. Khan, . Chock, R. Stadtman

, Proc. Natl. Acad. Sci. U.S.A. 102, 17326 (2005).

Abstract/FREE Full Text

  1. E. Lee, Brunette, G. Puente, A. Megeney

, Proc. Natl. Acad. Sci. U.S.A. 107, 13348(2010).

 

Abstract/FREE Full Text

  1. E. Balch, I. Morimoto, Dillin, W. Kelly

, Science 319, 916 (2008).

 

Abstract/FREE Full Text

  1. Aguilaniu, Gustafsson, Rigoulet, Nyström

, Science 299, 1751 (2003).

Abstract/FREE Full Text

  1. Vilchez et al

., Nature 489, 304 (2012).

 

CrossRefMedlineWeb of ScienceGoogle Scholar

  1. A. Rujano et al

., PLOS Biol. 4, e417 (2006).

 

CrossRefMedlineGoogle Scholar

  1. H. Kampinga, A. Craig

, Nat. Rev. Mol. Cell Biol. 11, 579 (2010).

 

CrossRefMedlineWeb of ScienceGoogle Scholar

  1. R. Glover, Lindquist

, Cell 94, 73 (1998).

 

CrossRefMedlineWeb of ScienceGoogle Scholar

 the following Related Report

Life-span extension by a metacaspase in the yeast Saccharomyces cerevisiae

Sandra Malmgren Hill, Xinxin Hao, Beidong Liu, and Thomas Nyström

Science 20 June 2014: 1389-1392.

 

Synthetic biology: the many facets of T7 RNA polymerase

David L Shis, Matthew R Bennett
Molecular Systems Biology(2014)10:745   30.07.2014
http://dx.doi.org:/10.15252/msb.20145492

 

Added 8-2-2014

Split T7 RNA polymerase provides new avenues for creating synthetic gene circuits that are decoupled from host regulatory processes—but how many times can this enzyme be split, yet retain function? New research by Voigt and colleagues (SegallShapiro et al, 2014) indicates that it may be more than you think.

See also: TH Segall‐Shapiro et al (July 2014)

Synthetic gene circuits have become an invaluable tool for studying the design principles of native gene networks and facilitating new biotechnologies (Wayet al2014). Synthetic biologists often strive to build circuits within a framework that enables their consistent and robust operation across a range of hosts and conditions. Currently, however, each circuit must be fastidiously tuned and retuned in order to properly function within a particular host, leading to costly design cycles and esoteric conclusions. As a result, researchers have invested a great deal in developing strategies that

  • decouple synthetic gene circuits from host metabolism and regulation.

In their recent work, Segall‐Shapiro et al (2014) address this problem by

  • expanding the capabilities of orthogonal transcriptional systems in Escherichia coli using fragmented mutants of bacteriophage‐T7 RNA polymerase (T7 RNAP).

T7 RNAP has had a long relationship with biotechnology and

  • is renowned for its compactness and transcriptional activity.

This single subunit polymerase strongly

  • drives transcription from a miniscule 17‐bp promoter
  • that is orthogonally regulated inE. coli.

In this context, orthogonal means that

  • T7 RNAP will not transcribe genes driven by native E. coli promoters, and
  • native polymerases in E. coli will not recognize T7 RNAP’s special promoter—that is
  • the two transcriptional systems leave each other alone.

Interestingly, T7 RNAP drives transcription so strongly that,

  • if left unregulated, it can quickly exhaust cellular resources and lead to cell death.

Because of this, T7 RNAP

  • has been leveraged in many situations calling for protein over‐expression (Studier & Moffatt, 1986).

Additionally, studies examining the binding of T7 RNAP to its promoter have identified

  • a specificity loop within the enzyme that makes direct contact with the promoter
  • between base pairs −11 and −8.

This has led to a number of efforts that have generated T7 RNAP mutants

  • with modified specificities to promoters orthogonal to the original (Chelliserrykattil et al2001).

Given the growing interest in the development of synthetic gene circuits, researchers have taken a renewed interest in T7 RNAP. The orthogonality,

  • transcriptional activity and promoter malleability of T7 RNAP make the enzyme uniquely suited for use in synthetic gene circuits. Importantly,
  • any modifications made to the enzyme increase the possible functionality of circuits. For instance, we recently utilized
  • a split version of T7 RNAP in conjunction with promoter specificity mutants to create a library of transcriptional AND gates (Shis & Bennett, 2013).

The split version of T7 RNAP was originally discovered during purification and shown to be active in vitro (Ikeda & Richardson, 1987). While the catalytic core and DNA‐binding domain

  • are both located on the C‐terminal fragment of split T7 RNAP,
  • the N‐terminal fragment is needed for transcript elongation.

Therefore, if the two halves of split T7 RNAP are placed behind two different inducible promoters,

  1. both inputs must be active in order to form a functional enzyme and
  2. activate a downstream gene.

When the split mutant is combined with promoter specificity mutants,

  • a library of transcriptional AND gates is created.

Segall‐Shapiro et al take the idea of splitting T7 RNAP for novel regulatory architectures one step further. Instead of settling for the one split site already discovered,

  • the authors first streamlined a transposon mutagenesis strategy (Segall‐Shapiro et al2011) to identify four novel cut sites within T7 RNAP.

By expressing T7 RNAP split at two different sites,

  • they create a tripartite T7 RNAP—a polymerase
  • that requires all three subunits for activity.

The authors suggestively designate the fragments of the tripartite enzyme as ‘core’, ‘alpha’, and ‘sigma’ (Fig 1) and they go on to show that

  • tripartite T7 RNAP can not only be used to create 3‐input AND gates, but
  • it also works as a ‘resource allocator’.

In other words, the transcriptional activity of the split polymerase can be regulated

  • by limiting the availability of core and/or alpha fragment, or
  • by expressing additional sigma fragments.

The authors demonstrate strategies to account for common pitfalls in synthetic gene networks

  • such as host toxicity and plasmid copy number variability.

 

Figure 1. Segall‐Shapiro et al extend previous efforts to engineer split T7 RNAP by fragmenting the enzyme at two novel locations to create a tripartite transcription complex.

Co‐expressing different sigma fragments with the alpha and core fragments enables a network of multi‐input transcriptional AND gates.

The tripartite T7 RNAP presented by Segall‐Shapiro et al

  • expands the utility of T7 RNAP in orthogonal gene circuits.

Until now, while T7 RNAP has been attractive for use in synthetic gene circuits,

  • the inability to regulate its activity has often prevented its use.

Splitting the protein into fragments and regulating the transcription complex by fragment availability

  • brings the regulation of T7 RNAP closer to the regulation of multi‐subunit prokaryotic RNA polymerases.

Sigma fragments direct the activity of the transcription complex much like σ‐factors, and the alpha fragment helps activate transcription

  • in the same way as α‐fragments of prokaryotic polymerases.

For additional regulation, the authors note that the tripartite T7 RNAP can be further split at the previously discovered split site to create a four‐fragment enzyme.

More nuanced regulation using split T7 RNAP may be possible

  • with the addition of heterodimerization domains
  • that can drive the specific association of fragments.

This strategy has been successfully applied to engineer specificity and signal diversity

  • in two‐component signaling pathways (Whitaker et al2012).

The activity of T7 RNAP might also be directed to various promoters

  • by using multiple sigma fragments simultaneously,
  • just as σ‐factors do in E. coli.

Finally, synthetic gene circuits driven primarily by T7 RNAP create the possibility of easily transplantable gene circuits. A synthetic gene circuit driven entirely by fragmented T7 RNAP

  • would depend more on fragment availability than unknown interactions with host metabolism.

This would enable rapid prototyping of synthetic gene circuits in laboratory‐friendly strains or cell‐free systems (Shin & Noireaux, 2012) before transplantation into the desired host.

References

  1. Chelliserrykattil J, Cai G, Ellington AD (2001) A combined in vitro/in vivo selection for polymerases with novel promoter specificities. BMC Biotechnol 1: 13

CrossRefMedline

  1. Ikeda RA, Richardson CC (1987) Interactions of a proteolytically nicked RNApolymerase of bacteriophageT7 with its promoter. J Biol Chem 262: 3800–3808

Abstract/FREE Full Text

  1. SegallShapiro TH, Meyer AJ, Ellington AD, Sontag ED, Voigt CA (2014) A “resource allocator” for transcription based on a highly fragmented T7 RNA polymerase.Mol Syst Biol 10: 742

Abstract/FREE Full Text

  1. SegallShapiro TH, Nguyen PQ, Dos Santos ED, Subedi S, Judd J, Suh J, Silberg JJ(2011) Mesophilic and hyperthermophilic adenylate kinases differ in their tolerance to random fragmentation. J Mol Biol 406: 135–148

CrossRefMedline

  1. Shin J, Noireaux V (2012) An  coli cellfree expression toolbox: application to synthetic gene circuits and artificial cells. Acs Synth Biol 1: 29–41

CrossRefMedlineWeb of Science

  1. Shis DL, Bennett MR (2013) Library of synthetic transcriptional AND gates built with split T7 RNA polymerase mutants. Proc Natl Acad Sci USA 110: 5028–5033

Abstract/FREE Full Text

  1. Studier FW, Moffatt BA (1986) Use of bacteriophageT7 RNApolymerase to direct selective highlevel expression of cloned genes. J Mol Biol 189: 113–130

CrossRefMedlineWeb of Science

  1. Way JC, Collins JJ, Keasling JD, Silver PA (2014) Integrating biological redesign: where synthetic biology came from and where it needs to go. Cell 157: 151–161
  2. Whitaker WR, Davis SA, Arkin AP, Dueber JE (2012) Engineering robust control of twocomponent system phosphotransfer using modular scaffolds. Proc Natl Acad Sci USA 109: 18090–18095

Abstract/FREE Full Text

© 2014 The Authors. Published under the terms of the CC BY 4.0 license

 

 

MicroRNA References

Plasma microRNAs serve as biomarkers of therapeutic efficacy and disease progression in hypertension-induced heart failure. Dickinson BA, Semus HM, Montgomery RL, Stack C, Latimer PA, et al. Eur J Heart Fail. 2013 Jun; 15(6):650-9.  http://dx.doi.org:/10.1093/eurjhf/hft018

Circulating microRNAs – Biomarkers or mediators of cardiovascular disease?  S Fichtlscherer, AM Zeiher, S Dimmeler. Arteriosclerosis, Thrombosis, and Vascular Biology. 2011; 31:2383-2390.
http://dx.doi.org:/10.1161/​ATVBAHA.111.226696

Circulating microRNAs as diagnostic biomarkers for cardiovascular diseases. AJ Tijsen, YM Pinto, and EE Creemers. Am J Physiol Heart Circ Physiol 303: H1085–H1095, 2012.  http://dx.doi.org:/10.1152/ajpheart.00191.2012.

MicroRNAs in Patients on Chronic Hemodialysis (MINOS Study). Emilian C, Goretti E, Prospert F, Pouthier D, Duhoux P, et al. Clin J Am Soc Nephrol  (CJASN)2012;  7: 619-623. http://dx.doi.org:/10.2215/CJN.10471011

Plasma microRNAs serve as biomarkers of therapeutic efficacy and disease progression in hypertension-induced heart failure.  BA Dickinson, HM Semus, RL Montgomery, C Stack, PA Latimer, et al. Eur J Heart Fail 2013 Jun 6;15(6):650-9. http://www.pubfacts.com/detail/23388090/Plasma-microRNAs-serve-as-biomarkers-of-therapeutic-efficacy-and-disease-progression-in-hypertension

Circulating MicroRNAs: Novel Biomarkers and Extracellular Communicators in Cardiovascular Disease?  Esther E. Creemers, Anke J. Tijsen, Yigal M. Pinto.  Circulation Research. 2012; 110: 483-495    http://dx.doi.org:/10.1161/​CIRCRESAHA.111.247452

Novel techniques and targets in cardiovascular microRNA research.  Dangwal S, Bang C, Thum T. Cardiovasc Res. 2012 Mar 15; 93(4):545-54.  http://dx.doi.org:/10.1093/cvr/cvr297

Microparticles: major transport vehicles for distinct microRNAs in circulation. Diehl P, Fricke A, Sander L, Stamm J, Bassler N, Htun N, et al.  Cardiovasc Res. 2012 Mar 15; 93(4):633-44. http://dx.doi.org:/10.1093/cvr/cvs007.

Profiling of circulating microRNAs: from single biomarkers to re-wired networks. A  ZampetakiP Willeit, I Drozdov, S Kiechl and M Mayr. Cardiovasc Res 2012; 93 (4): 555-562.  http://dx.doi.org:/10.1093/cvr/cvr266

Small but smart–microRNAs in the centre of inflammatory processes during cardiovascular diseases, the metabolic syndrome, and ageing. Schroen B, Heymans S.
Cardiovasc Res. 2012; 93(4):605-613http://dx.doi.org:/10.1093/cvr/cvr268

Therapeutic Inhibition of miR-208a Improves Cardiac Function and Survival During Heart Failure. RL Montgomery, TG Hullinger, HM Semus, BA Dickinson, AG Seto, et al.
http://dx.doi.org:/10.1161/​CIRCULATIONAHA.111.030932

Circulating microRNAs to identify human heart failure.  Seto AG, van Rooij E.
Eur J Heart Fail. 2012;14(2):118-119http://dx.doi.org:/10.1093/eurjhf/hfr179.

Use of Circulating MicroRNAs to Diagnose Acute Myocardial Infarction. Y Devaux,
M Vausort, E Goretti, PV Nazarov, F Azuaje. Clin Chem. 2012; 58:559-567. http://dx.doi.org:/10.1373/clinchem.2011.173823

Next Steps in Cardiovascular Disease Genomic Research–Sequencing, Epigenetics, and Transcriptomics  RB Schnabel, A Baccarelli, H Lin, PT Ellinor, and EJ Benjamin.
Clin Chem . 2012 Jan; 58(1): 113–126.  http://dx.doi.org:/10.1373/clinchem.2011.170423

MicroRNA-133 Modulates the {beta}1-Adrenergic Receptor Transduction Cascade.  A Castaldi, T Zaglia, V Di Mauro, P Carullo, G Viggiani, et al.  Circ. Res.. 2014; 115:273-283.
http://dx.doi.org:/10.1161/​CIRCRESAHA.115.303252

Development of microRNA therapeutics is coming of age.  E van Rooij, S Kauppinen.  EMBO Mol Med.. 2014; 6:851-864.  http://dx.doi.org:/10.15252/emmm.201100899

Pitx2-microRNA pathway that delimits sinoatrial node development and inhibits predisposition to atrial fibrillation.   J Wang, Y Bai, N Li, W Ye, M Zhang,et al. PNAS 2014; 111: 9181-9186.

MicroRNA-126 modulates endothelial SDF-1 expression and mobilization of Sca-1+/Lin- progenitor cells in ischaemia  Cardiovasc Res. 2011; 92:449-455,

The use of genomics for treatment is another matter, and has several factors, e.g., age, residual function after AMI, comorbidities

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A Primer on DNA and DNA Replication

Reporter and Curator: Larry H. Bernstein, MD, FCAP 

 

 

This is the FIRST discussion of a several part series leading from the genome, to protein synthesis (1), posttranslational modification of proteins (2), examples of protein effects on metabolism and signaling pathways (3), and leading to disruption of signaling pathways in disease (4), and effects leading to mutagenesis.

1.  A Primer on DNAand DNA Replication

 

dna-replication-primer-synthesis

 

dna-replication-unwinding

 

dna-replication-ligation

 

dna-replication-primer-removal

 

dna-replication-leading-strand

 

dna-replication-lagging-strand

 

dna-replication-termination

 

 

Polymerase Chain Reaction

Polymerase Chain Reaction

 

 

 

 

 

2. Overview of translational medicine

3. Genes, proteomes, and their interaction

4. Regulation of somatic stem cell Function

5.  Proteomics – The Pathway to Understanding and Decision-making in Medicine

6.  Genomics, Proteomics and standards

7.  Long Non-coding RNAs Can Encode Proteins After All

8.  Proteins and cellular adaptation to stress

9.  Loss of normal growth regulation

 

 

A Primer on DNA and DNA Replication

 

DNA Replication

DNA carries the information for making all of the cell’s proteins. These pro­teins implement all of the functions of a living organism and determine the organism’­s characteristics. When the cell reproduces, it has to pass all of this information on to the daughter cells.

Before a cell can reproduce, it must first replicate, or make a copy of, its DNA. Where DNA replication occurs depends upon whether the cells is a prokaryote or a eukaryote (see the RNA sidebar on the previous page for more about the types of cells). DNA replication occurs in the cytoplasm of prokaryotes and in the nucleus of eukaryotes. Regardless of where DNA replication occurs, the basic process is the same.

The structure of DNA lends itself easily to DNA replication. Each side of the double helix runs in opposite (anti-parallel) directions. The beauty of this structure is that it can unzip down the middle and each side can serve as a pattern or template for the other side (called semi-conservative replication). However, DNA does not unzip entirely. It unzips in a small area called a replication fork, which then moves down the entire length of the molecule.

Eukaryotic DNA replication (Wikipedia), is a conserved mechanism that restricts DNA replication to only once per cell cycle. Eukaryotic DNA replication of chromosomal DNA is central for the duplication of a cell and is necessary for the maintenance of the eukaryotic genome.

DNA replication is the action of DNA polymerases synthesizing a DNA strand complementary to the original template strand. To synthesize DNA, the double-stranded DNA is unwound by DNA helicases ahead of polymerases, forming a replication fork containing two single-stranded templates.

Replication processes permit the copying of a single DNA double helix into two DNA helices, which are divided into the daughter cells at mitosis. The major enzymatic functions carried out at the replication fork are well conserved from prokaryotes to eukaryotes, but the replication machinery in eukaryotic DNA replication is a much larger complex, coordinating many proteins at the site of replication, forming the replisome.[1]

The replisome is responsible for copying the entirety of genomic DNA in each proliferative cell. This process allows for the high-fidelity passage of hereditary/genetic information from parental cell to daughter cell and is thus essential to all organisms. Much of the cell cycle is built around ensuring that DNA replication occurs without errors.[1]

In G1 phase of the cell cycle, many of the DNA replication regulatory processes are initiated. In eukaryotes, the vast majority of DNA synthesis occurs during S phase of the cell cycle, and the entire genome must be unwound and duplicated to form two daughter copies. During G2, any damaged DNA or replication errors are corrected. Finally, one copy of the genomes is segregated to each daughter cell at mitosis or M phase.[2] These daughter copies each contain one strand from the parental duplex DNA and one nascent antiparallel strand.

This mechanism is conserved from prokaryotes to eukaryotes and is known as semiconservative DNA replication. The process of semiconservative replication for the site of DNA replication is a fork-like DNA structure, the replication fork, where the DNA helix is open, or unwound, exposing unpaired DNA nucleotides for recognition and base pairing for the incorporation of free nucleotides into double-stranded DNA.[3]

 

Let’s look at the details:

  1. An enzyme called DNA gyrase makes a nick in the double helix and each side separates
  2. An enzyme called helicase unwinds the double-stranded DNA
  3. Several small proteins called single strand binding proteins(SSB) temporarily bind to each side and keep them separated
  4. An enzyme complex called DNA polymerase“walks” down the DNA strands and adds new nucleotides to each strand. The nucleotides pair with the complementary nucleotides on the existing stand (A with T, G with C).
  5. A subunit of the DNA polymerase proofreads the new DNA
  6. An enzyme called DNA ligaseseals up the fragments into one long continuous strand
  7. The new copies automatically wind up again

Different types of cells replicated their DNA at different rates. Some cells constantly divide, like those in your hair and fingernails and bone marrow cells. Other cells go through several rounds of cell division and stop (including specialized cells, like those in your brainmuscle and heart). Finally, some cells stop dividing, but can be induced to divide to repair injury (such as skin cells and liver cells). In cells that do not constantly divide, the cues for DNA replication/cell division come in the form of chemicals. These chemicals can come from other parts of the body (hormones) or from the environment.

 

 

Pre-replicative_complex

Pre-replicative_complex

 

 

 

 

Diagram of the formation of the pre-replicative complex transforming into an active replisomeMcm 2-7 complex loads onto DNA at replication origins during G1 and unwinds DNA ahead of replicative polymerases.Cdc6 and Cdt1 bring Mcm complexes to replication origins. CDK/DDK-dependent phosphorylation of pre-replicative proteins leads toreplisome assembly and origin firing. Cdc6 and Cdt1 are no longer required and are removed from the nucleus or degraded. Mcms and associated proteins, GINS and Cdc45, unwind DNA to expose template DNA. At this point replisome assembly is completed and replication is initiated. “P” represents phosphorylation.

 

Minichromosome Maintenance Protein Complex[edit]

Main article: Minichromosome maintenance

The assembly of the minichromosome maintenance (Mcm) proteins function together as a complex in the cell. The assembly of the Mcm proteins onto chromatin requires the coordinated function of the Origin Recognition Complex (ORC), Cdc6, and Cdt1.[18] Once the Mcm proteins have been loaded onto the chromatin, ORC and Cdc6 can be removed from the chromatin without preventing subsequent DNA replication. This suggests that the primary role of the pre-replication complex is to correctly load the Mcm proteins.[19]

The Mcm proteins support roles both in the initiation and elongation steps of DNA synthesis.[20] Each Mcm protein is highly related to all others, but unique sequences distinguishing each of the subunit types are conserved across eukaryotes. All eukaryotes have exactly six Mcm protein analogs that each fall into one of the existing classes (Mcm2-7), which suggests that each Mcm protein has a unique and important function.[21]

Minichromosome maintenance proteins have been found to be required for DNA helicase activity and inactivation of any of the six Mcm proteins prevents further progression of the replication fork. This is consistent with the requirement of ORC, Cdc6, and Cdt1 function to assemble the Mcm proteins at the origin of replication.[22] The complex containing all six Mcm proteins creates a hexameric, doughnut like structure with a central cavity.[23] The helicase activity of the Mcm protein complex raises the question of how the ring-like complex is loaded onto the single-stranded DNA. One possibility is that the helicase activity of the Mcm protein complex can oscillate between an open and a closed ring formation to allow single-stranded DNA loading.[6]

Along with the minichromosome maintenance protein complex helicase activity, the complex also has associated ATPase activity.[24] A mutation in any one of the six Mcm proteins reduces the conserved ATP binding sites, which indicates that ATP hydrolysis is a coordinated event involving all six subunits of the Mcm complex.[25] Studies have shown that within the Mcm protein complex are specific catalytic pairs of Mcm proteins that function together to coordinate ATP hydrolysis. For example, Mcm3 but not Mcm6 can activate Mcm6 activity. These studies suggest that the structure for the Mcm complex is a hexamer with Mcm3 next to Mcm7Mcm2 next to Mcm6, and Mcm4 next to Mcm5. Both members of the catalytic pair contribute to the conformation that allows ATP binding and hydrolysis and the mixture of active and inactive subunits create a coordinated ATPase activity that allows the Mcm protein complex to complete ATP binding and hydrolysis as a whole.[26]

The nuclear localization of the minichromosome maintenance proteins is regulated in budding yeast cells. The Mcm proteins are present in the nucleus in G1 stage and S phase of the cell cycle, but are exported to the cytoplasm during the G2 stage and M phase. A complete and intact six subunit Mcm complex is required to enter into the cell nucleus.[27] InS. cerevisiaenuclear export is promoted by cyclin-dependent kinase (CDK) activity. Mcm proteins that are associated with chromatin are protected from CDK export machinery due to the lack of accessibility to CDK.[28]

 

Initiation Complex[edit]

During the G1 stage of the cell cycle, the replication initiation factors, origin recognition complex (ORC), Cdc6, Cdt1, and minichromosome maintenance (Mcm) protein complex, bind sequentially to DNA to form the pre-replication complex (pre-RC). At the transition of the G1 stage to the S phase of the cell cycle, S phase–specific cyclin-dependent protein kinase (CDK) and Cdc7/Dbf4 kinase (DDK) transform the pre-RC into an active replication fork. During this transformation, the pre-RC is disassembled with the loss of Cdc6, creating the initiation complex. In addition to the binding of the Mcm proteins, cell division cycle 45 (Cdc45) protein is also essential for initiating DNA replication.[29][30] Studies have shown that Mcm is critical for the loading of Cdc45 onto chromatin and this complex containing both Mcm and Cdc45 is formed at the onset of the S phase of the cell cycle.[31][32] Cdc45 targets the Mcm protein complex, which has been loaded onto the chromatin, as a component of the pre-RC at the origin of replication during the G1 stage of the cell cycle.[20]

GINS[edit]

The six minichromosome maintenance proteins and Cdc45 are essential during initiation and elongation for the movement of replication forks and for unwinding of the DNA. GINS are essential for the interaction of Mcm and Cdc45 at the origins of replication during initiation and then at DNA replication forks as the replisome progresses.[37][38] The GINS complex is composed of four small proteins Sld5 (Cdc105), Psf1 (Cdc101), Psf2 (Cdc102) and Psf3 (Cdc103), GINS represents ‘go, ichi, ni, san’ which means ‘5, 1, 2, 3’ in Japanese.[39]

Mcm10[edit]

Main article: MCM10

Mcm10 is essential for chromosome replication and interacts with the minichromosome maintenance 2-7 helicase that is loaded in an inactive form at origins of DNA replication. Mcm10 chaperones the catalytic DNA polymerase α and helps stabilize the polymerase.[40]

DDK and CDK Kinases[edit]

Main article: Cyclin-dependent kinase

At the onset of S phase, the pre-replicative complex must be activated by two S phase-specific kinases in order to form an initiation complex at an origin of replication. One kinase is the Cdc7-Dbf4 kinase called Dbf4-dependent kinase (DDK) and the other is cyclin-dependent kinase (CDK).[41] Chromatin-binding assays of Cdc45 in yeast and Xenopus have shown that a downstream event of CDK action is loading of Cdc45 onto chromatin.[30][31] Cdc6 has been speculated to be a target of CDK action, because of the association between Cdc6 and CDK, and the CDK-dependent phosphorylation of Cdc6. The CDK-dependent phosphorylation of Cdc6 has been considered to be required for entry into the S phase.[42]

Elongation[edit]

Eukaryotic_replisome_complex

 

 

 

 

Eukaryotic replisome complex and associated proteins.

The formation of the pre-replicative complex (pre-RC) marks the potential sites for the initiation of DNA replication. Consistent with the minichromosome maintenance complex encircling double stranded DNA, formation of the pre-RC does not lead to the immediate unwinding of origin DNA or the recruitment of DNA polymerases. Instead, the pre-RC that is formed during the G1 of the cell cycle is only activated to unwind the DNA and initiate replication after the cells pass from the G1 to the S phase of the cell cycle.[2]

Once the initiation complex is formed and the cells pass into the S phase, the complex then becomes a replisome. The eukaryotic replisome complex is responsible for coordinating DNA replication. Replication on the leading and lagging strands is performed by DNA polymerase ε and DNA polymerase δ. Many replisome factors including Claspin, And1, replication factor C clamp loader and the fork protection complex are responsible for regulating polymerase functions and coordinating DNA synthesis with the unwinding of the template strand by Cdc45-Mcm-GINS complex. As the DNA is unwound the twist number decreases. To compensate for this the writhe number increases, introducing positive supercoils in the DNA. These supercoils would cause DNA replication to halt if they were not removed. Topoisomerases are responsible for removing these supercoils ahead of the replication fork.

Replication Fork[edit]

The replication fork is the junction the between the newly separated template strands, known as the leading and lagging strands, and the double stranded DNA. Since duplex DNA is antiparallel, DNA replication occurs in opposite directions between the two new strands at the replication fork, but all DNA polymerases synthesize DNA in the 5′ to 3′ direction with respect to the newly synthesized strand. Further coordination is required during DNA replication. Two replicative polymerases synthesize DNA in opposite orientations. Polymerase ε synthesizes DNA on the “leading” DNA strand continuously as it is pointing in the same direction as DNA unwinding by the replisome. In contrast, polymerase δ synthesizes DNA on the “lagging” strand, which is the opposite DNA template strand, in a fragmented or discontinuous manner.

The discontinuous stretches of DNA replication products on the lagging strand are known as Okazaki fragments and are about 100 to 200 bases in length at eukaryotic replication forks. The lagging strand usually contains longer stretches of single-stranded DNA that is coated with single-stranded binding proteins, which help stabilize the single-stranded templates by preventing a secondary structure formation. In eukaryotes, these single-stranded binding proteins are a heterotrimeric complex known as replication protein A(RPA).[56]

Each Okazaki fragment is preceded by an RNA primer, which is displaced by the procession of the next Okazaki fragment during synthesis. RNAse H recognizes the DNA:RNA hybrids that are created by the use of RNA primers and is responsible for removing these from the replicated strand, leaving behind a primer:template junction. DNA polymerase α, recognizes these sites and elongates the breaks left by primer removal. In eukaryotic cells,

Replication_fork.svg

 

 

Depiction of DNA replication at replication fork. a: template strands, b: leading strand, c: lagging strand, d: replication fork, e: RNA primer, f: Okazaki fragment

Leading Strand

Lagging Strand

Replicative DNA Polymerases

 

After the replicative helicase has unwound the parental DNA duplex, exposing two single-stranded DNA templates, replicative polymerases are needed to generate two copies of the parental genome. DNA polymerase function is highly specialized and accomplish replication on specific templates and in narrow localizations. At the eukaryotic replication fork, there are three distinct replicative polymerase complexes that contribute to DNA replication: Polymerase α, Polymerase δ, and Polymerase ε. These three polymerases are essential for viability of the cell.[66]

Because DNA polymerases require a primer on which to begin DNA synthesis, polymerase α (Pol α) acts as a replicative primase. Pol α is associated with an RNA primase and this complex accomplishes the priming task by synthesizing a primer that contains a short 10 nucleotide stretch of RNA followed by 10 to 20 DNA bases.[3] Importantly, this priming action occurs at replication initiation at origins to begin leading-strand synthesis and also at the 5′ end of each Okazaki fragment on the lagging strand.

However, Pol α is not able to continue DNA replication and must be replaced with another polymerase to continue DNA synthesis.[67] Polymerase switching requires clamp loaders and it has been proven that normal DNA replication requires the coordinated actions of all three DNA polymerases: Pol α for priming synthesis, Pol ε for leading-strand replication, and the Pol δ, which is constantly loaded, for generating Okazaki fragments during lagging-strand synthesis.[68]

Cdc45–Mcm–GINS Helicase Complex[edit]

The DNA helicases and polymerases must remain in close contact at the replication fork. If unwinding occurs too far in advance of synthesis, large tracts of single-stranded DNA are exposed. This can activate DNA damage signaling or induce DNA repair processes. To thwart these problems, the eukaryotic replisome contains specialized proteins that are designed to regulate the helicase activity ahead of the replication fork. These proteins also provide docking sites for physical interaction between helicases and polymerases, thereby ensuring that duplex unwinding is coupled with DNA synthesis.[73]

Proliferating Cell Nuclear Antigen[edit]

Main article: proliferating cell nuclear antigen

To strengthen the interaction between the polymerase and the template DNA, DNA sliding clamps associate with the polymerase to promote the processivity of the replicative polymerase. In eukaryotes, the sliding clamp is a homotrimer ring structure known as the proliferating cell nuclear antigen (PCNA). The PCNA ring has polarity with surfaces that interact with DNA polymerases and tethers them securely to the DNA template. PCNA-dependent stabilization of DNA polymerases has a significant effect on DNA replication because PCNAs are able to enhance the polymerase processivity up to 1,000-fold.[85][86] PCNA is an essential cofactor and has the distinction of being one of the most common interaction platforms in the replisome to accommodate multiple processes at the replication fork, and so PCNA is also viewed as a regulatory cofactor for DNA polymerases.[87)

PCNA loading is accomplished by the replication factor C (RFC) complex. The RFC complex is composed of five ATPases: Rfc1, Rfc2, Rfc3, Rfc4 and Rfc5.[88] RFC recognizes primer-template junctions and loads PCNA at these sites.[89][90] The PCNA homotrimer is opened by RFC by ATP hydrolysis and is then loaded onto DNA in the proper orientation to facilitate its association with the polymerase.[91][92] Clamp loaders can also unload PNCA from DNA; a mechanism needed when replication must be terminated.[92]

Termination

The end replication problem is handled in eukaryotic cells by telomere regions and telomerase. Telomeres extend the 3′ end of the parental chromosome beyond the 5′ end of the daughter strand. This single-stranded DNA structure can act as an origin of replication that recruits telomerase. Telomerase is a specialized DNA polymerase that consists of multiple protein subunits and an RNA component. The RNA component of telomerase anneals to the single stranded 3′ end of the template DNA and contains 1.5 copies of the telomeric sequence.[60] Telomerase contains a protein subunit that is a reverse transcriptase called telomerase reverse transcriptase or TERT. TERT synthesizes DNA until the end of the template telomerase RNA and then disengages.[60] This process can be repeated as many times as needed with the extension of the 3′ end of the parental DNA molecule. This 3′ addition provides a template for extension of the 5′ end of the daughter strand by lagging strand DNA synthesis. Regulation of telomerase activity is handled by telomere-binding proteins.

-Working_principle_of_telomerase

 

A depiction of telomerase progressively elongating telomeric DNA.

 

DNA replication is a tightly orchestrated process that is controlled within the context of the cell cycle. Progress through the cell cycle and in turn DNA replication is tightly regulated by the formation and activation of pre-replicative complexs (pre-RCs) which is achieved through the activation and inactivation of cyclin-dependent kinases (Cdks). Specifically it is the interactions of cyclins and cyclin dependent kinases that are responsible for the transition from G1 into S-phase.

 

 

 

 

-Cell_Cycle_

Cell_Cycle_

 

 

 

 

 

 

 

Bhatt et al., GA, 6-26-12

 

Revised_definition_of_gene_and_flow_of_genetic_information

 

 

 

 

 

 

 

 

 

 

Epigenetic_mechanisms

 

– G-quadruplex

It will be exactly 60 years ago in February that James Watson and Francis Crick famously burst into the pub next to their Cambridge laboratory to announce the discovery of the “secret of life”.

What they had actually done was describe the way in which two long chemical chains wound up around each other to encode the information cells need to build and maintain our bodies.

Today, the pair’s modern counterparts in the university city continue to work on DNA’s complexities.

Balasubramanian’s group has been pursuing a four-stranded version of the molecule that scientists have produced in the test tube now for a number of years.

It is called the G-quadruplex. The “G” refers to guanine, one of the four chemical groups, or “bases”, that hold DNA together and which encode our genetic information (the others being adenine, cytosine, and thymine).

The G-quadruplex seems to form in DNA where guanine exists in substantial quantities.

And although ciliates, relatively simple microscopic organisms, have displayed evidence for the incidence of such DNA, the new research is said to be the first to firmly pinpoint the quadruple helix in human cells.

‘Funny target’

The team, led by Giulia Biffi, a researcher in Balasubramaninan’s lab, produced antibody proteins that were designed specifically to track down and bind to regions of human DNA that were rich in the quadruplex structure. The antibodies were tagged with a fluorescence marker so that the time and place of the structures’ emergence in the cell cycle could be noted and imaged.

This revealed the four-stranded DNA arose most frequently during the so-called “s-phase” when a cell copies its DNA just prior to dividing.

Prof Balasubramaninan said that was of key interest in the study of cancers, which were usually driven by genes, or oncogenes, that had mutated to increase DNA replication.

If the G-quadruplex could be implicated in the development of some cancers, it might be possible, he said, to make synthetic molecules that contained the structure and blocked the runaway cell proliferation at the root of tumours.

 

 

John Berger

Founder at Novagon DNA

If the first and core mission of the genetic code is to faithfully replicate the “genetic material” encoded in the DNA and RNA nucleic acids, then every metabolic process must be functioning in a synchronous 24/7 manner. The only way to do this is to use all the purine and pyrmidine nucleotide, nucleoside and bases (ATUIXGC) =7 necessary and sufficient to make RNA first and then with the assistance of Thioredoxin i.e. ferredoxin purple sulphur bacteria to oxidize rna to dna.

In regards to purine metabolism which is my major area of focus. The two purine nucleotides left out of the current genetic code i.e. IMP and XMP have the following functions through their enzymes.1. Begin purine nucleotide synthesis de novo by IMPDH cyclodehydrogenase the last step in closing the purine ring and the current foundation molecular structure for DNA and RNA; 2. HPRT is the main enzyme is purine salvage for IMP and GMP; APRT provides same service for AMP; 3. Finally the last step in purine metabolism is by xanthine oxidase with the assistance of FES and molybendum. In essence the IMP and XMP families were the first to build the nucleic acid molecular structure; design a process to recycle functional side groups while keeping the purine ring intact and finally developing the biochemical pathway to eliminate toxic ammonia NH3 from the CNS and liver/kidneys.

I believe the 7 nucleotide Novagon DNA triplex genetic code should be called the epigenetic code since it works not only in protein metabolism which is 2% of the genome but noncoding intronic regions ie. rna editing, RNAi, piRNA, snMRN, long noncoding RNA and many other small rnas which operate above the level of the dna and rna base pair i.e. epigenesis suppressing or enhancing whole genes and networks of genes which control protein,lipid,carbohydrate and nucleic acid metabolism.

I am in the process of deveoping a 7 code epigenetic primer to control the gene switches which in turn allows the genetic material to be inherited from generation to generation as the species constantly adapts to external and internal stressors and competitive antagonist.

A Conserved Structural Core in Type II Restriction Enzymes.

A Conserved Structural Core in Type II Restriction Enzymes.

 

 

 

 

Dna triplex pic

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Agents that Damage DNA

  • Certain wavelengths of radiation
    • ionizing radiation such as gamma rays and X-rays
    • ultraviolet rays, especially the UV-C rays (~260 nm) that are absorbed strongly by DNA but also the longer-wavelength UV-B that penetrates the ozone shield [Link].
  • Highly-reactive oxygen radicals produced during normal cellular respiration as well as by other biochemical pathways. [Link to further discussion.]
  • Chemicals in the environment
    • many hydrocarbons, including some found in cigarette smoke

  Aflatoxin structures

 

 

 

 

 

Link to description of a test measuring the mutations caused by the hydrocarbon benzopyrene.
    • some plant and microbial products, e.g. the aflatoxins produced in moldy peanuts
  • Chemicals used in chemotherapy, especially chemotherapy of cancers

Types of DNA Damage

  1. All four of the bases in DNA(A, T, C, G)can be covalently modified at various positions.
    • One of the most frequent is the loss of an amino group(“deamination”) — resulting, for example, in a C being converted to a U.
  2. Mismatchesof the normal bases because of a failure of proofreading during DNA replication.
    • Common example: incorporation of the pyrimidineU (normally found only in RNA) instead of T.
  3. Breaksin the backbone.
    • Can be limited to one of the two strands (a single-stranded break, SSB) or
    • on both strands(a double-stranded break (DSB).
    • Ionizing radiation is a frequent cause, but some chemicals produce breaks as well.
  4. CrosslinksCovalent linkagescan be formed between bases
    • on the same DNA strand (“intrastrand”) or
    • on the opposite strand (“interstrand”).

Several chemotherapeutic drugs used against cancers crosslink DNA [Link].

Repairing Damaged Bases

Damaged or inappropriate bases can be repaired by several mechanisms:

  • Direct chemical reversal of the damage
  • Excision Repair, in which the damaged base or bases are removed and then replaced with the correct ones in a localized burst of DNA synthesis. There are three modes of excision repair, each of which employs specialized sets of enzymes.
    1. Base Excision Repair (BER)
    2. Nucleotide Excision Repair (NER)
    3. Mismatch Repair (MMR)

 

 

Gene expression profiles associated with acute myocardial infarction and risk of cardiovascular death

J Kim,  N Ghasemzadeh,  DJ Eapen, NC Chung, JD Storey, AA Quyyumi and G Gibson
Kim et al. Genome Medicine 2014, 6:40
http://genomemedicine.com/content/6/5/40

Background: Genetic risk scores have been developed for coronary artery disease and atherosclerosis, but are not predictive of adverse cardiovascular events. We asked whether peripheral blood expression profiles may be predictive of acute myocardial infarction (AMI) and/or cardiovascular death.

Methods: Peripheral blood samples from 338 subjects aged 62 ± 11 years with coronary artery disease (CAD) were analyzed in two phases (discovery N = 175, and replication N = 163), and followed for a mean 2.4 years for cardiovascular death. Gene expression was measured on Illumina HT-12 microarrays with two different normalization procedures to control technical and biological covariates. Whole genome genotyping was used to support comparative genome-wide association studies of gene expression. Analysis of variance was combined with receiver operating curve and survival analysis to define a transcriptional signature of cardiovascular death.

Results: In both phases, there was significant differential expression between healthy and AMI groups with overall down-regulation of genes involved in T-lymphocyte signaling and up-regulation of inflammatory genes. Expression quantitative trait loci analysis provided evidence for altered local genetic regulation of transcript abundance in AMI samples. On follow-up there were 31 cardiovascular deaths. A principal component (PC1) score capturing covariance of 238 genes that were differentially expressed between deceased and survivors in the discovery phase significantly predicted risk of cardiovascular death in the replication and combined samples (hazard ratio = 8.5, P< 0.0001) and improved the C-statistic (area under the curve 0.82 to 0.91, P= 0.03) after adjustment for traditional covariates.

Conclusions: A specific blood gene expression profile is associated with a significant risk of death in Caucasian subjects with CAD. This comprises a subset of transcripts that are also altered in expression during acute myocardial infarction.

 

Lecture Contents delivered at Koch Institute for Integrative Cancer Research, Summer Symposium 2014: RNA Biology, Cancer and Therapeutic Implications, June 13, 2014 @MIT

Curator of Lecture Contents: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.wordpress.com/wp-admin/post.php?post=23174&action=edit
3:15 – 3:45, 6/13/2014, Laurie Boyer “Long non-coding RNAs: molecular regulators of cell fate”     https://pharmaceuticalintelligence.com/2014/06/13/315-345-2014-laurie-boyer-long-non-coding-rnas-molecular-regulators-of-cell-fate/

 

TAR DNA-binding protein 43

TDP-43 is a transcriptional repressor that binds to chromosomally integrated TAR DNA and represses HIV-1 transcription. In addition, this protein regulates alternate splicing of the CFTR gene. In particular, TDP-43 is a splicing factor binding to the intron8/exon9 junction of the CFTR gene and to the intron2/exon3 region of the apoA-II gene.[2] A similar pseudogene is present on chromosome 20.[3]

TDP-43 has been shown to bind both DNA and RNA and have multiple functions in transcriptional repression, pre-mRNA splicing and translational regulation.

TDP-43 was originally identified as a transcriptional repressor that binds to chromosomally integrated trans-activation response element (TAR) DNA and represses HIV-1 transcription.[1] It was also reported to regulate alternate splicing of theCFTR gene and the apoA-II gene.

In spinal motor neurons TDP-43 has also been shown in humans to be a low molecular weight microfilament (hNFL) mRNA-binding protein.[4] It has also shown to be a neuronal activity response factor in the dendrites of hippocampal neurons suggesting possible roles in regulating mRNA stability, transport and local translation in neurons.[5]

Clinical significance[edit]

Hyper-phosphorylatedubiquitinated and cleaved form of TDP-43, known as pathologic TDP43, is the major disease protein in ubiquitin-positive, tau-, and alpha-synuclein-negative frontotemporal dementia (FTLD-TDP, previously referred to as FTLD-U[6]) and in Amyotrophic lateral sclerosis (ALS).[7] Elevated levels of the TDP-43 protein have also been identified in individuals diagnosed with chronic traumatic encephalopathy, a condition that often mimics ALS and that has been associated with athletes who have experienced multiple concussions and other types of head injury.[8]

HIV-1, the causative agent of acquired immunodeficiency syndrome (AIDS), contains an RNA genome that produces a chromosomally integrated DNA during the replicative cycle. Activation of HIV-1 gene expression by the transactivator “Tat” is dependent on an RNA regulatory element (TAR) located “downstream” (i.e. to-be transcribed at a later point in time) of the transcription initiation site.

Mutations in the TARDBP gene are associated with neurodegenerative disorders including frontotemporal lobar degeneration and amyotrophic lateral sclerosis (ALS).[9] In particular, the TDP-43 mutants M337V and Q331K are being studied for their roles in ALS.[10][11] Cytoplasmic TDP-43 pathology is the dominant histopathological feature of multisystem proteinopathy.[12]

 

 

General annotation (Comments)

Function DNA and RNA-binding protein which regulates transcription and
splicing. Involved in the regulation of CFTR splicing. It promotes
CFTR exon 9 skipping by binding to the UG repeated motifs in the
polymorphic region near the 3′-splice site of this exon. The resulting
aberrant splicing is associated with pathological features typical of
cystic fibrosis. May also be involved in microRNA biogenesis,
apoptosis and cell division. Can repress HIV-1 transcription by
binding to the HIV-1 long terminal repeat. Stabilizes the low
molecular weight neurofilament (NFL) mRNA through a direct
interaction with the 3′ UTR. Ref.2 Ref.12
Subunit structure Homodimer. Interacts with BRDT By similarity. Binds specifically to
pyrimidine-rich motifs of TAR DNA and to single stranded TG
repeated sequences. Binds to RNA, specifically to UG repeated
sequences with a minimun of six contiguous repeats. Interacts with
ATNX2; the interaction is RNA-dependent. Ref.16
Subcellular location Nucleus. Note: In patients with frontotemporal lobar degeneration
and amyotrophic lateral sclerosis, it is absent from the nucleus of
affected neurons but it is the primary component of cytoplasmic
ubiquitin-positive inclusion bodies. Ref.2 Ref.11
Tissue specificity Ubiquitously expressed. In particular, expression is high in pancreas,
placenta, lung, genital tract and spleen.
Domain The RRM domains can bind to both DNA and RNA By similarity.
Post-translational modification Hyperphosphorylated in hippocampus, neocortex, and spinal cord
from individuals affected with ALS and FTLDU. Ref.11Ubiquitinated in hippocampus, neocortex, and spinal cord from
individuals affected with ALS and FTLDU. Ref.2 Ref.11  Cleaved to
generate C-terminal fragments in hippocampus, neocortex, and
spinal cord from individuals affected with ALS and FTLDU.
Involvement in disease Amyotrophic lateral sclerosis 10 (ALS10) [MIM:612069]: A
neurodegenerative disorder affecting upper motor neurons in the
brain and lower motor neurons in the brain stem and spinal cord,
resulting in fatal paralysis. Sensory abnormalities are absent. The
pathologic hallmarks of the disease include pallor of the corticospinal
tract due to loss of motor neurons, presence of ubiquitin-positive
inclusions within surviving motor neurons, and deposition of
pathologic aggregates. The etiology of amyotrophic lateral sclerosis is likely to be multifactorial, involving both genetic and environmental factors. The disease is inherited in 5-10% of the cases.  Note: The disease is caused by mutations affecting the gene represented in this
entry.

  1. 16Ref.21 Ref.22 Ref.23 Ref.24 Ref.25 Ref.26 Ref.27 Ref.28 Ref.29 Ref.30 Ref.31Ref.32
Sequence similarities Contains 2 RRM (RNA recognition motif) domains.

 

 

How DNA is made?

Deoxyribonucleic acid (DNA) synthesis is a process by which copies of nucleic acid strands are made. In nature, DNA synthesis takes place in cells by a mechanism known as DNA replication. Using genetic engineering and enzyme chemistry, scientists have developed man-made methods for synthesizing DNA. The most important of these is poly-merase chain reaction (PCR). First developed in the early 1980s, PCR has become a multi-billion dollar industry with the original patent being sold for $300 million dollars.

History

DNA was discovered in 1951 by Francis Crick, James Watson, and Maurice Wilkins. Using x-ray crystallography data generated by Rosalind Franklin, Watson and Crick determined that the structure of DNA was that of a double helix. For this work, Watson, Crick, and Wilkins received the Nobel Prize in Physiology or Medicine in 1962. Over the years, scientists worked with DNA trying to figure out the “code of life.” They found that DNA served as the instruction code for protein sequences. They also found that every organism has a unique DNA sequence and it could be used for screening, diagnostic, and identification purposes. One thing that proved limiting in these studies was the amount of DNA available from a single source.

After the nature of DNA was determined, scientists were able to examine the composition of the cellular genes. A gene is a specific sequence of DNA base pairs that provide the code for the construction of a protein. These proteins determine the traits of an organism, such as eye color or blood type. When a certain gene was isolated, it became desirable to synthesize copies of that molecule. One of the first ways in which a large amount of a specific DNA was synthesized was though genetic engineering.

Genetic engineering begins by combining a gene of interest with a bacterial plasmid. A plasmid is a small stretch of DNA that is found in many bacteria. The resulting hybrid DNA is called recombinant DNA. This new recombinant DNA plasmid is then injected into bacterial cells. The cells are then cloned by allowing it to grow and multiply in a culture. As the cells multiply so do copies of the inserted gene. When the bacteria has multiplied enough, the multiple copies of the inserted gene can then be isolated. This method of DNA synthesis can produce billions of copies of a gene in a couple of weeks.

In 1983, the time required to produce copies of DNA was significantly reduced when Kary Mullis developed a process for synthesizing DNA called polymerase chain reaction (PCR). This method is much faster than previous known methods producing billions of copies of a DNA strand in just a few hours. It begins by putting a small section of double stranded DNA in a solution containing DNA polymerase, nucleotides and primers. The solution is heated to separate the DNA strands. When it is cooled, the polymerase creates a copy of each strand. The process is repeated every five minutes until the desired amount of DNA is produced. In 1993, Mullis’s development of PCR earned him the Nobel Prize in Chemistry.

Background

The key to understanding DNA synthesis is understanding its structure. Typically, DNA exists as two chains of chemically linked nucleotides. These links follow specific patterns dictated by the base pairing rules. Each nucleotide is made up of a deoxyribose sugar molecule, a phosphate group, and one of four nitrogen containing bases. The bases include the pyrimidines thymine (T) and cytosine (C)and the purines adenine (A) and guanine (G). In DNA, adenine generally links with thymine and guanine with cytosine. The molecule is arranged in a structure called a double helix which can be imagined by picturing a twisted ladder or spiral staircase. The bases make up the rungs of the ladder while the sugar and phosphate portions make up the ladder sides. The order in which the nucleotides are linked, called the sequence, is determined by a process known as DNA sequencing.

In a eukaryotic cell, DNA synthesis occurs just prior to cell division through a process called replication. When replication begins the two strands of DNA are separated by a variety of enzymes. Thus opened, each strand serves as a template for producing new strands. This whole process is catalyzed by an enzyme called DNA polymerase. This molecule brings corresponding, or complementary, nucleotides in line with each of the DNA strands. The nucleotides are then chemically linked to form new DNA strands which are exact copies of the original strand. These copies, called the daughter strands, contain half of the parent DNA molecule and half of a whole new molecule. Replication by this method is known as semiconservative replication.

Raw Materials

The primary raw materials used for DNA synthesis include DNA starting materials, taq DNA polymerase, primers, nucleotides, and the buffer solution. Each of these play an important role in the production of millions of DNA molecules.

Controlled DNA synthesis begins by identifying a small segment of DNA to copy. This is typically a specific sequence of DNA that contains the code for a desired protein. Called template DNA, this material must be highly purified.

While the process of DNA replication was known before 1980, PCR was not possible because there were no known heat stable DNA polymerases.  In the early 1980s, scientists found bacteria living around natural steam vents. It turned out that these organisms, called thermus aquaticus, had a DNA polymerase that was stable and functional at extreme levels of heat. This taq DNA polymerase became the cornerstone for modern DNA synthesis techniques. During a typical PCR process, 2-3 micrograms of taq DNA polymerase is needed.

The polymerase builds the DNA strands by combining corresponding nucleotides on each DNA strand. Chemically speaking, nucleotides are made up of three types of molecular groups including a sugar structure, a phosphate group, and a cyclic base. The sugar portion provides the primary structure for all nucleotides. In general, the sugars are composed of five carbon atoms with a number of hydroxy (-OH) groups attached. For DNA, the sugar is 2-deoxy-D-ribose. The defining part of a nucleotide is the hetero-cyclic base that is covalently bound to the sugar. These bases are either pyrimidine or purine groups, and they form the basis for the nucleic acid code. Two types of purine bases are found including adenine and guanine. In DNA, two types of pyrimidine bases are present, thymine and cytosine. A phosphate group makes up the final portion of a nucleotide. This group is derived from phosphoric acid and is covalently bonded to the sugar structure on the fifth carbon.

cost of oligo and gene synthesis

 

 

 

 

The first phase of polymerase chain reaction (PCR) involves the denaturation of DNA. This “opening up” of the DNA molecule provides the template for the next DNA molecule from which to be produced. With the DNA split into separate strands, the temperature is lowered—the primer annealing step. During the next phase, the DNA polymerase interacts with the strands and adds complementary nucleotides along the entire length. The time required at this phase is about one minute for every 1,000 base pairs.

To initiate DNA synthesis, short primer sections of DNA must be used. These primer sections, called oligo fragments, are about 18-25 nucleotides in length and correspond to a section on the template DNA. They typically have a C and G nucleotide concentration of about 60% with even distribution. This provides the maximum efficiency in the synthesis process.

The buffer solution provides the medium in which DNA synthesis can occur. This is an aqueous solution which contains MgCl2, HCI, EDTA, and KCI. The MgCl2 concentration is important because the Mg2+ ions interact with the DNA and the primers creating crucial complexes for DNA synthesis. The pH of this system is critical so it may also be buffered with ammonium sulfate. To energize the reaction, various energy molecules are added such as ATP, GTP, and NTP.

DNA synthesis involves three distinct processes, typically done in separate areas to avoid contamination, including sample preparation, DNA synthesis reaction cycle and DNA isolation. Following these procedures scientists are able to convert a few strands of DNA into millions and millions of exact copies.

Preparation of the samples

  • 1 Typically, all of the starting solutions except the primers, polymerases and the dNTPs are put in an autoclave to kill off any contaminating organism. Two separate solutions are made. One contains the buffer, primers and the polymerase. The other contains the MgCl2 and the template DNA. These solutions are all put into small tubes to begin the reaction.

 

Kary Banks Mullis.

Kary Banks Mullis was born in Lenoir, North Carolina, in 1944. Upon graduation from Georgia Tech in 1966 with a B.S. in chemistry, Muilis entered the biochemistry doctoral program at the University of California, Berkeley. Earning his Ph.D. in 1973, he accepted a teaching position at the University of Kansas Medical School in Kansas City. In 1977, he assumed a postdoctoral fellowship at the University of California, San Francisco.

Muilis accepted a position as a research scientist in 1979 with a growing biotech firm—Cetus Corporation, in Emeryville, California—that synthesized chemicals used by other scientists in genetic cloning. While there, he designed polymerase chain reaction (PCR), a fast and effective technique for reproducing specific genes or DNA (deoxyribonucleic acid) fragments that can create billions of copies in a few hours. The most effective way to reproduce DNA was by cloning, but it was problematic. It took time to convince Mullis’s colleagues of the importance of this discovery but soon PCR became the focus of intensive research. Scientists at Cetus developed a commercial version of the process and a machine called the Thermal Cycler (with the addition of the chemical building blocks of DNA [nucleotides] and a biochemical catalyst [polymerase], the machine would perform the process automatically on a target piece of DNA).

Read more: http://www.madehow.com/Volume-6/DNA-Synthesis.html#ixzz38sovuX5n

types_RNAi_Q3_used_in_research

 

lncRNA-s   A summary of the various functions described for lncRNA

 

Additional References to Leaders in Pharmaceutical Intelligence

Content Consultant: Larry H Bernstein, MD, FCAP

Series C: e-Books on Cancer & Oncology

Content Consultant: Larry H Bernstein, MD, FCAP.

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