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

http://pharmaceuticalintelligence.com/7/17/2014/Genes, proteomes, and their interaction

This is the third 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

• http://pharmaceuticalintelligence.com/2014-28-07/A_Primer_on_DNA_and_DNA_Replication

2. Overview of translational medicine

• http://pharmaceuticalintelligence.com/2014-07-28/larryhbern/overview-of-posttranslational-medicine

3. Genes, proteomes, and their interaction

•  http://pharmaceuticalintelligence.com/7/17/2014/Genes, proteomes, and their interaction

4. Regulation of somatic stem cell Function

•  http://pharmaceuticalintelligence.com/2013-01-23/larryhbern/Regulation-of-somatic-stem-cell-function/

5.  Proteomics – The Pathway to Understanding and Decision-making in Medicine

• http://pharmaceuticalintelligence.com/2014/06/24/proteomics-the-pathway-to-understanding-and-decision-making-in-medicine/

6.  Genomics, Proteomics and standards

• http://pharmaceuticalintelligence.com/2014/07/06/genomics-proteomics-and-standards/

7.  Long Non-coding RNAs Can Encode Proteins After All

• http://pharmaceuticalintelligence.com/2014/06/29/long-non-coding-rnas-can-encode-proteins-after-all/
• 3:15 – 3:45, 2014, Laurie Boyer “Long non-coding RNAs: molecular regulators of cell fate”; http://pharmaceuticalintelligence.com/2014/06/04/koch-institute-for-integrative-cancer-research-mit-summer-symposium-2014-rna-biology-cancer-and-therapeutic-implications-june-13-2014-830am-430pm-kresge-auditorium-mit/

8.  Proteins and cellular adaptation to stress

• http://pharmaceuticalintelligence.com/2014/07/08/proteins-and-cellular-adaptation-to-stress/

9.  Loss of normal growth regulation

• http://pharmaceuticalintelligence.com/2014/07/06/loss-of-normal-growth-regulation/

This discussion is the beginning of a diversion away from the routine discussion of a specific sequence and pairing of nucleotides in the classic model, to explore the interaction between proteins, or folded proteins and RNA or hidtones that reside in the nucleus and contribute to induction or inactivation of gene expression.  The basic text document is rigid, inflexible, and resides in all cells.  Yet, in bacteria, yeast, and eukaryotic cells, there are models of gene expression, and in eukaryotes, there is the development of expressed organ systems.  These systems have similar proteins or enzymes that are functionally identical, but they have isoforms that bind with proteins, membranes, lipopolysaccharides, and lipoproteins – which has an impact on the catabolic and anabolic activity of the cells, and they are affected by oxidative stress, and they are often dependent on the energy of binding with metal ions,i.e., Mn, Cu, Cd, Zn,..,Fe, and in other cases anionic ligands, such as I, and they may transiently act through a nucleotide or influenced by a hormone.

This will be presented as a group of predetermined articles to follow:

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

2.  Thyroid Hormone Key to Lipid Kinase Regulation

3.  Mammalian Target of Rapamycin Complex 1 Orchestrates Invariant NKT Cell Differentiation and Effector Function

4   The E3 ligase PARC mediates the degradation of cytosolic cytochrome c to promote survival in neurons and cancer cells

5.  Nf k-beta signaling pathway

6.  P181 cAMP-mediated Rac1 activation regulates the re-establishment of endothelial adherens junctions and barrier restoration during inflammation.

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

8.  Protein misfolding, congophilia, oligomerization, and defective amyloid processing in preeclampsia

9.  Removing parts of shape-shifting protein explains how blood clots

1.  Added Layers of Proteome Complexity

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

By Anna Azvolinsky | July 17, 2014

added layers of proteome

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.

Tags  tRNA, proteomics, protein synthesis and human proteome project

2. Thyroid Hormone Key to Lipid Kinase Regulation

Published: Jul 16, 2014 | Updated: Jul 17, 2014
By Salynn Boyles, Contributing Writer, MedPage Today
Reviewed by Zalman S. Agus, MD; Emeritus Professor, Perelman School of Medicine at the University of Pennsylvania and
Dorothy Caputo, MA, BSN, RN, Nurse Planner

Action Points

• Thyroid hormone is an essential regulator of human growth, brain maturation, and adult cognition and metabolism.
• This study provides evidence that cytoplasmic thyroid hormone signaling through phosphatidylinositol 3-kinase appears to be an essential mechanism underlying normal synaptic maturation and plasticity in the postnatal mouse hippocampus

Thyroid hormones are key for brain development and synaptic maturation, and researchers have identified a specific molecular mechanism for rapid lipid kinase activation by the thyroid hormone receptor beta (TR-beta) that involves a cytoplasmic complex of the gene.

Many effects of the thyroid hormone on mammalian cells in vitro have been shown to be mediated by the phosphatidylinositol 3-kinase (PI3K), but the molecular mechanism of PI3K regulation and its relevance to brain development have not been clear, according to David L. Armstrong, PhD, of the National Institute of Environmental Health and Development in Research Triangle, N.C., and colleagues.

They identified a specific molecular mechanism for rapid PI3 kinase activation by TR-beta which involves a cytoplasmic complex of TR-beta, the p85 regulatory subunit of PI3 kinase and the Src family kinase, Lyn, they wrote in Endocrinology.Armstrong’s co-authors are from Duke University and Loyola University in Chicago.

This complex provides a unique mechanism for integrating growth signals through thyroid hormone and receptor tyrosine kinases, they explained.

“Most everyone agrees that thyroid hormones are essential for brain development and synaptic maturation, but we didn’t know how exactly,” Armstrong told MedPage Today. “We show that nongenomic signaling in TR-beta through PI3 kinase is essential for one of its physiological actions.”

The Role of T3 Hormone

The recognition that many hormones regulate gene expression through receptor proteins that bind to DNA is a major biological discovery over the past 50 years, the researchers noted.

“More recently, it has become clear that in many cases the same hormones produce rapid effects on cell physiology though the same receptors signaling in the cytoplasm,” they wrote. “However, testing the relative importance of the genomic and nongenomic mechanisms in vivo has been prevented by the absence of specific molecular mechanisms for the nongenomic effects that could be blocked by mutation of the receptor without disrupting its direct effects on gene expression.”

The thyroid hormone T3 has been shown to be a regulator of many physiological effects, including human growth, brain maturation, and adult cognition and metabolism.

Many of these effects have been found to be mediated through the regulation of gene expression by zinc-finger nuclear receptor proteins that are encoded by the THRA and THRB genes. But many in vitro effects of T3 are too rapid to be explained by transcriptional regulation, Armstrong and colleagues noted.

In earlier work, they identified PI3 kinase as a key player in these rapid effects. Like thyroid hormone, PI3 kinase activity has been identified as essential for growth, metabolism, and brain development.

PI3 kinase is regulated primarily by receptor tyrosine kinases, and an integrin receptor has been identified that mediates some of the PI3 kinase-dependent effects of thyroxine (T4), the widely circulated precursor of T3.

Both TR-alpha and TR-beta have also been reported to associate with PI3 kinase and stimulate its activity in many cell types. In a 2006 study in the Proceedings of the National Academy of Sciences, Armstrong and colleagues demonstrated that TRis required to reconstitute T3 and PI3 kinase-dependent regulation of Kv11.1 channels in cell-free membrane patches from Chinese hamster ovary (CHO) cells.

Based on that research, they concluded that TR-beta signaling through PI3K “provides a molecular explanation for the essential role of thyroid hormone in human brain development and adult lipid metabolism.”

Measuring PIP3 Production

In the newly reported series of experiments, the researchers used fluorescent PIP₃ indicator to directly measure PIP₃ production in response to thyroid hormone on the same time scale as the electrophysiological measurements in the CHO cells expressing recombinant human thyroid hormone receptors.

The research revealed that, in the absence of hormone, the nuclear receptor TR-beta forms a cytoplasmic complex with the p85 subunit of PI3 kinase and the Src family tyrosine kinase, Lyn, which depends on two canonical phosphotyrosine motifs in the second zinc finger of TR that are not conserved in  TR-beta

“When hormone is added, [TR-beta] dissociates and moves to the nucleus, and PIP₃production goes up rapidly,” the researchers wrote. “Mutating either tyrosine to a phenylalanine prevents rapid signaling through PI3 kinase but does not prevent hormone-dependent transcription of genes with a thyroid hormone response element.”

“It is only when you have both thyroid hormone and phosphotyrosine signaling that you get maximal stimulation of PI3 kinase,” Armstrong said, adding that the novel methodology of the study, which involved serum from thyroidectomized animals, led to the finding.

These experiments led to in vivo research to test the physiological relevance of thyroid hormone signaling through PI3 kinase for brain development in a novel mouse line created by the researchers.

“We reasoned that blocking binding of TR-beta to p85 by mutating Y171 might eliminate any dominant negative effect of the mutant, in much the same way that receptor knockdown proved much less deleterious to the organism than hormone withdrawal, presumably because many of the effects of the receptor on gene expression are mediated by binding of the unliganded receptor,” they wrote.

They created a novel mouse line with a targeted mutation knocked into the THRB gene to substitute phenylalanine for tyrosine at residue 147 of TR-beta-1, which prevents Lyn binding to the mutant receptor.

They confirmed that the mutation did not alter total circulating levels of thyroxine (T4) or T3 by mass spectrometry of serum samples from 4-month-old mice.

“When the rapid signaling mechanism was blocked chronically throughout development in mice by a targeted point mutation in both alleles of THRB, circulating hormone levels, TR-betaexpression, and direct gene regulation by TR-beta in the brain and liver were all unaffected,” the researchers wrote. “The mutation did significantly impair maturation and plasticity of the Schaffer collateral synapses on CA1 pyramidal neurons in the postnatal hippocampus. Thus, phosphotyrosine-dependent association of TR-betawith PI3K provides a potential mechanism for integrating regulation of development and metabolism by thyroid hormone and receptor tyrosine kinases.”

A Novel Finding

The finding that thyroid hormone signaling through PI3 kinase appears to be an essential mechanism underlying normal synaptic maturation and plasticity in the postnatal mouse hippocampus is novel.

The researchers noted that they could not formally exclude some more subtle effects of the mutation on the regulation of an unknown gene that plays as central a role in synaptic development as PI3K, but the added that “our results do categorically rule out a role for other thyroid hormone receptors in this particular aspect of synaptic maturation in the mouse hippocampus.

“In either case, given the importance of thyroid hormone signaling for human brain development and adult metabolism, future studies will need to investigate whether PI₃ kinase stimulation by thyroid hormone is also susceptible to disruption by environmental toxicants,” they wrote.

Armstrong also pointed out that the tyrosine motifs in TR-beta, which were shown to be essential for signaling through PI3 kinase, are present in all mammals, but not in other species with known genome data, with the exception of the gecko and the axolotl (Mexican salamander).

“Mammals evolved from reptiles, and the thinking is that they survived by adopting a nocturnal niche,” he said. “This is exactly what thyroid hormone does, so it may be that this mutation contributed to the (evolutionary) success of mammals.”

Primary source: Endocrinology
Source reference: Martin NP, et al “A rapid cytoplasmic mechanism for PI3 kinase regulation by the nuclear thyroid hormone receptor, TR beta, and genetic evidence for its role in the maturation of mouse hippocampal synapses in vivo”

Endocrinology 2014;         http://dx.doi.org:/10.1210/en.2013-2058.

3.  Mammalian Target of Rapamycin Complex 1 Orchestrates Invariant NKT Cell Differentiation and Effector Function.

Lianjun Zhang, Benjamin O Tschumi, Stéphanie Corgnac, Markus A Rüegg,Michael N Hall, Jean-Pierre Mach, Pedro Romero, Alena Donda

Journal of immunology (Baltimore, Md. : 1950) 07/2014;     http://dx.doi.org:/10.4049/jimmunol.1400769

Source: PubMed

ABSTRACT Invariant NKT (iNKT) cells play critical roles in bridging innate and adaptive immunity. The Raptor containing mTOR complex 1 (mTORC1) has been well documented to control peripheral CD4 or CD8 T cell effector or memory differentiation. However, the role of mTORC1 in iNKT cell development and function remains largely unknown. By using mice with T cell-restricted deletion of Raptor, we show that mTORC1 is selectively required for iNKT but not for conventional T cell development. Indeed, Raptor-deficient iNKT cells are mostly blocked at thymic stage 1-2, resulting in a dramatic decrease of terminal differentiation into stage 3 and severe reduction of peripheral iNKT cells. Moreover, residual iNKT cells in Raptor knockout mice are impaired in their rapid cytokine production upon αGalcer challenge. Bone marrow chimera studies demonstrate that mTORC1 controls iNKT differentiation in a cell-intrinsic manner. Collectively, our data provide the genetic evidence that iNKT cell development and effector functions are under the control of mTORC1 signaling.

4.  PARC

The E3 ligase PARC mediates the degradation of cytosolic cytochrome c to promote survival in neurons and cancer cells

Vivian Gama^1,2, Vijay Swahari^1,2, Johanna Schafer^1*, Adam J. Kole², Allyson Evans², Yolanda Huang², Anna Cliffe^1,2, Brian Golitz^3,4, Noah Sciaky^3,4, Xin-Hai Pei^5,6, Yue Xiong^5,6, and Mohanish Deshmukh^1,2,5

¹ Neuroscience Center, ² Department of Cell Biology and Physiology, ³ UNC RNAi Screening Facility,⁴ Department of Pharmacology, ⁵ Lineberger Comprehensive Cancer Center, ⁶ Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA.^* Present address: Vanderbilt University, Nashville, TN 37232, USA.  Present address: Cell Press, Cambridge, MA 02139, USA.  Present address: Department of Anesthesiology, Columbia University Medical Center, New York, NY 10032, USA.

Abstract: The ability to withstand mitochondrial damage is especially critical for the survival of postmitotic cells, such as neurons. Likewise, cancer cells can also survive mitochondrial stress. We found that cytochrome c (Cyt c), which induces apoptosis upon its release from damaged mitochondria, is targeted for proteasome-mediated degradation in mouse neurons, cardiomyocytes, and myotubes and in human glioma and neuroblastoma cells, but not in proliferating human fibroblasts. In mouse neurons, apoptotic protease-activating factor 1 (Apaf-1) prevented the proteasome-dependent degradation of Cyt c in response to induced mitochondrial stress. An RNA interference screen in U-87 MG glioma cells identified p53-associated Parkin-like cytoplasmic protein (PARC, also known as CUL9) as an E3 ligase that targets Cyt c for degradation. The abundance of PARC positively correlated with differentiation in mouse neurons, and overexpression of PARC reduced the abundance of mitochondrially-released cytosolic Cyt c in various cancer cell lines and in mouse embryonic fibroblasts. Conversely, neurons from Parc-deficient mice had increased sensitivity to mitochondrial damage, and neuroblastoma or glioma cells in which PARC or ubiquitin was knocked down had increased abundance of mitochondrially-released cytosolic Cyt c and decreased viability in response to stress. These findings suggest that PARC-mediated ubiquitination and degradation of Cyt c is a strategy engaged by both neurons and cancer cells to prevent apoptosis during conditions of mitochondrial stress.
Sci. Signal., 15 July 2014   Vol. 7, Issue 334, p. ra67
http://dx.doi.org:/10.1126/scisignal.2005309

Citation: V. Gama, V. Swahari, J. Schafer, A. J. Kole, A. Evans, Y. Huang, A. Cliffe, B. Golitz, N. Sciaky, X.-H. Pei, Y. Xiong, M. Deshmukh, The E3 ligase PARC mediates the degradation of cytosolic cytochrome c to promote survival in neurons and cancer cells. Sci. Signal. 7, ra67 (2014).

Killing the Killer: PARC/CUL9 Promotes Cell Survival by Destroying Cytochrome c

Jonathan Lopez and Stephen W. G. Tait^*
Cancer Research UK Beatson Institute, Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK.

Abstract: Balanced amounts of apoptotic cell death are essential for health; its deregulation plays key roles in neurodegeneration, autoimmunity, and cancer. Mitochondria orchestrate apoptosis through a process called mitochondrial outer-membrane permeabilization (MOMP). After MOMP, mitochondrial cytochrome c is released into the cytoplasm, where it binds the adaptor molecule APAF1, triggering caspase protease activation and cell death. In this issue of Science Signaling, Deshmukh and colleagues define a new survival mechanism downstream of mitochondrial permeabilization. Specifically, they identify proteasomal degradation of cytochrome c as a major determinant of cell survival. In an unbiased approach, PARC (also known as CUL9) was found to be the ubiquitin ligase responsible for the ubiquitination and proteasomal degradation of cytochrome c. The consequences of this survival process may be double-edged because both cancer cells and postmitotic cells use PARC/CUL9–mediated cytochrome c degradation to ensure cell survival. Ultimately, differential targeting of this process may promote survival of postmitotic tissue or enhance tumor-specific killing.

Citation: J. Lopez, S. W. G. Tait, Killing the Killer: PARC/CUL9 Promotes Cell Survival by Destroying Cytochrome c. Sci. Signal. 7, pe17 (2014).

Sci. Signal., 15 July 2014  Vol. 7, Issue 334, p. pe17
http://dx.doi.org:/10.1126/scisignal.2005619

4. The WNK-SPAK/OSR1 pathway: Master regulator of cation-chloride cotransporters

Dario R. Alessi¹, Jinwei Zhang¹, Arjun Khanna², Thomas Hochdörfer¹, Yuze Shang³, and Kristopher T. Kahle^2,3*
1 MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland.
² Department of Neurosurgery, Massachusetts General Hospital, and Harvard Medical School, ³ Manton Center for Orphan Disease Research, Boston Children’s Hospital, Boston, MA 02115, USA.

Abstract: The WNK-SPAK/OSR1 kinase complex is composed of the kinases WNK (with no lysine) and SPAK (SPS1-related proline/alanine-rich kinase) or the SPAK homolog OSR1 (oxidative stress–responsive kinase 1). The WNK family senses changes in intracellular Cl^– concentration, extracellular osmolarity, and cell volume and transduces this information to sodium (Na⁺), potassium (K⁺), and chloride (Cl^–) cotransporters [collectively referred to as CCCs (cation-chloride cotransporters)] and ion channels to maintain cellular and organismal homeostasis and affect cellular morphology and behavior. Several genes encoding proteins in this pathway are mutated in human disease, and the cotransporters are targets of commonly used drugs. WNKs stimulate the kinases SPAK and OSR1, which directly phosphorylate and stimulate Cl^–-importing, Na⁺-driven CCCs or inhibit the Cl^–-extruding, K⁺-driven CCCs. These coordinated and reciprocal actions on the CCCs are triggered by an interaction between RFXV/I motifs within the WNKs and CCCs and a conserved carboxyl-terminal docking domain in SPAK and OSR1. This interaction site represents a potentially druggable node that could be more effective than targeting the cotransporters directly. In the kidney, WNK-SPAK/OSR1 inhibition decreases epithelial NaCl reabsorption and K⁺ secretion to lower blood pressure while maintaining serum K⁺. In neurons, WNK-SPAK/OSR1 inhibition could facilitate Cl^–extrusion and promote -aminobutyric acidergic (GABAergic) inhibition. Such drugs could have efficacy as K⁺-sparing blood pressure–lowering agents in essential hypertension, nonaddictive analgesics in neuropathic pain, and promoters of GABAergic inhibition in diseases associated with neuronal hyperactivity, such as epilepsy, spasticity, neuropathic pain, schizophrenia, and autism.
Citation: D. R. Alessi, J. Zhang, A. Khanna, T. Hochdörfer, Y. Shang, K. T. Kahle, The WNK-SPAK/OSR1 pathway: Master regulator of cation-chloride cotransporters. Sci. Signal. 7, re3 (2014).

Sci. Signal., 15 July 2014  Vol. 7, Issue 334, p. re3
http://dx.doi.org:/10.1126/scisignal.2005365

5. Nf k-beta signaling pathway

Cracking the NF-B Code

Karen E. Tkach, Jennifer E. Oyler, and Grégoire Altan-Bonnet^*
ImmunoDynamics Group, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

Abstract: The discovery of feedback loops between signaling and gene expression is ushering in new quantitative models of cellular regulation. In a recent issue of Science Signaling, Sung et al. showed how positive feedback downstream of nuclear factor B (NF-B) signaling enhances the capacity of macrophages to scale their antimicrobial responses to the dose of pathogen-associated molecular cues. This finding stemmed from analysis of cell-to-cell variability and computational modeling of time integration between signaling and transcriptional responses. Ultimately, such quantitative approaches challenge the oft-assumed time separation of “fast” signal transduction followed by “slow” gene expression, and they provide a better understanding of complex biological regulation over long time scales.

Citation: K. E. Tkach, J. E. Oyler, G. Altan-Bonnet, Cracking the NF-B Code. Sci. Signal. 7, pe5 (2014).

Sci. Signal., 18 February 2014  Vol. 7, Issue 313, p. pe5
http://dx.doi.org:/10.1126/scisignal.2005108

Switching of the Relative Dominance Between Feedback Mechanisms in Lipopolysaccharide-Induced Nfk-B Signaling

Myong-Hee Sung^1*, Ning Li², Qizong Lao¹, Rachel A. Gottschalk², Gordon L. Hager^1*, and Iain D. C. Fraser^2*
1 Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, ² Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

Abstract: A fundamental goal in biology is to gain a quantitative understanding of how appropriate cell responses are achieved amid conflicting signals that work in parallel. Through live, single-cell imaging, we monitored both the dynamics of nuclear factor B (NF-B) signaling and inflammatory cytokine transcription in macrophages exposed to the bacterial product lipopolysaccharide (LPS). Our analysis revealed a previously uncharacterized positive feedback loop involving induction of the expression of Rela, which encodes the RelA (p65) NF-B subunit. This positive feedback loop rewired the regulatory network when cells were exposed to LPS above a distinct concentration. Paradoxically, this rewiring of NF-B signaling in macrophages (a myeloid cell type) required the transcription factor Ikaros, which promotes the development of lymphoid cells. Mathematical modeling and experimental validation showed that the RelA positive feedback overcame existing negative feedback loops and enabled cells to discriminate between different concentrations of LPS to mount an effective innate immune response only at higher concentrations. We suggest that this switching in the relative dominance of feedback loops (“feedback dominance switching”) may be a general mechanism in immune cells to integrate opposing feedback on a key transcriptional regulator and to set a response threshold for the host.

Citation: M.-H. Sung, N. Li, Q. Lao, R. A. Gottschalk, G. L. Hager, I. D. C. Fraser, Switching of the Relative Dominance Between Feedback Mechanisms in Lipopolysaccharide-Induced NF-B Signaling. Sci. Signal. 7, ra6 (2014).

Sci. Signal., 14 January 2014  Vol. 7, Issue 308, p. ra6
http://dx.doi.org:/10.1126/scisignal.2004764

Drug development in the Alzheimer’s field has been riddled with failures, and most research efforts have focused on pinpointing genetic and environmental factors responsible for causing or accelerating the progression of the disease.

Now, researchers from Montreal’s Douglas Mental Health Institute and McGill University have identified a relatively frequent genetic variant that may provide protection against the devastating neurodegenerative disease.

“We found that specific genetic variants in a gene called HMG CoA reductase which normally regulates cholesterol production and mobilization in the brain can interfere with, and delay the onset of Alzheimer’s disease by nearly four years. This is an exciting breakthrough in a field where successes have been scarce these past few years,” said Dr. Judes Poirier, whose previous research led to the discovery that a genetic variant was formally associated with the common form of Alzheimer’s disease.

This variant may explain why some people who are carriers of predisposing genetic factors for the common form of Alzheimer’s do not develop the disease, living long lives without memory problems until their nineties.

6.  P181 cAMP-mediated Rac1 activation regulates the re-establishment of endothelial adherens junctions and barrier restoration during inflammation.

M Aslam, H Nef, C Troidl, R Schulz, T Noll, C Hamm, D Guenduez

Cardiovascular research 07/2014; 103(suppl 1):S32.
http://dx.doi.org:/10.1093/cvr/cvu082.117
Source: PubMed

ABSTRACT Inflammatory mediators like thrombin and TNFα disrupt endothelial junctions and barrier integrity, leading to edema formation. This increase in endothelial permeability is followed by slow restoration of the endothelial barrier, which is critical for the maintenance of basal endothelial permeability. However, the molecular mechanism of recovery of the endothelial barrier in response to inflammatory mediators has not yet been well delineated. The aim of the present study was to explore the mechanism of this barrier restoration. Specific emphasis was given to the role of Rac1 GTPase activation, which is an important regulator of endothelial adherens junction (AJ) integrity.

7.  Thalidomide

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

Eric S. Fischer, Kerstin Böhm, John R. Lydeard, Haidi Yang, Michael B. Stadler, et al.
Nature (2014)     http://dx.doi.org:/10.1038/nature13527

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 CRL4^CRBN) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4^CRBN. 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 CRL4^CRBN and enantioselectively binds IMiDs. Using an unbiased screen, we identified the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4^CRBN. Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4^CRBN 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.

Figure 1: The overall structure of the DDB1–CRBN complex.

http://www.nature.com/nature/journal/vaop/ncurrent/carousel/nature13527-f1.jpg

a, Cartoon representation of the structure of the complex of human DDB1, G. gallus CRBN and thalidomide: DDB1, highlighting the domains BPA (red), BPB (magenta), BPC (orange) and DDB1-CTD (grey); G. gallus CRBN, highlighting the domain…

Figure 2: IMiD binding to CRBN.

http://www.nature.com/nature/journal/vaop/ncurrent/carousel/nature13527-f2.jpg

a, Chemical structure of lenalidomide. b, Chemical structure of pomalidomide. c, Sketch of thalidomide and its interactions with G. gallus CRBN. Hydrogen bonds are shown as dashed lines, and hydrophobic interactions are indicated as gr

Figure 3: CRBN is a substrate receptor in the ligase CRL4^CRBN.

http://www.nature.com/nature/journal/vaop/ncurrent/carousel/nature13527-f3.jpg

a, Architecture of the CRL4^DDB2 complex bound to DNA (PDB ID 4A0K). b, Model of CRL4^CRBN bound to thalidomide. c, Firefly luciferase (Fluc) to Renillaluciferase (Rluc) ratios (Fluc:Rluc) of IKZF1-reporter-plasmid-transfected HEK 293T…

Figure 5: Molecular model of IMiD function.

http://www.nature.com/nature/journal/vaop/ncurrent/carousel/nature13527-f5.jpg

a, Thalidomide binds to CRBN at the canonical substrate-binding site. b, The potent anti-myeloma drug thalidomide and its derivatives lenalidomide and pomalidomide occupy the same site but with different solvent-exposed moieties. c, Bi…

8. Preeclampsia of pregnancyand protein misfolding

Protein misfolding, congophilia, oligomerization, and defective amyloid processing in preeclampsia

Irina A. Buhimschi^1,2,*, Unzila A. Nayeri², Guomao Zhao¹, Lydia L. Shook², Anna Pensalfini³, et al.
¹Center for Perinatal Research, The Research Institute at Nationwide Children’s Hospital and Department of Pediatrics, ⁴Depart of ObGyn, The Ohio State University College of Medicine, Columbus, OH
²Depart of ObGyn and Reproductive Sciences, Yale University School of Medicine, New Haven, CT

³Center for Dementia Research, Nathan Kline Institute for Psychiatric Research and Department of Psychiatry, New York University School of Medicine, New York, NY
⁵Depart of ObGyn and Reproductive Sciences, University of Vermont College of Medicine, Burlington, VT .
⁶Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA 92617, USA.
⁷Department of Biochemistry and Experimental Biochemistry Unit, King Abdulaziz Univ, Jeddah , Saudi Arabia.

Preeclampsia is a pregnancy-specific disorder of unknown etiology and a leading contributor to maternal and perinatal morbidity and mortality worldwide. Because there is no cure other than delivery, preeclampsia is the leading cause of iatrogenic preterm birth. We show that preeclampsia shares pathophysiologic features with recognized protein misfolding disorders. These features include urine congophilia (affinity for the amyloidophilic dye Congo red), affinity for conformational state–dependent antibodies, and dysregulation of prototype proteolytic enzymes involved in amyloid precursor protein (APP) processing. Assessment of global protein misfolding load in pregnancy based on urine congophilia (Congo red dot test) carries diagnostic and prognostic potential for preeclampsia. We used conformational state–dependent antibodies to demonstrate the presence of generic supramolecular assemblies (prefibrillar oligomers and annular protofibrils), which vary in quantitative and qualitative representation with preeclampsia severity. In the first attempt to characterize the preeclampsia misfoldome, we report that the urine congophilic material includes proteoforms of ceruloplasmin, immunoglobulin free light chains, SERPINA1, albumin, interferon-inducible protein 6-16, and Alzheimer’s β-amyloid. The human placenta abundantly expresses APP along with prototype APP-processing enzymes, of which the α-secretase ADAM10, the β-secretases BACE1 and BACE2, and the γ-secretase presenilin-1 were all up-regulated in preeclampsia. The presence of β-amyloid aggregates in placentas of women with preeclampsia and fetal growth restriction further supports the notion that this condition should join the growing list of protein conformational disorders. If these aggregates play a pathophysiologic role, our findings may lead to treatment for preeclampsia.

Citation: I. A. Buhimschi, U. A. Nayeri, G. Zhao, L. L. Shook, A. Pensalfini, E. F. Funai, I. M. Bernstein, C. G. Glabe, C. S. Buhimschi,Protein misfolding, congophilia, oligomerization, and defective amyloid processing in preeclampsia. Sci. Transl. Med. 6, 245ra92 (2014).

9. Blood Clotting

Removing parts of shape-shifting protein explains how blood clots

prothrombin (FII)

Using x-ray crystallography, SLU researchers published the first image of the important blood-clotting protein prothrombin (coagulation factor II). The protein’s flexible structure is key to the development of blood-clotting.In results recently published in Proceedings of the National Academy of Sciences (PNAS), Saint Louis University scientists have discovered that removal of disordered sections of a protein’s structure reveals the molecular mechanism of a key reaction that initiates blood clotting.

Enrico Di Cera, M.D., chair of the Edward A. Doisy department of biochemistry and molecular biology at Saint Louis University, studies thrombin, a key vitamin K-dependent blood-clotting protein, and its inactive precursor prothrombin (or coagulation factor II).

“Prothrombin is essential for life and is the most important clotting factor,” Di Cera said. “We are proud to report that our lab here at SLU has finally succeeded in crystallizing prothrombin for the first time.”

Blood-clotting has long ensured our survival, stopping blood loss after an injury. However, when triggered in the wrong circumstances, clotting can lead to debilitating or fatal conditions such as a heart attack, stroke or deep vein thrombosis.

Before thrombin becomes active, it circulates throughout the blood in the inactive (zymogen) form called prothrombin. When the active enzyme is needed (after a vascular injury, for example), the coagulation cascade is initiated and prothrombin is converted into the active enzyme thrombin that causes blood to clot.

X-ray crystallography is one tool in scientists’ toolbox for understanding processes at the molecular level. It offers a way to obtain a “snap shot” of a protein’s structure.

In this technique, scientists grow crystals of the protein they want to study, shoot x-rays at them and record data about the way the rays are scattered by crystals. Then they use computer programs to create an image of the protein based on that data.

Once scientists can visualize the three dimensional structure of a molecule, they can begin to piece together the way in which the protein functions and interacts with other molecules in the body, or with drugs.

Last year, Di Cera and colleagues published the first structure of prothrombin. This first structure lacked a domain responsible for interaction with membranes and certain other sections were not detected by x-ray analysis. Though the scientists were able to crystallize the protein, there were disordered regions in the structure that they could not see.

Within prothrombin there are two kringle domains (looped sections of a protein named after the Scandinavian pastry) connected by a “linker” region that intrigued the SLU investigators because of its intrinsic disorder.

“We deleted this linker and crystals grew in a few days instead of months, revealing for the first time the full architecture of prothrombin,” Di Cera said.

In addition to this remarkable discovery, Di Cera and colleagues found that the deleted version of prothrombin is activated to thrombin much faster than the intact prothrombin. The structure without the disordered linker is in fact optimized for conversion to thrombin and reveals key information on the mechanism of prothrombin activation.

For over four decades, scientists have tried to crystallize prothrombin but without success.

“It took us almost two years to discover that the disordered linker was the key,” Di Cera said.  “Finally, prothrombin revealed its secrets and with that the molecular mechanism of a key reaction of blood clotting finally becomes amenable to rational drug design for therapeutic intervention.”

SLU researchers Nicola Pozzi, Ph.D., Zhiwei Chen, Leslie Pelc and Daniel Shropshire also are authors on the paper.

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