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Posts Tagged ‘Protein folding’


Growth Factors, Suppressors and Receptors in Tumorigenesis

Writer and Curator: Larry H Bernstein, MD, FCAP

7.1 Growth Factors, Suppressors and Receptors in Tumorigenesis

7.1.1 Friend or Foe: Endoplasmic reticulum protein 29 (ERp29) in epithelial cancer

7.1.2 Putting together structures of epidermal growth factor receptors

7.1.3 Complex Relationship between Ligand Binding and Dimerization in the Epidermal Growth Factor Receptor

7.1.4 IGFBP-2.PTEN- A critical interaction for tumors and for general physiology

7.1.5 Emerging-roles-for-the-Ph-sensing-G-protein-coupled-receptor

7.1.6 Protein amino-terminal modifications and proteomic approaches for N-terminal profiling

7.1.7 Protein homeostasis networks in physiology and disease

7.1.8 Proteome sequencing goes deep

7.1.1 Friend or Foe: Endoplasmic reticulum protein 29 (ERp29) in epithelial cancer

Chen S1Zhang D2
FEBS Open Bio. 2015 Jan 30; 5:91-8
http://dx.doi.org:/10.1016/j.fob.2015.01.004

The endoplasmic reticulum (ER) protein 29 (ERp29) is a molecular chaperone that plays a critical role in protein secretion from the ER in eukaryotic cells. Recent studies have also shown that ERp29 plays a role in cancer. It has been demonstrated that ERp29 is inversely associated with primary tumor development and functions as a tumor suppressor by inducing cell growth arrest in breast cancer. However, ERp29 has also been reported to promote epithelial cell morphogenesis, cell survival against genotoxic stress and distant metastasis. In this review, we summarize the current understanding on the biological and pathological functions of ERp29 in cancer and discuss the pivotal aspects of ERp29 as “friend or foe” in epithelial cancer.

The endoplasmic reticulum (ER) is found in all eukaryotic cells and is complex membrane system constituting of an extensively interlinked network of membranous tubules, sacs and cisternae. It is the main subcellular organelle that transports different molecules to their subcellular destinations or to the cell surface [10,85].

The ER contains a number of molecular chaperones involved in protein synthesis and maturation. Of the ER chaperones, protein disulfide isomerase (PDI)-like proteins are characterized by the presence of a thioredoxin domain and function as oxido-reductases, isomerases and chaperones [33]. ERp29 lacks the active-site double-cysteine (CxxC) motif and does not belong to the redox-active PDIs [5,47]. ERp29 is recognized as a characterized resident of the cellular ER, and it is expressed ubiquitously and abundantly in mammalian tissues [50]. Protein structural analysis showed that ERp29 consists of N-terminal and C-terminal domains [5]: N-terminal domain involves dimerization whereas the C-terminal domain is essential for substrate binding and secretion [78]. The biological function of ERp29 in protein secretion has been well established in cells [8,63,67].

ERp9 is proposed to be involved in the unfolded protein response (UPR) as a factor facilitating transport of synthesized secretory proteins from the ER to Golgi [83]. The expression of ERp29 was demonstrated to be increased in cells exposed to radiation [108], sperm cells undergoing maturation [42,107], and in certain cell types both under the pharmacologically induced UPR and under the physiological conditions (e.g., lactation, differentiation of thyroid cells) [66,82]. Under ER stress, ERp29 translocates the precursor protein p90ATF6 from the ER to Golgi where it is cleaved to be a mature and active form p50ATF by protease (S1P and S2P) [48]. In most cases, ERp29 interacts with BiP/GRP78 to exert its function under ER stress [65].

ERp29 is considered to be a key player in both viral unfolding and secretion [63,67,77,78] Recent studies have also demonstrated that ERp29 is involved in intercellular communication by stabilizing the monomeric gap junction protein connexin43 [27] and trafficking of cystic fibrosis transmembrane conductance regulator to the plasma membrane in cystic fibrosis and non-cystic fibrosis epithelial cells [90]. It was recently reported that ERp29 directs epithelial Na(+) channel (ENaC) toward the Golgi, where it undergoes cleavage during its biogenesis and trafficking to the apical membrane [40]. ERp29 expression protects axotomized neurons from apoptosis and promotes neuronal regeneration [111]. These studies indicate a broad biological function of ERp29 in cells.

Recent studies demonstrated a tumor suppressive function of ERp29 in cancer. It was found that ERp29 expression inhibited tumor formation in mice [4,87] and the level of ERp29 in primary tumors is inversely associated with tumor development in breast, lung and gallbladder cancer [4,29].

However, its expression is also responsible for cancer cell survival against genotoxic stress induced by doxorubicin and radiation [34,76,109]. The most recent studies demonstrate other important roles of ERp29 in cancer cells such as the induction of mesenchymal–epithelial transition (MET) and epithelial morphogenesis [3,4]. MET is considered as an important process of transdifferentiation and restoration of epithelial phenotype during distant metastasis [23,52]. These findings implicate ERp29 in promoting the survival of cancer cells and also metastasis. Hence, the current review focuses on the novel functions of ERp29 and discusses its pathological importance as a “friend or foe” in epithelial cancer.

ERp29 regulates mesenchymal–epithelial transition

Epithelial–mesenchymal transition (EMT) and MET

The EMT is an essential process during embryogenesis [6] and tumor development [43,96]. The pathological conditions such as inflammation, organ fibrosis and cancer progression facilitate EMT [16]. The epithelial cells after undergoing EMT show typical features characterized as: (1) loss of adherens junctions (AJs) and tight junctions (TJs) and apical–basal polarity; (2) cytoskeletal reorganization and distribution; and (3) gain of aggressive phenotype of migration and invasion [98]. Therefore, EMT has been considered to be an important process in cancer progression and its pathological activation during tumor development induces primary tumor cells to metastasize [95]. However, recent studies showed that the EMT status was not unanimously correlated with poorer survival in cancer patients examined [92].

In addition to EMT in epithelial cells, mesenchymal-like cells have capability to regain a fully differentiated epithelial phenotype via the MET [6,35]. The key feature of MET is defined as a process of transdifferentiation of mesenchymal-like cells to polarized epithelial-like cells [23,52] and mediates the establishment of distant metastatic tumors at secondary sites [22]. Recent studies demonstrated that distant metastases in breast cancer expressed an equal or stronger E-cadherin signal than the respective primary tumors and the re-expression of E-cadherin was independent of the E-cadherin status of the primary tumors [58]. Similarly, it was found that E-cadherin is re-expressed in bone metastasis or distant metastatic tumors arising from E-cadherin-negative poorly differentiated primary breast carcinoma [81], or from E-cadherin-low primary tumors [25]. In prostate and bladder cancer cells, the nonmetastatic mesenchymal-like cells were interacted with metastatic epithelial-like cells to accelerate their metastatic colonization [20]. It is, therefore, suggested that the EMT/MET work co-operatively in driving metastasis.

Molecular regulation of EMT/MET

E-cadherin is considered to be a key molecule that provides the physical structure for both cell–cell attachment and recruitment of signaling complexes [75]. Loss of E-cadherin is a hallmark of EMT [53]. Therefore, characterizing transcriptional regulators of E-cadherin expression during EMT/MET has provided important insights into the molecular mechanisms underlying the loss of cell–cell adhesion and the acquisition of migratory properties during carcinoma progression [73].

Several known signaling pathways, such as those involving transforming growth factor-β (TGF-β), Notch, fibroblast growth factor and Wnt signaling pathways, have been shown to trigger epithelial dedifferentiation and EMT [28,97,110]. These signals repress transcription of epithelial genes, such as those encoding E-cadherin and cytokeratins, or activate transcription programs that facilitate fibroblast-like motility and invasion [73,97].

The involvement of microRNAs (miRNAs) in controlling EMT has been emphasized [11,12,18]. MiRNAs are small non-coding RNAs (∼23 nt) that silence gene expression by pairing to the 3′UTR of target mRNAs to cause their posttranscriptional repression [7]. MiRNAs can be characterized as “mesenchymal miRNA” and “epithelial miRNA” [68]. The “mesenchymal miRNA” plays an oncogenic role by promoting EMT in cancer cells. For instance, the well-known miR-21, miR-103/107 are EMT inducer by repressing Dicer and PTEN [44].

The miR-200 family has been shown to be major “epithelial miRNA” that regulate MET through silencing the EMT-transcriptional inducers ZEB1 and ZEB2 [13,17]. MiRNAs from this family are considered to be predisposing factors for cancer cell metastasis. For instance, the elevated levels of the epithelial miR-200 family in primary breast tumors associate with poorer outcomes and metastasis [57]. These findings support a potential role of “epithelial miRNAs” in MET to promote metastatic colonization [15].

ERp29 promotes MET in breast cancer

The role of ERp29 in regulating MET has been established in basal-like MDA-MB-231 breast cancer cells. It is known that myosin light chain (MLC) phosphorylation initiates to myosin-driven contraction, leading to reorganization of the actin cytoskeleton and formation of stress fibers [55,56]. ERp29 expression in this type of cells markedly reduced the level of phosphorylated MLC [3]. These results indicate that ERp29 regulates cortical actin formation through a mechanism involved in MLC phosphorylation (Fig. 1). In addition to the phenotypic change, ERp29 expression leads to: expression and membranous localization of epithelial cell marker E-cadherin; expression of epithelial differentiation marker cytokeratin 19; and loss of the mesenchymal cell marker vimentin and fibronectin [3] (Fig. 1). In contrast, knockdown of ERp29 in epithelial MCF-7 cells promotes acquisition of EMT traits including fibroblast-like phenotype, enhanced cell spreading, decreased expression of E-cadherin and increased expression of vimentin [3,4]. These findings further substantiate a role of ERp29 in modulating MET in breast cancer cells.

Fig. 1  ERp29 triggers mesenchymal–epithelial transition. Exogenous expression of ERp29 in mesenchymal MDA-MB-231 breast cancer cells inhibits stress fiber formation by suppressing MLC phosphorylation. In addition, the overexpressed ERp29 decreases the 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329646/bin/gr1.gif

ERp29 targets E-cadherin transcription repressors

The transcription repressors such as Snai1, Slug, ZEB1/2 and Twist have been considered to be the main regulators for E-cadherin expression [19,26,32]. Mechanistic studies revealed that ERp29 expression significantly down-regulated transcription of these repressors, leading to their reduced nuclear expression in MDA-MB-231 cells [3,4] (Fig. 2). Consistent with this, the extracellular signal-regulated kinase (ERK) pathway which is an important up-stream regulator of Slug and Ets1 was highly inhibited [4]. Apparently, ERp29 up-regulates the expressions of E-cadherin transcription repressors through repressing ERK pathway. Interestingly, ERp29 over-expression in basal-like BT549 cells resulted in incomplete MET and did not significantly affect the mRNA or protein expression of Snai1, ZEB2 and Twist, but increased the protein expression of Slug [3]. The differential regulation of these transcriptional repressors of E-cadherin by ERp29 in these two cell-types may occur in a cell-context-dependent manner.

Fig. 2  ERp29 decreases the expression of EMT inducers to promote MET. Exogenous expression of ERp29 in mesenchymal MDA-MB-231 breast cancer cells suppresses transcription and protein expression of E-cadherin transcription repressors (e.g., ZEB2, SNAI1 and Twist), ..

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ERp29 antagonizes Wnt/ β-catenin signaling

Wnt proteins are a family of highly conserved secreted cysteine-rich glycoproteins. The Wnt pathway is activated via a binding of a family member to a frizzled receptor (Fzd) and the LDL-Receptor-related protein co-receptor (LRP5/6). There are three different cascades that are activated by Wnt proteins: namely canonical/β-catenin-dependent pathway and two non-canonical/β-catenin-independent pathways that include Wnt/Ca2+ and planar cell polarity [84]. Of note, the Wnt/β-catenin pathway has been extensively studied, due to its important role in cancer initiation and progression [79]. The presence of Wnt promotes formation of a Wnt–Fzd–LRP complex, recruitment of the cytoplasmic protein Disheveled (Dvl) to Fzd and the LRP phosphorylation-dependent recruitment of Axin to the membrane, thereby leading to release of β-catenin from membrane and accumulation in cytoplasm and nuclei. Nuclear β-catenin replaces TLE/Groucho co-repressors and recruits co-activators to activate expression of Wnt target genes. The most important genes regulated are those related to proliferation, such as Cyclin D1 and c-Myc [46,94], which are over-expressed in most β-catenin-dependent tumors. When β-catenin is absent in nucleus, the transcription factors T-cell factor/lymphoid enhancer factors (TCF/LEF) recruits co-repressors of the TLE/Groucho family and function as transcriptional repressors.

β-catenin is highly expressed in the nucleus of mesenchymal MDA-MB-231 cells. ERp29 over-expression in this type of cells led to translocation of nuclear β-catenin to membrane where it forms complex with E-cadherin [3] (Fig. 3). This causes a disruption of β-catenin/TCF/LEF complex and abolishes its transcription activity. Indeed, ERp29 significantly decreased the expression of cyclin D1/D2 [36], one of the downstream targets of activated Wnt/β-catenin signaling [94], indicating an inhibitory effect of ERp29 on this pathway. Meanwhile, expression of ERp29 in this cell type increased the nuclear expression of TCF3, a transcription factor regulating cancer cell differentiation while inhibiting self-renewal of cancer stem cells [102,106]. Hence, ERp29 may play dual functions in mesenchymal MDA-MB-231 breast cancer cells by: (1) suppressing activated Wnt/β-catenin signaling via β-catenin translocation; and (2) promoting cell differentiation via activating TCF3 (Fig. 3). Because β-catenin serves as a signaling hub for the Wnt pathway, it is particularly important to focus on β-catenin as the target of choice in Wnt-driven cancers. Though the mechanism by which ERp29 expression promotes the disassociation of β-catenin/TCF/LEF complex in MDA-MB-231 cells remains elusive, activating ERp29 expression may exert an inhibitory effect on the poorly differentiated, Wnt-driven tumors.

Fig. 3  ERp29 over-expression “turns-off” activated Wnt/β-catenin signaling. In mesenchymal MDA-MB-231 cells, high expression of nuclear β-catenin activates its downstream signaling involved in cell cycles and cancer stem cell 

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ERp29 regulates epithelial cell integrity

Cell adherens and tight junctions

Adherens junctions (AJs) and tight junctions (TJs) are composed of transmembrane proteins that adhere to similar proteins in the adjacent cell [69]. The transmembrane region of the TJs is composed mainly of claudins, tetraspan proteins with two extracellular loops [1]. AJs are mediated by Ca2+-dependent homophilic interactions of cadherins [71] which interact with cytoplasmic catenins that link the cadherin/catenin complex to the actin cytoskeleton [74].

The cytoplasmic domain of claudins in TJs interacts with occludin and several zona occludens proteins (ZO1-3) to form the plaque that associates with the cytoskeleton [99]. The AJs form and maintain intercellular adhesion, whereas the TJs serve as a diffusion barrier for solutes and define the boundary between apical and basolateral membrane domains [21]. The AJs and TJs are required for integrity of the epithelial phenotype, as well as for epithelial cells to function as a tissue [75].

The TJs are closely linked to the proper polarization of cells for the establishment of epithelial architecture[86]. During cancer development, epithelial cells lose the capability to form TJs and correct apico–basal polarity [59]. This subsequently causes the loss of contact inhibition of cell growth [91]. In addition, reduction of ZO-1 and occludin were found to be correlated with poorly defined differentiation, higher metastatic frequency and lower survival rates [49,64]. Hence, TJs proteins have a tumor suppressive function in cancer formation and progression.

Apical–basal cell polarity

The apical–basal polarity of epithelial cells in an epithelium is characterized by the presence of two specialized plasma membrane domains: namely, the apical surface and basolateral surface [30]. In general, the epithelial cell polarity is determined by three core complexes. These protein complexes include: (1) the partitioning-defective (PAR) complex; (2) the Crumbs (CRB) complex; and (3) the Scribble complex[2,30,45,51]. PAR complex is composed of two scaffold proteins (PAR6 and PAR3) and an atypical protein kinase C (aPKC) and is localized to the apical junction domain for the assembly of TJs [31,39]. The Crumbs complex is formed by the transmembrane protein Crumbs and the cytoplasmic scaffolding proteins such as the homologue of Drosophila Stardust (Pals1) and Pals-associated tight junction protein (Patj) and localizes to the apical [38]. The Scribble complex is comprised of three proteins, Scribble, Disc large (Dlg) and Lethal giant larvae (Lgl) and is localized in the basolateral domain of epithelial cells [100].

Fig. 4  ERp29 regulates epithelial cell morphogenesis. Over-expression of ERp29 in breast cancer cells induces the transition from a mesenchymal-like to epithelial-like phenotype and the restoration of tight junctions and cell polarity. Up-regulation and membrane 

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The current data from breast cancer cells supports the idea that ERp29 can function as a tumor suppressive protein, in terms of suppression of cell growth and primary tumor formation and inhibition of signaling pathways that facilitate EMT. Nevertheless, the significant role of ERp29 in cell survival against drugs, induction of cell differentiation and potential promotion of MET-related metastasis may lead us to re-assess its function in cancer progression, particularly in distant metastasis. Hence, it is important to explore in detail the ERp29’s role in cancer as a “friend or foe” and to elucidate its clinical significance in breast cancer and other epithelial cancers. Targeting ERp29 and/or its downstream molecules might be an alternative molecular therapeutic approach for chemo/radio-resistant metastatic cancer treatment

7.1.2 Putting together structures of epidermal growth factor receptors

Bessman NJ1Freed DM2Lemmon MA3
Curr Opin Struct Biol. 2014 Dec; 29:95-101
http://dx.doi.org:/10.1016/j.sbi.2014.10.002

Highlights

  • Several studies suggest flexible linkage between extracellular and intracellular regions.
  • Others imply more rigid connections, required for allosteric regulation of dimers.
  • Interactions with membrane lipids play important roles in EGFR regulation.
  • Cellular studies suggest half-of-the-sites negative cooperativity for human EGFR.

Numerous crystal structures have been reported for the isolated extracellular region and tyrosine kinase domain of the epidermal growth factor receptor (EGFR) and its relatives, in different states of activation and bound to a variety of inhibitors used in cancer therapy. The next challenge is to put these structures together accurately in functional models of the intact receptor in its membrane environment. The intact EGFR has been studied using electron microscopy, chemical biology methods, biochemically, and computationally. The distinct approaches yield different impressions about the structural modes of communication between extracellular and intracellular regions. They highlight possible differences between ligands, and also underline the need to understand how the receptor interacts with the membrane itself.

http://ars.els-cdn.com/content/image/1-s2.0-S0959440X14001304-gr1.sml

http://ars.els-cdn.com/content/image/1-s2.0-S0959440X14001304-gr2.sml

7.1.3 Complex Relationship between Ligand Binding and Dimerization in the Epidermal Growth Factor Receptor

Bessman NJ1Bagchi A2Ferguson KM2Lemmon MA3.
Cell Rep. 2014 Nov 20; 9(4):1306-17.
http://dx.doi.org/10.1016/j.celrep.2014.10.010

Highlights

  • Preformed extracellular dimers of human EGFR are structurally heterogeneous
  • EGFR dimerization does not stabilize ligand binding
  • Extracellular mutations found in glioblastoma do not stabilize EGFR dimerization
  • Glioblastoma mutations in EGFR increase ligand-binding affinity

Summary

The epidermal growth factor receptor (EGFR) plays pivotal roles in development and is mutated or overexpressed in several cancers. Despite recent advances, the complex allosteric regulation of EGFR remains incompletely understood. Through efforts to understand why the negative cooperativity observed for intact EGFR is lost in studies of its isolated extracellular region (ECR), we uncovered unexpected relationships between ligand binding and receptor dimerization. The two processes appear to compete. Surprisingly, dimerization does not enhance ligand binding (although ligand binding promotes dimerization). We further show that simply forcing EGFR ECRs into preformed dimers without ligand yields ill-defined, heterogeneous structures. Finally, we demonstrate that extracellular EGFR-activating mutations in glioblastoma enhance ligand-binding affinity without directly promoting EGFR dimerization, suggesting that these oncogenic mutations alter the allosteric linkage between dimerization and ligand binding. Our findings have important implications for understanding how EGFR and its relatives are activated by specific ligands and pathological mutations.

http://www.cell.com/cms/attachment/2020816777/2040986303/fx1.jpg

X-ray crystal structures from 2002 and 2003 (Burgess et al., 2003) yielded the scheme for ligand-induced epidermal growth factor receptor (EGFR) dimerization shown in Figure 1. Binding of a single ligand to domains I and III within the same extracellular region (ECR) stabilizes an “extended” conformation and exposes a dimerization interface in domain II, promoting self-association with a KD in the micromolar range (Burgess et al., 2003, Dawson et al., 2005, Dawson et al., 2007). Although this model satisfyingly explains ligand-induced EGFR dimerization, it fails to capture the complex ligand-binding characteristics seen for cell-surface EGFR, with concave-up Scatchard plots indicating either negative cooperativity (De Meyts, 2008, Macdonald and Pike, 2008) or distinct affinity classes of EGF-binding site with high-affinity sites responsible for EGFR signaling (Defize et al., 1989). This cooperativity or heterogeneity is lost when the ECR from EGFR is studied in isolation, as also described for the insulin receptor (De Meyts, 2008).

ligand-induced-dimerization-of-the-hegfr-ecr

ligand-induced-dimerization-of-the-hegfr-ecr

Figure 1

Structural View of Ligand-Induced Dimerization of the hEGFR ECR

(A) Surface representation of tethered, unliganded, sEGFR from Protein Data Bank entry 1NQL (Ferguson et al., 2003). Ligand-binding domains I and III are green and cysteine-rich domains II and IV are cyan. The intramolecular domain II/IV tether is circled in red.

(B) Hypothetical model for an extended EGF-bound sEGFR monomer based on SAXS studies of an EGF-bound dimerization-defective sEGFR variant (Dawson et al., 2007) from PDB entry 3NJP (Lu et al., 2012). EGF is blue, and the red boundary represents the primary dimerization interface.

(C) 2:2 (EGF/sEGFR) dimer, from PDB entry 3NJP (Lu et al., 2012), colored as in (B). Dimerization arm contacts are circled in red.

http://www.cell.com/cms/attachment/2020816777/2040986313/gr1.sml

Here, we describe studies of an artificially dimerized ECR from hEGFR that yield useful insight into the heterogeneous nature of preformed ECR dimers and into the origins of negative cooperativity. Our data also argue that extracellular structures induced by ligand binding are not “optimized” for dimerization and conversely that dimerization does not optimize the ligand-binding sites. We also analyzed the effects of oncogenic mutations found in glioblastoma patients (Lee et al., 2006), revealing that they affect allosteric linkage between ligand binding and dimerization rather than simply promoting EGFR dimerization. These studies have important implications for understanding extracellular activating mutations found in EGFR/ErbB family receptors in glioblastoma and other cancers and also for understanding specificity of ligand-induced ErbB receptor heterodimerization

Predimerizing the EGFR ECR Has Modest Effects on EGF Binding

To access preformed dimers of the hEGFR ECR (sEGFR) experimentally, we C-terminally fused (to residue 621 of the mature protein) either a dimerizing Fc domain (creating sEGFR-Fc) or the dimeric leucine zipper from S. cerevisiae GCN4 (creating sEGFR-Zip). Size exclusion chromatography (SEC) and/or sedimentation equilibrium analytical ultracentrifugation (AUC) confirmed that the resulting purified sEGFR fusion proteins are dimeric (Figure S1). To measure KD values for ligand binding to sEGFR-Fc and sEGFR-Zip, we labeled EGF with Alexa-488 and monitored binding in fluorescence anisotropy (FA) assays. As shown in Figure 2A, EGF binds approximately 10-fold more tightly to the dimeric sEGFR-Fc or sEGFR-Zip proteins than to monomeric sEGFR (Table 1). The curves obtained for EGF binding to sEGFR-Fc and sEGFR-Zip showed no signs of negative cooperativity, with sEGFR-Zip actually requiring a Hill coefficient (nH) greater than 1 for a good fit (nH = 1 for both sEGFRWT and sEGFR-Fc). Thus, our initial studies argued that simply dimerizing human sEGFR fails to restore the negatively cooperative ligand binding seen for the intact receptor in cells.

One surprise from these data was that forced sEGFR dimerization has only a modest (≤10-fold) effect on EGF-binding affinity. Under the conditions of the FA experiments, isolated sEGFR (without zipper or Fc fusion) remains monomeric; the FA assay contains just 60 nM EGF, so the maximum concentration of EGF-bound sEGFR is also limited to 60 nM, which is over 20-fold lower than the KD for dimerization of the EGF/sEGFR complex (Dawson et al., 2005, Lemmon et al., 1997). This ≤10-fold difference in affinity for dimeric and monomeric sEGFR seems small in light of the strict dependence of sEGFR dimerization on ligand binding (Dawson et al., 2005,Lax et al., 1991, Lemmon et al., 1997). Unliganded sEGFR does not dimerize detectably even at millimolar concentrations, whereas liganded sEGFR dimerizes with KD ∼1 μM, suggesting that ligand enhances dimerization by at least 104– to 106-fold. Straightforward linkage of dimerization and binding equilibria should stabilize EGF binding to dimeric sEGFR similarly (by 5.5–8.0 kcal/mol). The modest difference in EGF-binding affinity for dimeric and monomeric sEGFR is also significantly smaller than the 40- to 100-fold difference typically reported between high-affinity and low-affinity EGF binding on the cell surface when data are fit to two affinity classes of binding site (Burgess et al., 2003, Magun et al., 1980).

Mutations that Prevent sEGFR Dimerization Do Not Significantly Reduce Ligand-Binding Affinity

The fact that predimerizing sEGFR only modestly increased ligand-binding affinity led us to question the extent to which domain II-mediated sEGFR dimerization is linked to ligand binding. It is typically assumed that the domain II conformation stabilized upon forming the sEGFR dimer in Figure 1C optimizes the domain I and III positions for EGF binding. To test this hypothesis, we introduced a well-characterized pair of domain II mutations into sEGFRs that block dimerization: one at the tip of the dimerization arm (Y251A) and one at its “docking site” on the adjacent molecule in a dimer (R285S). The resulting (Y251A/R285S) mutation abolishes sEGFR dimerization and EGFR signaling (Dawson et al., 2005, Ogiso et al., 2002). Importantly, we chose isothermal titration calorimetry (ITC) for these studies, where all interacting components are free in solution. Previous surface plasmon resonance (SPR) studies have indicated that dimerization-defective sEGFR variants bind immobilized EGF with reduced affinity (Dawson et al., 2005), and we were concerned that this reflects avidity artifacts, where dimeric sEGFR binds more avidly than monomeric sEGFR to sensor chip-immobilized EGF.

Surprisingly, our ITC studies showed that the Y251A/R285S mutation has no significant effect on ligand-binding affinity for sEGFR in solution (Table 1). These experiments employed sEGFR (with no Fc fusion) at 10 μM—ten times higher than KD for dimerization of ligand-saturated WT sEGFR (sEGFRWT) (KD ∼1 μM). Dimerization of sEGFRWT should therefore be complete under these conditions, whereas the Y251A/R285S-mutated variant (sEGFRY251A/R285S) does not dimerize at all (Dawson et al., 2005). The KD value for EGF binding to dimeric sEGFRWT was essentially the same (within 2-fold) as that for sEGFRY251A/R285S (Figures 2B and 2C; Table 1), arguing that the favorable Gibbs free energy (ΔG) of liganded sEGFR dimerization (−5.5 to −8 kcal/mol) does not contribute significantly (<0.4 kcal/mol) to enhanced ligand binding. …

Thermodynamics of EGF Binding to sEGFR-Fc

If there is no discernible positive linkage between sEGFR dimerization and EGF binding, why do sEGFR-Fc and sEGFR-Zip bind EGF ∼10-fold more strongly than wild-type sEGFR? To investigate this, we used ITC to compare EGF binding to sEGFR-Fc and sEGFR-Zip (Figures 3A and 3B ) with binding to isolated (nonfusion) sEGFRWT. As shown in Table 1, the positive (unfavorable) ΔH for EGF binding is further elevated in predimerized sEGFR compared with sEGFRWT, suggesting that enforced dimerization may actually impair ligand/receptor interactions such as hydrogen bonds and salt bridges. The increased ΔH is more than compensated for, however, by a favorable increase in TΔS. This favorable entropic effect may reflect an “ordering” imposed on unliganded sEGFR when it is predimerized, such that it exhibits fewer degrees of freedom compared with monomeric sEGFR. In particular, since EGF binding does induce sEGFR dimerization, it is clear that predimerization will reduce the entropic cost of bringing two sEGFR molecules into a dimer upon ligand binding, possibly underlying this effect.

Possible Heterogeneity of Binding Sites in sEGFR-Fc

Close inspection of EGF/sEGFR-Fc titrations such as that in Figure 3A suggested some heterogeneity of sites, as evidenced by the slope in the early part of the experiment. To investigate this possibility further, we repeated titrations over a range of temperatures. We reasoned that if there are two different types of EGF-binding sites in an sEGFR-Fc dimer, they might have different values for heat capacity change (ΔCp), with differences that might become more evident at higher (or lower) temperatures. Indeed, ΔCp values correlate with the nonpolar surface area buried upon binding (Livingstone et al., 1991), and we know that this differs for the two Spitz-binding sites in the asymmetric Drosophila EGFR dimer (Alvarado et al., 2010). As shown in Figure 3C, the heterogeneity was indeed clearer at higher temperatures for sEGFR-Fc—especially at 25°C and 30°C—suggesting the possible presence of distinct classes of binding sites in the sEGFR-Fc dimer. We were not able to fit the two KD values (or ΔH values) uniquely with any precision because the experiment has insufficient information for unique fitting to a model with four variables. Whereas binding to sEGFRWT could be fit confidently with a single-site binding model throughout the temperature range, enforced sEGFR dimerization (by Fc fusion) creates apparent heterogeneity in binding sites, which may reflect negative cooperativity of the sort seen with dEGFR. …

Ligand Binding Is Required for Well-Defined Dimerization of the EGFR ECR

To investigate the structural nature of the preformed sEGFR-Fc dimer, we used negative stain electron microscopy (EM). We hypothesized that enforced dimerization might cause the unliganded ECR to form the same type of loose domain II-mediated dimer seen in crystals of unliganded Drosophila sEGFR (Alvarado et al., 2009). When bound to ligand (Figure 4A), the Fc-fused ECR clearly formed the characteristic heart-shape dimer seen by crystallography and EM (Lu et al., 2010, Mi et al., 2011). Figure 4B presents a structural model of an Fc-fused liganded sEGFR dimer, and Figure 4C shows a calculated 12 Å resolution projection of this model. The class averages for sEGFR-Fc plus EGF (Figure 4A) closely resemble this model, yielding clear densities for all four receptor domains, arranged as expected for the EGF-induced domain II-mediated back-to-back extracellular dimer shown in Figure 1 (Garrett et al., 2002, Lu et al., 2010). In a subset of classes, the Fc domain also appeared well resolved, indicating that these particular arrangements of the Fc domain relative to the ECR represent highly populated states, with the Fc domains occupying similar positions to those of the kinase domain in detergent-solubilized intact receptors (Mi et al., 2011). …

Our results and those of Lu et al. (2012)) argue that preformed extracellular dimers of hEGFR do not contain a well-defined domain II-mediated interface. Rather, the ECRs in these dimers likely sample a broad range of positions (and possibly conformations). This conclusion argues against recent suggestions that stable unliganded extracellular dimers “disfavor activation in preformed dimers by assuming conformations inconsistent with” productive dimerization of the rest of the receptor (Arkhipov et al., 2013). The ligand-free inactive dimeric ECR species modeled by Arkhipov et al. (2013) in their computational studies of the intact receptor do not appear to be stable. The isolated ECR from EGFR has a very low propensity for self-association without ligand, with KD in the millimolar range (or higher). Moreover, sEGFR does not form a defined structure even when forced to dimerize by Fc fusion. It is therefore difficult to envision how it might assume any particular autoinhibitory dimeric conformation in preformed dimers. …

Extracellular Oncogenic Mutations Observed in Glioblastoma May Alter Linkage between Ligand Binding and sEGFR Dimerization

Missense mutations in the hEGFR ECR were discovered in several human glioblastoma multiforme samples or cell lines and occur in 10%–15% of glioblastoma cases (Brennan et al., 2013, Lee et al., 2006). Several elevate basal receptor phosphorylation and cause EGFR to transform NIH 3T3 cells in the absence of EGF (Lee et al., 2006). Thus, these are constitutively activating oncogenic mutations, although the mutated receptors can be activated further by ligand (Lee et al., 2006, Vivanco et al., 2012). Two of the most commonly mutated sites in glioblastoma, R84 and A265 (R108 and A289 in pro-EGFR), are in domains I and II of the ECR, respectively, and contribute directly in inactive sEGFR to intramolecular interactions between these domains that are thought to be autoinhibitory (Figure 5). Domains I and II become separated from one another in this region upon ligand binding to EGFR (Alvarado et al., 2009), as illustrated in the lower part of Figure 5. Interestingly, analogous mutations in the EGFR relative ErbB3 were also found in colon and gastric cancers (Jaiswal et al., 2013).

We hypothesized that domain I/II interface mutations might activate EGFR by disrupting autoinhibitory interactions between these two domains, possibly promoting a domain II conformation that drives dimerization even in the absence of ligand. In contrast, however, sedimentation equilibrium AUC showed that sEGFR variants harboring R84K, A265D, or A265V mutations all remained completely monomeric in the absence of ligand (Figure 6A) at a concentration of 10 μM, which is similar to that experienced at the cell surface (Lemmon et al., 1997). As with WT sEGFR, however, addition of ligand promoted dimerization of each mutated sEGFR variant, with KD values that were indistinguishable from those of WT. Thus, extracellular EGFR mutations seen in glioblastoma do not simply promote ligand-independent ECR dimerization, consistent with our finding that even dimerized sEGFR-Fc requires ligand binding in order to form the characteristic heart-shaped dimer. …

We suggest that domain I is normally restrained by domain I/II interactions so that its orientation with respect to the ligand is compromised. When the domain I/II interface is weakened with mutations, this effect is mitigated. If this results simply in increased ligand-binding affinity of the monomeric receptor, the biological consequence might be to sensitize cells to lower concentrations of EGF or TGF-α (or other agonists). However, cellular studies of EGFR with glioblastoma-derived mutations (Lee et al., 2006, Vivanco et al., 2012) clearly show ligand-independent activation, arguing that this is not the key mechanism. The domain I/II interface mutations may also reduce restraints on domain II so as to permit dimerization of a small proportion of intact receptor, driven by the documented interactions that promote self-association of the transmembrane, juxtamembrane, and intracellular regions of EGFR (Endres et al., 2013, Lemmon et al., 2014, Red Brewer et al., 2009).

Setting out to test the hypothesis that simply dimerizing the EGFR ECR is sufficient to recover the negative cooperativity lost when it is removed from the intact receptor, we were led to revisit several central assumptions about this receptor. Our findings suggest three main conclusions. First, we find that enforcing dimerization of the hEGFR ECR does not drive formation of a well-defined domain II-mediated dimer that resembles ligand-bound ECRs or the unliganded ECR from Drosophila EGFR. Our EM and SAXS data show that ligand binding is necessary for formation of well-defined heart-shaped domain II-mediated dimers. This result argues that the unliganded extracellular dimers modeled by Arkhipov et al. (2013)) are not stable and that it is improbable that stable conformations of preformed extracellular dimers disfavor receptor activation by assuming conformations that counter activating dimerization of the rest of the receptor. Recent work from the Springer laboratory employing kinase inhibitors to drive dimerization of hEGFR (Lu et al., 2012) also showed that EGF binding is required to form heart-shaped ECR dimers. These findings leave open the question of the nature of the ECR in preformed EGFR dimers but certainly argue that it is unlikely to resemble the crystallographic dimer seen for unligandedDrosophila EGFR (Alvarado et al., 2009) or that suggested by computational studies (Arkhipov et al., 2013).

This result argues that ligand binding is required to permit dimerization but that domain II-mediated dimerization may compromise, rather than enhance, ligand binding. Assuming flexibility in domain II, we suggest that this domain serves to link dimerization and ligand binding allosterically. Optimal ligand binding may stabilize one conformation of domain II in the scheme shown in Figure 1 that is then distorted upon dimerization of the ECR, in turn reducing the strength of interactions with the ligand. Such a mechanism would give the appearance of a lack of positive linkage between ligand binding and ECR dimerization, and a good test of this model would be to determine the high-resolution structure of a liganded sEGFR monomer (which we expect to differ from a half dimer). This model also suggests a mechanism for selective heterodimerization over homodimerization of certain ErbB receptors. If a ligand-bound EGFR monomer has a domain II conformation that heterodimerizes with ErbB2 in preference to forming EGFR homodimers, this could explain several important observations. It could explain reports that ErbB2 is a preferred heterodimerization partner of EGFR (Graus-Porta et al., 1997) and might also explain why EGF binds more tightly to EGFR in cells where it can form heterodimers with ErbB2 than in cells lacking ErbB2, where only EGFR homodimers can form (Li et al., 2012).

7.1.4 IGFBP-2.PTEN- A critical interaction for tumors and for general physiology

Li ZengClaire M. PerksJeff M.P. Holly
Growth Hormone & IGF Research online 7 February 2015
http://dx.doi.org/10.1016/j.ghir.2015.01.003

Highlights

  • IGFBP-2 is the second most abundant of the IGFBPs in the circulation.
  • IGFBP2 levels are increased in a variety of tumors and associated with progression and poor prognosis.
  • PTEN is a phosphatase that returns the PI3K/AKT/mTOR pathway to its inactivated state.
  • PTEN is the second most commonly mutated gene in a variety of common cancers.
  • Recent evidence indicates that IGFBP-2 regulates PTEN in a variety of normal and malignant cell types.
  • This review summarizes the evidence that these extracellular and intracellular modulators of the IGF-system are linked.

Abstract

IGFBP-2 is an important modulator of IGF availability and activity. It is the second most abundant of the IGFBPs in the circulation and its levels are increased in a variety of tumors and associated with progression and poor prognosis. PTEN is a phosphatase that returns the PI3K/AKT/mTOR pathway to its inactivated state and is therefore a critical modulator of one of the main intracellular signaling pathways activated by the IGFs. Recent evidence has indicated that IGFBP-2 regulates PTEN in a variety of normal and malignant cell types. This review summarizes the recent evidence that these extracellular and intracellular modulators are linked to provide a synchronous system for cell regulation with coordinated control of both the ‘accelerator’ and the ‘brake’.

IGFBP-2.PTEN

IGFBP-2.PTEN

http://ars.els-cdn.com/content/image/1-s2.0-S1096637415000167-gr1.sml

7.1.5 Emerging-roles-for-the-Ph-sensing-G-protein-coupled-receptor

Sanderlin EJ, Justus CR, Krewson EA, Yang LV
CHC March 2015 Volume 2015:7 Pages 99—109

http://www.dovepress.com/emerging-roles-for-the-ph-sensing-g-protein-coupled-receptors-in-respo-peer-reviewed-fulltext-article-CHC#

Protons (hydrogen ions) are the simplest form of ions universally produced by cellular metabolism including aerobic respiration and glycolysis. Export of protons out of cells by a number of acid transporters is essential to maintain a stable intracellular pH that is critical for normal cell function. Acid products in the tissue interstitium are removed by blood perfusion and excreted from the body through the respiratory and renal systems. However, the pH homeostasis in tissues is frequently disrupted in many pathophysiologic conditions such as in ischemic tissues and tumors where protons are overproduced and blood perfusion is compromised. Consequently, accumulation of protons causes acidosis in the affected tissue. Although acidosis has profound effects on cell function and disease progression, little is known about the molecular mechanisms by which cells sense and respond to acidotic stress. Recently a family of pH-sensing G protein-coupled receptors (GPCRs), including GPR4, GPR65 (TDAG8), and GPR68 (OGR1), has been identified and characterized. These GPCRs can be activated by extracellular acidic pH through the protonation of histidine residues of the receptors. Upon activation by acidosis the pH-sensing GPCRs can transduce several downstream G protein pathways such as the Gs, Gq/11, and G12/13 pathways to regulate cell behavior. Studies have revealed the biological roles of the pH-sensing GPCRs in the immune, cardiovascular, respiratory, renal, skeletal, endocrine, and nervous systems, as well as the involvement of these receptors in a variety of pathological conditions such as cancer, inflammation, pain, and cardiovascular disease. As GPCRs are important drug targets, small molecule modulators of the pH-sensing GPCRs are being developed and evaluated for potential therapeutic applications in disease treatment.

Cellular metabolism produces acid as a byproduct. Metabolism of each glucose molecule by glycolysis generates two pyruvate molecules. Under anaerobic conditions the metabolism of pyruvate results in the production of the glycolytic end product lactic acid, which has a pKa of 3.9. Lactic acid is deprotonated at the carboxyl group and results in one lactate ion and one proton at the physiological pH. Under aerobic conditions pyruvate is converted into acetyl-CoA and CO2 in the mitochondria. CO2in water forms a chemical equilibrium of carbonic acid and bicarbonate, an important physiological pH buffering system. The body must maintain suitable pH for proper physiological functions. Some regulatory mechanisms to control systemic pH are respiration, renal excretion, bone buffering, and metabolism.14 The respiratory system can buffer the blood by excreting carbonic acid as CO2 while the kidney responds to decreased circulatory pH by excreting protons and electrolytes to stabilize the physiological pH. Bone buffering helps maintain systemic pH by Ca2+ reabsorption and mineral dissolution. Collectively, it is clear that several biological systems require tight regulation to maintain pH for normal physiological functions. Cells utilize vast varieties of acid-base transporters for proper pH homeostasis within each biological context.58 Some such transporters are H+-ATPase, Na+/H+exchanger, Na+-dependent HCO3/C1 exchanger, Na+-independent anion exchanger, and monocarboxylate transporters. Cells can also maintain short-term pH homeostasis of the intracellular pH by rapid H+ consuming mechanisms. Some such mechanisms utilize metabolic conversions that move acids from the cytosol into organelles. Despite these cellular mechanisms that tightly maintain proper pH homeostasis, there are many diseases whereby pH homeostasis is disrupted. These pathological conditions are characterized by either local or systemic acidosis. Systemic acidosis can occur from respiratory, renal, and metabolic diseases and septic shock.14,9 Additionally, local acidosis is characterized in ischemic tissues, tumors, and chronically inflamed conditions such as in asthma and arthritis caused by deregulated metabolism and hypoxia.1015

Acidosis is a stress for the cell. The ability of the cell to sense and modulate activity for adaptation to the stressful environment is critical. There are several mechanisms whereby cells sense acidosis and modulate cellular functions to facilitate adaptation. Cells can detect extracellular pH changes by acid sensing ion channels (ASICs) and transient receptor potential (TRP) channels.16 Apart from ASIC and TRP channels, extracellular acidic pH was shown to stimulate inositol polyphosphate formation and calcium efflux.17,18 This suggested the presence of an unknown cell surface receptor that may be activated by a certain functional group, namely the imidazole of a histidine residue. The identity of the acid-activated receptor was later unmasked by Ludwig et al as a family of proton-sensing G protein-coupled receptors (GPCRs). This group identified human ovarian cancer GPCR 1 (OGR1) which upon activation will produce inositol phosphate and calcium efflux through the Gq pathway.19 These pH-sensing GPCR family members, including GPR4, GPR65 (TDAG8), and GPR68 (OGR1), will be discussed in this review (Figure 1). The proton-sensing GPCRs sense extracellular pH by protonation of several histidine residues on their extracellular domain. The activation of these proton-sensing GPCRs facilitates the downstream signaling through the Gq/11, Gs, and G12/13 pathways. Their expression varies in different cell types and play critical roles in sensing extracellular acidity and modulating cellular functions in several biological systems.

Figure 1 Biological roles and G protein coupling of the pH-sensing GPCRs

Biological roles and G protein coupling of the pH-sensing GPCRs

Biological roles and G protein coupling of the pH-sensing GPCRs

http://www.dovepress.com/cr_data/article_fulltext/s60000/60508/img/fig1small.jpg

Cells encounter acidotic stress in many pathophysiologic conditions such as inflammation, cancer, and ischemia. Intricate molecular mechanisms, including a large array of acid/base transporters and acid sensors, have evolved for cells to sense and respond to acidotic stress. Emerging evidence has demonstrated that a family of the pH-sensing GPCRs can be activated by extracellular acidotic stress and regulate the function of multiple physiological systems (Table 1). The pH-sensing GPCRs also play important roles in various pathological disorders. Agonists, antagonists and other modulators of the pH-sensing GPCRs are being actively developed and evaluated as potential novel treatment for acidosis-related diseases.

Table 1 The main biological functions of the pH-sensing GPCRs
Table1 The main biological functions of the pH-sensing GPCRs

Table1 The main biological functions of the pH-sensing GPCRs

http://www.dovepress.com/cr_data/article_fulltext/s60000/60508/img/Table1small.jpg

7.1.6 Protein amino-terminal modifications and proteomic approaches for N-terminal profiling

Lai ZW1Petrera A2Schilling O3.
Curr Opin Chem Biol. 2015 Feb; 24:71-9
http://dx.doi.org/10.1016/j.cbpa.2014.10.026

Highlights

  • N-terminal acetylation, pyroglutamate formation, N-degrons and proteolysis are reviewed.
  • N-terminomics provide comprehensive profiling of modification at protein N-termini in a proteome-wide manner.
  • We outline a number of established methodologies for the enrichment of protein N-termini through positive and negative selection strategies.
  • Peptidomics-based approach is beneficial for the study of post-translational processing of protein N-termini.

Amino-/N-terminal processing is a crucial post-translational modification affecting almost all proteins. In addition to altering the chemical properties of the N-terminus, these modifications affect protein activation, conversion, and degradation, which subsequently lead to diversified biological functions. The study of N-terminal modifications is of increasing interest; especially since modifications such as proteolytic truncation or pyroglutamate formation have been linked to disease processes. During the past decade, mass spectrometry has played an important role in facilitating the investigation of N-terminal modifications. Continuous progress is being made in the development and application of robust methods for the dedicated analysis of native and modified protein N-termini in a proteome-wide manner. Here we highlight recent progress in our understanding of protein N-terminal biology as well as outlining present enrichment strategies for mass spectrometry-based studies of protein N-termini

7.1.7 Protein homeostasis networks in physiology and disease

Claudio Hetz1,2,3,* and Laurie H. Glimcher3,4,*
Curr Opin Cell Biol. 2011 Apr; 23(2): 123–125.
http://dx.doi.org/10.1016%2Fj.ceb.2011.01.004

Although most text books of biochemistry describe the process of protein folding to a three dimensional native state as an intrinsic property of the primary sequence, it is becoming increasingly clear that this process can go wrong in an almost infinite number of ways. In fact, many different diseases are caused by the misfolding and aggregation of certain proteins without genetic mutations in the primary sequence. An integrative view of the mechanisms that maintain protein folding homeostasis is emerging, which could be thought as a balanced and dynamic network of interconnected processes tightly regulated by a series of quality control mechanisms. This protein homeostasis network involves families of folding catalysts, co-factors under specific environmental and metabolic conditions. Maintaining protein homeostasis is particularly challenging in specialized secretory cells where the high demand for protein synthesis generates a constant source of stress that could lead to proteotoxicity.

Protein folding is assisted and monitored by diverse interconnected processes that follow a sequential pattern over time. The calnexin/calreticulin cycle ensures the proper folding of glycosylated proteins through the secretory pathway, which establishes the final pattern of disulfide bond formation through interactions with the disulfide isomerase ERp57. Coupled to this cycle is the ER-associated degradation (ERAD) pathway, which translocates terminally misfolded proteins to the cytosol for degradation by proteasomes. In addition, macroautophagy is becoming a relevant mechanism for the clearance of damaged proteins and abnormal protein aggregates through lysosomal hydrolysis, a process also referred to as ERAD-II. The folding status at the ER is constantly monitored by the Unfolded Protein Response (UPR), a specialized signaling pathway initiated by the activation of three types of stress sensors. The process underlying the surveillance of protein folding stress by the UPR is not fully understood, but it may require coupling to key folding mediators such as BiP or the direct recognition of the misfolded peptides by stress sensors. The UPR regulates genes and processs related to almost every folding step in the secretory pathway to reduce the load of misfolded proteins, including protein translation into the ER, translocation, folding, quality control, ERAD, the redox status, and many other related functions. Protein folding stress is observed in many disease conditions such as cancer, diabetes, and neurodegeneration. For example, abnormal protein aggregation and the accumulation of protein inclusions is associated with Parkinson’s and Alzheimer’s Disease, and amyotrophic lateral sclerosis. In those diseases and many others, neuronal dysfunction and disease progression correlates with the presence of a strong ER stress response; however, the direct in vivo role of the UPR in the disease process has been experimentally defined in only a few cases. Therapeutic strategies are currently being developed to increase protein folding and clearance of misfolded proteins, with the goal of alleviating ER stress.

In this issue of Current Opinion in Cell Biology we present a series of focused reviews from recognized experts in the field, that provide an overview of mechanisms underlying protein folding and quality control, and how balance of protein homeostasis is maintained in physiology and deregulated in diseases. Daniela Roth and William Balch integrate the concept of protein homeostasis networks into an interesting model termed FoldFx, showing how the interconnection between different pathways in the context of the cellular proteome determines the energetic barrier required to generate a functional folded peptide. The authors have previously proposed the term Proteostasis to refer to the set of interacting activities that maintain the health of the proteome and the organism (protein homeostasis). The ER is a central subcellular compartment for protein synthesis and quality control in the secretory pathway. Yukio Kimata and Kenji Kohno give an overview of the signaling pathways that control adaptation to ER stress and maintenance of protein folding homeostasis. The authors summarize the models proposed so far for the activation of UPR stress sensors, and discuss how this directly or indirectly relates to the accumulation of unfolded proteins in the ER lumen. Chronic or irreversible ER stress triggers cell death by apoptosis. Gordon Shore, Feroz Papa, and Scott Oakes summarize the complex signaling pathways initiating apoptosis by ER stress, where cross talk between the ER and the mitochondria play a central role. The authors focus on addressing the role of the BCL-2 protein family on the activation of intrinsic mitochondrial apoptosis pathways, highlighting different cytosolic and transcriptional events that determine the transition between adaptive responses to apoptosis programmed by the UPR to eliminate irreversibly injured cells.

Although diverse families of chaperones, foldases and co-factors are expressed at the ER, only a few protein folding networks have been well defined. However, molecular explanations for specific substrate recognition and quality control mechanisms are poorly defined. Here we present a series of reviews covering different aspects of protein maturation. Amy Lee summarizes what is known about the biology of the key ER folding chaperone BiP/Grp78, and its emerging role in diverse pathological conditions including cancer. In two reviews, David B. Williams and Linda M. Hendershot describe the best characterized mechanism of protein quality control at the ER, the calnexin cycle. In addition, they give an overview of the function of a family of ER foldases, the protein disulfide isomerases (PDIs), in folding, quality control and degradation of abnormally folded proteins. PDIs are also becoming key factors in establishing the redox tone of the ER. Riccardo Bernasconi and Maurizio Molinari overview the ERAD process and how this pathway affects the efficiency of the protein folding process at the ER and its relation to pathological conditions.

Lysosomal-mediated degradation is becoming a fundamental process for the control of the haft-life of proteins and the degradation of misfolded, aggregate prone proteins. Ana Maria Cuervo reviews the relevance of Chaperone-mediated autophagy in the selective degradation of soluble cytosolic proteins in lysosomes, and also points out a key role for Chaperone-mediated autophagy in the cellular defense against proteotoxicity. David Rubinsztein and Guido Kroemer present two reviews highlighting the emerging relevance of macroautophagy in maintaining the homeostasis of the nervous system. They also discuss the actual impact of macroautophagy in the clearance of protein aggregates related to neurodegenerative diseases, including Parkinson’s disease, amyotrophic lateral sclerosis, Huntington’s disease among others. In addition, recent evidence suggesting an actual impairment of macroautophagy as a causative factor in aging-related disorders is also discussed.

Strategies to increase the efficiency of quality control mechanisms, to reduce protein aggregation and to enhance folding are suggested to be beneficial in the setting of diseases associated with the disruption of protein homeostasis.  Jeffery Kelly reviews recent chemical and biological therapeutic strategies to restore protein homeostasis, which could be achieved by enhancing the biological capacity of the proteostasis network or through small molecule to stabilize misfolding-prone proteins. In summary, this volume of Current Opinion in Cell Biology compiles the most recent advances in understanding the impact of protein folding stress in physiology and disease, and integrates a variety of complex mechanisms that evolved to maintain protein homeostasis in a dynamic way in the context of a changing environment. The biomedical applications of developing strategies to cope with protein folding stress have profound implications for the treatment of the most prevalent diseases in the human population.

7.1.8 Proteome sequencing goes deep

Richards AL1Merrill AE2Coon JJ3.
Curr Opin Chem Biol. 2015 Feb; 24:11-7
http://dx.doi.org/10.1016/j.cbpa.2014.10.017

Highlights

  • Recent MS advances have transformed the depth of coverage of the human proteome.
  • Expression of half the estimated human protein coding genes can be verified by MS.
  • MS sample preparation, instrumentation, and data analysis techniques are highlighted.

Advances in mass spectrometry (MS) have transformed the scope and impact of protein characterization efforts. Identifying hundreds of proteins from rather simple biological matrices, such as yeast, was a daunting task just a few decades ago. Now, expression of more than half of the estimated ∼20 000 human protein coding genes can be confirmed in record time and from minute sample quantities. Access to proteomic information at such unprecedented depths has been fueled by strides in every stage of the shotgun proteomics workflow — from sample processing to data analysis — and promises to revolutionize our understanding of the causes and consequences of proteome variation.

  1. Advances in proteomic sample preparation
  2. Advances in peptide separation and MS instrumentation
  3. Advances in computational proteomics
  4. Conclusions and outlook

Mg²+ is critical for maintaining the positional integrity of closely clustered phosphate groups. These clusters appear in numerous and distinct parts of the cell nucleus and cytoplasm. The Mg²+ ion maintains the integrity of nucleic acids, ribosomes and proteins. In addition, this ion acts as an oligo-element with role in energy catalysis. [6] Biological cell membranes and cell walls exhibit poly-anionic charges on the surface. This finding has important implications for the transport of ions, particularly because different membranes preferentially bind different ions. Both Mg²+ and Ca²+ regularly stabilize membranes by cross-linking the carboxylated and phosphorylated head groups of lipids.

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Introduction to Protein Synthesis and Degradation

Curator: Larry H. Bernstein, MD, FCAP

Updated 8/31/2019

 

Introduction to Protein Synthesis and Degradation

This chapter I made to follow signaling, rather than to precede it. I had already written much of the content before reorganizing the contents. The previous chapters on carbohydrate and on lipid metabolism have already provided much material on proteins and protein function, which was persuasive of the need to introduce signaling, which entails a substantial introduction to conformational changes in proteins that direct the trafficking of metabolic pathways, but more subtly uncovers an important role for microRNAs, not divorced from transcription, but involved in a non-transcriptional role.  This is where the classic model of molecular biology lacked any integration with emerging metabolic concepts concerning regulation. Consequently, the science was bereft of understanding the ties between the multiple convergence of transcripts, the selective inhibition of transcriptions, and the relative balance of aerobic and anaerobic metabolism, the weight of the pentose phosphate shunt, and the utilization of available energy source for synthetic and catabolic adaptive responses.

The first subchapter serves to introduce the importance of transcription in translational science.  The several subtitles that follow are intended to lay out the scope of the transcriptional activity, and also to direct attention toward the huge role of proteomics in the cell construct.  As we have already seen, proteins engage with carbohydrates and with lipids in important structural and signaling processes.  They are integrasl to the composition of the cytoskeleton, and also to the extracellular matrix.  Many proteins are actually enzymes, carrying out the transformation of some substrate, a derivative of the food we ingest.  They have a catalytic site, and they function with a cofactor – either a multivalent metal or a nucleotide.

The amino acids that go into protein synthesis include “indispensable” nutrients that are not made for use, but must be derived from animal protein, although the need is partially satisfied by plant sources. The essential amino acids are classified into well established groups. There are 20 amino acids commonly found in proteins.  They are classified into the following groups based on the chemical and/or structural properties of their side chains :

  1. Aliphatic Amino Acids
  2. Cyclic Amino Acid
  3. AAs with Hydroxyl or Sulfur-containing side chains
  4. Aromatic Amino Acids
  5. Basic Amino Acids
  6. Acidic Amino Acids and their Amides

Examples include:

Alanine                  aliphatic hydrophobic neutral
Arginine                 polar hydrophilic charged (+)
Cysteine                polar hydrophobic neutral
Glutamine             polar hydrophilic neutral
Histidine                aromatic polar hydrophilic charged (+)
Lysine                   polar hydrophilic charged (+)
Methionine            hydrophobic neutral
Serine                   polar hydrophilic neutral
Tyrosine                aromatic polar hydrophobic

Transcribe and Translate a Gene

  1. For each RNA base there is a corresponding DNA base
  2. Cells use the two-step process of transcription and translation to read each gene and produce the string of amino acids that makes up a protein.
  3. mRNA is produced in the nucleus, and is transferred to the ribosome
  4. mRNA uses uracil instead of thymine
  5. the ribosome reads the RNA sequence and makes protein
  6. There is a sequence combination to fit each amino acid to a three letter RNA code
  7. The ribosome starts at AUG (start), and it reads each codon three letters at a time
  8. Stop codons are UAA, UAG and UGA

 

protein synthesis

protein synthesis

http://learn.genetics.utah.edu/content/molecules/transcribe/images/TandT.png

mcell-transcription-translation

mcell-transcription-translation

http://www.vcbio.science.ru.nl/images/cellcycle/mcell-transcription-translation_eng_zoom.gif

transcription_translation

transcription_translation

 

http://www.biologycorner.com/resources/transcription_translation.JPG

 

What about the purine inosine?

Inosine triphosphate pyrophosphatase – Pyrophosphatase that hydrolyzes the non-canonical purine nucleotides inosine triphosphate (ITP), deoxyinosine triphosphate (dITP) as well as 2′-deoxy-N-6-hydroxylaminopurine triposphate (dHAPTP) and xanthosine 5′-triphosphate (XTP) to their respective monophosphate derivatives. The enzyme does not distinguish between the deoxy- and ribose forms. Probably excludes non-canonical purines from RNA and DNA precursor pools, thus preventing their incorporation into RNA and DNA and avoiding chromosomal lesions.

Gastroenterology. 2011 Apr;140(4):1314-21.  http://dx.doi.org:/10.1053/j.gastro.2010.12.038. Epub 2011 Jan 1.

Inosine triphosphate protects against ribavirin-induced adenosine triphosphate loss by adenylosuccinate synthase function.

Hitomi Y1, Cirulli ET, Fellay J, McHutchison JG, Thompson AJ, Gumbs CE, Shianna KV, Urban TJ, Goldstein DB.

Genetic variation of inosine triphosphatase (ITPA) causing an accumulation of inosine triphosphate (ITP) has been shown to protect patients against ribavirin (RBV)-induced anemia during treatment for chronic hepatitis C infection by genome-wide association study (GWAS). However, the biologic mechanism by which this occurs is unknown.

Although ITP is not used directly by human erythrocyte ATPase, it can be used for ATP biosynthesis via ADSS in place of guanosine triphosphate (GTP). With RBV challenge, erythrocyte ATP reduction was more severe in the wild-type ITPA genotype than in the hemolysis protective ITPA genotype. This difference also remains after inhibiting adenosine uptake using nitrobenzylmercaptopurine riboside (NBMPR).

ITP confers protection against RBV-induced ATP reduction by substituting for erythrocyte GTP, which is depleted by RBV, in the biosynthesis of ATP. Because patients with excess ITP appear largely protected against anemia, these results confirm that RBV-induced anemia is due primarily to the effect of the drug on GTP and consequently ATP levels in erythrocytes.

Ther Drug Monit. 2012 Aug;34(4):477-80.  http://dx.doi.org:/10.1097/FTD.0b013e31825c2703.

Determination of inosine triphosphate pyrophosphatase phenotype in human red blood cells using HPLC.

Citterio-Quentin A1, Salvi JP, Boulieu R.

Thiopurine drugs, widely used in cancer chemotherapy, inflammatory bowel disease, and autoimmune hepatitis, are responsible for common adverse events. Only some of these may be explained by genetic polymorphism of thiopurine S-methyltransferase. Recent articles have reported that inosine triphosphate pyrophosphatase (ITPase) deficiency was associated with adverse drug reactions toward thiopurine drug therapy. Here, we report a weak anion exchange high-performance liquid chromatography method to determine ITPase activity in red blood cells and to investigate the relationship with the occurrence of adverse events during azathioprine therapy.

The chromatographic method reported allows the analysis of IMP, inosine diphosphate, and ITP in a single run in <12.5 minutes. The method was linear in the range 5-1500 μmole/L of IMP. Intraassay and interassay precisions were <5% for red blood cell lysates supplemented with 50, 500, and 1000 μmole/L IMP. Km and Vmax evaluated by Lineweaver-Burk plot were 677.4 μmole/L and 19.6 μmole·L·min, respectively. The frequency distribution of ITPase from 73 patients was investigated.

The method described is useful to determine the ITPase phenotype from patients on thiopurine therapy and to investigate the potential relation between ITPase deficiency and the occurrence of adverse events.

 

System wide analyses have underestimated protein abundances and the importance of transcription in mammals

Jingyi Jessica Li1, 2, Peter J Bickel1 and Mark D Biggin3

PeerJ 2:e270; http://dx.doi.org:/10.7717/peerj.270

Using individual measurements for 61 housekeeping proteins to rescale whole proteome data from Schwanhausser et al. (2011), we find that the median protein detected is expressed at 170,000 molecules per cell and that our corrected protein abundance estimates show a higher correlation with mRNA abundances than do the uncorrected protein data. In addition, we estimated the impact of further errors in mRNA and protein abundances using direct experimental measurements of these errors. The resulting analysis suggests that mRNA levels explain at least 56% of the differences in protein abundance for the 4,212 genes detected by Schwanhausser et al. (2011), though because one major source of error could not be estimated the true percent contribution should be higher.We also employed a second, independent strategy to determine the contribution of mRNA levels to protein expression.We show that the variance in translation rates directly measured by ribosome profiling is only 12% of that inferred by Schwanhausser et al. (2011), and that the measured and inferred translation rates correlate poorly (R2 D 0.13). Based on this, our second strategy suggests that mRNA levels explain 81% of the variance in protein levels. We also determined the percent contributions of transcription, RNA degradation, translation and protein degradation to the variance in protein abundances using both of our strategies. While the magnitudes of the two estimates vary, they both suggest that transcription plays a more important role than the earlier studies implied and translation a much smaller role. Finally, the above estimates only apply to those genes whose mRNA and protein expression was detected. Based on a detailed analysis by Hebenstreit et al. (2012), we estimat that approximately 40% of genes in a given cell within a population express no mRNA. Since there can be no translation in the ab-sence of mRNA, we argue that differences in translation rates can play no role in determining the expression levels for the 40% of genes that are non-expressed.

 

Related studies that reveal issues that are not part of this chapter:

  1. Ubiquitylation in relationship to tissue remodeling
  2. Post-translational modification of proteins
    1. Glycosylation
    2. Phosphorylation
    3. Methylation
    4. Nitrosylation
    5. Sulfation – sulfotransferases
      cell-matrix communication
    6. Acetylation and histone deacetylation (HDAC)
      Connecting Protein Phosphatase to 1α (PP1α)
      Acetylation complexes (such as CBP/p300 and PCAF)
      Sirtuins
      Rel/NF-kB Signal Transduction
      Homologous Recombination Pathway of Double-Strand DNA Repair
    7. Glycination
    8. cyclin dependent kinases (CDKs)
    9. lyase
    10. transferase

 

This year, the Lasker award for basic medical research went to Kazutoshi Mori (Kyoto University) and Peter Walter (University of California, San Francisco) for their “discoveries concerning the unfolded protein response (UPR) — an intracellular quality control system that

detects harmful misfolded proteins in the endoplasmic reticulum and signals the nucleus to carry out corrective measures.”

About UPR: Approximately a third of cellular proteins pass through the Endoplasmic Reticulum (ER) which performs stringent quality control of these proteins. All proteins need to assume the proper 3-dimensional shape in order to function properly in the harsh cellular environment. Related to this is the fact that cells are under constant stress and have to make rapid, real time decisions about survival or death.

A major indicator of stress is the accumulation of unfolded proteins within the Endoplasmic Reticulum (ER), which triggers a transcriptional cascade in order to increase the folding capacity of the ER. If the metabolic burden is too great and homeostasis cannot be achieved, the response shifts from

damage control to the induction of pro-apoptotic pathways that would ultimately cause cell death.

This response to unfolded proteins or the UPR is conserved among all eukaryotes, and dysfunction in this pathway underlies many human diseases, including Alzheimer’s, Parkinson’s, Diabetes and Cancer.

 

The discovery of a new class of human proteins with previously unidentified activities

In a landmark study conducted by scientists at the Scripps Research Institute, The Hong Kong University of Science and Technology, aTyr Pharma and their collaborators, a new class of human proteins has been discovered. These proteins [nearly 250], called Physiocrines belong to the aminoacyl tRNA synthetase gene family and carry out novel, diverse and distinct biological functions.

The aminoacyl tRNA synthetase gene family codes for a group of 20 ubiquitous enzymes almost all of which are part of the protein synthesis machinery. Using recombinant protein purification, deep sequencing technique, mass spectroscopy and cell based assays, the team made this discovery. The finding is significant, also because it highlights the alternate use of a gene family whose protein product normally performs catalytic activities for non-catalytic regulation of basic and complex physiological processes spanning metabolism, vascularization, stem cell biology and immunology

 

Muscle maintenance and regeneration – key player identified

Muscle tissue suffers from atrophy with age and its regenerative capacity also declines over time. Most molecules discovered thus far to boost tissue regeneration are also implicated in cancers.  During a quest to find safer alternatives that can regenerate tissue, scientists reported that the hormone Oxytocin is required for proper muscle tissue regeneration and homeostasis and that its levels decline with age.

Oxytocin could be an alternative to hormone replacement therapy as a way to combat aging and other organ related degeneration.

Oxytocin is an age-specific circulating hormone that is necessary for muscle maintenance and regeneration (June 2014)

 

Proc Natl Acad Sci U S A. 2014 Sep 30;111(39):14289-94.   http://dx.doi.org:/10.1073/pnas.1407640111. Epub 2014 Sep 15.

Role of forkhead box protein A3 in age-associated metabolic decline.

Ma X1, Xu L1, Gavrilova O2, Mueller E3.

Aging is associated with increased adiposity and diminished thermogenesis, but the critical transcription factors influencing these metabolic changes late in life are poorly understood. We recently demonstrated that the winged helix factor forkhead box protein A3 (Foxa3) regulates the expansion of visceral adipose tissue in high-fat diet regimens; however, whether Foxa3 also contributes to the increase in adiposity and the decrease in brown fat activity observed during the normal aging process is currently unknown. Here we report that during aging, levels of Foxa3 are significantly and selectively up-regulated in brown and inguinal white fat depots, and that midage Foxa3-null mice have increased white fat browning and thermogenic capacity, decreased adipose tissue expansion, improved insulin sensitivity, and increased longevity. Foxa3 gain-of-function and loss-of-function studies in inguinal adipose depots demonstrated a cell-autonomous function for Foxa3 in white fat tissue browning. Furthermore, our analysis revealed that the mechanisms of Foxa3 modulation of brown fat gene programs involve the suppression of peroxisome proliferator activated receptor γ coactivtor 1 α (PGC1α) levels through interference with cAMP responsive element binding protein 1-mediated transcriptional regulation of the PGC1α promoter.

 

Asymmetric mRNA localization contributes to fidelity and sensitivity of spatially localized systems

RJ Weatheritt, TJ Gibson & MM Babu
Nature Structural & Molecular Biology 24 Aug, 2014; 21: 833–839 http://dx.do.orgi:/10.1038/nsmb.2876

Although many proteins are localized after translation, asymmetric protein distribution is also achieved by translation after mRNA localization. Why are certain mRNA transported to a distal location and translated on-site? Here we undertake a systematic, genome-scale study of asymmetrically distributed protein and mRNA in mammalian cells. Our findings suggest that asymmetric protein distribution by mRNA localization enhances interaction fidelity and signaling sensitivity. Proteins synthesized at distal locations frequently contain intrinsically disordered segments. These regions are generally rich in assembly-promoting modules and are often regulated by post-translational modifications. Such proteins are tightly regulated but display distinct temporal dynamics upon stimulation with growth factors. Thus, proteins synthesized on-site may rapidly alter proteome composition and act as dynamically regulated scaffolds to promote the formation of reversible cellular assemblies. Our observations are consistent across multiple mammalian species, cell types and developmental stages, suggesting that localized translation is a recurring feature of cell signaling and regulation.

 

An overview of the potential advantages conferred by distal-site protein synthesis, inferred from our analysis.

 

An overview of the potential advantages conferred by distal-site protein synthesis

An overview of the potential advantages conferred by distal-site protein synthesis

 

Turquoise and red filled circle represents off-target and correct interaction partners, respectively. Wavy lines represent a disordered region within a distal site synthesis protein. Grey and red line in graphs represents profiles of t…

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F5.jpg

 

Tweaking transcriptional programming for high quality recombinant protein production

Since overexpression of recombinant proteins in E. coli often leads to the formation of inclusion bodies, producing properly folded, soluble proteins is undoubtedly the most important end goal in a protein expression campaign. Various approaches have been devised to bypass the insolubility issues during E. coli expression and in a recent report a group of researchers discuss reprogramming the E. coli proteostasis [protein homeostasis] network to achieve high yields of soluble, functional protein. The premise of their studies is that the basal E. coli proteostasis network is insufficient, and often unable, to fold overexpressed proteins, thus clogging the folding machinery.

By overexpressing a mutant, negative-feedback deficient heat shock transcription factor [σ32 I54N] before and during overexpression of the protein of interest, reprogramming can be achieved, resulting in high yields of soluble and functional recombinant target protein. The authors explain that this method is better than simply co-expressing/over-expressing chaperones, co-chaperones, foldases or other components of the proteostasis network because reprogramming readies the folding machinery and up regulates the essential folding components beforehand thus  maintaining system capability of the folding machinery.

The Heat-Shock Response Transcriptional Program Enables High-Yield and High-Quality Recombinant Protein Production in Escherichia coli (July 2014)

 

 Unfolded proteins collapse when exposed to heat and crowded environments

Proteins are important molecules in our body and they fulfil a broad range of functions. For instance as enzymes they help to release energy from food and as muscle proteins they assist with motion. As antibodies they are involved in immune defence and as hormone receptors in signal transduction in cells. Until only recently it was assumed that all proteins take on a clearly defined three-dimensional structure – i.e. they fold in order to be able to assume these functions. Surprisingly, it has been shown that many important proteins occur as unfolded coils. Researchers seek to establish how these disordered proteins are capable at all of assuming highly complex functions.

Ben Schuler’s research group from the Institute of Biochemistry of the University of Zurich has now established that an increase in temperature leads to folded proteins collapsing and becoming smaller. Other environmental factors can trigger the same effect.

Measurements using the “molecular ruler”

“The fact that unfolded proteins shrink at higher temperatures is an indication that cell water does indeed play an important role as to the spatial organisation eventually adopted by the molecules”, comments Schuler with regard to the impact of temperature on protein structure. For their studies the biophysicists use what is known as single-molecule spectroscopy. Small colour probes in the protein enable the observation of changes with an accuracy of more than one millionth of a millimetre. With this “molecular yardstick” it is possible to measure how molecular forces impact protein structure.

With computer simulations the researchers have mimicked the behaviour of disordered proteins.
(Courtesy of Jose EDS Roselino, PhD.

 

MLKL compromises plasma membrane integrity

Necroptosis is implicated in many diseases and understanding this process is essential in the search for new therapies. While mixed lineage kinase domain-like (MLKL) protein has been known to be a critical component of necroptosis induction, how MLKL transduces the death signal was not clear. In a recent finding, scientists demonstrated that the full four-helical bundle domain (4HBD) in the N-terminal region of MLKL is required and sufficient to induce its oligomerization and trigger cell death.

They also found a patch of positively charged amino acids on the surface of the 4HBD that bound to phosphatidylinositol phosphates (PIPs) and allowed the recruitment of MLKL to the plasma membrane that resulted in the formation of pores consisting of MLKL proteins, due to which cells absorbed excess water causing them to explode. Detailed knowledge about how MLKL proteins create pores offers possibilities for the development of new therapeutic interventions for tolerating or preventing cell death.

MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates (May 2014)

 

Mitochondrial and ER proteins implicated in dementia

Mitochondria and the endoplasmic reticulum (ER) form tight structural associations that facilitate a number of cellular functions. However, the molecular mechanisms of these interactions aren’t properly understood.

A group of researchers showed that the ER protein VAPB interacted with mitochondrial protein PTPIP51 to regulate ER-mitochondria associations and that TDP-43, a protein implicated in dementia, disturbs this interaction to regulate cellular Ca2+ homeostasis. These studies point to a new pathogenic mechanism for TDP-43 and may also provide a potential new target for the development of new treatments for devastating neurological conditions like dementia.

ER-mitochondria associations are regulated by the VAPB-PTPIP51 interaction and are disrupted by ALS/FTD-associated TDP-43. Nature (June 2014)

 

A novel strategy to improve membrane protein expression in Yeast

Membrane proteins play indispensable roles in the physiology of an organism. However, recombinant production of membrane proteins is one of the biggest hurdles facing protein biochemists today. A group of scientists in Belgium showed that,

by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis,

enhanced expression of recombinant membrane proteins in yeast is achieved.

Specifically, they engineered the oleotrophic yeast, Yarrowia lipolytica, by

deleting the phosphatidic acid phosphatase, PAH1 gene,

which led to massive proliferation of endoplasmic reticulum (ER) membranes.

For all 8 tested representatives of different integral membrane protein families, they obtained enhanced protein accumulation.

 

An unconventional method to boost recombinant protein levels

MazF is an mRNA interferase enzyme in E.coli that functions as and degrades cellular mRNA in a targeted fashion, at the “ACA” sequence. This degradation of cellular mRNA causes a precipitous drop in cellular protein synthesis. A group of scientists at the Robert Wood Johnson Medical School in New Jersey, exploited the degeneracy of the genetic code to modify all “ACA” triplets within their gene of interest in a way that the corresponding amino acid (Threonine) remained unchanged. Consequently, induction of MazF toxin caused degradation of E.coli cellular mRNA but the recombinant gene transcription and protein synthesis continued, causing significant accumulation of high quality target protein. This expression system enables unparalleled signal to noise ratios that could dramatically simplify structural and functional studies of difficult-to-purify, biologically important proteins.

 

Tandem fusions and bacterial strain evolution for enhanced functional membrane protein production

Membrane protein production remains a significant challenge in its characterization and structure determination. Despite the fact that there are a variety of host cell types, E.coli remains the popular choice for producing recombinant membrane proteins. A group of scientists in Netherlands devised a robust strategy to increase the probability of functional membrane protein overexpression in E.coli.

By fusing Green Fluorescent Protein (GFP) and the Erythromycin Resistance protein (ErmC) to the C-terminus of a target membrane protein they wer e able to track the folding state of their target protein while using Erythromycin to select for increased expression. By increasing erythromycin concentration in the growth media and testing different membrane targets, they were able to identify four evolved E.coli strains, all of which carried a mutation in the hns gene, whose product is implicated in genome organization and transcriptional silencing. Through their experiments the group showed that partial removal of the transcriptional silencing mechanism was related to production of proteins that were essential for functional overexpression of membrane proteins.

 

The role of an anti-apoptotic factor in recombinant protein production

In a recent study, scientists at the Johns Hopkins University and Frederick National Laboratory for Cancer Research examined an alternative method of utilizing the benefits of anti-apoptotic gene expression to enhance the transient expression of biotherapeutics, specifically, through the co-transfection of Bcl-xL along with the product-coding target gene.

Chinese Hamster Ovary(CHO) cells were co-transfected with the product-coding gene and a vector containing Bcl-xL, using Polyethylenimine (PEI) reagent. They found that the cells co-transfected with Bcl-xL demonstrated reduced apoptosis, increased specific productivity, and an overall increase in product yield.

B-cell lymphoma-extra-large (Bcl-xL) is a mitochondrial transmembrane protein and a member of the Bcl-2 family of proteins which are known to act as either pro- or anti-apoptotic proteins. Bcl-xL itself acts as an anti-apoptotic molecule by preventing the release of mitochondrial contents such as cytochrome c, which would lead to caspase activation. Higher levels of Bcl-xL push a cell toward survival mode by making the membranes pores less permeable and leaky.

Introduction to Protein Synthesis and Degradation Updated 8/31/2019

N-Terminal Degradation of Proteins: The N-End Rule and N-degrons

In both prokaryotes and eukaryotes mitochondria and chloroplasts, the ribosomal synthesis of proteins is initiated with the addition of the N-formyl methionine residue.  However in eukaryotic cytosolic ribosomes, the N terminal was assumed to be devoid of the N-formyl group.  The unformylated N-terminal methionine residues of eukaryotes is then  often N-acetylated (Ac) and creates specific degradation signals, the Ac N-end rule.  These N-end rule pathways are proteolytic systems which recognize these N-degrons resulting in proteosomal degradation or autophagy.  In prokaryotes this system is stimulated by certain amino acid deficiencies and in eukaryotes is dependent on the Psh1 E3 ligase.

Two papers in the journal Science describe this N-degron in more detail.

Structured Abstract
INTRODUCTION

In both bacteria and eukaryotic mitochondria and chloroplasts, the ribosomal synthesis of proteins is initiated with the N-terminal (Nt) formyl-methionine (fMet) residue. Nt-fMet is produced pretranslationally by formyltransferases, which use 10-formyltetrahydrofolate as a cosubstrate. By contrast, proteins synthesized by cytosolic ribosomes of eukaryotes were always presumed to bear unformylated N-terminal Met (Nt-Met). The unformylated Nt-Met residue of eukaryotic proteins is often cotranslationally Nt-acetylated, a modification that creates specific degradation signals, Ac/N-degrons, which are targeted by the Ac/N-end rule pathway. The N-end rule pathways are a set of proteolytic systems whose unifying feature is their ability to recognize proteins containing N-degrons, thereby causing the degradation of these proteins by the proteasome or autophagy in eukaryotes and by the proteasome-like ClpAP protease in bacteria. The main determinant of an N‑degron is a destabilizing Nt-residue of a protein. Studies over the past three decades have shown that all 20 amino acids of the genetic code can act, in cognate sequence contexts, as destabilizing Nt‑residues. The previously known eukaryotic N-end rule pathways are the Arg/N-end rule pathway, the Ac/N-end rule pathway, and the Pro/N-end rule pathway. Regulated degradation of proteins and their natural fragments by the N-end rule pathways has been shown to mediate a broad range of biological processes.

RATIONALE

The chemical similarity of the formyl and acetyl groups and their identical locations in, respectively, Nt‑formylated and Nt-acetylated proteins led us to suggest, and later to show, that the Nt-fMet residues of nascent bacterial proteins can act as bacterial N-degrons, termed fMet/N-degrons. Here we wished to determine whether Nt-formylated proteins might also form in the cytosol of a eukaryote such as the yeast Saccharomyces cerevisiae and to determine the metabolic fates of Nt-formylated proteins if they could be produced outside mitochondria. Our approaches included molecular genetic techniques, mass spectrometric analyses of proteins’ N termini, and affinity-purified antibodies that selectively recognized Nt-formylated reporter proteins.

RESULTS

We discovered that the yeast formyltransferase Fmt1, which is imported from the cytosol into the mitochondria inner matrix, can generate Nt-formylated proteins in the cytosol, because the translocation of Fmt1 into mitochondria is not as efficacious, even under unstressful conditions, as had previously been assumed. We also found that Nt‑formylated proteins are greatly up-regulated in stationary phase or upon starvation for specific amino acids. The massive increase of Nt-formylated proteins strictly requires the Gcn2 kinase, which phosphorylates Fmt1 and mediates its retention in the cytosol. Notably, the ability of Gcn2 to retain a large fraction of Fmt1 in the cytosol of nutritionally stressed cells is confined to Fmt1, inasmuch as the Gcn2 kinase does not have such an effect, under the same conditions, on other examined nuclear DNA–encoded mitochondrial matrix proteins. The Gcn2-Fmt1 protein localization circuit is a previously unknown signal transduction pathway. A down-regulation of cytosolic Nt‑formylation was found to increase the sensitivity of cells to undernutrition stresses, to a prolonged cold stress, and to a toxic compound. We also discovered that the Nt-fMet residues of Nt‑formylated cytosolic proteins act as eukaryotic fMet/N-degrons and identified the Psh1 E3 ubiquitin ligase as the recognition component (fMet/N-recognin) of the previously unknown eukaryotic fMet/N-end rule pathway, which destroys Nt‑formylated proteins.

CONCLUSION

The Nt-formylation of proteins, a long-known pretranslational protein modification, is mediated by formyltransferases. Nt-formylation was thought to be confined to bacteria and bacteria-descended eukaryotic organelles but was found here to also occur at the start of translation by the cytosolic ribosomes of a eukaryote. The levels of Nt‑formylated eukaryotic proteins are greatly increased upon specific stresses, including undernutrition, and appear to be important for adaptation to these stresses. We also discovered that Nt-formylated cytosolic proteins are selectively destroyed by the eukaryotic fMet/N-end rule pathway, mediated by the Psh1 E3 ubiquitin ligase. This previously unknown proteolytic system is likely to be universal among eukaryotes, given strongly conserved mechanisms that mediate Nt‑formylation and degron recognition.

The eukaryotic fMet/N-end rule pathway.

(Top) Under undernutrition conditions, the Gcn2 kinase augments the cytosolic localization of the Fmt1 formyltransferase, and possibly also its enzymatic activity. Consequently, Fmt1 up-regulates the cytosolic fMet–tRNAi (initiator transfer RNA), and thereby increases the levels of cytosolic Nt-formylated proteins, which are required for the adaptation of cells to specific stressors. (Bottom) The Psh1 E3 ubiquitin ligase targets the N-terminal fMet-residues of eukaryotic cytosolic proteins, such as Cse4, Pgd1, and Rps22a, for the polyubiquitylation-mediated, proteasome-dependent degradation.

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The eukaryotic fMet/N-end rule pathway.

(Top) Under undernutrition conditions, the Gcn2 kinase augments the cytosolic localization of the Fmt1 formyltransferase, and possibly also its enzymatic activity. Consequently, Fmt1 up-regulates the cytosolic fMet–tRNAi (initiator transfer RNA), and thereby increases the levels of cytosolic Nt-formylated proteins, which are required for the adaptation of cells to specific stressors. (Bottom) The Psh1 E3 ubiquitin ligase targets the N-terminal fMet-residues of eukaryotic cytosolic proteins, such as Cse4, Pgd1, and Rps22a, for the polyubiquitylation-mediated, proteasome-dependent degradation.

 

A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation. Richard T. Timms1,2Zhiqian Zhang1,2David Y. Rhee3J. Wade Harper3Itay Koren1,2,*,Stephen J. Elledge1,2

Science  05 Jul 2019: Vol. 365, Issue 6448

The second paper describes a glycine specific N-degron pathway in humans.  Specifically the authors set up a screen to identify specific N-terminal degron motifs in the human.  Findings included an expanded repertoire for the UBR E3 ligases to include substrates with arginine and lysine following an intact initiator methionine and a glycine at the extreme N-terminus, which is a potent degron.

Glycine N-degron regulation revealed

For more than 30 years, N-terminal sequences have been known to influence protein stability, but additional features of these N-end rule, or N-degron, pathways continue to be uncovered. Timms et al. used a global protein stability (GPS) technology to take a broader look at these pathways in human cells. Unexpectedly, glycine exposed at the N terminus could act as a potent degron; proteins bearing N-terminal glycine were targeted for proteasomal degradation by two Cullin-RING E3 ubiquitin ligases through the substrate adaptors ZYG11B and ZER1. This pathway may be important, for example, to degrade proteins that fail to localize properly to cellular membranes and to destroy protein fragments generated during cell death.

Science, this issue p. eaaw4912

Structured Abstract

INTRODUCTION

The ubiquitin-proteasome system is the major route through which the cell achieves selective protein degradation. The E3 ubiquitin ligases are the major determinants of specificity in this system, which is thought to be achieved through their selective recognition of specific degron motifs in substrate proteins. However, our ability to identify these degrons and match them to their cognate E3 ligase remains a major challenge.

RATIONALE

It has long been known that the stability of proteins is influenced by their N-terminal residue, and a large body of work over the past three decades has characterized a collection of N-end rule pathways that target proteins for degradation through N-terminal degron motifs. Recently, we developed Global Protein Stability (GPS)–peptidome technology and used it to delineate a suite of degrons that lie at the extreme C terminus of proteins. We adapted this approach to examine the stability of the human N terminome, allowing us to reevaluate our understanding of N-degron pathways in an unbiased manner.

RESULTS

Stability profiling of the human N terminome identified two major findings: an expanded repertoire for UBR family E3 ligases to include substrates that begin with arginine and lysine following an intact initiator methionine and, more notably, that glycine positioned at the extreme N terminus can act as a potent degron. We established human embryonic kidney 293T reporter cell lines in which unstable peptides that bear N-terminal glycine degrons were fused to green fluorescent protein, and we performed CRISPR screens to identify the degradative machinery involved. These screens identified two Cul2 Cullin-RING E3 ligase complexes, defined by the related substrate adaptors ZYG11B and ZER1, that act redundantly to target substrates bearing N-terminal glycine degrons for proteasomal degradation. Moreover, through the saturation mutagenesis of example substrates, we defined the composition of preferred N-terminal glycine degrons specifically recognized by ZYG11B and ZER1.

We found that preferred glycine degrons are depleted from the native N termini of metazoan proteomes, suggesting that proteins have evolved to avoid degradation through this pathway, but are strongly enriched at annotated caspase cleavage sites. Stability profiling of N-terminal peptides lying downstream of all known caspase cleavages sites confirmed that Cul2ZYG11Band Cul2ZER1 could make a substantial contribution to the removal of proteolytic cleavage products during apoptosis. Last, we identified a role for ZYG11B and ZER1 in the quality control of N-myristoylated proteins. N-myristoylation is an important posttranslational modification that occurs exclusively on N-terminal glycine. By profiling the stability of the human N-terminome in the absence of the N-myristoyltransferases NMT1 and NMT2, we found that a failure to undergo N-myristoylation exposes N-terminal glycine degrons that are otherwise obscured. Thus, conditional exposure of glycine degrons to ZYG11B and ZER1 permits the selective proteasomal degradation of aberrant proteins that have escaped N-terminal myristoylation.

CONCLUSION

These data demonstrate that an additional N-degron pathway centered on N-terminal glycine regulates the stability of metazoan proteomes. Cul2ZYG11B– and Cul2ZER1-mediated protein degradation through N-terminal glycine degrons may be particularly important in the clearance of proteolytic fragments generated by caspase cleavage during apoptosis and in the quality control of protein N-myristoylation.

The glycine N-degron pathway.

Stability profiling of the human N-terminome revealed that N-terminal glycine acts as a potent degron. CRISPR screening revealed two Cul2 complexes, defined by the related substrate adaptors ZYG11B and ZER1, that recognize N-terminal glycine degrons. This pathway may be particularly important for the degradation of caspase cleavage products during apoptosis and the removal of proteins that fail to undergo N-myristoylation.

” data-icon-position=”” data-hide-link-title=”0″>

The glycine N-degron pathway.

Stability profiling of the human N-terminome revealed that N-terminal glycine acts as a potent degron. CRISPR screening revealed two Cul2 complexes, defined by the related substrate adaptors ZYG11B and ZER1, that recognize N-terminal glycine degrons. This pathway may be particularly important for the degradation of caspase cleavage products during apoptosis and the removal of proteins that fail to undergo N-myristoylation.

 

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Larry H Bernstein, MD, FCAP, Curator

http://pharmaceuticalintelligence/7/8/2014/Proteins and cellular adaptation to stress

There are two recent articles that are, if not interesting, possibly important in the direction of cellular regulation, adaptation, and decline.  One deals with apoptosis, or cell death, which is synchronized with recovery of membrane and protein breakdown for reuse in synthesis and maintenance.  The other is a new perspective to Alzhemier’s Disease, for which there is no effective pharmacotherapy. In both cases, the stresses of the cell are critical to the responce to the environment.  This is not just about the classical transcriptomics story. This is a perfect followup to the just posted research on the regulatory role of a small RNA that is related to, but distinct from silencing RNA, and also the revelations about lncRNA.

Protein Helps Cells Adapt—or Die

Scientists show how cell stress both prevents and promotes cell suicide in a study that’s equally divisive.

By Ruth Williams | July 3, 2014

A cellular stress pathway called the unfolded-protein-response (UPR) both activates and degrades death receptor 5 protein (DR5), which can promote or prevent cell suicide, according to a paper published in Science today (July 3). The theory is that initial stress blocks cell suicide, or apoptosis, to give the cell a chance to adapt, but that if the stress persists, it eventually triggers apoptosis.

“This work has made the most beautiful simplification of all this big complex mess. Basically, they identified and pinpointed the specific protein involved in the switching decision and explain how the decision is made,” said Alexei Korennykh, a professor of molecular biology at Princeton University, who was not involved in the work.

But Randal Kaufman of the Sanford-Burnham Medical Research Institute in La Jolla, California, was not impressed. He questioned the physiological relevance of the experiments supporting the authors’ main conclusions about this key cellular process.

Protein folding in a cell takes place largely in the endoplasmic reticulum (ER), but if the process goes awry, unfolded proteins accumulate, stressing the ER. This triggers the UPR, which shuts down translation, degrades unfolded proteins, and increases production of protein-folding machinery. If ER stress is not resolved, however, the UPR can also induce apoptosis.

Two main factors control the UPR—IRE1a and PERK. IRE1a promotes cell survival by activating the transcription factor XBP1, which drives expression of cell-survival genes. PERK, on the other hand, activates a transcription factor called CHOP, which in turn drives expression of the proapoptotic factor DR5.

Peter Walter of the University of California, San Francisco, and his colleagues have now confirmed that CHOP activates DR5, showing that it is a cell-autonomous process. But they have also found that IRE1a suppresses DR5, directly degrading its mRNA through a process called regulated IRE1a-dependent degradation (RIDD). Inhibition of IRE1a in a human cancer cell line undergoing ER stress both prevented DR5 mRNA decay and increased apoptosis.

However, in an e-mail to The Scientist, Kaufman expressed concern that “the significance of RIDD has not been demonstrated in a physiologically-relevant context.”

Walter insisted that the evidence for RIDD’s existence is “crystal clear.” His only concession was that “the effects aren’t 100 percent,” he said, because “RIDD degrades mRNA by a few-fold,” making it difficult to measure.

This RIDD debate aside, the researchers have also sparked a rumpus with their finding that IRE1a expression switches off just 24 hours after ER stress initiation, leaving PERK to drive the cell toward apoptosis. “We and others have evidence that suggests another model,” said Scott Oakes, a professor of pathology at the University of California, San Francisco, “which is that both PERK and IRE1a under high stress will send out death signals.”

Whether IRE1a promotes or inhibits apoptosis under extreme stress “is controversial,” said Ira Tabas, a professor at Columbia University in New York City. But it’s essential that scientists figure it out. Cell death from ER stress is a pathological process in many major diseases, Tabas said, and there are IRE1a inhibitors in pharmaceutical development. “It is very important because under high stress you have two different views here,” said Oakes. “One is that you want to keep IRE1a on, the other is that you want to shut it off.”

Because ER stress is central to many diseases, “a lot of people are passionate about it,” said Tabas, explaining the polemic views. “Who’s right? . . . I think it depends on the context in which the experiments are done—one pathway may be important in some settings, and another pathway may be important in different settings,” he suggested. What might help to resolve the issues, he said, will be “in vivo causation studies using actual disease models.”

Researchers will continue to debate. So, said Walter, “we’ll have to see what holds-up five years from now.”

M. Lu et al., “Opposing unfolded-protein-response signals converge on death receptor 5 to control apoptosis,” Science, 345:98-101, 2014.

Tags stress responseprotein foldingdisease/medicinecell & molecular biology and apoptosis

 

Protein May Hold the Key to Who Gets Alzheimer’s

 

By PAM BELLUCK     MARCH 19, 2014

 

It is one of the big scientific mysteries of Alzheimer’s disease: Why do some people whose brains accumulate the plaques and tangles so strongly associated with Alzheimer’s not develop the disease?

 

Now, a series of studies by Harvard scientists suggests a possible answer, one that could lead to new treatments if confirmed by other research.

 

The memory and thinking problems of Alzheimer’s disease and other dementias, which affect an estimated seven million Americans, may be related to a failure in the brain’s stress response system, the new research suggests. If this system is working well, it can protect the brain from abnormal Alzheimer’s proteins; if it gets derailed, critical areas of the brain start degenerating.

“This is an extremely important study,” said Li-Huei Tsai, director of the Picower Institute for Learning and Memory at the Massachusetts Institute of Technology, who was not involved in the research but wrote a commentary accompanying the study. “This is the first study that is really starting to provide a plausible pathway to explain why some people are more vulnerable to Alzheimer’s than other people.”

An image of tau tangles in the brain, often a hallmark of Alzheimer’s disease.

An image of tau tangles in the brain, often a hallmark of Alzheimer’s disease.

 

 

 

The research, published on Wednesday in the journal Nature, focuses on a protein previously thought to act mostly in the brains of developing fetuses. The scientists found that the protein also appears to protect neurons in healthy older people from aging-related stresses. But in people with Alzheimer’s and other dementias, the protein is sharply depleted in key brain regions.

Experts said if other scientists could replicate and expand upon the findings, the role of the protein, called REST, could spur development of new drugs for dementia, which has so far been virtually impossible to treat. But they cautioned that much more needed to be determined, including whether the decline of REST was a cause, or an effect, of brain deterioration, and whether it was specific enough to neurological diseases that it could lead to effective therapies.

“You’re going to see a lot of papers now following up on it,” said Dr. Eric M. Reiman, executive director of the Banner Alzheimer’s Institute in Phoenix, who was not involved in the study. “While it’s a preliminary finding, it raises an avenue that hasn’t been considered before. And if this provides a handle on which to understand normal brain aging, that will be great, too.”

REST, a regulator that switches off certain genes, is primarily known to keep fetal neurons in an immature state until they develop to perform brain functions, said Dr. Bruce A. Yankner, a professor of genetics at Harvard Medical School and the lead author of the new study. By the time babies are born, REST becomes inactive, he said, except in some areas outside the brain like the colon, where it seems to suppress cancer.

While investigating how different genes in the brain change as people age, Dr. Yankner’s team was startled to find that REST was the most active gene regulator in older brains. The researchers have found that this protein, normally active in fetuses, may also protect the neurons in older people.  It is not yet possible to measure the levels of this protein that is a gene regulator called REST, in living people.

“Why should a fetal gene be coming on in an aging brain?” he wondered. He hypothesized that it was because in aging, as in birth, brains encounter great stress, threatening neurons that cannot regenerate if harmed.

His team discovered that REST appears to switch off genes that promote cell death, protecting neurons from normal aging processes like energy decrease, inflammation and oxidative stress.

Analyzing brains from brain banks and dementia studies, the researchers found that brains of young adults ages 20 to 35 contained little REST, while healthy adults between the ages of 73 and 106 had plenty. REST levels grew the older people got, so long as they did not develop dementia, suggesting that REST is related to longevity.

But in people with Alzheimer’s, mild cognitive impairment, frontotemporal dementia and Lewy body dementia, the brain areas affected by these diseases contained much less REST than healthy brains.

This was true only in people who actually had memory and thinking problems. People who remained cognitively healthy, but whose brains had the same accumulation of amyloid plaques and tau tangles as people with Alzheimer’s, had three times more REST than those suffering Alzheimer’s symptoms. About a third of people who have such plaques will not develop Alzheimer’s symptoms, studies show.

REST levels dropped as symptoms worsened, so people with mild cognitive impairment had more REST than Alzheimer’s patients. And only key brain regions were affected. In Alzheimer’s, REST steeply declined in the prefrontal cortex and hippocampus, areas critical to learning, memory and planning. Other areas of the brain not involved in Alzheimer’s showed no REST drop-off.

It is not yet possible to analyze REST levels in the brains of living people, and several Alzheimer’s experts said that fact limited what the new research could prove.

John Hardy, an Alzheimer’s researcher at University College London, cautioned in an email that information from post-mortem brains could not prove that a decline in REST caused dementia because death might produce unrelated damage to brain cells.

To investigate further, the team conducted what both Dr. Tsai and Dr. Reiman called a “tour de force” of research, examining REST in mice, roundworms and cells in the lab.

“We wanted to make sure the story was right,” Dr. Yankner said. “It was difficult to believe at first, to be honest with you.”

Especially persuasive was that mice genetically engineered to lack REST lost neurons as they aged in brain areas afflicted in Alzheimer’s.

Dr. Yankner said REST appeared to work by traveling to a neuron’s nucleus when the brain was stressed. In dementia, though, REST somehow gets diverted, traveling with toxic dementia-related proteins to another part of the neuron where it is eventually destroyed.

Experts said the research, while intriguing, left many unanswered questions. Bradley Wise of the National Institute on Aging’s neuroscience division, which helped finance the studies, said REST’s role needed further clarification. “I don’t think you can really say if it’s a cause of Alzheimer’s or a consequence of Alzheimer’s” yet, he said.

Dr. Samuel E. Gandy, an Alzheimer’s researcher at Mount Sinai Medical Center, wondered if REST figured only in neurodegenerative diseases or in other diseases, too, which could make it difficult to use REST to develop specific treatments or diagnostic tests for dementia.

“My ambivalence is, is this really a way that advances our understanding of the disease or does this just tell us this is even more complicated than we thought?” he said.

Dr. Yankner’s team is looking at REST in other neurological diseases, like Parkinson’s. He also has thoughts about a potential treatment, lithium, which he said appears to stimulate REST function, and is considered relatively safe.

But he and other experts said it was too early. “I would hesitate to start rushing into lithium treatment” unless rigorous studies showed that it could forestall dementia, said Dr. John C. Morris, an Alzheimer’s researcher at Washington University in St. Louis.

Still, Dr. Morris said, the REST research the team conducted so far is “very well done, and certainly helps support this idea that we’ve all tried to understand about why Alzheimer’s is age-associated and why, while amyloid is necessary for the development of Alzheimer’s disease, it certainly is not sufficient.”

He added, “There have to be some other processes and triggers that result in Alzheimer’s.”

Correction: March 19, 2014 
Because of an editing error, an earlier version of this article misstated the gender of Dr. Li-Huei Tsai. Dr. Tsai is a woman.

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Larry H Bernstein, MD, FCAP, Author and Curator

https://pharmaceuticalintelligence.com/2014/06/22/Proteomics – The Pathway to Understanding and Decision-making in Medicine

This dialogue is a series of discussions introducing several perspective on proteomics discovery, an emerging scientific enterprise in the -OMICS- family of disciplines that aim to clarify many of the challenges toward the understanding of disease and aiding in the diagnosis as well as guiding treatment decisions. Beyond that focus, it will contribute to personalized medical treatment in facilitating the identification of treatment targets for the pharmaceutical industry. Despite enormous advances in genomics research over the last two decades, there is a still a problem in reaching anticipated goals for introducing new targeted treatments that has seen repeated failures in stage III of clinical trials, and even when success has been achieved, it is temporal.  The other problem has been toxicity of agents widely used in chemotherapy.  Even though the genomic approach brings relieve to the issues of toxicity found in organic chemistry derivative blocking reactions, the specificity for the target cell without an effect on normal cells has been elusive.

This is not confined to cancer chemotherapy, but can also be seen in pain medication, and has been a growing problem in antimicrobial therapy.  The stumbling block has been inability to manage a multiplicity of reactions that also have to be modulated in a changing environment based on 3-dimension structure of proteins, pH changes, ionic balance, micro- and macrovascular circulation, and protein-protein and protein- membrane interactions. There is reason to consider that the present problems can be overcome through a much better modification of target cellular metabolism as we peel away the confounding and blinding factors with a multivariable control of these imbalances, like removing the skin of an onion.

This is the first of a series of articles, and for convenience we shall here  only emphasize the progress of application of proteomics to cardiovascular disease.

growth in funding proteomics 1990-2010

growth in funding proteomics 1990-2010

Part I.

Panomics: Decoding Biological Networks  (Clinical OMICs 2014; 5)

Technological advances such as high-throughput sequencing are transforming medicine from symptom-based diagnosis and treatment to personalized medicine as scientists employ novel rapid genomic methodologies to gain a broader comprehension of disease and disease progression. As next-generation sequencing becomes more rapid, researchers are turning toward large-scale pan-omics, the collective use of all omics such as genomics, epigenomics, transcriptomics, proteomics, metabolomics, lipidomics and lipoprotein proteomics, to better understand, identify, and treat complex disease.

Genomics has been a cornerstone in understanding disease, and the sequencing of the human genome has led to the identification of numerous disease biomarkers through genome-wide association studies (GWAS). It was the goal of these studies that these biomarkers would serve to predict individual disease risk, enable early detection of disease, help make treatment decisions, and identify new therapeutic targets. In reality, however, only a few have gone on to become established in clinical practice. For example in human GWAS studies for heart failure at least 35 biomarkers have been identified but only natriuretic peptides have moved into clinical practice, where they are limited primarily for use as a diagnostic tool.

Proteomics Advances Will Rival the Genetics Advances of the Last Ten Years

Seventy percent of the decisions made by physicians today are influenced by results of diagnostic tests, according to N. Leigh Anderson, founder of the Plasma Proteome Institute and CEO of SISCAPA Assay Technologies. Imagine the changes that will come about when future diagnostics tests are more accurate, more useful, more economical, and more accessible to healthcare practitioners. For Dr. Anderson, that’s the promise of proteomics, the study of the structure and function of proteins, the principal constituents of the protoplasm of all cells.

In explaining why proteomics is likely to have such a major impact, Dr. Anderson starts with a major difference between the genetic testing common today, and the proteomic testing that is fast coming on the scene. “Most genetic tests are aimed at measuring something that’s constant in a person over his or her entire lifetime. These tests provide information on the probability of something happening, and they can help us understand the basis of various diseases and their potential risks. What’s missing is, a genetic test is not going to tell you what’s happening to you right now.”

Mass Spec-Based Multiplexed Protein Biomarkers

Clinical proteomics applications rely on the translation of targeted protein quantitation technologies and methods to develop robust assays that can guide diagnostic, prognostic, and therapeutic decision-making. The development of a clinical proteomics-based test begins with the discovery of disease-relevant biomarkers, followed by validation of those biomarkers.

“In common practice, the discovery stage is performed on a MS-based platform for global unbiased sampling of the proteome, while biomarker qualification and clinical implementation generally involve the development of an antibody-based protocol, such as the commonly used enzyme linked ELISA assays,” state López et al. in Proteome Science (2012; 10: 35–45). “Although this process is potentially capable of delivering clinically important biomarkers, it is not the most efficient process as the latter is low-throughput, very costly, and time-consuming.”

Part II.  Proteomics for Clinical and Research Use: Combining Protein Chips, 2D Gels and Mass Spectrometry in 

The next Step: Exploring the Proteome: Translation and Beyond

N. Leigh Anderson, Ph.D., Chief Scientific Officer, Large Scale Proteomics Corporation

Three streams of technology will play major roles in quantitative (expression) proteomics over the coming decade. Two-dimensional electrophoresis and mass spectrometry represent well-established methods for, respectively, resolving and characterizing proteins, and both have now been automated to enable the high-throughput generation of data from large numbers of samples.

These methods can be powerfully applied to discover proteins of interest as diagnostics, small molecule therapeutic targets, and protein therapeutics. However, neither offers a simple, rapid, routine way to measure many proteins in common samples like blood or tissue homogenates.

Protein chips do offer this possibility, and thus complete the triumvirate of technologies that will deliver the benefits of proteomics to both research and clinical users. Integration of efforts in all three approaches are discussed, highlighting the application of the Human Protein Index® database as a source of protein leads.

leighAnderson

leighAnderson

N. Leigh Anderson, Ph D. is Chief Scientific Officer of the Proteomics subsidiary of Large Scale Biology Corporation (LSBC).
Dr. Anderson obtained his B.A. in Physics with honors from Yale and a Ph.D. in Molecular Biology from Cambridge University
(England) where he worked with M. F. Perutz as a Churchill Fellow at the MRC Laboratory of Molecular Biology. Subsequently
he co-founded the Molecular Anatomy Program at the Argonne National Laboratory (Chicago) where his work in the development
of 2D electrophoresis and molecular database technology earned him, among other distinctions, the American Association for
Clinical Chemistry’s Young Investigator Award for 1982, the 1983 Pittsburgh Analytical Chemistry Award, 2008 AACC Outstanding
Research Award, and 2013 National Science Medal..

In 1985 Dr. Anderson co-founded LSBC in order to pursue commercial development and large scale applications of 2-D electro-
phoretic protein mapping technology. This effort has resulted in a large-scale proteomics analytical facility supporting research
work for LSBC and its pharmaceutical industry partners. Dr. Anderson’s current primary interests are in the automation of proteomics
technologies, and the expansion of LSBC’s proteomics databases describing drug effects and disease processes in vivo and in vitro.
Large Scale Biology went public in August 2000.

Part II. Plasma Proteomics: Lessons in Biomarkers and Diagnostics

Exposome Workshop
N Leigh Anderson
Washington 8 Dec 2011

QUESTIONS AND LESSONS:

CLINICAL DIAGNOSTICS AS A MODEL FOR EXPOSOME INDICATORS
TECHNOLOGY OPTIONS FOR MEASURING PROTEIN RESPONSES TO EXPOSURES
SCALE OF THE PROBLEM: EXPOSURE SIGNALS VS POPULATION NOISE

The Clinical Plasma Proteome
• Plasma and serum are the dominant non-invasive clinical sample types
– standard materials for in vitro diagnostics (IVD)
• Proteins measured in clinically-available tests in the US
– 109 proteins via FDA-cleared or approved tests
• Clinical test costs range from $9 (albumin) to $122 (Her2)
• 90% of those ever approved are still in use
– 96 additional proteins via laboratory-developed tests (not FDA
cleared or approved)
– Total 205 proteins (≅ products of 211genes, excluding Ig’s)
• Clinically applied proteins thus account for
– About 1% of the baseline human proteome (1 gene :1 protein)
– About 10% of the 2,000+ proteins observed in deep discovery
plasma proteome datasets

“New” Protein Diagnostics Are FDA-Cleared at a Rate of ~1.5/yr:
Insufficient to Meet Dx or Rx Development Needs

FDA clearance of protein diagnostics

FDA clearance of protein diagnostics

A  Major Technology Gulf Exists Between Discovery

Proteomics and Routine Diagnostic Platforms

Two Streams of Proteomics
A.  Problem Technology
Basic biology: maximum proteome coverage (including PTM’s, splices) to
provide unbiased discovery of mechanistic information
• Critical: Depth and breadth
• Not critical: Cost, throughput, quant precision

B.  Discovery proteomics
Specialized proteomics field,
large groups,
complex workflows and informatics

Part III.  Addressing the Clinical Proteome with Mass Spectrometric Assays

N. Leigh Anderson, PhD, SISCAPA Assay Technologies, Inc.

protein changes in biological mechanisms

protein changes in biological mechanisms

No Increase in FDA Cleared Protein Tests in 20 yr

“New” Protein Tests in Plasma Are FDA-Cleared at a Rate of ~1.5/yr:
Insufficient to Meet Dx or Rx Development Needs

See figure above

An Explanation: the Biomarker Pipeline is Blocked at the Verification Step

Immunoassay Weaknesses Impact Biomarker Verification

1) Specificity: what actually forms the immunoassay sandwich – or prevents its
formation – is not directly visualized

2) Cost: an assay developed to FDA approvable quality costs $2-5M per
protein

Major_Plasma_Proteins

Major_Plasma_Proteins

Immunoassay vs Hybrid MS-based assays

Immunoassay vs Hybrid MS-based assays

MASS SPECTROMETRY: MRM’s provide what is missing in..IMMUNOASSAYS:

– SPECIFICITY
– INTERNAL STANDARDIZATION
– MULTIPLEXING
– RAPID CONFIGURATION PROVIDED A PROTEIN CAN ACT LIKE A SMALL
MOLECULE

MRM of Proteotypic Tryptic Peptides Provides Highly Specific Assays for Proteins > 1ug/ml in Plasma

Peptide-Level MS Provides High Structural Specificity
Multiple Reaction Monitoring (MRM) Quantitation

ADDRESSING MRM LIMITATIONS VIA SPECIFIC ENRICHMENT OF ANALYTE  PEPTIDES: SISCAPA

– SENSITIVITY
– THROUGHPUT (LC-MS/MS CYCLE TIME)

SISCAPA combines best features of immuno and MS

SISCAPA combines best features of immuno and MS

SISCAPA Process Schematic Diagram
Stable Isotope-labeled Standards with Capture on Anti-Peptide Antibodies

An automated process for SISCAPA targeted protein quantitation utilizes high affinity capture antibodies that are immobilized on magnetic beads

An automated process for SISCAPA targeted protein quantitation utilizes high affinity capture antibodies that are immobilized on magnetic beads

Antibodies sequence specific peptide binding

Antibodies sequence specific peptide binding

SISCAP target enrichmant

SISCAP target enrichmant

Multiple reaction monitoring (MRM) quantitation

Multiple reaction monitoring (MRM) quantitation

protein-quantitation-via-signature-peptides.png

protein-quantitation-via-signature-peptides.png

First SISCAP Assay - thyroglobulin

First SISCAP Assay – thyroglobulin

personalized reference range within population range

Glycemic control in DM

Glycemic control in DM

Part IV. National Heart, Lung, and Blood Institute Clinical

Proteomics Working Group Report
Christopher B. Granger, MD; Jennifer E. Van Eyk, PhD; Stephen C. Mockrin, PhD;
N. Leigh Anderson, PhD; on behalf of the Working Group Members*
Circulation. 2004;109:1697-1703 doi: 10.1161/01.CIR.0000121563.47232.2A
http://circ.ahajournals.org/content/109/14/1697

Abstract—The National Heart, Lung, and Blood Institute (NHLBI) Clinical Proteomics Working Group
was charged with identifying opportunities and challenges in clinical proteomics and using these as a
basis for recommendations aimed at directly improving patient care. The group included representatives
of clinical and translational research, proteomic technologies, laboratory medicine, bioinformatics, and
2 of the NHLBI Proteomics Centers, which form part of a program focused on innovative technology development.

This report represents the results from a one-and-a-half-day meeting on May 8 and 9, 2003. For the purposes
of this report, clinical proteomics is defined as the systematic, comprehensive, large-scale identification of
protein patterns (“fingerprints”) of disease and the application of this knowledge to improve patient care
and public health through better assessment of disease susceptibility, prevention of disease, selection of
therapy for the individual, and monitoring of treatment response. (Circulation. 2004;109:1697-1703.)
Key Words: proteins diagnosis prognosis genetics plasma

Part V.  Overview: The Maturing of Proteomics in Cardiovascular Research

Jennifer E. Van Eyk
Circ Res. 2011;108:490-498  doi: 10.1161/CIRCRESAHA.110.226894
http://circres.ahajournals.org/content/108/4/490

Abstract: Proteomic technologies are used to study the complexity of proteins, their roles, and biological functions.
It is based on the premise that the diversity of proteins, comprising their isoforms, and posttranslational modifications
(PTMs) underlies biology.

Based on an annotated human cardiac protein database, 62% have at least one PTM (phosphorylation currently dominating),
whereas 25% have more than one type of modification.

The field of proteomics strives to observe and quantify this protein diversity. It represents a broad group of technologies
and methods arising from analytic protein biochemistry, analytic separation, mass spectrometry, and bioinformatics.
Since the 1990s, the application of proteomic analysis has been increasingly used in cardiovascular research.

prevalence-of-cardiovascular-diseases-in-adults-by-age-and-sex-u-s-2007-2010.

prevalence-of-cardiovascular-diseases-in-adults-by-age-and-sex-u-s-2007-2010.

Technology development and adaptation have been at the heart of this progress. Technology undergoes a maturation,

becoming routine and ultimately obsolete, being replaced by newer methods. Because of extensive methodological
improvements, many proteomic studies today observe 1000 to 5000 proteins.

Only 5 years ago, this was not feasible. Even so, there are still road blocks. Nowadays, there is a focus on obtaining
better characterization of protein isoforms and specific PTMs. Consequentl, new techniques for identification and
quantification of modified amino acid residues are required, as is the assessment of single-nucleotide polymorphisms
in addition to determination of the structural and functional consequences.

In this series, 4 articles provide concrete examples of how proteomics can be incorporated into cardiovascular
research and address specific biological questions. They also illustrate how novel discoveries can be made and
how proteomic technology has continued to evolve. (Circ Res. 2011;108:490-498.)
Key Words: proteomics technology protein isoform posttranslational modification polymorphism

Part VI.   The -omics era: Proteomics and lipidomics in vascular research

Athanasios Didangelos, Christin Stegemann, Manuel Mayr∗

King’s British Heart Foundation Centre, King’s College London, UK

Atherosclerosis 2012; 221: 12– 17     http://dx.doi.org/10.1016/j.atherosclerosis.2011.09.043

a b s t r a c t

A main limitation of the current approaches to atherosclerosis research is the focus on the investigation of individual
factors, which are presumed to be involved in the pathophysiology and whose biological functions are, at least in part, understood.

These molecules are investigated extensively while others are not studied at all. In comparison to our detailed
knowledge about the role of inflammation in atherosclerosis, little is known about extracellular matrix remodelling
and the retention of individual lipid species rather than lipid classes in early and advanced atherosclerotic lesions.

The recent development of mass spectrometry-based methods and advanced analytical tools are transforming
our ability to profile extracellular proteins and lipid species in animal models and clinical specimen with the goal
of illuminating pathological processes and discovering new biomarkers.

Fig. 1. ECM in atherosclerosis

Fig. 1. ECM in atherosclerosis. The bulk of the vascular ECM is synthesised by smooth muscle cells and composed primarily of collagens, proteoglycans and glycoproteins.During the early stages of atherosclerosis, LDL binds to the proteoglycans of the vessel wall, becomes modified, i.e. by oxidation (ox-LDL), and sustains a proinflammatory cascade that is proatherogenic

Lipidomics of atherosclerotic plaques

Lipidomics of atherosclerotic plaques

Fig. 2. Lipidomics of atherosclerotic plaques. Lipids were separated by ultra performance reverse phase
liquid chromatography on a Waters® ACQUITY UPLC® (HSS T3 Column, 100 mm × 2.1 mm i.d., 1.8 _m
particle size, 55 ◦C, flow rate 400 _L/min, Waters, Milford MA, USA) and analyzed on a quadrupole time-of-flight
mass spectrometer (Waters® SYNAPTTM HDMSTM system) in both positive (A) and negative ion mode (C).
In positive MS mode, lysophosphatidyl-cholines (lPCs) and lysophosphatidylethanolamines (lPEs) eluted first;
followed by phosphatidylcholines (PCs), sphingomyelin (SMs), phosphatidylethanol-amines (PEs) and cholesteryl
esters (CEs); diacylglycerols (DAGs) and triacylglycerols (TAGs) had the longest retention times. In negative MS mode,
fatty acids (FA) were followed by phosphatidyl-glycerols (PGs), phosphatidyl-inositols (PIs), phosphatidylserines (PS)
and PEs. The chromatographic peaks corresponding to the different classes were detected as retention time-mass to
charge ratio (m/z) pairs and their areas were recorded. Principal component analyses on 629 variables from triplicate
analysis (C1, 2, 3 = control 1, 2, 3; P1, 2, 3 = endarterectomy patient 1, 2, 3) demonstrated a clear separation of
atherosclerotic plaques and control radial arteries in positive (B) and negative (D) ion mode. The clustering of the
technical replicates and the central projection of the pooled sample within the scores plot confirm the reproducibility
of the analyses, and the Goodness of Fit test returned a chi-squared of 0.4 and a R-squared value of 0.6.

Challenges in mass spectrometry

Mass spectrometry is an evolving technology and the technological advances facilitate the detection and quantification
of scarce proteins. Nonetheless, the enrichment of specific subproteomes using differential solubilityor isolation of cellular
organelleswill remain important to increase coverage and, at least partially, overcome the inhomogeneity of diseased tissue,
one of the major factors affecting sample-to-sample variation.

Proteomics is also the method of choice for the identification of post-translational modifications, which play an essential
role in protein function, i.e. enzymatic activation, binding ability and formation of ECM structures. Again, efficient enrichment
is essential to increase the likelihood of identifying modified peptides in complex mixtures. Lipidomics faces similar challenges.
While the extraction of lipids is more selective, new enrichment methods are needed for scarce lipids as well as labile lipid
metabolites, that may have important bioactivity. Another pressing issue in lipidomics is data analysis, in particular the lack
of automated search engines that can analyze mass spectra obtained from instruments of different vendors. Efforts to
overcome this issue are currently underway.

Conclusions

Proteomics and lipidomics offer an unbiased platform for the investigation of ECM and lipids within atherosclerosis. In
combination, these innovative technologies will reveal key differences in proteolytic processes responsible for plaque rupture
and advance our understanding of ECM – lipoprotein interactions in atherosclerosis.

references

Virtualization in Proteomics: ‘Sakshat’ in India, at IIT Bombay(tginnovations.wordpress.com)

Proteome Portraits (the-scientist.com)

A Protease for ‘Middle-down’ Proteomics(pharmaceuticalintelligence.com)

Intrinsic Disorder in the Human Spliceosomal Proteome(ploscompbiol.org)

proteome

proteome

active site of eNOS (PDB_1P6L) and nNOS (PDB_1P6H).

active site of eNOS (PDB_1P6L) and nNOS (PDB_1P6H).

Table - metabolic  targets

Table – metabolic targets

HK-II Phosphorylation

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Prologue to Cancer – e-book Volume One – Where are we in this journey?


Prologue to Cancer – e-book Volume One – Where are we in this journey?

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

Consulting Reviewer and Contributor:  Jose Eduardo de Salles Roselino, MD

 

LH Bernstein

LH Bernstein

Jose Eduardo de Salles Roselino

LES Roselino

 

 

This is a preface to the fourth in the ebook series of Leaders in Pharmaceutical Intelligence, a collaboration of experienced doctorate medical and pharmaceutical professionals.  The topic is of great current interest, and it entails a significant part of current medical expenditure by a group of neoplastic diseases that may develop at different periods in life, and have come to supercede infections or even eventuate in infectious disease as an end of life event.  The articles presented are a collection of the most up-to-date accounts of the state of a now rapidly emerging field of medical research that has benefitted enormously by progress in immunodiagnostics,  radiodiagnostics, imaging, predictive analytics, genomic and proteomic discovery subsequent to the completion of the Human Genome Project, advances in analytic methods in qPCR, gene sequencing, genome mapping, signaling pathways, exome identification, identification of therapeutic targets in inhibitors, activators, initiators in the progression of cell metabolism, carcinogenesis, cell movement, and metastatic potential.  This story is very complicated because we are engaged in trying to evoke from what we would like to be similar clinical events, dissimilar events in their expression and classification, whether they are within the same or different anatomic class.  Thus, we are faced with constructing an objective evidence-based understanding requiring integration of several disciplinary approaches to see a clear picture.  The failure to do so creates a high risk of failure in biopharmaceutical development.

The chapters that follow cover novel and important research and development in cancer related research, development, diagnostics and treatment, and in balance, present a substantial part of the tumor landscape, with some exceptions.  Will there ever be a unifying concept, as might be hoped for? I certainly can’t see any such prediction on the horizon.  Part of the problem is that disease classification is a human construct to guide us, and so are treatments that have existed and are reexamined for over 2,000 years.  In that time, we have changed, our afflictions have been modified, and our environment has changed with respect to the microorganisms within and around us, viruses, the soil, and radiation exposure, and the impacts of war and starvation, and access to food.  The outline has been given.  Organic and inorganic chemistry combined with physics has given us a new enterprise in biosynthetics that is and will change our world.  But let us keep in mind that this is a human construct, just as drug target development is such a construct, workable with limitations.

What Molecular Biology Gained from Physics

We need greater clarity and completeness in defining the carcinogenetic process.  It is the beginning, but not the end.  But we must first examine the evolution of the scientific structure that leads to our present understanding. This was preceded by the studies of anatomy, physiology, and embryology that had to occur as a first step, which was followed by the researches into bacteriology, fungi, sea urchins and the evolutionary creatures that could be studied having more primary development in scale.  They are still major objects of study, with the expectation that we can derive lessons about comparative mechanisms that have been passed on through the ages and have common features with man.  This became the serious intent of molecular biology, the discipline that turned to find an explanation for genetics, and to carry out controlled experiments modelled on the discipline that already had enormous success in physics, mathematics, and chemistry. In 1900, when Max Planck hypothesized that the frequency of light emitted by the black body depended on the frequency of the oscillator that emitted it, it had important ramifications for chemistry and biology (See Appendix II and Footnote 1, Planck equation, energy and oscillation).  The leading idea is to search below the large-scale observations of classical biology.

The central dogma of molecular biology where genetic material is transcribed into RNA and then translated into protein, provides a starting point, but the construct is undergoing revision in light of emerging novel roles for RNA and signaling pathways.   The term, coined by Warren Weaver (director of Natural Sciences for the Rockefeller Foundation), who observed an emergence of significant change given recent advances in fields such as X-ray crystallography. Molecular biology also plays important role in understanding formations, actions, regulations of various parts of cellswhich can be used efficiently for targeting new drugs, diagnosis of disease, physiology of the Cell. The Nobel Prize in Physiology or Medicine in 1969 was shared by Max Delbrück, Alfred D. Hershey, Salvador E. Luria, whose work with viral replication gave impetus to the field.  Delbruck was a physicist who trained in Copenhagen under Bohr, and specifically committed himself to a rigor in biology, as was in physics.

Dorothy Hodgkin  protein crystallography

Dorothy Hodgkin protein crystallography

Rosalind Franlin crystallographer double helix

Rosalind Franlin
crystallographer
double helix

 Max Delbruck         molecular biology

Max Delbruck        
molecular biology

Max Planck

Max Planck Quantum Physics

 

 

 

We then stepped back from classical (descriptive) physiology, with the endless complexity, to molecular biology.  This led us to the genetic code, with a double helix model.  It has recently been found insufficiently explanatory, with the recent construction of triplex and quadruplex models. They have a potential to account for unaccounted for building blocks, such as inosine, and we don’t know whether more than one model holds validity under different conditions .  The other major field of development has been simply unaccounted for in the study of proteomics, especially in protein-protein interactions, and in the energetics of protein conformation, first called to our attention by the work of Jacob, Monod, and Changeux (See Footnote 2).  Proteins are not just rigid structures stamped out by the monotonously simple DNA to RNA to protein concept.  Nothing is ever quite so simple. Just as there are epigenetic events, there are posttranslational events, and yet more.

JPChangeux-150x170

JP Changeux

 

 

 

 

 

 

 

 

The Emergence of Molecular Biology

I now return the discussion to the topic of medicine, the emergence of molecular biology and the need for convergence with biochemistry in the mid-20th century. Jose Eduardo de Salles Roselino recalls “I was previously allowed to make of the conformational energy as made by R Marcus in his Nobel lecture revised (J. of Electroanalytical  Chemistry 438:(1997) p251-259. (See Footnote 1) His description of the energetic coordinates of a landscape of a chemical reaction is only a two-dimensional cut of what in fact is a volcano crater (in three dimensions) (each one varies but the sum of the two is constant. Solvational+vibrational=100% in ordinate) nuclear coordinates in abcissa. In case we could represent it by research methods that allow us to discriminate in one by one degree of different pairs of energy, we would most likely have 360 other similar representations of the same phenomenon. The real representation would take into account all those 360 representations together. In case our methodology was not that fine, for instance it discriminates only differences of minimal 10 degrees in 360 possible, will have 36 partial representations of something that to be perfectly represented will require all 36 being taken together. Can you reconcile it with ATGC?  Yet, when complete genome sequences were presented they were described as though we will know everything about this living being. The most important problems in biology will be viewed by limited vision always and the awareness of this limited is something we should acknowledge and teach it. Therefore, our knowledge is made up of partial representations. If we had the entire genome data for the most intricate biological problems, they are still not amenable to this level of reductionism. But going from general views of signals andsymptoms we could get to the most detailed molecular view and in this case genome provides an anchor.”

“Warburg Effect” describes the preference of glycolysis and lactic acid fermentation rather than oxidative phosphorylation for energy production in cancer cells. Mitochondrial metabolism is an important and necessary component in the functioning and maintenance of the cell, and accumulating evidence suggests that dysfunction of mitochondrial metabolism plays a role in cancer. Progress has demonstrated the mechanisms of the mitochondrial metabolism-to-glycolysis switch in cancer development and how to target this metabolic switch.

 

 

Glycolysis

glycolysis

 

Otto Heinrich Warburg (1883-  )

Otto Warburg

435px-Louis_Pasteur,_foto_av_Félix_Nadar_Crisco_edit

Louis Pasteur

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The expression “Pasteur effect” was coined by Warburg when inspired by Pasteur’s findings in yeast cells, when he investigated this metabolic observation (Pasteur effect) in cancer cells. In yeast cells, Pasteur had found that the velocity of sugar used was greatly reduced in presence of oxygen. Not to be confused, in the “Crabtree effect”, the velocity of sugar metabolism was greatly increased, a reversal, when yeast cells were transferred from the aerobic to an anaerobic condition. Thus, the velocity of sugar metabolism of yeast cells was shown to be under metabolic regulatory control in response to change in environmental oxygen conditions in growth. Warburg had to verify whether cancer cells and tissue related normal mammalian cells also have a similar control mechanism. He found that this control was also found in normal cells studied, but was absent in cancer cells. Strikingly, cancer cells continue to have higher anaerobic gycolysis despite the presence of oxygen in their culture media (See Footnote 3).

Taking this a step further, food is digested and supplied to cells In vertebrates mainly in the form of glucose, which is metabolized producing Adenosine Triphosphate (ATP) by two pathways. Glycolysis, occurs via anaerobic metabolism in the cytoplasm, and is of major significance for making ATP quickly, but in a minuscule amount (2 molecules).  In the presence of oxygen, the breakdown process continues in the mitochondria via the Krebs’s cycle coupled with oxidative phosphorylation, which is more efficient for ATP production (36 molecules). Cancer cells seem to depend on glycolysis. In the 1920s, Otto Warburg first proposed that cancer cells show increased levels of glucose consumption and lactate fermentation even in the presence of ample oxygen (known as “Warburg Effect”). Based on this theory, oxidative phosphorylation switches to glycolysis which promotes the proliferation of cancer cells. Many studies have demonstrated glycolysis as the main metabolic pathway in cancer cells.

Albert Szent Gyogy (Warburg’s student) and Otto Meyerhof both studied striated skeletal muscle metabolism invertebrates, and they found those changes observed in yeast by Pasteur. The description of the anaerobic pathway was largely credited to Emden and Meyerhof. Whenever there is increase in muscle work, energy need is above what can be provided by blood supply, the cell metabolism changes from aerobic (where  Acetyl CoA  provides the chemical energy for aerobic production of ATP) to anaerobic metabolism of glucose. In this condition, glucose is obtained directly from its muscle glycogen stores (not from hepatic glycogenolysis).  This is the sole source of chemical energy that is independent of oxygen supplied to the cell. It is a physiological change on muscle metabolism that favors autonomy. It does not depend upon the blood oxygen for aerobic metabolim or blood sources of carbon metabolites borne out from adipose tissue (free fatty acids) or muscle proteins (branched chain amino acids), or vascular delivery of glucose. On that condition, the muscle can perform contraction by its internal source of ATP and uses conversion of pyruvate to lactate in order to regenerate much-needed NAD (by hydride transfer from pyruvate) as a replacement for this mitochondrial function. This regulatory change, keeps glycolysis going at fast rate in order to meet ATP needs of the cell under low yield condition (only two or three ATP for each glucose converted into two lactate molecules). Therefore, it cannot last for long periods of time. This regulatory metabolic change is made in seconds, minutes and therefore happens with the proteins that are already presented in the cell. It does not requires the effect of transcription factors and/or changes in gene expression (See Footnote 1, 2).

In other types mammalian cells, like those from the lens of the eye (86% gycolysis + pentose shunt),  and red blood cells (RBC)[both lacking mitochondria], and also in the deep medullary layer of the kidneys, for lack of mitochondria in the first two cases and normally reduced blood perfusion in the third – A condition required for the counter current mechanism and our ability to concentrate urine also have, permanent higher anaerobic metabolism. In the case of RBC, it includes the ability to produce in a shunt of glycolytic pathway 2,3 diphospho- glycerate that is required to place the hemogloblin macromolecule in an unstable equilibrium between its two forms (R and T – Here presented as simplified accordingly to the model of Monod, Wyman and Changeux. The final model would be even much complex (see for instance, H-W and K review Nature 2007 vol 450: p 964-972 )

Any tissue under a condition of ischemia that is required for some medical procedures (open heart surgery, organ transplants, etc) displays this fast regulatory mechanism (See Footnote 1, 2). A display of these regulatory metabolic changes can be seen in: Cardioplegia: the protection of the myocardium during open heart surgery: a review. D. J. Hearse J. Physiol., Paris, 1980, 76, 751-756 (Fig 1).  The following points are made:

1-       It is a fast regulatory response. Therefore, no genetic mechanism can be taken into account.

2-       It moves from a reversible to an irreversible condition, while the cells are still alive. Death can be seen at the bottom end of the arrow. Therefore, it cannot be reconciled with some of the molecular biology assumptions:

A-       The gene and genes reside inside the heart muscle cells but, in order to preserve intact, the source of coded genetic information that the cell reads and transcribes, DNA must be kept to a minimal of chemical reactivity.

B-       In case sequence determines conformation, activity and function , elevated potassium blood levels could not cause cardiac arrest.

In comparison with those conditions here presented, cancer cells keep the two metabolic options for glucose metabolism at the same time. These cells can use glucose that our body provides to them or adopt temporarily, an independent metabolic form without the usual normal requirement of oxygen (one or another form for ATP generation).  ATP generation is here, an over-simplification of the metabolic status since the carbon flow for building blocks must also be considered and in this case oxidative metabolism of glucose in cancer cells may be viewed as a rich source of organic molecules or building blocks that dividing cells always need.

JES Roselino has conjectured that “most of the Krebs cycle reaction works as ideal reversible thermodynamic systems that can supply any organic molecule that by its absence could prevent cell duplication.” In the vision of Warburg, cancer cells have a defect in Pasteur-effect metabolic control. In case it was functioning normally, it will indicate which metabolic form of glucose metabolism is adequate for each condition. What more? Cancer cells lack differentiated cell function. Any role for transcription factors must be considered as the role of factors that led to the stable phenotypic change of cancer cells. The failure of Pasteur effect must be searched for among the fast regulatory mechanisms that aren’t dependent on gene expression (See Footnote 3).

Extending the thoughts of JES Roselino (Hepatology 1992;16: 1055-1060), reduced blood flow caused by increased hydrostatic pressure in extrahepatic cholestasis decreases mitochondrial function (quoted in Hepatology) and as part of Pasteur effect normal response, increased glycolysis in partial and/or functional anaerobiosis and therefore blocks the gluconeogenic activity of hepatocytes that requires inhibited glycolysis. In this case, a clear energetic link can be perceived between the reduced energetic supply and the ability to perform differentiated hepatic function (gluconeogenesis). In cancer cells, the action of transcription factors that can be viewed as different ensembles of kaleidoscopic pieces (with changing activities as cell conditions change) are clearly linked to the new stable phenotype. In relation to extrahepatic cholestasis mentioned above it must be reckoned that in case a persistent chronic condition is studied a secondary cirrhosis is installed as an example of persistent stable condition, difficult to be reversed and without the requirement for a genetic mutation. (See Footnote 4).

 The Rejection of Complexity

Most of our reasoning about genes was derived from scientific work in microorganisms. These works have provided great advances in biochemistry.

250px-DNA_labeled  DNA diagram showing base pairing

double helix

 

hgp_hubris_220x288_72  genome cartoon

Dna triplex pic

Triple helix

 

formation of a triplex DNA structure

formation of triple helix

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1-      The “Gelehrter idea”: No matter what you are doing you will always be better off, in case you have a gene (In chapter 7 Principles of Medical Genetics Gelehrter and Collins Williams & Wilkins 1990).

2-      The idea that everything could be found following one gene one enzyme relationship that works fine for our understanding of the metabolism, in all biological problems.

3-      The idea that everything that explains biochemistry in microorganisms explains also for every living being (J Nirenberg).

4-      The idea that biochemistry may not require that time should be also taken into account. Time must be considered only for genetic and biological evolution studies (S Luria. In Life- The unfinished experiment 1977 C Scribner´s sons NY).

5-      Finally, the idea that everything in biology, could be found in the genome. Since all information in biology goes from DNA through RNA to proteins. Alternatively, are in the DNA, in case the strict line that includes RNA is not included.

This last point can be accepted in case it is considered that ALL GENETIC information is in our DNA. Genetics as part of life and not as its total expression.

For example, when our body is informed that the ambient temperature is too low or alternatively is too high, our body is receiving an information that arrives from our environment. This external information will affect our proteins and eventually, in case of longer periods in a new condition will cause adaptive response that may include conformational changes in transcription factors (proteins) that will also, produce new readings on the DNA. However, it is an information that moves from outside, to proteins and not from DNA to proteins. The last pathway, when transcription factors change its conformation and change DNA reading will follow the dogmatic view as an adaptive response (See Footnotes 1-3).

However, in case, time is taken into account, the first reactions against cold or warmer temperatures will be the ones that happen through change in protein conformation, activities and function before any change in gene expression can be noticed at protein level. These fast changes, in seconds, minutes cannot be explained by changes in gene expression and are strongly linked to what is needed for the maintenance of life.

“It is possible”, says Roselino, “desirable, to explain all these fast biochemical responses to changes in a living being condition as the sound foundation of medical practices without a single mention to DNA. In case a failure in any mechanism necessary to life is found to be genetic in its origin, the genome in context with with this huge set of transcription factors must be taken into account. This is the biochemical line of reasoning that I have learned with Houssay and Leloir. It would be an honor to see it restored in modern terms.”

More on the Mechanism of Metabolic Control

It was important that genomics would play such a large role in medical research for the last 70 years. There is also good reason to rethink the objections of the Nobelists James Watson and Randy Schekman in the past year, whatever discomfort it brings.  Molecular biology has become a tautology, and as a result deranged scientific rigor inside biology.

Crick & Watson with their DNA model, 1953

Eatson and Crick

Randy-Schekman Berkeley

Randy-Schekman Berkeley

 

 

According to JES Roselino, “consider that glycolysis is oscillatory thanks to the kinetic behavior of Phosphofructokinase. Further, by its effect upon Pyruvate kinase through Fructose 1,6 diphosphate oscillatory levels, the inhibition of gluconeogenesis is also oscillatory. When the carbon flow through glycolysis is led to a maximal level gluconeogenesis will be almost completely blocked. The reversal of the Pyruvate kinase step in liver requires two enzymes (Pyruvate carboxylase (maintenance of oxaloacetic levels) + phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32)) and energy requiring reactions that most likely could not as an ensemble, have a fast enough response against pyruvate kinase short period of inhibition during high frequency oscillatory periods of glycolytic flow. Only when glycolysis oscillates at low frequency the opposite reaction could enable gluconeogenic carbon flow.”

In case it can be shown in a rather convincing way, the same reasoning could be applied to understand how simple replicative signals inducing Go to G1 transition in cells, could easily overcome more complex signals required for cell differentiation and differentiated function.

Perhaps the problem of overextension of the equivalence of the DNA and what happens to the organism is also related to the initial reliance on a single cell model to relieve the complexity (which isn’t fully the case).

For instance, consider this fragment:
“Until only recently it was assumed that all proteins take on a clearly defined three-dimensional structure – i.e. they fold in order to be able to assume these functions.”
Cold Spring Harbour Symp. Quant. Biol. 1973  p 187-193 J.C Seidel and J Gergely – Investigation of conformational changes in Spin-Labeled Myosin Model for muscle contraction:
Huxley, A. F. 1971 Proc. Roy. Soc (London) (B) 178:1
Huxley, A.F and R. M. Simmons,1971. Nature 233:633
J.C Haselgrove X ray Evidence for a conformational Change in the Actin-containing filaments…Cold Spring Harbour Symp Quant Biol.1972 v 37: p 341-352

Only a very small sample indicating otherwise. Proteins were held as interacting macromolecules, changing their conformation in regulatory response to changes in the microenvironment (See Footnote 2). DNA was the opposite, non-interacting macromolecules to be as stable as a library must be.

The dogma held that the property of proteins could be read in DNA alone. Consequenly, the few examples quoted above, must be ignored and all people must believe that DNA alone, without environmental factors roles, controls protein amino acid sequence (OK), conformation (not true), activity (not true) and function (not true).

It appeared naively to be correct from the dogma to conclude from interpreting your genome: You have a 50% increased risk of developing the following disease (deterministic statement).  The correct form must be: You belong to a population that has a 50% increase in the risk of….followed by –  what you must do to avoid increase in your personal risk and the care you should take in case you want to have longer healthy life.  Thus, genetics and non-genetic diseases were treated as the same and medical foundations were reinforced by magical considerations (dogmas) in a very profitable way for those involved besides the patient.

 Footnotes:

  1. There is a link of electricity with ions in biology and the oscillatory behavior of some electrical discharges.  In addition, the oscillatory form of electrical discharged may have allowed Planck to relate high energy content with higher frequencies and conversely, low energy content in low frequency oscillatory events.  One may think of high density as an indication of great amount of matter inside a volume in space.  This helps the understanding of Planck’s idea as a high-density-energy in time for a high frequency phenomenon.
  1. Take into account a protein that may have its conformation restricted by an S-S bridge. This protein also, may move to another more flexible conformation in case it is in HS HS condition when the S-S bridge is broken. Consider also that, it takes some time for a protein to move from one conformation for instance, the restricted conformation (S-S) to other conformations. Also, it takes a few seconds or minutes to return to the S-S conformation (This is the Daniel Koshland´s concept of induced fit and relaxation time used by him in order to explain allosteric behavior of monomeric proteins- Monod, Wyman and Changeux requires tetramer or at least, dimer proteins).
  1. In case you have glycolysis oscillating in a frequency much higher than the relaxation time you could lead to the prevalence of high NADH effect leading to high HS /HS condition and at low glycolytic frequency, you could have predominance of S-S condition affecting protein conformation. In case you have predominance of NAD effect upon protein S-S you would get the opposite results.  The enormous effort to display the effect of citrate and over Phosphofructokinase conformation was made by others. Take into account that ATP action as an inhibitor in this case, is a rather unusual one. It is a substrate of the reaction, and together with its action as activator  F1,6 P (or its equivalent F2,6 P) is also unusual. However, it explains oscillatory behaviour of glycolysis. (Goldhammer , A.R, and Paradies: PFK structure and function, Curr. Top Cell Reg 1979; 15:109-141).
  1. The results presented in our Hepatology work must be viewed in the following way: In case the hepatic (oxygenated) blood flow is preserved, the bile secretory cells of liver receive well-oxygenated blood flow (the arterial branches bath secretory cells while the branches originated from portal vein irrigate the hepatocytes.  During extra hepatic cholestasis the low pressure, portal blood flow is reduced and the hepatocytes do not receive enough oxygen required to produce ATP that gluconeogenesis demands. Hepatic artery do not replace this flow since, its branches only join portal blood fluxes after the previous artery pressure  is reduced to a low pressure venous blood – at the point where the formation of hepatic vein is. Otherwise, the flow in the portal vein would be reversed or, from liver to the intestine. It is of no help to take into account possible valves for this reasoning since minimal arterial pressure is well above maximal venous pressure and this difference would keep this valve in permanent close condition. In low portal blood flow condition, the hepatocyte increases pyruvate kinase activity and with increased pyruvate kinase activity Gluconeogenesis is forbidden (See Walsh & Cooper revision quoted in the Hepatology as ref 23). For the hemodynamic considerations, role of artery and veins in hepatic portal system see references 44 and 45 Rappaport and Schneiderman and Rappapaport.

 

 Appendix I.

metabolic pathways

metabolic pathways

Signals Upstream and Targets Downstream of Lin28 in the Lin28 Pathway

Signals Upstream and Targets Downstream of Lin28 in the Lin28 Pathway

 

 

 

 

 

 

 

 

1.  Functional Proteomics Adds to Our Understanding

Ben Schuler’s research group from the Institute of Biochemistry of the University of Zurich has now established that an increase in temperature leads to folded proteins collapsing and becoming smaller. Other environmental factors can trigger the same effect. The crowded environments inside cells lead to the proteins shrinking. As these proteins interact with other molecules in the body and bring other proteins together, understanding of these processes is essential “as they play a major role in many processes in our body, for instance in the onset of cancer”, comments study coordinator Ben Schuler.

Measurements using the “molecular ruler”

“The fact that unfolded proteins shrink at higher temperatures is an indication that cell water does indeed play an important role as to the spatial organisation eventually adopted by the molecules”, comments Schuler with regard to the impact of temperature on protein structure. For their studies the biophysicists use what is known as single-molecule spectroscopy. Small colour probes in the protein enable the observation of changes with an accuracy of more than one millionth of a millimetre. With this “molecular yardstick” it is possible to measure how molecular forces impact protein structure.

With computer simulations the researchers have mimicked the behaviour of disordered proteins. They want to use them in future for more accurate predictions of their properties and functions.

Correcting test tube results

That’s why it’s important, according to Schuler, to monitor the proteins not only in the test tube but also in the organism. “This takes into account the fact that it is very crowded on the molecular level in our body as enormous numbers of biomolecules are crammed into a very small space in our cells”, says Schuler. The biochemists have mimicked this “molecular crowding” and observed that in this environment disordered proteins shrink, too.

Given these results many experiments may have to be revisited as the spatial organisation of the molecules in the organism could differ considerably from that in the test tube according to the biochemist from the University of Zurich. “We have, therefore, developed a theoretical analytical method to predict the effects of molecular crowding.” In a next step the researchers plan to apply these findings to measurements taken directly in living cells.

Explore further: Designer proteins provide new information about the body’s signal processesMore information: Andrea Soranno, Iwo Koenig, Madeleine B. Borgia, Hagen Hofmann, Franziska Zosel, Daniel Nettels, and Benjamin Schuler. Single-molecule spectroscopy reveals polymer effects of disordered proteins in crowded environments. PNAS, March 2014. DOI: 10.1073/pnas.1322611111

 

Effects of Hypoxia on Metabolic Flux

  1. Glucose-6-phosphate dehydrogenase regulation in the hepatopancreas of the anoxia-tolerantmarinemollusc, Littorina littorea

JL Lama , RAV Bell and KB Storey

Glucose-6-phosphate dehydrogenase (G6PDH) gates flux through the pentose phosphate pathway and is key to cellular antioxidant defense due to its role in producing NADPH. Good antioxidant defenses are crucial for anoxia-tolerant organisms that experience wide variations in oxygen availability. The marine mollusc, Littorina littorea, is an intertidal snail that experiences daily bouts of anoxia/hypoxia with the tide cycle and shows multiple metabolic and enzymatic adaptations that support anaerobiosis. This study investigated the kinetic, physical and regulatory properties of G6PDH from hepatopancreas of L. littorea to determine if the enzyme is differentially regulated in response to anoxia, thereby providing altered pentose phosphate pathway functionality under oxygen stress conditions.

Several kinetic properties of G6PDH differed significantly between aerobic and 24 h anoxic conditions; compared with the aerobic state, anoxic G6PDH (assayed at pH 8) showed a 38% decrease in K G6P and enhanced inhibition by urea, whereas in pH 6 assays Km NADP and maximal activity changed significantly.

All these data indicated that the aerobic and anoxic forms of G6PDH were the high and low phosphate forms, respectively, and that phosphorylation state was modulated in response to selected endogenous protein kinases (PKA or PKG) and protein phosphatases (PP1 or PP2C). Anoxia-induced changes in the phosphorylation state of G6PDH may facilitate sustained or increased production of NADPH to enhance antioxidant defense during long term anaerobiosis and/or during the transition back to aerobic conditions when the reintroduction of oxygen causes a rapid increase in oxidative stress.

Lama et al.  Peer J 2013.   http://dx.doi.org/10.7717/peerj.21

 

  1. Structural Basis for Isoform-Selective Inhibition in Nitric Oxide Synthase

    TL. Poulos and H Li

In the cardiovascular system, the important signaling molecule nitric oxide synthase (NOS) converts L-arginine into L-citrulline and releases nitric oxide (NO). NO produced by endothelial NOS (eNOS) relaxes smooth muscle which controls vascular tone and blood pressure. Neuronal NOS (nNOS) produces NO in the brain, where it influences a variety of neural functions such as neural transmitter release. NO can also support the immune system, serving as a cytotoxic agent during infections. Even with all of these important functions, NO is a free radical and, when overproduced, it can cause tissue damage. This mechanism can operate in many neurodegenerative diseases, and as a result the development of drugs targeting nNOS is a desirable therapeutic goal.

However, the active sites of all three human isoforms are very similar, and designing inhibitors specific for nNOS is a challenging problem. It is critically important, for example, not to inhibit eNOS owing to its central role in controlling blood pressure. In this Account, we summarize our efforts in collaboration with Rick Silverman at Northwestern University to develop drug candidates that specifically target NOS using crystallography, computational chemistry, and organic synthesis. As a result, we have developed aminopyridine compounds that are 3800-fold more selective for nNOS than eNOS, some of which show excellent neuroprotective effects in animal models. Our group has solved approximately 130 NOS-inhibitor crystal structures which have provided the structural basis for our design efforts. Initial crystal structures of nNOS and eNOS bound to selective dipeptide inhibitors showed that a single amino acid difference (Asp in nNOS and Asn in eNOS) results in much tighter binding to nNOS. The NOS active site is open and rigid, which produces few large structural changes when inhibitors bind. However, we have found that relatively small changes in the active site and inhibitor chirality can account for large differences in isoform-selectivity. For example, we expected that the aminopyridine group on our inhibitors would form a hydrogen bond with a conserved Glu inside the NOS active site. Instead, in one group of inhibitors, the aminopyridine group extends outside of the active site where it interacts with a heme propionate. For this orientation to occur, a conserved Tyr side chain must swing out of the way. This unanticipated observation taught us about the importance of inhibitor chirality and active site dynamics. We also successfully used computational methods to gain insights into the contribution of the state of protonation of the inhibitors to their selectivity. Employing the lessons learned from the aminopyridine inhibitors, the Silverman lab designed and synthesized symmetric double-headed inhibitors with an aminopyridine at each end, taking advantage of their ability to make contacts both inside and outside of the active site. Crystal structures provided yet another unexpected surprise. Two of the double-headed inhibitor molecules bound to each enzyme subunit, and one molecule participated in the generation of a novel Zn site that required some side chains to adopt alternate conformations. Therefore, in addition to achieving our specific goal, the development of nNOS selective compounds, we have learned how subtle differences in and structure can control proteinligand interactions and often in unexpected ways.

 

300px-Nitric_Oxide_Synthase

Nitric oxide synthase

arginine-NO-citulline cycle

arginine-NO-citulline cycle

active site of eNOS (PDB_1P6L) and nNOS (PDB_1P6H).

active site of eNOS (PDB_1P6L) and nNOS (PDB_1P6H).

 

 

NO - muscle, vasculature, mitochondria

NO – muscle, vasculature, mitochondria

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure:  (A) Structure of one of the early dipeptide lead compounds, 1, that exhibits excellentisoform selectivity. (B, C) show the crystal structures of the dipeptide inhibitor 1 in the active site of eNOS (PDB: 1P6L) and nNOS (PDB: 1P6H). In nNOS, the inhibitor “curls” which enables the inhibitor R-amino group to interact with both Glu592 and Asp597. In eNOS, Asn368 is the homologue to nNOS Asp597.

Accounts in Chem Res 2013; 46(2): 390-98.

  1. Jamming a Protein Signal

Interfering with a single cancer-promoting protein and its receptor can open this resistance mechanism by initiating autophagy of the affected cells,  according to researchers at The University of Texas MD Anderson Cancer Center  in the journal Cell Reports.  According to Dr. Anil Sood and Yunfei Wen, lead and first authors, blocking  prolactin, a potent growth factor for ovarian cancer, sets off downstream events that result in cell by autophagy, the process  recycles damaged organelles and proteins for new use by the cell through the phagolysozome. This in turn, provides a clinical rationale for blocking prolactin and its receptor to initiate sustained autophagy as an alternative strategy for treating cancers.

Steep reductions in tumor weight

Prolactin (PRL) is a hormone previously implicated in ovarian, endometrial and other cancer development andprogression. When PRL binds to its cell membrane receptor, PRLR, activation of cancer-promoting cell signaling pathways follows.  A variant of normal prolactin called G129R blocks the reaction between prolactin and its receptor. Sood and colleagues treated mice that had two different lines of human ovarian cancer, both expressing the prolactin receptor, with G129R. Tumor weights fell by 50 percent for mice with either type of ovarian cancer after 28 days of treatment with G129R, and adding the taxane-based chemotherapy agent paclitaxel cut tumor weight by 90 percent. They surmise that higher doses of G129R may result in even greater therapeutic benefit.

 

3D experiments show death by autophagy

 

[video width=”1280″ height=”720″ mp4=”https://pharmaceuticalintelligence.files.wordpress.com/2014/04/1741-7007-11-65-s1-macromolecular-juggling-by-ubiquitylation-enzymes1.mp4″][/video]

 

Next the team used the prolactin-mimicking peptide to treat cultures of cancer spheroids which sharply reduced their numbers, and blocked the activation of JAK2 and STAT signaling pathways.

Protein analysis of the treated spheroids showed increased presence of autophagy factors and genomic analysis revealed increased expression of a number of genes involved in autophagy progression and cell death.  Then a series of experiments using fluorescence and electron microscopy showed that the cytosol of treated cells had large numbers of cavities caused by autophagy.

The team also connected the G129R-induced autophagy to the activity of PEA-15, a known cancer inhibitor. Analysis of tumor samples from 32 ovarian cancer patients showed that tumors express higher levels of the prolactin receptor and lower levels of phosphorylated PEA-15 than normal ovarian tissue. However, patients with low levels of the prolactin receptor and higher PEA-15 had longer overall survival than those with high PRLR and low PEA-15.

Source: MD Anderson Cancer Center

 

  1. Chemists’ Work with Small Peptide Chains of Enzymes

Korendovych and his team designed seven simple peptides, each containing seven amino acids. They then allowed the molecules of each peptide to self-assemble, or spontaneously clump together, to form amyloids. (Zinc, a metal with catalytic properties, was introduced to speed up the reaction.) What they found was that four of the seven peptides catalyzed the hydrolysis of molecules known as esters, compounds that react with water to produce water and acids—a feat not uncommon among certain enzymes.

“It was the first time that a peptide this small self-assembled to produce an enzyme-like catalyst,” says Korendovych. “Each enzyme has to be an exact fit for its respective substrate,” he says, referring to the molecule with which an enzyme reacts. “Even after millions of years, nature is still testing all the possible combinations of enzymes to determine which ones can catalyze metabolic reactions. Our results make an argument for the design of self-assembling nanostructured catalysts.”

Source: Syracuse University

Here are three articles emphasizing the value of combinatorial analysis, which can be formed from genomic, clinical, and proteomic data sets.

 

  1. Comparative analysis of differential network modularity in tissue specific normal and cancer protein interaction networks

    F Islam , M Hoque , RS Banik , S Roy , SS Sumi, et al.

As most biological networks show modular properties, the analysis of differential modularity between normal and cancer protein interaction networks can be a good way to understand cancer more significantly. Two aspects of biological network modularity e.g. detection of molecular complexes (potential modules or clusters) and identification of crucial nodes forming the overlapping modules have been considered in this regard.

The computational analysis of previously published protein interaction networks (PINs) has been conducted to identify the molecular complexes and crucial nodes of the networks. Protein molecules involved in ten major cancer signal transduction pathways were used to construct the networks based on expression data of five tissues e.g. bone, breast, colon, kidney and liver in both normal and cancer conditions.

Cancer PINs show higher level of clustering (formation of molecular complexes) than the normal ones. In contrast, lower level modular overlapping is found in cancer PINs than the normal ones. Thus a proposition can be made regarding the formation of some giant nodes in the cancer networks with very high degree and resulting in reduced overlapping among the network modules though the predicted molecular complex numbers are higher in cancer conditions.

Islam et al. Journal of Clinical Bioinformatics 2013, 3:19-32

  1. A new 12-gene diagnostic biomarker signature of melanoma revealed by integrated microarray analysis

    Wanting Liu , Yonghong Peng and Desmond J. Tobin
    PeerJ 1:e49;        http://dx.doi.org/10.7717/peerj.49

Here we present an integrated microarray analysis framework, based on a genome-wide relative significance (GWRS) and genome-wide global significance (GWGS) model. When applied to five microarray datasets on melanoma published between 2000 and 2011, this method revealed a new signature of 200 genes. When these were linked to so-called ‘melanoma driver’ genes involved in MAPK, Ca2+, and WNT signaling pathways we were able to produce a new 12-gene diagnostic biomarker signature for melanoma (i.e., EGFR, FGFR2, FGFR3, IL8, PTPRF, TNC, CXCL13, COL11A1, CHP2, SHC4, PPP2R2C, andWNT4).We have begun to experimentally validate a subset of these genes involved inMAPK signaling at the protein level, including CXCL13, COL11A1, PTPRF and SHC4 and found these to be overexpressed inmetastatic and primarymelanoma cells in vitro and in situ compared to melanocytes cultured from healthy skin epidermis and normal healthy human skin.

 

catalytic amyloid forming particle

catalytic amyloid forming particle

 

 

 

 

 

 

 

        8.    PanelomiX: A threshold-based algorithm to create panels of biomarkers

X Robin , N Turck , A Hainard , N Tiberti, et al.
               Translational Proteomics 2013.    http://dx.doi.org/10.1016/j.trprot.2013.04.003

The PanelomiX toolbox combines biomarkers and evaluates the performance of panels to classify patients better than singlemarkers or other classifiers. The ICBTalgorithm proved to be an efficient classifier, the results of which can easily be interpreted.

Here are two current examples of the immense role played by signaling pathways in carcinogenic mechanisms and in treatment targeting, which is also confounded by acquired resistance.

 

  1. Triple-Negative Breast Cancer

  1. epidermal growth factor receptor (EGFR or ErbB1) and
  2. high activity of the phosphatidylinositol 3-kinase (PI3K)–Akt pathway

are both targeted in triple-negative breast cancer (TNBC).

  • activation of another EGFR family member [human epidermal growth factor receptor 3 (HER3) (or ErbB3)] may limit the antitumor effects of these drugs.

This study found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3.

The phosphorylation of HER3 and EGFR in response to these treatments

  1. was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A).
  2. MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and
  3. the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors.

In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3

  1. decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone.
  2. Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and
  3. this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody.
  4. After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction).

Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting.

Reference: Antagonism of EGFR and HER3 Enhances the Response to Inhibitors of the PI3K-Akt Pathway in Triple-Negative Breast Cancer. JJ Tao, P Castel, N Radosevic-Robin, M Elkabets, et al.  Sci. Signal., 25 March 2014;
7(318), p. ra29   http://dx.doi.org/10.1126/scisignal.2005125

 

                  10.   Metastasis in RAS Mutant or Inhibitor-Resistant Melanoma Cells

The protein kinase BRAF is mutated in about 40% of melanomas, and BRAF inhibitors improve progression-free and overall survival in these patients. However, after a relatively short period of disease control, most patients develop resistance because of reactivation of the RAF–ERK (extracellular signal–regulated kinase) pathway, mediated in many cases by mutations in RAS. We found that BRAF inhibition induces invasion and metastasis in RAS mutant melanoma cells through a mechanism mediated by the reactivation of the MEK (mitogen-activated protein kinase kinase)–ERK pathway.

Reference: BRAF Inhibitors Induce Metastasis in RAS Mutant or Inhibitor-Resistant Melanoma Cells by Reactivating MEK and ERK Signaling. B Sanchez-Laorden, A Viros, MR Girotti, M Pedersen, G Saturno, et al., Sci. Signal., 25 March 2014;  7(318), p. ra30  http://dx.doi.org/10.1126/scisignal.2004815

Appendix II.

The world of physics in the twentieth century saw the end of determinism established by Newton. This is characterized by discrete laws that describe natural observations. These are in gravity and in eletricity. In an early phase of investigation, an era of galvanic or voltaic electricity represented a revolutionary break from the historical focus on frictional electricity. Alessandro Voltadiscovered that chemical reactions could be used to create positively charged anodes and negatively charged cathodes.  In 1790, Prof. Luigi Alyisio Galvani of Bologna, while conducting experiments on “animal electricity“, noticed the twitching of a frog’s legs in the presence of an electric machine. He observed that a frog’s muscle, suspended on an iron balustrade by a copper hook passing through its dorsal column, underwent lively convulsions without any extraneous cause, the electric machine being at this time absent.  Volta communicated a description of his pile to the Royal Society of London and shortly thereafter Nicholson and Cavendish (1780) produced the decomposition of water by means of the electric current, using Volta’s pile as the source of electromotive force.

Siméon Denis Poisson attacked the difficult problem of induced magnetization, and his results provided  a first approximation. His innovation required the application of mathematics to physics.  His memoirs on the theory of electricity and magnetism created a new branch of mathematical physics.  The discovery of electromagnetic induction was made almost simultaneously and independently by Michael Faraday and Joseph Henry. Michael Faraday, the successor of Humphry Davy, began his epoch-making research relating to electric and electromagnetic induction in 1831. In his investigations of the peculiar manner in which iron filings arrange themselves on a cardboard or glass in proximity to the poles of a magnet, Faraday conceived the idea of magnetic “lines of force” extending from pole to pole of the magnet and along which the filings tend to place themselves. On the discovery being made that magnetic effects accompany the passage of an electric current in a wire, it was also assumed that similar magnetic lines of force whirled around the wire. He also posited that iron, nickel, cobalt, manganese, chromium, etc., are paramagnetic (attracted by magnetism), whilst other substances, such as bismuth, phosphorus, antimony, zinc, etc., are repelled by magnetism or are diamagnetic.

Around the mid-19th century, Fleeming Jenkin‘s work on ‘ Electricity and Magnetism ‘ and Clerk Maxwell’s ‘ Treatise on Electricity and Magnetism ‘ were published. About 1850 Kirchhoff published his laws relating to branched or divided circuits. He also showed mathematically that according to the then prevailing electrodynamic theory, electricity would be propagated along a perfectly conducting wire with the velocity of light. Herman Helmholtz investigated the effects of induction on the strength of a current and deduced mathematical equations, which experiment confirmed. In 1853 Sir William Thomson (later Lord Kelvin) predicted as a result of mathematical calculations the oscillatory nature of the electric discharge of a condenser circuit.  Joseph Henry, in 1842 discerned  the oscillatory nature of the Leyden jardischarge.

In 1864 James Clerk Maxwell announced his electromagnetic theory of light, which was perhaps the greatest single step in the world’s knowledge of electricity. Maxwell had studied and commented on the field of electricity and magnetism as early as 1855/6 when On Faraday’s lines of force was read to the Cambridge Philosophical Society. The paper presented a simplified model of Faraday’s work, and how the two phenomena were related. He reduced all of the current knowledge into a linked set of differential equations with 20 equations in 20 variables. This work was later published as On Physical Lines of Force in1861. In order to determine the force which is acting on any part of the machine we must find its momentum, and then calculate the rate at which this momentum is being changed. This rate of change will give us the force. The method of calculation which it is necessary to employ was first given by Lagrange, and afterwards developed, with some modifications, by Hamilton’s equations. Now Maxwell logically showed how these methods of calculation could be applied to the electro-magnetic field. The energy of a dynamical systemis partly kinetic, partly potential. Maxwell supposes that the magnetic energy of the field is kinetic energy, the electric energy potential.  Around 1862, while lecturing at King’s College, Maxwell calculated that the speed of propagation of an electromagnetic field is approximately that of the speed of light.   Maxwell’s electromagnetic theory of light obviously involved the existence of electric waves in free space, and his followers set themselves the task of experimentally demonstrating the truth of the theory. By 1871, he presented the Remarks on the mathematical classification of physical quantities.

A Wave-Particle Dilemma at the Century End

In 1896 J.J. Thomson performed experiments indicating that cathode rays really were particles, found an accurate value for their charge-to-mass ratio e/m, and found that e/m was independent of cathode material. He made good estimates of both the charge e and the mass m, finding that cathode ray particles, which he called “corpuscles”, had perhaps one thousandth of the mass of the least massive ion known (hydrogen). He further showed that the negatively charged particles produced by radioactive materials, by heated materials, and by illuminated materials, were universal.  In the late 19th century, the Michelson–Morley experiment was performed by Albert Michelson and Edward Morley at what is now Case Western Reserve University. It is generally considered to be the evidence against the theory of a luminiferous aether. The experiment has also been referred to as “the kicking-off point for the theoretical aspects of the Second Scientific Revolution.” Primarily for this work, Albert Michelson was awarded theNobel Prize in 1907.

Wave–particle duality is a theory that proposes that all matter exhibits the properties of not only particles, which have mass, but also waves, which transfer energy. A central concept of quantum mechanics, this duality addresses the inability of classical concepts like “particle” and “wave” to fully describe the behavior of quantum-scale objects. Standard interpretations of quantum mechanics explain this paradox as a fundamental property of the universe, while alternative interpretations explain the duality as an emergent, second-order consequence of various limitations of the observer. This treatment focuses on explaining the behavior from the perspective of the widely used Copenhagen interpretation, in which wave–particle duality serves as one aspect of the concept of complementarity, that one can view phenomena in one way or in another, but not both simultaneously.  Through the work of Max PlanckAlbert EinsteinLouis de BroglieArthur Compton, Niels Bohr, and many others, current scientific theory holds that all particles also have a wave nature (and vice versa).

Beginning in 1670 and progressing over three decades, Isaac Newton argued that the perfectly straight lines of reflection demonstrated light’s particle nature, but Newton’s contemporaries Robert Hooke and Christiaan Huygens—and later Augustin-Jean Fresnel—mathematically refined the wave viewpoint, showing that if light traveled at different speeds in different, refraction could be easily explained. The resulting Huygens–Fresnel principle was supported by Thomas Young‘s discovery of double-slit interference, the beginning of the end for the particle light camp.  The final blow against corpuscular theory came when James Clerk Maxwell discovered that he could combine four simple equations, along with a slight modification to describe self-propagating waves of oscillating electric and magnetic fields. When the propagation speed of these electromagnetic waves was calculated, the speed of light fell out. While the 19th century had seen the success of the wave theory at describing light, it had also witnessed the rise of the atomic theory at describing matter.

Matter and Light

In 1789, Antoine Lavoisier secured chemistry by introducing rigor and precision into his laboratory techniques. By discovering diatomic gases, Avogadro completed the basic atomic theory, allowing the correct molecular formulae of most known compounds—as well as the correct weights of atoms—to be deduced and categorized in a consistent manner. The final stroke in classical atomic theory came when Dimitri Mendeleev saw an order in recurring chemical properties, and created a table presenting the elements in unprecedented order and symmetry.   Chemistry was now an atomic science.

Black-body radiation, the emission of electromagnetic energy due to an object’s heat, could not be explained from classical arguments alone. The equipartition theorem of classical mechanics, the basis of all classical thermodynamic theories, stated that an object’s energy is partitioned equally among the object’s vibrational modes. This worked well when describing thermal objects, whose vibrational modes were defined as the speeds of their constituent atoms, and the speed distribution derived from egalitarian partitioning of these vibrational modes closely matched experimental results. Speeds much higher than the average speed were suppressed by the fact that kinetic energy is quadratic—doubling the speed requires four times the energy—thus the number of atoms occupying high energy modes (high speeds) quickly drops off. Since light was known to be waves of electromagnetism, physicists hoped to describe this emission via classical laws. This became known as the black body problem. The Rayleigh–Jeans law which, while correctly predicting the intensity of long wavelength emissions, predicted infinite total energy as the intensity diverges to infinity for short wavelengths.

The solution arrived in 1900 when Max Planck hypothesized that the frequency of light emitted by the black body depended on the frequency of the oscillator that emitted it, and the energy of these oscillators increased linearly with frequency (according to his constant h, where E = hν). By demanding that high-frequency light must be emitted by an oscillator of equal frequency, and further requiring that this oscillator occupy higher energy than one of a lesser frequency, Planck avoided any catastrophe; giving an equal partition to high-frequency oscillators produced successively fewer oscillators and less emitted light. And as in the Maxwell–Boltzmann distribution, the low-frequency, low-energy oscillators were suppressed by the onslaught of thermal jiggling from higher energy oscillators, which necessarily increased their energy and frequency. Planck had intentionally created an atomic theory of the black body, but had unintentionally generated an atomic theory of light, where the black body never generates quanta of light at a given frequency with energy less than .

In 1905 Albert Einstein took Planck’s black body model in itself and saw a wonderful solution to another outstanding problem of the day: the photoelectric effect, the phenomenon where electrons are emitted from atoms when they absorb energy from light.   Only by increasing the frequency of the light, and thus increasing the energy of the photons, can one eject electrons with higher energy. Thus, using Planck’s constant h to determine the energy of the photons based upon their frequency, the energy of ejected electrons should also increase linearly with frequency; the gradient of the line being Planck’s constant. These results were not confirmed until 1915, when Robert Andrews Millikan, produced experimental results in perfect accord with Einstein’s predictions. While  the energy of ejected electrons reflected Planck’s constant, the existence of photons was not explicitly proven until the discovery of the photon antibunching effect  When Einstein received his Nobel Prizein 1921, it was  for the photoelectric effect, the suggestion of quantized light. Einstein’s “light quanta” represented the quintessential example of wave–particle duality. Electromagnetic radiation propagates following  linear wave equations, but can only be emitted or absorbed as discrete elements, thus acting as a wave and a particle simultaneously.

Radioactivity Changes the Scientific Landscape

The turn of the century also features radioactivity, which later came to the forefront of the activities of World War II, the Manhattan Project, the discovery of the chain reaction, and later – Hiroshima and Nagasaki.

Marie Curie

Marie Curie

 

 

 

Marie Skłodowska-Curie was a Polish and naturalized-French physicist and chemist who conducted pioneering research on radioactivity. She was the first woman to win a Nobel Prize, the only woman to win in two fields, and the only person to win in multiple sciences. She was also the first woman to become a professor at the University of Paris, and in 1995 became the first woman to be entombed on her own merits in the Panthéon in Paris. She shared the 1903 Nobel Prize in Physics with her husband Pierre Curie and with physicist Henri Becquerel. She won the 1911 Nobel Prize in Chemistry.  Her achievements included a theory of radioactivity (a term that she coined, techniques for isolating radioactive isotopes, and the discovery of polonium and radium. She named the first chemical element that she discovered – polonium, which she first isolated in 1898 – after her native country. Under her direction, the world’s first studies were conducted into the treatment of neoplasms using radioactive isotopes. She founded the Curie Institutes in Paris and in Warsaw, which remain major centres of medical research today. During World War I, she established the first military field radiological centres.  Curie died in 1934 due to aplastic anemia brought on by exposure to radiation – mainly, it seems, during her World War I service in mobile X-ray units created by her.

 

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Reporter: Aviva Lev-Ari, PhD, RN

UPDATED 9/16/2013

VIDEO CLIPS
Enzymes That Are Not Proteins: The Discovery of Ribozymes
Listen to past HHMI President Dr. Thomas Cech discussing his Nobel Prize-winning discovery of RNA’s catalytic properties.

http://www.hhmi.org/biointeractive/enzymes-are-not-proteins-discovery-ribozymes

Stanford Report, March 15, 2013

Long-term evolution is ‘surprisingly predictable,’ Stanford experiment shows

A protein-folding simulation shows that the debated theory of long-term evolution is not only possible, but that the outcomes are predictable. The Stanford experiment provides a framework for testing evolutionary outcomes in living organisms.

BY BJORN CAREY

L.A. CiceroVisiting scholar Mike Palmer left, and Professor Marcus FeldmanDr. Michael Palmer, left, and Professor Marcus Feldman, with co-author Arnav Moudgil (not pictured), found that the long-term evolutionary dynamics were surprisingly predictable in a model of protein folding and binding.

Two birds are vying for food. One bird’s beak is shaped, by virtue of a random mutation, such that it’s slightly more adept at cracking seeds. This sets the bird on the road toward acquiring more food, a better chance of scoring a mate and, most important, passing on its genetic endowment.

This individual’s success is an example of short-term evolution, the widely accepted Darwinian process of natural selection by which individual organisms that have better adapted to their surroundings prevail.

In recent years, however, some scientists have argued that natural selection occurs not just at the individual organism level, but also between lineages over the course of many generations. In a new study, Stanford biologists have demonstrated that not only is this long-term evolution possible, but that long-term evolutionary outcomes can be surprisingly predictable.

The group set up a computer simulation in which 128 lineages of proteins continuously folded into new shapes, competing to bind with other molecules, called ligands, in each new configuration. The better each protein could attach itself to the ligands, the more ligands it would scoop up, and the higher its fitness – that is, its average number of “offspring” – would be. The simulation was run for 10,000 generations.

Although the chaos of 128 lineages – a total of more than 16,000 individual proteins – mutating over thousands of generations might seem unpredictable, and that it would be nearly impossible for the same thing to happen twice, it’s actually the opposite.

“Even though things look complicated, the possible evolutionary trajectories are quite constrained,” said lead author Michael Palmer, a computational biologist at Stanford. “There are only a few viable mutations at any point, which makes the dynamics predictable and repeatable, even over the long term.”

The study, co-authored by Marcus Feldman, a biology professor at Stanford, and Stanford research biologist Arnav Moudgil, was recently published in the Journal of the Royal Society Interface.

In some experiments, the lineages that consistently came out on top in the long term were not initially the best adapted at binding to ligands. “The immediate fitness is not the only important thing,” Palmer said. “Yes, a lineage does have to survive in the short term. But just as important is how it is able to adapt to new and potentially variable environments over the longer term.”

A good example of this scenario is Darwin’s famous finches. It’s thought that individuals – perhaps just a single pair of birds – from a South American species ended up on the Galápagos Islands about 1 million years ago. Today their descendants have diversified into about 15 modern species. Some eat seeds, some eat insects, or flowers. Some eat ticks, or even drink the blood of other birds.

“If there was some catastrophe that removed one of those food sources, it might wipe out one or more of the 15 species, but the rest of the lineage – the descendants of that initial pair of birds – would persist,” Palmer said. “Now say there was a competing lineage that was great at cracking seeds, but unable to evolve to other diets due to some prior genetic constraint. The same catastrophe could wipe it out.”

The finding, and others like it, could represent a significant shift in viewpoint for biologists. For one thing, it means that in certain situations, scientists should look beyond the details at the level of the individual organism, as the evolutionary dynamics can be accurately understood as lineage selection.

It also has implications on a species’ genomic architecture, or how a genome is organized on the lineage level. While a lineage’s genome might primarily select for a particular set of traits in order for individuals to survive in the short term, in order to out-compete other lineages, it must also be able to adapt to new conditions over the long term.

“An individual can have a lucky mutation that produces an immediate adaptation,” said Palmer. “Or a lineage can have a lucky mutation that happens to position it to adapt to the range of environments it will experience over the next thousand generations. A single mutation can have a distinct short-term and long-term fitness.”

The authors believe that the work can be replicated in microorganisms, and are now hoping that microbiologists will apply the new metrics of selection in vitro.

“There is already some evidence in vitro that there is a lot of constraint on evolutionary trajectories,” Palmer said, “and we think we’ve come up with a good framework to quantify evolutionary predictability and long-term fitness.”

Media Contact

Michael Palmer, Biology: (415) 867-3653, mepalmer@charles.stanford.edu

Bjorn Carey, Stanford News Service: (650) 725-1944, bccarey@stanford.edu

SOURCE:

http://news.stanford.edu/news/2013/march/long-term-evolution-031513.html

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Reporter: Aviva Lev-Ari, PhD, RN
July 25, 2012
Insights into protein folding may lead to better flu vaccine
folding proteins

S.B. Qian
This image shows shows mRNA (purple) with ribosomes (beige) bearing nascent protein chains (pink) in different stages of folding.

A new method for looking at how proteins fold inside mammal cells could one day lead to better flu vaccines, among other practical applications, say Cornell researchers.

The method, described online in the Proceedings of the National Academy of Sciences July 16, allows researchers to take snapshots of the cell’s protein-making machinery — called ribosomes — in various stages of protein production. The scientists then pieced together the snapshots to reconstruct how proteins fold during their synthesis.

Proteins are made up of long chains of amino acids called polypeptides, and folding gives each protein its characteristic structure, which determines its function. Though researchers have used synthetic and purified proteins to study protein folding, this study looks at proteins from their inception, providing a truer picture for how partially synthesized polypeptides can fold in cells.

Proteins fold so quickly — in microseconds — that it has been a longtime mystery just how polypeptide chains fold to create the protein’s structure.

“The speed is very fast, so it’s very hard to capture certain steps, but our approach can look at protein folding at the same time as it is being synthesized by the ribosomes,” said Shu-Bing Qian, assistant professor of nutritional sciences and the corresponding author on the paper. Yan Han, a postdoctoral associate in Qian’s lab, is the paper’s first author.

In a nutshell, messenger RNA (mRNA) carries the coding information for proteins from the DNA to ribosomes, which translate those codes into chains of amino acids that make up proteins. Previously, other researchers had developed a technique to localize the exact position of the ribosomes on the mRNA. Qian and colleagues further advanced this technique to selectively enrich only a certain portion of the protein-making machinery, basically taking snapshots of different stages of the protein synthesis process.

“Like a magnifier, we enrich a small pool from the bigger ocean and then paint a picture from early to late stages of the process,” Qian said.

In the paper, the researchers also describe applying this technique to better understanding a protein called hemagglutinin (HA), located on the surface of the influenza A virus; HA’s structure (folding) allows it to infect the cell.

Flu vaccines are based on antibodies that recognize such proteins as HA. But viruses have high mutation rates to escape antibody detection. Often, flu vaccines lose their effectiveness because surface proteins on the virus mutate. HA, for example, has the highest mutation rate of the flu virus’ surface proteins.

The researchers proved that their technique can identify how the folding process changes when HA mutates.

“If people know the folding picture of how a mutation changes, it will be helpful for designing a better vaccine,” Qian said.

“Folding is a very fundamental issue in biology,” Qian added. “It’s been a long-term mystery how the cell achieves this folding successfully, with such speed and with such a great success rate.”

Co-authors include researchers at the National Institute of Allergy and Infectious Diseases.

The research was funded by the National Institute of Allergy and Infectious Diseases Division of Intramural Research, National Institutes of Health Grant, Ellison Medical Foundation Grant and U.S. Department of Defense Exploration-Hypothesis Development Award.

 http://www.news.cornell.edu/stories/July12/ProteinFoldingQian.html

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