Posts Tagged ‘Endoplasmic reticulum’

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

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

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), ..

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

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

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


  • 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.

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.


  • 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


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.

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).



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.

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


  • 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.


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’.



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

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

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

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


  • 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.

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


  • 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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Cytoskeleton and Cell Membrane Physiology


Curator: Larry H Bernstein, MD, FCAP




early evolution of lipid membranes and the three domains of life

early evolution of lipid membranes and the three domains of life

Definition and Function

The cytoskeleton is a series of intercellular proteins that help a cell with

  1. shape,
  2. support, and
  3. movement.

Cytoskeleton has three main structural components:

  1. microfilaments,
  2. intermediate filaments, and
  3. movement

The cytoskeleton mediates movement by

  • helping the cell move in its environment and
  • mediating the movement of the cell’s components.

Thereby it provides an important structural framework for the cell –

  • the framework for the movement of organelles, contiguous with the cell membrane, around the cytoplasm. By the activity of
  • the network of protein microfilaments, intermediate filaments, and microtubules.

The structural framework supports cell function as follows:

Cell shape. For cells without cell walls, the cytoskeleton determines the shape of the cell. This is one of the functions of the intermediate filaments.

Cell movement. The dynamic collection of microfilaments and microtubles can be continually in the process of assembly and disassembly, resulting in forces that move the cell. There can also be sliding motions of these structures. Audesirk and Audesirk give examples of white blood cells “crawling” and the migration and shape changes of cells during the development of multicellular organisms.

Organelle movement. Microtubules and microfilaments can help move organelles from place to place in the cell. In endocytosis a vesicle formed engulfs a particle abutting the cell. Microfilaments then attach to the vesicle and pull it into the cell. Much of the complex synthesis and distribution function of the endoplasmic reticulum and the Golgi complex makes use of transport vescicles,  associated with the cytoskeleton.

Cell division. During cell division, microtubules accomplish the movement of the chromosones to the daughter nucleus. Also, a ring of microfilaments helps divide two developing cells by constricting the central region between the cells (fission).

Hickman, et al. Ch 4 Hickman, Cleveland P., Roberts, Larry S., and Larson, Allan, Integrated Principles of Zoology, 9th. Ed., Wm C. Brown, 1995.
Audesirk & Audesirk Ch 6 Audesirk, Teresa and Audesirk, Gerald, Biology, Life on Earth, 5th Ed., Prentice-Hall, 1999.

Intermediate filaments are 8-12 nanometers in diameter and are twisted together in a cord shape. They are composed of keratin and keratin-like proteins.  These filaments are tough and resist tension.

Microtubules are composed of alpha and beta tubulin that form long, hollow cylinders.  These are fairly strong proteins and are the largest component of cytoskeleton at 25 nanometers. Tubular monomers can be lengthened or shortened from the positive end.

Microtubules have three different functions.

They make up the cell’s

  1. centriole
  2. the flagella and cilia of a cell, and
  3. they serve as “tracks” for transport vesicles to move along.

Key Points 


  1. help the cell resist compression,
  2. provide a track along which vesicles can move throughout the cell, and
  3. are the components of cilia and flagella.

Cilia and flagella are hair-like structures that

  1. assist with locomotion in some cells, as well as
  2. line various structures to trap particles.

The structures of cilia and flagella are a “9+2 array,” meaning that

  • a ring of nine microtubules is surrounded by two more microtubules.

Microtubules attach to replicated chromosomes

  • during cell division and
  • pull them apart to opposite ends of the pole,
  • allowing the cell to divide with a complete set of chromosomes in each daughter cell.

Microtubules are the largest element of the cytoskeleton.

The walls of the microtubule are made of

  • polymerized dimers of α-tubulin and β-tubulin, two globular proteins.

With a diameter of about 25 nm, microtubules are the widest components of the cytoskeleton.

They help the cell

  • resist compression,
  • provide a track along which vesicles move through the cell, and
  • pull replicated chromosomes to opposite ends of a dividing cell.

Like microfilaments, microtubules can dissolve and reform quickly.

Microtubules are also the structural elements of flagella, cilia, and centrioles (the latter are the two perpendicular bodies of the centrosome). In animal cells, the centrosome is the microtubule-organizing center. In eukaryotic cells, flagella and cilia are quite different structurally from their counterparts in prokaryotes.

Intermediate Filaments

Intermediate filaments (IFs) are cytoskeletal components found in animal cells. They are composed of a family of related proteins sharing common structural and sequence features.

epithelial cells

epithelial cells

flagella and cilia share a common structural arrangement of microtubules called a “9 + 2 array.” This is an appropriate name because a single flagellum or cilium is made of a ring of nine microtubule doublets surrounding a single microtubule doublet in the center.

9+2 array

9+2 array

The `Spectraplakins’: cytoskeletal giants with characteristics of both spectrin and plakin families

Katja Röper, Stephen L. Gregory and Nicholas H. Brown
J Cell Sci Nov 15, 2002; 115: 4215-4225​jcs.00157





The sequential endosymbiotic origins of eukaryotes: Compared to bacteria and archaea, the typical eukaryotic cell is much more structurally complex.

While the prokaryotes have a rigid cell wall, the ancestral eukaryote appears to have been wall-less (the walls of plant cells appear to represent a adaptation, and are not homologous to prokaryotic cell walls).

In addition to a nucleus (wherein the cell’s DNA is located, and which we will return to in the next section), there are cytoskeletal structures, including distinctive flagella (quite different from those found in prokaryotes), an active (motile) plasma membrane, capable of engulfing other cells, and multiple internal membrane systems. (A more complete description of cell structure is beyond this version of Biofundamentals).

In aerobic bacteria and cyanobacteria, the electron transport chains associated with ATP synthesis (through either photosynthesis or aerobic respiration) located within the plasma membrane (and in the case of cyanobacteria, internal membrane systems as well).

The same processes (aerobic respiration and photosynthesis) occur within eukaryotic cells. Animals have aerobic respiration, while plants have both).

However, these processes do not occur on the plasma membrane, but rather within distinct cytoplasmic organelles: mitochondria for aerobic respiration and chloroplasts for photosynthesis. All eukaryotic cells have mitochondria, plants (which are eukaryotic) have both mitochondria and chloroplasts.

An intriguing evolutionary question was, are these processes related, that is, are the processes of aerobic respiration and photosynthesis found in eukaryotes homologous to the processes found in bacteria and cyanobacteria, or did they originate independently.

The path to understanding that homologous nature of these processes began with studies of cell structure.

spectrin protein superfamily.large

spectrin protein superfamily.large

The role of secreted factors and extracellular matrix

The role of secreted factors and extracellular matrix

Focal Adhesions: Transmembrane Junctions Between the Extracellular Matrix and the Cytoskeleton

K Burridge, K Fath, T Kelly, G Nuckolls, and C Turner
Ann Rev Cell Biol Nov 1988; 4: 487-525

the extracellular matrix (ECM) is a collection of extracellular molecules secreted by cells that

  • provides structural and biochemical support to the surrounding cells.[1]

Because multicellularity evolved independently in different multicellular lineages, the composition of ECM varies between multicellular structures; however,

  • cell adhesion,
  • cell-to-cell communication and
  • differentiation

are common functions of the ECM.[2]

The animal extracellular matrix includes

  • the interstitial matrix and
  • the basement membrane.[3]

Interstitial matrix is present between various animal cells (i.e., in the intercellular spaces).

Gels of polysaccharides and fibrous proteins

  • fill the interstitial space and act as
  • a compression buffer against the stress placed on the ECM.[4]

Basement membranes are sheet-like depositions of ECM on which various epithelial cells rest.

The Extracellular Matrix (ECM)

Mechanical support to tissues

Organization of cells into tissues

  1. Activation of signaling pathways (cell growth, proliferation; development); examples:
  2. TGF-β, integrins
  3. specialized roles (tendon, bone; cartilage; cell movement during development; basal lamina in epithelia)


  1. proteoglycans
  2. collagen fibers (mechanical strength)
  3. multiadhesive matrix proteins (linking cell surface receptors to the (ECM)

Integrin connects the extracellular matrix with the actin cytoskeleton inside the cell

Fibronectin Integrin

Fibronectin Integrin

Continuous membrane-cytoskeleton adhesion requires continuous accommodation to lipid and cytoskeleton dynamics.

Sheetz MP, Sable JE, Döbereiner HG.
Annu Rev Biophys Struct Biomol. 2006;35:417-34.

The plasma membrane of most animal cells conforms to the cytoskeleton and only occasionally separates to form blebs. Previous studies indicated that

  • many weak interactions between cytoskeleton and the lipid bilayer
  • kept the surfaces together to counteract the normal outward pressure of cytoplasm.

Either the loss of adhesion strength or the formation of gaps in the cytoskeleton enables the pressure to form blebs. Membrane-associated cytoskeleton proteins, such as spectrin and filamin, can

  • control the movement and aggregation of membrane proteins and lipids,
    e.g., phosphoinositol phospholipids (PIPs), as well as blebbing.

At the same time, lipids (particularly PIPs) and membrane proteins affect

  • cytoskeleton and signaling dynamics.

We consider here the roles of the major phosphatidylinositol-4,5-diphosphate (PIP2) binding protein, MARCKS, and PIP2 levels in controlling cytoskeleton dynamics. Further understanding of dynamics will provide important clues about how membrane-cytoskeleton adhesion rapidly adjusts to cytoskeleton and membrane dynamics.

Interaction of membrane/lipid rafts with the cytoskeleton: impact on signaling and function: membrane/lipid rafts, mediators of cytoskeletal arrangement and cell signaling.

Head BP, Patel HH, Insel PA   Epub 2013 Jul 27.
Biochim Biophys Acta. 2014 Feb;1838(2):532-45.

The plasma membrane in eukaryotic cells contains microdomains that are

  • enriched in certain glycosphingolipids, gangliosides, and sterols (such as cholesterol) to form membrane/lipid rafts (MLR).

These regions exist as caveolae, morphologically observable flask-like invaginations, or as a less easily detectable planar form. MLR are scaffolds for many molecular entities, including

  • signaling receptors and ion channels that
  • communicate extracellular stimuli to the intracellular milieu.

Much evidence indicates that this organization and/or the clustering of MLR into more active signaling platforms

  • depends upon interactions with and dynamic rearrangement of the cytoskeleton.

Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to MLR and help

  • regulate lateral diffusion of membrane proteins and lipids in response to extracellular events
    (e.g., receptor activation, shear stress, electrical conductance, and nutrient demand).

MLR regulate

  • cellular polarity,
  • adherence to the extracellular matrix,
  • signaling events (including ones that affect growth and migration), and
  • are sites of cellular entry of certain pathogens, toxins and nanoparticles.

The dynamic interaction between MLR and the underlying cytoskeleton thus regulates many facets of the function of eukaryotic cells and their adaptation to changing environments. Here, we review general features of MLR and caveolae and their role in several aspects of cellular function, including

  • polarity of endothelial and epithelial cells,
  • cell migration,
  • mechanotransduction,
  • lymphocyte activation,
  • neuronal growth and signaling, and
  • a variety of disease settings.

This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

Cell control by membrane–cytoskeleton adhesion

Michael P. Sheetz
Nature Reviews Molecular Cell Biology 2, 392-396 (May 2001) | http://dx.doi.doi:/10.1038/35073095

The rates of mechanochemical processes, such as endocytosis, membrane extension and membrane resealing after cell wounding, are known to be controlled biochemically, through interaction with regulatory proteins. Here, I propose that these rates are also controlled physically, through an apparently continuous adhesion between plasma membrane lipids and cytoskeletal proteins.

Lipid Rafts, Signalling and the Cytoskeleton

Lipid rafts are specialised membrane domains enriched in certain lipids cholesterol and proteins. The existence of lipid rafts was first hypothesised in 1988 (Simons & van Meer, 1988; Simon & Ikonen, 1997), but what we know as “caveolae” were first observed  much earlier (Palade, 1953; Yamada, 1955).  Caveolae are flask shaped invaginations on the cell surface that are a type of membrane raft, these were named “caveolae intracellulare” (Yamada, 1955).  After a long argument (Jacobson & Dietrich, 1999), most now consider that these rafts actually exist, however, there is some confusion surrounding the classification of these rafts. It presently seems that there could be three types; caveolae, glycosphingolipid enriched membranes (GEM), and polyphospho inositol rich rafts. It may also be that there are inside rafts (PIP2 rich and caveolae) and outside rafts (GEM).

The fatty-acid chains of lipids within the rafts tend to be extended and so more tightly packed, creating domains with higher order. It is therefore thought that  rafts exist in a separate ordered phase that floats in a sea of poorly ordered lipids.  Glycosphingolipids, and other lipids with long, straight acyl chains are preferentially incorporated into the rafts.

Caveolae are similar in composition to GEMs that lack caveolae and in fact cells that lack caveolin-1 do not have morphologically identifiable caveolae but instead have extra GEM.  These cells can then be transfected with caveolin-1 cDNA and the caveolae then appear.  This suggests that GEM are merely caveolae without caveolin-1.  Caveolin-1 is a 21kDa integral membrane protein that binds cholesterol (Maruta et al, 1995). In cells lacking caveolin-1, caveolin-2 is synthesised but remains in the Golgi.  Caveolin 1 and 2 colocalise when expressed in the same cells and they may form hetero-dimers (Scherer et al, 1997). Caveolin-3 is expressed in muscle where it forms muscle-type caveolae.  Caveolin-3 is involved in certain types of muscular dystrophy (Galbiati et al, ). A slightly confusing finding is that caveolae are the reported site of integrin signalling ().  It is difficult to imagine integrins being available in the depths of membrane invaginations for binding extra-cellular ligands.

The function of rafts

Many functions have been attributed to rafts, from cholesterol transport, endocytosis and signal transduction.  The later is almost certainly the case. It has been suggested that the primary function of caveolae was in constitutive endocytic trafficking but recent data show that this is not the case, instead caveolae are very stable regions of membranes that are not involved in  endocytosis (Thompsen et al, 2002).

lipid raft

lipid raft

Rafts and the Cytoskeleton

Many actin binding proteins are known to bind to polyphosphoinositides and to be regulated by them (see PI and ABPs), by a series of protein domains such as PH, PX and ENTH (see Domains).  It is consequently scarcely surprising that some ABPs are suggested to link the actin cytoskeleton and PIP2-enriched rafts. One of these is gelsolin, a Ca2+, pH and polyphosphoinositide regulated actin capping and severing protein (see Gelsolin Family), that partitions into rafts isolated biochemically from brain (Fanatsu et al, 2000).

GEMs too are suggested to link to the actin cytoskeleton through ABPs particularly ERM proteins through EBP50, a protein that binds members of the ERM proteins through the ERM C-terminus (Brdickova et al, 2001).


Brdickova, N., Brdicka, T., Andrea, L., Spicka, J., Angelisova, P., Milgram, S. L. & Horejsi, V. (2001) Interaction between two adaptor proteins, PAG and EBP50: a possible link between membrane rafts and actin cytoskeleton.  FEBS letters. 507, 133-136.

Cary, L. A. & Cooper, J. A. (2000) Molecular switches in lipid rafts.  Nature. 404, 945-947.

Czarny, M., Fiucci, G., Lavie, Y., Banno, Y., Nozawa, Y. & Liscovitch, M. (2000) Phospholipase D2: functional interaction with caveolin in low-density membrane microdomains.,  FEBS letters.

Foger, N., Funatsu, N., Kumanogoh, H., Sokawa, Y. & Maekawa, S. (2000) Identification of gelsolin as an actin regulatory component in a Triton insoluble low density fraction (raft) of newborn bovine brain.  Neuroscience Research. 36, 311-317.

Galbiati, F., Engelman, J. A., Volonte, D., Zhang, X. L., Minetti, C., Li, M., Hou jr, H., Kneitz, B., Edelman, W. & Lisanti, M. P. (2001) Caveolin-3 null mice show a loss of caveolae, changes in the microdomain distribution of the dystrophin-glycoprotein complex, and T-tubule abnormalities.  J. Biol.Chem. 276, 21425-21433.

…  (more)

centralpore-small  Gating and Ion Conductivity

centralpore-small Gating and Ion Conductivity

Interaction of epithelial ion channels with the actin-based cytoskeleton.

Mazzochi C, Benos DJ, Smith PR.
Am J Physiol Renal Physiol. 2006 Dec;291(6):F1113-22. Epub 2006 Aug 22

The interaction of ion channels with the actin-based cytoskeleton in epithelial cells

  • not only maintains the polarized expression of ion channels within specific membrane domains,
  • it also functions in the intracellular trafficking and regulation of channel activity.

Initial evidence supporting an interaction between

  • epithelial ion channels and the
  • actin-based cytoskeleton

came from patch-clamp studies of the effects of cytochalasins on channel activity. Cytochalasins were shown to

  • either activate or inactivate epithelial ion channels.

An interaction between

  • the actin-based cytoskeleton and epithelial ion channels

was further supported by the fact that the addition of monomeric or filamentous actin to excised patches had an effect on channel activity comparable to that of cytochalasins. Through the recent application of molecular and proteomic approaches, we now know that

  • the interactions between epithelial ion channels and actin can either be direct or indirect,
  • the latter being mediated through scaffolding or actin-binding proteins
  • that serve as links between the channels and the actin-based cytoskeleton.

This review discusses recent advances in our understanding of the interactions between epithelial ion channels and the actin-based cytoskeleton, and the roles these interactions play in regulating the cell surface expression, activity, and intracellular trafficking of epithelial ion channels.

epithelial ion channels

epithelial ion channels

Actin cytoskeleton regulates ion channel activity in retinal neurons.

Maguire G, Connaughton V, Prat AG, Jackson GR Jr, Cantiello HF.
Neuroreport. 1998 Mar 9;9(4):665-70

The actin cytoskeleton is an important contributor to the integrity of cellular shape and responses in neurons. However, the molecular mechanisms associated with functional interactions between the actin cytoskeleton and neuronal ion channels are largely unknown. Whole-cell and single channel recording techniques were thus applied to identified retinal bipolar neurons of the tiger salamander (Ambystoma tigrinum) to assess the role of acute changes in actin-based cytoskeleton dynamics in the regulation of voltage-gated ion channels. Disruption of endogenous actin filaments after brief treatment (20-30 min) with cytochalasin D (CD) activated voltage-gated K+ currents in bipolar cells, which were largely prevented by intracellular perfusion with the actin filament-stabilizer agent, phalloidin. Either CD treatment under cell-attached conditions or direct addition of actin to excised, inside-out patches of bipolar cells activated and/or increased single K+ channels. Thus, acute changes in actin-based cytoskeleton dynamics regulate voltage-gated ion channel activity in bipolar cells.

Cytoskeletal Basis of Ion Channel Function in Cardiac Muscle

Matteo Vatta, Ph.D1,2 and Georgine Faulkner, Ph.D3

The publisher’s final edited version of this article is available at Future Cardiol

The heart is a force-generating organ that responds to self-generated electrical stimuli from specialized cardiomyocytes. This function is modulated by sympathetic and parasympathetic activity.

In order to contract and accommodate the repetitive morphological changes induced by the cardiac cycle,

  • cardiomyocytes depend on their highly evolved and specialized cytoskeletal apparatus.

Defects in components of the cytoskeleton, in the long term, affect

  • the ability of the cell to compensate at
  • both functional and structural levels.

In addition to the structural remodeling, the myocardium becomes

  • increasingly susceptible to altered electrical activity leading to arrhythmogenesis.

The development of arrhythmias secondary to structural remodeling defects has been noted, although the detailed molecular mechanisms are still elusive. Here I will review the current knowledge of the molecular and functional relationships between the cytoskeleton and ion channels and, I will discuss the future impact of new data on molecular cardiology research and clinical practice. 

Stretch-activated ion channel

Stretch-activated or stretch-gated ion channels are

  • ion channels which open their pores in response to
  • mechanical deformation of a neuron’s plasma membrane.

[Also see mechanosensitive ion channels and mechanosensitive channels, with which they may be synonymous]. Opening of the ion channels

  • depolarizes the afferent neuron producing an action potential with sufficient depolarization.[1]

Channels open in response to two different mechanisms: the prokaryotic model and the mammalian hair cell model.[2][3] Stretch-activated ion channels have been shown to detect vibration, pressure, stretch, touch, sounds, tastes, smell, heat, volume, and vision.[4][5][6] Stretch-activated ion channels have been categorized into

three distinct “superfamilies”:

  1. the ENaC/DEG family,
  2. the TRP family, and
  3. the K1 selective family.

These channels are involved with bodily functions such as blood pressure regulation.[7] They are shown to be associated with many cardiovascular diseases.[3] Stretch-activated channels were first observed in chick skeletal muscles by Falguni Guharay and Frederick Sachs in 1983 and the results were published in 1984.[8] Since then stretch-activated channels have been found in cells from bacteria to humans as well as plants.

Mechanosensitivity of cell membranes. Ion channels, lipid matrix and cytoskeleton.

Petrov AG, Usherwood PN.
Eur Biophys J. 1994;23(1):1-19

Physical and biophysical mechanisms of mechano-sensitivity of cell membranes are reviewed. The possible roles of

  • the lipid matrix and of
  • the cytoskeleton in membrane mechanoreception

are discussed. Techniques for generation of static strains and dynamic curvatures of membrane patches are considered. A unified model for

  • stress-activated and stress-inactivated ion channels

under static strains is described. A review of work on

  • stress-sensitive pores in lipid-peptide model membranes

is presented. The possible role of flexoelectricity in mechano-electric transduction, e.g. in auditory receptors is discussed. Studies of

  • flexoelectricity in model lipid membranes, lipid-peptide membranes and natural membranes containing ion channels

are reviewed. Finally, possible applications in molecular electronics of mechanosensors employing some of the recognized principles of mechano-electric transduction in natural membranes are discussed.Marhaba, R. & Zoller, M. (2001) Involvement of CD44 in cytoskeleton rearrangement and raft reorganization in T cells.  J.Cell Sci. 114, 1169-1178.

FIGURE 2 | The transient pore model.

peroxisomal matrix protein

peroxisomal matrix protein

Peroxisomal matrix protein import: the transient pore model

Ralf Erdmann & Wolfgang Schliebs
Nature Reviews Molecular Cell Biology 6, 738-742 (September 2005)

Peroxisomal matrix protein import: the transient pore model
The transient pore model

The peroxisomal import receptor peroxin-5 (Pex5) recognizes peroxisomal targeting signal-1 (PTS1)-containing cargo proteins in the cytosol. It then moves to the peroxisome where it inserts into the peroxisomal membrane to become an integral part of the protein-import apparatus. Pex14 and/or Pex13, which are associated with Pex17, are proposed to be involved in tethering the receptor to the membrane and in the assembly, stabilization and rearrangement of the translocon. Cargo release into the peroxisomal matrix is thought to be initiated by intraperoxisomal factors — for example, the competitive binding of the intraperoxisomal Pex8, which also has a PTS1. The disassembly and recycling of Pex5 is triggered by a cascade of protein–protein interactions at the peroxisomal membrane that results in the Pex1-, Pex6-driven, ATP-dependent dislocation of Pex5 from the peroxisomal membrane to the cytosol. Pex1 and Pex6 are AAA+ (ATPases associated with a variety of cellular activities) peroxins that are associated with the peroxisome membrane through Pex15 in yeast or its orthologue PEX26 in mammals. Pex4, which is membrane-anchored through Pex22, is a member of the E2 family of ubiquitin-conjugating enzymes, and Pex2, Pex10 and Pex12 contain the RING-finger motif that is a characteristic element of E3 ubiquitin ligases. Mono- or di-ubiquitylation are reversible steps that seem to be required for the efficient recycling of import receptors, whereas polyubiquitylation might signal the proteasome-dependent degradation of receptors when the physiological dislocation of receptors is blocked. Ub, ubiquitin.

Nature Reviews Molecular Cell Biology 6, 738-742 (September 2005) |


peroxisomal protein pore model

peroxisomal protein pore model

Peroxisomal matrix protein import: the transient pore model

Ralf Erdmann & Wolfgang Schliebs
Nature Reviews Molecular Cell Biology 6, 738-742 (September 2005)

Peroxisomal matrix protein import: the transient pore model

Peroxin-13 (Pex13), Pex14 and Pex17 are constituents of the docking complex for cycling peroxisomal import receptors. Another protein assembly in the peroxisomal membrane comprises the RING-finger-motif-containing peroxins Pex2, Pex10 and Pex12. This motif is a characteristic element of E3 ubiquitin ligases, and this subcomplex is linked to the docking complex by Pex8, which is peripherally attached to the lumenal side of the peroxisomal membrane. Pex4 is a member of the E2 family of ubiquitin-conjugating enzymes and is anchored to the peroxisomal membrane through the cytosolic domain of Pex22. Pex1 and Pex6 are interacting AAA+ proteins (ATPases associated with a variety of cellular activities), which are attached to the membrane through binding to Pex15 in yeast or to its mammalian counterpart PEX26.

Peroxisomal matrix protein import: the transient pore model

Ralf Erdmann & Wolfgang Schliebs

Peroxisomes import folded, even oligomeric, proteins, which distinguishes the peroxisomal translocation machinery from the well-characterized translocons of other organelles. How proteins are transported across the peroxisomal membrane is unclear. Here, we propose a mechanistic model that conceptually divides the import process into three consecutive steps: the formation of a

  • translocation pore by the import receptor,
  • the ubiquitylation of the import receptors, and
  • pore disassembly/receptor recycling.


Masoud Naderi Maralani

Identification of the phytosphingosine metabolic pathway leading to odd-numbered fatty acids

The long-chain base ​phytosphingosine is a component of sphingolipids and exists in yeast, plants and some mammalian tissues. ​Phytosphingosine is unique in that it possesses an additional hydroxyl group compared with other long-chain bases. However, its metabolism is unknown. Here we show that ​phytosphingosine is metabolized to odd-numbered fatty acids and is incorporated into glycerophospholipids both in yeast and mammalian cells. Disruption of the yeast gene encoding long-chain base 1-phosphate lyase, which catalyzes the committed step in the metabolism of ​phytosphingosine to glycerophospholipids, causes an ~40% reduction in the level of phosphatidylcholines that contain a C15 fatty acid. We also find that ​2-hydroxypalmitic acid is an intermediate of the phytosphingosine metabolic pathway. Furthermore, we show that the yeast ​MPO1 gene, whose product belongs to a large, conserved protein family of unknown function, is involved in ​phytosphingosine metabolism. Our findings provide insights into fatty acid diversity and identify a pathway by which hydroxyl group-containing lipids are metabolized.

About GPCRs

G-protein-coupled receptors (GPCRs) are a class of membrane proteins that allow the transmission of a wide variety of signals over the cell membrane, between different cells and over long distances inside the body. The molecular mechanisms of action of GPCRs were worked in great detail by Brian Kobilka and Robert Lefkowitz for which they were jointly awarded the Nobel Prize in Chemistry for 2012. Read More

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Liver Endoplasmic Reticulum Stress and Hepatosteatosis

Larry H Bernstein, MD, FCAP


1. Absence of adipose triglyceride lipase protects from hepatic endoplasmic reticulum stress in mice.

Fuchs CD, Claudel T, Kumari P, Haemmerle G, et al.
LabExpMol Hepatology, Medical Univ of Graz, Austria.
Hepatology. 2012 Jul;56(1):270-80. Epub 2012 May 29.

Nonalcoholic fatty liver disease (NAFLD) is characterized by

  • triglyceride (TG) accumulation and
  • endoplasmic reticulum (ER) stress.

Fatty acids (FAs) may trigger ER stress, therefore,

  •  the absence of adipose triglyceride lipase (ATGL/PNPLA2)-
    • the main enzyme for intracellular lipolysis,
  • releasing FAs, and
  • closest homolog to adiponutrin (PNPLA3)

recently implicated in the pathogenesis of NAFLD-

  • could protect against hepatic ER stress.

Wild-type (WT) and ATGL knockout (KO) mice

  •  were challenged with tunicamycin (TM) to induce ER stress.

Markers of hepatic

  •  lipid metabolism,
  • ER stress, and
  • inflammation were explored
    • for gene expression by
    •  serum biochemistry,
    • hepatic TG and FA profiles,
    • liver histology,
    • cell-culture experiments were performed in Hepa1.6 cells
  • after the knockdown of ATGL before FA and TM treatment.

TM increased hepatic TG accumulation in ATGL KO, but not in WT mice. Lipogenesis and β-oxidation
were repressed at the gene-expression level
(sterol regulatory element-binding transcription factor 1c,
fatty acid synthase, acetyl coenzyme A carboxylase 2, and carnitine palmitoyltransferase 1 alpha) in
both WT and ATGL KO mice. Genes for very-low-density lipoprotein (VLDL) synthesis (microsomal
triglyceride transfer protein and apolipoprotein B)

  •  were down-regulated by TM in WT
  • and even more in ATGL KO mice,
  • which displayed strongly reduced serum VLDL cholesterol levels.

ER stress markers were induced exclusively in TM-treated WT, but not ATGL KO, mice:

  •  glucose-regulated protein,
  • C/EBP homolog protein,
  • spliced X-box-binding protein,
  • endoplasmic-reticulum-localized DnaJ homolog 4, and
  • inflammatory markers Tnfα and iNos.

Total hepatic FA profiling revealed a higher palmitic acid/oleic acid (PA/OA) ratio in WT mice.
Phosphoinositide-3-kinase inhibitor-

  • known to be involved in FA-derived ER stress and
  • blocked by OA-
  • was increased in TM-treated WT mice only.

In line with this, in vitro OA protected hepatocytes from TM-induced ER stress. Lack of ATGL may protect from
hepatic ER stress through alterations in FA composition. ATGL could constitute a new therapeutic strategy
to target ER stress in NAFLD.
PMID: 22271167 Diabetes Obes Metab. 2010 Oct;12 Suppl 2:83-92.

2. Hepatic steatosis: a role for de novo lipogenesis and the transcription factor SREBP-1c.
Ferré P, Foufelle F. INSERM, and Université Pierre et Marie Curie-Paris, Paris, France.    PMID: 21029304

Excessive availability of plasma fatty acids and lipid synthesis from glucose (lipogenesis) are important determinants of steatosis.
Lipogenesis is an insulin- and glucose-dependent process that is under the control of specific transcription factors,

Insulin induces the maturation of SREBP-1c in the endoplasmic reticulum (ER).

  • SREBP-1c in turn activates glycolytic gene expression,
    • allowing glucose metabolism, and
    • lipogenic genes in conjunction with ChREBP.

Lipogenesis activation in the liver of obese markedly insulin-resistant steatotic rodents is then paradoxical.
It appears the activation of SREBP-1c and thus of lipogenesis is

  •  secondary in the steatotic liver to an ER stress.

The ER stress activates the

  •  cleavage of SREBP-1c independent of insulin,
  • explaining the paradoxical stimulation of lipogenesis
  • in an insulin-resistant liver.

Inhibition of the ER stress in obese rodents

  •  decreases SREBP-1c activation and lipogenesis and
  • improves markedly hepatic steatosis and insulin sensitivity.
  • ER is thus worth considering as a potential therapeutic target for steatosis and metabolic syndrome.

3. SREBP-1c transcription factor and lipid homeostasis: clinical perspective
Ferré P, Foufelle F
Inserm, Centre de Recherches Biomédicales des Cordeliers, Paris, France.
Horm Res. 2007;68(2):72-82. Epub 2007 Mar 5. PMID:17344645

Insulin has long-term effects on glucose and lipid metabolism through its control on the expression of specific genes.
In insulin sensitive tissues and particularly in the liver,

  •  the transcription factor sterol regulatory element binding protein-1c (SREBP-1c) transduces the insulin signal, which is
  • synthetized as a precursor in the membranes of the endoplasmic reticulum
  • which requires post-translational modification to yield its transcriptionally active nuclear form.

Insulin activates the transcription and the proteolytic maturation of SREBP-1c, which induces the

  •  expression of a family of genes
  • involved in glucose utilization and fatty acid synthesis and
  • can be considered as a thrifty gene.

Since a high lipid availability is

  •  deleterious for insulin sensitivity and secretion,
  • a role for SREBP-1c in dyslipidaemia and type 2 diabetes
  • has been considered in genetic studies.

SREBP-1c could also participate in

  •  hepatic steatosis observed in humans
  • related to alcohol consumption and
  • hyperhomocysteinemia
  • concomitant with a ER-stress and
  • insulin-independent SREBP-1c activation.

4. Hepatic steatosis: a role for de novo lipogenesis and the transcription factor SREBP-1c
Ferré P, Foufelle F
INSERM, Centre de Recherches des Cordeliers and Université Pierre et Marie Curie-Paris, Paris, France.
Diabetes Obes Metab. 2010 Oct;12 Suppl 2:83-92. PMID: 21029304

Lipogenesis in liver steatosis is

  •  an insulin- and glucose-dependent process
  • under the control of specific transcription factors,
  • sterol regulatory element binding protein 1c (SREBP-1c),
  • activated by insulin and carbohydrate response element binding protein (ChREBP)

Insulin induces the maturation of SREBP-1c in the endoplasmic reticulum (ER).
SREBP-1c in turn activates glycolytic gene expression, allowing –

  •  glucose metabolism in conjunction with ChREBP.

activation of SREBP-1c and lipogenesis is secondary in the steatotic liver to ER stress, which

  •  activates the cleavage of SREBP-1c independent of insulin,
  • explaining the stimulation of lipogenesis in an insulin-resistant liver.
  • Inhibition of the ER stress in obese rodents decreases SREBP-1c activation and improves
  • hepatic steatosis and insulin sensitivity.

ER is thus a new partner in steatosis and metabolic syndrome

5. Pharmacologic ER stress induces non-alcoholic steatohepatitis in an animal model
Jin-Sook Leea, Ze Zhenga, R Mendeza, Seung-Wook Hac, et al.
Wayne State University SOM, Detroit, MI
Toxicology Letters 20 May 2012; 211(1):29–38

Endoplasmic reticulum (ER) stress refers to a condition of

  •  accumulation of unfolded or misfolded proteins in the ER lumen, which is known to
  • activate an intracellular stress signaling termed
  • Unfolded Protein Response (UPR).

A number of pharmacologic reagents or pathophysiologic stimuli

  •  can induce ER stress and activation of the UPR signaling,
  • leading to alteration of cell physiology that is
  • associated with the initiation and progression of a variety of diseases.

Non-alcoholic steatohepatitis (NASH), characterized by hepatic steatosis and inflammation, has been considered the
precursor or the hepatic manifestation of metabolic disease. In this study, we delineated the

  • toxic effect and molecular basis
  • by which pharmacologic ER stress,
  • induced by a bacterial nucleoside antibiotic tunicamycin (TM),
  • promotes NASH in an animal model.

Mice of C57BL/6J strain background were challenged with pharmacologic ER stress by intraperitoneal injection of TM. Upon TM injection,

  •  mice exhibited a quick NASH state characterized by
  • hepatic steatosis and inflammation.

TM-treated mice exhibited an increase in –

  •  hepatic triglycerides (TG) and a –
  • decrease in plasma lipids, including
  • plasma TG,
  • plasma cholesterol,
  • high-density lipoprotein (HDL), and
  • low-density lipoprotein (LDL),

In response to TM challenge,

  •  cleavage of sterol responsive binding protein (SREBP)-1a and SREBP-1c,
  •  the key trans-activators for lipid and sterol biosynthesis,
  • was dramatically increased in the liver.

Consistent with the hepatic steatosis phenotype, expression of

  •  some key regulators and enzymes in de novo lipogenesis and lipid droplet formation was up-regulated,
  • while expression of those involved in lipolysis and fatty acid oxidation was down-regulated
  • in the liver of mice challenged with TM.

TM treatment also increased phosphorylation of NF-κB inhibitors (IκB),

  •  leading to the activation of NF-κB-mediated inflammatory pathway in the liver.

Our study not only confirmed that pharmacologic ER stress is a strong “hit” that triggers NASH, but also demonstrated

  •  crucial molecular links between ER stress,
  • lipid metabolism, and
  • inflammation in the liver in vivo.

► Pharmacologic ER stress induced by tunicamycin (TM) induces a quick NASH state in vivo.
► TM leads to dramatic increase in cleavage of sterol regulatory element-binding protein in the liver.
► TM up-regulates lipogenic genes, but down-regulates the genes in lipolysis and FA oxidation.
► TM activates NF-κB and expression of genes encoding pro-inflammatory cytokines in the liver.
ER, endoplasmic reticulum; TM, tunicamycin; NASH, non-alcoholic steatohepatitis; NAFLD,
non-alcoholic fatty liver disease; TG, triglycerides; SREBP, sterol responsive binding protein;
NF-κB, activation of nuclear factor-kappa B; IκB, NF-κB inhibitor
Keywords: ER stress; Non-alcoholic steatohepatitis; Tunicamycin; Lipid metabolism; Hepatic inflammation
Figures and tables from this article:

Fig. 1. TM challenge alters lipid profiles and causes hepatic steatosis in mice. (A) Quantitative real-time RT-PCR analysis of liver mRNA isolated from mice challenged with TM or vehicle control. Total liver mRNA was isolated at 8 h or 30 h after injection with vehicle or TM (2 μg/g body weight) for real-time RT-PCR analysis. Expression values were normalized to β-actin mRNA levels. Fold changes of mRNA are shown by comparing to one of the control mice. Each bar denotes the mean ± SEM (n = 4 mice per group); **P < 0.01. Edem1, ER degradation enhancing, mannosidase alpha-like 1. (B) Oil-red O staining of lipid droplets in the livers of the mice challenged with TM or vehicle control (magnification: 200×). (C) Levels of TG in the liver tissues of the mice challenged with TM or vehicle control. (D) Levels of plasma lipids in the mice challenged with TM or vehicle control. TG, triglycerides; TC, total plasma cholesterol; HDL, high-density lipoproteins; VLDL/LDL, very low and low density lipoproteins. For C and D, each bar denotes mean ± SEM (n = 4 mice per group); *P < 0.05; **P < 0.01.

 F options

Fig. 2. TM challenge leads to a quick NASH state in mice. (A) Histological examination of liver tissue sections of the mice challenged with TM (2 μg/g body weight) or vehicle control. Upper panel, hematoxylin–eosin (H&E) staining of liver tissue sections; the lower panel, Sirius staining of collagen deposition of liver tissue sections (magnification: 200×). (B) Histological scoring for NASH activities in the livers of the mice treated with TM or vehicle control. The grade scores were calculated based on the scores of steatosis, hepatocyte ballooning, lobular and portal inflammation, and Mallory bodies. The stage scores were based on the liver fibrosis. Number of mice examined is given in parentheses. Mean ± SEM values are shown. P-values were calculated by Mann–Whitney U-test.

Fig. 3. TM challenge significantly increases levels of cleaved/activated forms of SREBP1a and SREBP1c in the liver. Western blot analysis of protein levels of SREBP1a (A) and SREBP1c (B) in the liver tissues from the mice challenged with TM (2 μg/g body weight) or vehicle control. Levels of GAPDH were included as internal controls. For A and B, the values below the gels represent the ratios of mature/cleaved SREBP signal intensities to that of SREBP precursors. The graph beside the images showed the ratios of mature/cleaved SREBP to precursor SREBP in the liver of mice challenged with TM or vehicle. The protein signal intensities shown by Western blot analysis were quantified by NIH imageJ software. Each bar represents the mean ± SEM (n = 3 mice per group); **P < 0.01. SREBP-p, SREBP precursor; SREBP-m, mature/cleaved SREBP.

Fig. 4. TM challenge up-regulates expression of genes involved in lipogenesis but down-regulates expression of genes involved in lipolysis and FA oxidation. Quantitative real-time RT-PCR analysis of liver mRNAs isolated from the mice challenged with TM (2 μg/g body weight) or vehicle control, which encode regulators or enzymes in: (A) de novo lipogenesis: PGC1α, PGC1β, DGAT1 and DGAT2; (B) lipid droplet production: ADRP, FIT2, and FSP27; (C) lipolysis: ApoC2, Acox1, and LSR; and (D) FA oxidation: PPARα. Expression values were normalized to β-actin mRNA levels. Fold changes of mRNA are shown by comparing to one of the control mice. Each bar denotes the mean ± SEM (n = 4 mice per group); **P < 0.01. (E and F) Isotope tracing analysis of hepatic de novo lipogenesis. Huh7 cells were incubated with [1-14C] acetic acid for 6 h (E) or 12 h (F) in the presence or absence of TM (20 μg/ml). The rates of de novo lipogenesis were quantified by determining the amounts of [1-14C]-labeled acetic acid incorporated into total cellular lipids after normalization to cell numbers.

Fig. 5. TM activates the inflammatory pathway through NF-κB, but not JNK, in the liver. Western blot analysis of phosphorylated Iκ-B, total Iκ-B, phosphorylated JNK, and total JNK in the liver tissues from the mice challenged with TM (2 μg/g body weight) or vehicle control. Levels of GAPDH were included as internal controls. The values below the gels represent the ratios of phosphorylated protein signal intensities to that of total proteins.

Fig. 6. TM induces expression of pro-inflammatory cytokines and acute-phase responsive proteins in the liver. Quantitative real-time RT-PCR analyses of liver mRNAs isolated from the mice challenged with TM (2 μg/g body weight) or vehicle control, which encode: (A) pro-inflammatory cytokine TNFα and IL6; and (B) acute-phase protein SAP and SAA3. Expression values were normalized to β-actin mRNA levels. Fold changes of mRNA are shown by comparing to one of the control mice. (C–E) ELISA analyses of serum levels of TNFα, IL6, and SAP in the mice challenged with TM or vehicle control for 8 h ELISA. Each bar denotes the mean ± SEM (n = 4 mice per group); *P < 0.05, **P < 0.01.

Corresponding author at: Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, 540 E. Canfield Avenue, Detroit, MI 48201, USA. Tel.: +1 313 577 2669; fax: +1 313 577 5218.

The SREBP regulatory pathway. Brown MS, Goldst...

The SREBP regulatory pathway. Brown MS, Goldstein JL (1997). “The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor”. Cell 89 (3) : 331–340. doi:10.1016/S0092-8674(00)80213-5. PMID 9150132. (Photo credit: Wikipedia)

English: Structure of the SREBF1 protein. Base...

English: Structure of the SREBF1 protein. Based on PyMOL rendering of PDB 1am9. (Photo credit: Wikipedia)

The SREBP regulatory pathway

The SREBP regulatory pathway (Photo credit: Wikipedia)

English: Diagram of rough endoplasmic reticulu...

English: Diagram of rough endoplasmic reticulum by Ruth Lawson, Otago Polytechnic. (Photo credit: Wikipedia)

Micrograph demonstrating marked (macrovesicula...

Micrograph demonstrating marked (macrovesicular) steatosis in non-alcoholic fatty liver disease. Masson’s trichrome stain. (Photo credit: Wikipedia)


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