Protein-Folding Homeostasis in the Endoplasmic Reticulum and Nutritional Regulation

The flux of newly synthesized proteins entering the endoplasmic reticulum (ER) is under negative regulation by the ER-localized PKR-like ER kinase (PERK). PERK is activated by unfolded protein stress in the ER lumen and inhibits new protein synthesis by the phosphorylation of translation initiation factor eIF2α. This homeostatic mechanism, shared by all animal cells, has proven to be especially important to the well-being of professional secretory cells, notably the endocrine pancreas. PERK, its downstream effectors, and the allied branches of the unfolded protein response intersect broadly with signaling pathways that regulate nutrient assimilation, and ER stress and the response to it have been implicated in the development of the metabolic syndrome accompanying obesity in mammals. Here we review our current understanding of the cell biology underlying these relationships.Insulin was among the first proteins to be sequenced, among the first to have its structure solved, and therefore among the first to provide clues to the diversity of modifications that affect secreted proteins. The β cell of the pancreas, which produces insulin, is one of the best-studied secretory cells, and the role of the secretory pathway in insulin biosynthesis has been recognized from the dawn of modern cell biology. Years later, when the stress pathways that contribute to protein-folding homeostasis in the endoplasmic reticulum (the unfolded protein response, UPR) came under scrutiny (Gardner et al. 2013; Olzmann et al. 2013), it was revealed that their integrity is important to insulin metabolism and to the function of β cells.The precursor of insulin, prepro-insulin, is recruited to the ER membrane cotranslationally through its amino-terminal signal sequence (Mandon et al. 2013). Oxidative folding and signal sequence removal yield mature pro-insulin, whose tertiary structure is stabilized by three disulfide bonds (Bulleid 2012). Folded pro-insulin clears ER quality control (Braakman and Hebert 2013) and traffics distally (Lord et al. 2013).The peptidase involved in post-ER steps of pro-insulin maturation has long been recognized as playing a key role in its secretion, but the sensitivity of insulin biosynthesis to integrity of ER steps was not recognized until later. An early clue came from study of a naturally occurring mutation in mouse Ins2. The Akita mutation results in a Cys-92→Tyr substitution, disrupting an essential disulfide bond and leading to misfolding of proinsulin 2 (Wang et al. 1999). Interestingly, a single copy of the mutation is sufficient to compromise β cells, whereas homozygosity for a null mutation in Ins2 is without an obvious phenotype in mice (because of redundancy between a rodent’s two insulin genes) (Duvillie et al. 1997). The biochemical (and phenotypic) dominance of the Akita mutation in mice (Colombo et al. 2008) fit well with retention of the mutant pro-insulin in the ER, high levels of UPR signaling, and with a progressive decline in β-cell mass and insulin stores as the mutant mice age. Thus, a perturbation to ER protein-folding homeostasis induced by the misfolding-prone mutant pro-insulin has a long-term negative effect on β-cell function.Unbiased human genetics provided an additional clue to the importance of protein-folding homeostasis in the ER; the Wolcott–Rallison syndrome is a rare recessive monogenic form of hypoinsulinaemic neonatal diabetes associated with bone dysplasia and episodic liver failure (Julier and Nicolino 2010). Positional cloning revealed that the causative mutations in EIF2AK3 severely disrupted the expression or function of PERK (Delepine et al. 2000), an ER-localized stress-activated kinase that tunes rates of new protein synthesis to the unfolded protein load in the ER (Harding et al. 1999). Although known to be enriched in β cells, PERK expression is ubiquitous (Shi et al. 1998). Therefore, the prominence of diabetes in the phenotype associated with loss-of-function mutations in a ubiquitous component of the unfolded protein response (UPR) pointed to a special role for ER homeostasis in β-cell health.More surprising has been the link between chronic ER stress and the ability of insulin target tissues to respond to the hormone; it has emerged that nutrient excess and obesity are associated with higher levels of UPR signaling in the liver and fat and that steps that mitigate ER stress in these tissues ameliorate the insulin resistance that is part of the metabolic syndrome linked to nutrient excess. Thus, ER stress and the response to it affect both the insulin-producing β cell and the insulin-responsive tissues and may therefore influence the pathophysiology of the common, type II form of diabetes mellitus by limiting both the production of insulin and the body’s sensitivity to it.     

Maintenance of Calcium Homeostasis in the Endoplasmic Reticulum by Bcl-2

The oncogene bcl-2 encodes a 26-kD protein localized to intracellular membranes, including the ER, mitochondria, and perinuclear membrane, but its mechanism of action is unknown. We have been investigating the hypothesis that Bcl-2 regulates the movement of calcium ions (Ca²⁺) through the ER membrane. Earlier findings in this laboratory indicated that Bcl-2 reduces Ca²⁺ efflux from the ER lumen in WEHI7.2 lymphoma cells treated with the Ca²⁺-ATPase inhibitor thapsigargin (TG) but does not prevent capacitative entry of extracellular calcium. In this report, we show that sustained elevation of cytosolic Ca²⁺ due to capacitative entry is not required for induction of apoptosis by TG, suggesting that ER calcium pool depletion may trigger apoptosis. Bcl-2 overexpression maintains Ca²⁺ uptake in the ER of TG-treated cells and prevents a TG-imposed delay in intralumenal processing of the endogenous glycoprotein cathepsin D. Also, Bcl-2 overexpression preserves the ER Ca²⁺ pool in untreated cells when extracellular Ca²⁺ is low. However, low extracellular Ca²⁺ reduces the antiapoptotic action of Bcl-2, suggesting that cytosolic Ca²⁺ elevation due to capacitative entry may be required for optimal ER pool filling and apoptosis inhibition by Bcl-2. In summary, the findings suggest that Bcl-2 maintains Ca²⁺ homeostasis within the ER, thereby inhibiting apoptosis induction by TG.     

Depletion of Cyclophilins B and C Leads to Dysregulation of Endoplasmic Reticulum Redox Homeostasis

Protein folding within the endoplasmic reticulum is assisted by molecular chaperones and folding catalysts that include members of the protein-disulfide isomerase and peptidyl-prolyl isomerase families. In this report, we examined the contributions of the cyclophilin subset of peptidyl-prolyl isomerases to protein folding and identified cyclophilin C as an endoplasmic reticulum (ER) cyclophilin in addition to cyclophilin B. Using albumin and transferrin as models of cis-proline-containing proteins in human hepatoma cells, we found that combined knockdown of cyclophilins B and C delayed transferrin secretion but surprisingly resulted in more efficient oxidative folding and secretion of albumin. Examination of the oxidation status of ER protein-disulfide isomerase family members revealed a shift to a more oxidized state. This was accompanied by a >5-fold elevation in the ratio of oxidized to total glutathione. This “hyperoxidation” phenotype could be duplicated by incubating cells with the cyclophilin inhibitor cyclosporine A, a treatment that triggered efficient ER depletion of cyclophilins B and C by inducing their secretion to the medium. To identify the pathway responsible for ER hyperoxidation, we individually depleted several enzymes that are known or suspected to deliver oxidizing equivalents to the ER: Ero1αβ, VKOR, PRDX4, or QSOX1. Remarkably, none of these enzymes contributed to the elevated oxidized to total glutathione ratio induced by cyclosporine A treatment. These findings establish cyclophilin C as an ER cyclophilin, demonstrate the novel involvement of cyclophilins B and C in ER redox homeostasis, and suggest the existence of an additional ER oxidative pathway that is modulated by ER cyclophilins.     

Signaling Networks Converge on TORC1-SREBP Activity to Promote Endoplasmic Reticulum Homeostasis

The function and capacity of the endoplasmic reticulum (ER) is determined by multiple processes ranging from the local regulation of peptide translation, translocation, and folding, to global changes in lipid composition. ER homeostasis thus requires complex interactions amongst numerous cellular components. However, describing the networks that maintain ER function during changes in cell behavior and environmental fluctuations has, to date, proven difficult. Here we perform a systems-level analysis of ER homeostasis, and find that although signaling networks that regulate ER function have a largely modular architecture, the TORC1-SREBP signaling axis is a central node that integrates signals emanating from different sub-networks. TORC1-SREBP promotes ER homeostasis by regulating phospholipid biosynthesis and driving changes in ER morphology. In particular, our network model shows TORC1-SREBP serves to integrate signals promoting growth and G1-S progression in order to maintain ER function during cell proliferation.     

Endoplasmic Reticulum Stress Pathway Required for Immune Homeostasis Is Neurally Controlled by Arrestin-1

In response to pathogen infection, the host innate immune system activates microbial killing pathways and cellular stress pathways that need to be balanced because insufficient or excessive immune responses have deleterious consequences. Recent studies demonstrate that two G protein-coupled receptors (GPCRs) in the nervous system of Caenorhabditis elegans control immune homeostasis. To investigate further how GPCR signaling controls immune homeostasis at the organismal level, we studied arrestin-1 (ARR-1), which is the only GPCR adaptor protein in C. elegans. The results indicate that ARR-1 is required for GPCR signaling in ASH, ASI, AQR, PQR, and URX neurons, which control the unfolded protein response and a p38 mitogen-activated protein kinase signaling pathway required for innate immunity. ARR-1 activity also controlled immunity through ADF chemosensory and AFD thermosensory neurons that regulate longevity. Furthermore, we found that although ARR-1 played a key role in the control of immunity by AFD thermosensory neurons, it did not control longevity through these cells. However, ARR-1 partially controlled longevity through ADF neurons.     

Re-evaluation of the Role of Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP) in Cellular Calcium Signaling

Changes in cytoplasmic Ca²⁺ concentration, resulting from activation of intracellular Ca²⁺ channels within the endoplasmic reticulum, regulate several aspects of cellular growth and differentiation. Ca²⁺ homeostasis endoplasmic reticulum protein (CHERP) is a ubiquitously expressed protein that has been proposed as a regulator of both major families of endoplasmic reticulum Ca²⁺ channels, inositol 1,4,5-trisphosphate receptors (IP₃Rs) and ryanodine receptors (RyRs), with resulting effects on mitotic cycling. However, the manner by which CHERP regulates intracellular Ca²⁺ channels to impact cellular growth is unknown. Here, we challenge previous findings that CHERP acts as a direct cytoplasmic regulator of IP₃Rs and RyRs and propose that CHERP acts in the nucleus to impact cellular proliferation by regulating the function of the U2 snRNA spliceosomal complex. The previously reported effects of CHERP on cellular growth therefore are likely indirect effects of altered spliceosomal function, consistent with prior data showing that loss of function of U2 snRNP components can interfere with cell growth and induce cell cycle arrest.     

Glycoprotein Folding in the Endoplasmic Reticulum

Our understanding of eukaryotic protein folding in the endoplasmic reticulum has increased enormously over the last 5 years. In this review, we summarize some of the major research themes that have captivated researchers in this field during the last years of the 20th century. We follow the path of a typical protein as it emerges from the ribosome and enters the reticular environment. While many of these events are shared between different polypeptide chains, we highlight some of the numerous differences between proteins, between cell types, and between the chaperones utilized by different ER glycopro-teins. Finally, we consider the likely advances in this field as the new century unfolds and we address the prospect of a unified understanding of how protein folding, degradation, and translation are coordinated within a cell.     

Phosphatase 2A Inhibition Affects Endoplasmic Reticulum and Mitochondria Homeostasis Via Cytoskeletal Alterations in Brain Endothelial Cells

The loss of endothelial cells (ECs) homeostasis is a trigger for cerebrovascular dysfunction that is a common event in several neurodegenerative disorders such as Alzheimer’s disease (AD). The present work addressed the role of phosphatase 2A (PP2A) in cytoskeleton rearrangement, endoplasmic reticulum (ER) homeostasis, ER–mitochondria communication and mitochondrial dynamics in brain ECs. For this purpose, rat brain endothelial (RBE4) cells were exposed to okadaic acid, a well-known inhibitor of PP2A activity. An increase in the levels of tau phosphorylated on Ser396 and Thr181 residues was observed upon PP2A inhibition, concomitantly with the rearrangement of microtubules and actin cytoskeleton. Under these conditions, an increase in the levels of ER stress markers, namely GRP78, XBP1, p-eIF2α^Ser51, and ERO1α, was observed. Moreover, PP2A inhibition upregulated the Sigma-1 receptor, an ER chaperone located at the ER–mitochondria interface, and enhanced inter-organelle Ca²⁺ transfer, culminating in mitochondrial Ca²⁺ overload and activation of mitochondria-dependent apoptosis. The inhibition of PP2A activity also promoted an alteration of the structural and spatial mitochondria network due to upregulation of mitochondrial fission (Drp1 and Fis1) and fusion (Mfn1, Mfn2 and OPA1) proteins, suggesting detrimental changes in mitochondrial dynamics. In accordance with our in vitro observations, brain vessels from 3xTg-AD mice showed a significant decrease in PP2A protein levels accompanied by an increase in tau phosphorylated on Ser396 and GRP78 protein levels. Collectively, these results suggest that the loss of cerebrovascular homeostasis that occurs in AD might be a downstream event of the compromised activity and/or expression of PP2A, which is observed in the brain of individuals affected with this devastating neurodegenerative disorder.     

TMTC1 and TMTC2 Are Novel Endoplasmic Reticulum Tetratricopeptide Repeat-containing Adapter Proteins Involved in Calcium Homeostasis

The endoplasmic reticulum (ER) is organized in part by adapter proteins that nucleate the formation of large protein complexes. Tetratricopeptide repeats (TPR) are well studied protein structural motifs that support intermolecular protein-protein interactions. TMTC1 and TMTC2 were identified by an in silico search as TPR-containing proteins possessing N-terminal ER targeting signal sequences and multiple hydrophobic segments, suggestive of polytopic membrane proteins that are targeted to the secretory pathway. A variety of cell biological and biochemical assays was employed to demonstrate that TMTC1 and TMTC2 are both ER resident integral membrane proteins with multiple clusters of TPR domains oriented within the ER lumen. Proteomic analysis followed by co-immunoprecipitation verification found that both proteins associated with the ER calcium uptake pump SERCA2B, and TMTC2 also bound to the carbohydrate-binding chaperone calnexin. Live cell calcium measurements revealed that overexpression of either TMTC1 or TMTC2 caused a reduction of calcium released from the ER following stimulation, whereas the knockdown of TMTC1 or TMTC2 increased the stimulated calcium released. Together, these results implicate TMTC1 and TMTC2 as ER proteins involved in ER calcium homeostasis.     

Psychostimulant-Induced Endoplasmic Reticulum Stress and Neurodegeneration

The endoplasmic reticulum (ER) is a subcellular organelle that ensures proper protein folding process. The ER stress is defined as cellular conditions that disturb the ER homeostasis, resulting in accumulation of unfolded and/or misfolded proteins in the lumen of the ER. The presence of these proteins within the ER activates the ER stress response, known as unfolded protein response (UPR), to restore normal functions of the ER. However, under the severe and/or prolonged ER stress, UPR initiates apoptotic cell death. Psychostimulants such as cocaine, amphetamine, and methamphetamine cause the ER stress and/or apoptotic cell death in regions of the brain related to drug addiction. Recent studies have shown that the ER stress in response to psychostimulants is linked to behavioral sensitization and that the psychostimulant-induced ER stress signaling cascades are closely associated with the pathogenesis of the neurodegenerative diseases. Therefore, this review was conducted to improve understanding of the functional role of the ER stress in the addiction as well as neurodegenerative diseases. This would be helpful to facilitate development of new therapeutic strategies for the drug addiction and/or neurodegenerative diseases caused or exacerbated by exposure to psychostimulants.     

内质网应激与肝脏疾病

内质网在细胞内分布广泛,是细胞内蛋白质、脂类和糖类合成的重要场所,是细胞内钙离子的储存场所,与物质运输、交换等作用密切相关。内质网稳态失衡会诱导内质网应激(Endoplasmic reticulum stress,ERS),持久应激会导致细胞凋亡。多项研究显示内质网应激与多种肝脏疾病密切相关。本文就内质网应激与肝脏疾病发病机制作一综述。     

细胞内质网应激与糖脂代谢紊乱的机制关联

在真核细胞中,内质网是蛋白质合成、折叠、加工及其质量监控的重要场所。当内质网难以承担蛋白折叠的高负荷时则引发内质网应激(ER stress),激活细胞的未折叠蛋白响应(unfoldedprotein response,UPR)。细胞通过内质网跨膜蛋白ATF6、PERK和IRE1介导的三条极为关键的UPR信号通路,调控下游相关基因的表达,以增强内质网对蛋白折叠的处理能力。因此,UPR通路在细胞的稳态平衡中具有举足轻重的作用,而这一动态过程的调控对于维持机体的正常生理功能至关重要。近来大量研究表明,在哺乳动物中内质网应激与机体的营养感应和糖脂代谢的调控过程密切相关。在肝脏、脂肪、胰岛以及下丘脑等不同的组织器官中,内质网应激均影响代谢通路的调节机制,因此在糖脂代谢紊乱的发生发展中扮演重要的角色。综上所述,进一步深入了解内质网应激引发代谢异常的生理学机制,可以为肥胖、脂肪肝及2型糖尿病等相关代谢性疾病的防治提供新的潜在药物靶点和重要的理论线索。     

Nonvesicular Lipid Transfer from the Endoplasmic Reticulum

The transport of lipids from their synthesis site at the endoplasmic reticulum (ER) to different target membranes could be mediated by both vesicular and nonvesicular transport mechanisms. Nonvesicular lipid transport appears to be the major transport route of certain lipid species, and could be mediated by either spontaneous lipid transport or by lipid-transfer proteins (LTPs). Although nonvesicular lipid transport has been extensively studied for more than four decades, its underlying mechanism, advantage and regulation, have not been fully explored. In particular, the function of LTPs and their involvement in intracellular lipid movement remain largely controversial. In this article, we describe the pathways by which lipids are synthesized at the ER and delivered to different cellular membranes, and discuss the role of LTPs in lipid transport both in vitro and in intact cells.The endoplasmic reticulum (ER) is a large interconnected membrane network that plays a major role in lipid biosynthesis in eukaryotic cells (Borgese et al. 2006). Newly synthesized lipids at the ER are then delivered to different cellular membranes or organelles, each of which shows unique lipid and protein composition and executes distinct cellular function (Holthuis et al. 2003). The transport of lipids from the ER can be mediated by both vesicular and nonvesicular transport mechanisms. Vesicular transport, as opposed to nonvesicular lipid transport, requires metabolic energy, intact cytoskeleton, and connection to the vesicular transport machinery (Kaplan and Simoni 1985a; Voelker 1990; Vance et al. 1991). Although vesicular lipid transport mediates the bulk transport of many lipids, increasing lines of evidence suggest that nonvesicular lipid transport is the major transport route for certain lipid types (Lev 2010). Nonvesicular lipid transport between membranes could be mediated by spontaneous lipid transport, in which a lipid monomer is diffused through the cytosol from a donor to the acceptor membrane. Given that most cellular lipids are highly hydrophobic, their diffusion through an aqueous phase is very slow and insufficient to support substantial transport of most lipids (Jones and Thompson 1989; Mesmin and Maxfield 2009). Nevertheless, spontaneous lipid transport can be greatly facilitated at membrane contact sites (MCSs) (Levine 2004; Holthuis and Levine 2005) and/or by lipid-transfer proteins (LTPs) (Lev 2010). MCSs are defined as small cytosolic gaps of 10–20 nm between the ER membranes and virtually all cellular organelles (Levine 2004; Lebiedzinska et al. 2009), whereas LTPs are intracellular proteins that can carry a lipid monomer in a hydrophobic pocket and transfer it between membranes through an aqueous phase. LTPs were initially discovered as soluble factors that accelerate the exchange or net transfer of different lipid species between membranes in vitro (Wirtz and Zilversmit 1968). Subsequently, many LTPs have been isolated, cloned, and crystallized. LTPs have been identified in all eukaryotes, in plants, and in bacteria, and according to their sequence and structure similarity have been subdivided into different protein families including SEC14, PITP (phosphatidylinositol-transfer protein), START (StAR-related lipid transfer), GLTP (glycolipid transfer protein), SCP-2 (nonspecific LTPs), and OSBP (oxysterol-binding protein)/ORP (OSBP-related proteins) (D’Angelo et al. 2008). In general, LTPs show specificity for one or more lipid types, and may contain only a single lipid-transfer domain (LTD), or additional structural domains with varying functions (Lev 2010). Over the past 40 years, LTPs have been extensively studied and the major principles of their action mode have been established from both biophysical measurements in vitro and structural data (Lev 2010). Nevertheless, the precise function of LTPs in intact cells remains controversial and a subject of an active field of research. In this article, we briefly describe how lipids are synthesized in the ER and delivered to different target membranes, and discuss how LTPs influence lipid transport in vitro and in intact cells.     

Lipid Transport between the Endoplasmic Reticulum and Mitochondria

Mitochondria are partially autonomous organelles that depend on the import of certain proteins and lipids to maintain cell survival and membrane formation. Although phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine are synthesized by mitochondrial enzymes, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and sterols need to be imported from other organelles. The origin of most lipids imported into mitochondria is the endoplasmic reticulum, which requires interaction of these two subcellular compartments. Recently, protein complexes that are involved in membrane contact between endoplasmic reticulum and mitochondria were identified, but their role in lipid transport is still unclear. In the present review, we describe components involved in lipid translocation between the endoplasmic reticulum and mitochondria and discuss functional as well as regulatory aspects that are important for lipid homeostasis.Biological membranes are major structural components of all cell types. They protect the cell from external influences, organize the interior in distinct compartments and allow balanced flux of components. Besides their specific proteome, organelles exhibit unique lipid compositions, which influence their shape, physical properties, and function. Major lipid classes found in biological membranes are phospholipids, sterols, and sphingolipids.The major “lipid factory” within the cell is the endoplasmic reticulum (ER). It is able to synthesize the bulk of structural phospholipids, sterols, and storage lipids such as triacylglycerols and steryl esters (van Meer et al. 2008). Furthermore, initial steps of ceramide synthesis occur in the ER providing precursors for the formation of complex sphingolipids in other organelles (Futerman 2006). Besides the export of ceramides, the ER supplies a large portion of lipids to other organelles, which cannot produce their own lipids or have a limited capacity to do so. Organelle interaction and transport of lipids require specific carrier proteins, membrane contact sites, tethering complexes, and/or vesicle flux. These processes are highly important for the maintenance of cell structure and survival but are still a matter of dispute. Most prominent organelle interaction partners are the ER and mitochondria. A subfraction of the ER named mitochondria-associated membrane (MAM) (Vance 1990) was described to be involved in lipid translocation to mitochondria. MAM is part of the ER network, which was shown to be in contact or close proximity to the outer mitochondrial membrane (OMM). Contact sites between MAM and mitochondria were assumed to facilitate exchange of components between the two compartments. Interestingly, MAM harbor a number of lipid synthesizing enzymes (Gaigg et al. 1994). Recently, molecular components governing membrane contact between the two organelles were identified (Dolman et al. 2005; Csordás et al. 2006; de Brito and Scorrano 2008; Kornmann et al. 2009; Friedman et al. 2010; Lavieu et al. 2010), although the specific role of these components in lipid translocation is not yet clear.     

内质网应激与阿尔茨海默病

内质网是蛋白质合成、修饰以及折叠的重要场所。内质网内未折叠蛋白堆积,钙离子失稳等可触发内质网应激,通过非折叠蛋白应答纠正这些异常变化。过度的内质网应激或内质网应激机制失常将导致细胞损害和死亡。近年来的研究发现阿尔茨海默病的神经系统损害与内质网应激异常有关。深入研究内质网应激将为进一步探索阿尔茨海默病发病机制和防治基础提供新的方向。