Protein quality control in the endoplasmic reticulum

Protein folding and quality control in the endoplasmic reticulum (ER) are synchronized mechanisms ensuring that only properly folded proteins are integrated in the plasma membrane or secreted from the cell. These mechanisms act in close collaboration with the molecular machinery involved in retrograde-translocation and degradation of non-native proteins and with the ER-stress activated signalling systems. The common goal of these mechanisms is to prevent expression and secretion of misfolded proteins. Protein misfolding can be detrimental to the cell and contributes to the disease mechanism in several inherited disorders, e.g. cystic fibrosis, familial hypercholesterolemia and diabetes insipidus. This review outlines the molecular mechanisms in protein quality control occurring in the ER, signalling caused by ER stress, and finally ER associated protein degradation.     

Protein quality control at the plasma membrane

Cellular proteostasis (or protein homeostasis) depends on the timely folding and disposal of conformationally damaged polypeptides during their life span at all subcellular locations. This process is particularly important for membrane proteins confined to the cell surface with crucial regulatory role in cellular homoeostasis and intercellular communication. Accumulating evidences indicate that membrane proteins exported from the endoplasmic reticulum (ER) are subjected to peripheral quality control (QC) along the late secretory and endocytic pathways, as well as at the plasma membrane (PM). Recently identified components of the PM QC recognition and effector mechanisms responsible for ubiquitination and lysosomal degradation of conformationally damaged PM proteins uncovered striking similarities to and differences from that of the ER QC machinery. Possible implications of the peripheral protein QC activity in phenotypic modulation of conformational diseases are also outlined.     

Pathogenesis of ER Storage Disorders: Modulating Russell Body Biogenesis by Altering Proximal and Distal Quality Control

In many protein storage diseases, detergent‐insoluble proteins accumulate in the early secretory compartment (ESC). Protein condensation reflects imbalances between entry into (synthesis/translocation) and exit from (secretion/degradation) ESC, and can be also a consequence of altered quality control (QC) mechanisms. Here we exploit the inducible formation of Russell bodies (RB), dilated ESC cisternae containing mutant Ig‐µ chains, as a model to mechanistically dissect protein condensation. Depending on the presence or absence of Ig‐L chains, mutant Ig‐µ chains lacking their first constant domain (Ch 1) accumulate in rough or smooth RB (rRB and sRB), dilations of the endoplasmic reticulum (ER) and ER‐Golgi intermediate compartment (ERGIC), respectively, reflecting the proximal and distal QC stations in the stepwise biogenesis of polymeric IgM. Either weakening ERp44‐dependent distal QC or facilitating ER‐associated degradation (ERAD) inhibits RB formation. Overexpression of PDI or ERp44 inhibits µΔCh 1 secretion. However, PDI inhibits while ERp44 promotes µΔCh 1 condensation. Both Ero1α silencing and overexpression prevent RB formation, demonstrating a strict redox dependency of the phenomenon. Altogether, our findings identify key controllers of protein condensation along the ESC as potential targets to handle certain storage disorders.     

Misfolded proteins are sorted by a sequential checkpoint mechanism of ER quality control

Misfolded proteins retained in the endoplasmic reticulum (ER) are degraded by the ER-associated degradation pathway. The mechanisms used to sort them from correctly folded proteins remain unclear. Analysis of substrates with defined folded and misfolded domains has revealed a system of sequential checkpoints that recognize topologically distinct domains of polypeptides. The first checkpoint examines the cytoplasmic domains of membrane proteins. If a lesion is detected, it is retained statically in the ER and rapidly degraded without regard to the state of its other domains. Proteins passing this test face a second checkpoint that monitors domains localized in the ER lumen. Proteins detected by this pathway are sorted from folded proteins and degraded by a quality control mechanism that requires ER-to-Golgi transport. Although the first checkpoint is obligatorily directed at membrane proteins, the second monitors both soluble and membrane proteins. Our data support a model whereby "properly folded" proteins are defined biologically as survivors that endure a series of distinct checkpoints.     

Protein folding and the order/disorder paradox

Most proteins encoded by the nuclear genome are synthesized in the cytoplasm and fold into precise 3D structures. During synthesis, the nascent polypeptide begins to fold as it traverses the large subunit of the ribosome and is assisted by molecular chaperones in attaining its precise folded/highly ordered state efficiently and in a biologically relevant timescale. Proteins that are misfolded are culled, re-routed, and marked by mechanisms such as ubiquitinylation for degradation ensuring strict quality control (QC). In addition to the highly ordered "globular" proteins, emerging evidence indicates that a large fraction of the proteome also comprises the so-called "Intrinsically Disordered Proteins" (IDPs). IDPs are proteins that lack rigid 3D structures and instead, exist as dynamic ensembles. The dynamic structures in the IDPs have many similarities with "normal" globular proteins such as the native (ordered), and non-native (molten globule, pre-molten globule, and coil-like) states seen during folding of "normal" globular proteins. However, unlike the case of the nascent globular proteins, IDPs evade being detected as "misfolded" and degraded by the cell's QC system. We refer to this paradox as the order/disorder paradox and postulate that the IDPs capitalize on their intrinsic promiscuity and ability to undergo disorder-to-order transitions upon binding to biological targets (coupled folding and binding) to escape the cell's surveillance machinery. Understanding the mechanism by which the IDPs evade the quality check has wide implications from protein folding to disease biology since the aggregation of misfolded proteins underlies several debilitating illnesses such as many neurodegenerative diseases and cancer.     

E3 Ubiquitin Ligases in Protein Quality Control Mechanism

In living cells, polypeptide chains emerging from ribosomes and preexisting polypeptide chains face constant threat of misfolding and aggregation. To prevent protein aggregation and to fulfill their biological activity, generally, protein must fold into its proper three-dimensional structure throughout their lifetimes. Eukaryotic cell possesses a quality control (QC) system to contend the problem of protein misfolding and aggregation. Cells achieve this functional QC system with the help of molecular chaperones and ubiquitin-proteasome system (UPS). The well-conserved UPS regulates the stability of various proteins and maintains all essential cellular function through intracellular protein degradation. E3 ubiquitin ligase enzyme determines specificity for degradation of certain substrates via UPS. New emerging evidences have provided considerable information that various E3 ubiquitin ligases play a major role in cellular QC mechanism and principally designated as QC E3 ubiquitin ligases. Nevertheless, very little is known about how E3 ubiquitin ligase maintains QC mechanism against abnormal proteins under various stress conditions. Here in this review, we highlight and discuss the functions of various E3 ubiquitin ligases implicated in protein QC mechanism. Improving our knowledge about such processes may provide opportunities to modulate protein QC mechanism in age-of-onset diseases that are caused by protein aggregation.     

未折叠蛋白质应答

内质网是真核细胞中蛋白质合成、折叠与分泌的重要细胞器.细胞进化出一套完整的机制来监督和帮助内质网内蛋白质的折叠与修饰.而当错误折叠的蛋白质累积时,细胞通过一系列信号转导途径,对其进行应答,包括增强蛋白质折叠能力、停滞大多数蛋白质的翻译、加速蛋白质的降解等.如果内质网功能素乱持续,细胞将最终启动凋亡程序.这些反应被统称为未折叠蛋白质应答(unfolded protein response,UPR).UPR是多个信号转导通路的总称,包括IRE1-XBP1、PERK-ATF4以及ATF6等信号途径.除了应激条件外,UPR还被用于正常生理条件下的调节,例如胆固醇合成代谢的负反馈调控.     

ER stress and diseases

Proteins synthesized in the endoplasmic reticulum (ER) are properly folded with the assistance of ER chaperones. Malfolded proteins are disposed of by ER-associated protein degradation (ERAD). When the amount of unfolded protein exceeds the folding capacity of the ER, human cells activate a defense mechanism called the ER stress response, which induces expression of ER chaperones and ERAD components and transiently attenuates protein synthesis to decrease the burden on the ER. It has been revealed that three independent response pathways separately regulate induction of the expression of chaperones, ERAD components, and translational attenuation. A malfunction of the ER stress response caused by aging, genetic mutations, or environmental factors can result in various diseases such as diabetes, inflammation, and neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and bipolar disorder, which are collectively known as 'conformational diseases'. In this review, I will summarize recent progress in this field. Molecules that regulate the ER stress response would be potential candidates for drug targets in various conformational diseases.     

Structures and functions of protein disulfide isomerase family members involved in proteostasis in the endoplasmic reticulum

The endoplasmic reticulum (ER) is an essential cellular compartment in which an enormous number of secretory and cell surface membrane proteins are synthesized and subjected to cotranslational or posttranslational modifications, such as glycosylation and disulfide bond formation. Proper maintenance of ER protein homeostasis (sometimes termed proteostasis) is essential to avoid cellular stresses and diseases caused by abnormal proteins. Accumulating knowledge of cysteine-based redox reactions catalyzed by members of the protein disulfide isomerase (PDI) family has revealed that these enzymes play pivotal roles in productive protein folding accompanied by disulfide formation, as well as efficient ER-associated degradation accompanied by disulfide reduction. Each of PDI family members forms a protein–protein interaction with a preferential partner to fulfill a distinct function. Multiple redox pathways that utilize PDIs appear to function synergistically to attain the highest quality and productivity of the ER, even under various stress conditions. This review describes the structures, physiological functions, and cooperative actions of several essential PDIs, and provides important insights into the elaborate proteostatic mechanisms that have evolved in the extremely active and stress-sensitive ER.     

Endoplasmic reticulum dysfunction – a common denominator for cell injury in acute and degenerative diseases of the brain?

Various physiological, biochemical and molecular biological disturbances have been put forward as mediators of neuronal cell injury in acute and chronic pathological states of the brain such as ischemia, epileptic seizures and Alzheimer's or Parkinson's disease. These include over-activation of glutamate receptors, a rise in cytoplasmic calcium activity and mitochondrial dysfunction. The possible involvement of the endoplasmic reticulum (ER) dysfunction in this process has been largely neglected until recently, although the ER plays a central role in important cell functions. Not only is the ER involved in the control of cellular calcium homeostasis, it is also the subcellular compartment in which the folding and processing of membrane and secretory proteins takes place. The fact that blocking of these processes is sufficient to cause cell damage indicates that they are crucial for normal cell functioning. This review presents evidence that ER function is disturbed in many acute and chronic diseases of the brain. The complex processes taken place in this subcellular compartment are however, affected in different ways in various disorders; whereas the ER-associated degradation of misfolded proteins is affected in Parkinson's disease, it is the unfolded protein response which is down-regulated in Alzheimer's disease and the ER calcium homeostasis that is disturbed in ischemia. Studying the consequences of the observed deteriorations of ER function and identifying the mechanisms causing ER dysfunction in these pathological states of the brain will help to elucidate whether neurodegeneration is indeed caused by these disturbances, and will help to facilitate the search for drugs capable of blocking the pathological process directly at an early stage.     

自噬与泛素化蛋白降解途径的分子机制及其功能

细胞内所有的蛋白质和大多数的细胞外蛋白都在不断的进行更新,即它们在不断地被降解,并被新合成的蛋白质取代。细胞内蛋白的降解主要通过两个途径,即自噬和泛素蛋白酶体系统。自噬是一种由溶酶体介导的细胞内过多或异常蛋白质的降解机制。在细胞内主要有3种类型的自噬,即分子伴侣介导的自噬、微自噬和巨自噬。泛素蛋白酶体系统是由泛素介导的一种高度复杂的蛋白降解机制,它参与降解细胞内许多蛋白质并且这个过程具有高度特异性。细胞内蛋白质的降解参与调节许多细胞过程,包括细胞周期、DNA修复、细胞生长和分化、细胞质量的控制、病原生物的感染反应和细胞凋亡等。许多严重的人类疾病被认为是由于蛋白质降解系统的紊乱而引起的。文章综述了自噬和泛素化途径及其分子机制,以及蛋白质降解系统紊乱的病理学意义。     

Herp enhances ER-associated protein degradation by recruiting ubiquilins

ER-associated protein degradation (ERAD) is a protein quality control system of ER, which eliminates misfolded proteins by proteasome-dependent degradation and ensures export of only properly folded proteins from ER. Herp, an ER membrane protein upregulated by ER stress, is implicated in regulation of ERAD. In the present study, we show that Herp interacts with members of the ubiquilin family, which function as a shuttle factor to deliver ubiquitinated substrates to the proteasome for degradation. Knockdown of ubiquilin expression by small interfering RNA stabilized the ERAD substrate CD3δ, whereas it did not alter or increased degradation of non-ERAD substrates tested. CD3δ was stabilized by overexpressed Herp mutants which were capable of binding to ubiquilins but were impaired in ER membrane targeting by deletion of the transmembrane domain. Our data suggest that Herp binding to ubiquilin proteins plays an important role in the ERAD pathway and that ubiquilins are specifically involved in degradation of only a subset of ubiquitinated targets, including Herp-dependent ERAD substrates.     

ER stress and its functional link to mitochondria: role in cell survival and death

The endoplasmic reticulum (ER) is the primary site for synthesis and folding of secreted and membrane-bound proteins. Proteins are translocated into ER lumen in an unfolded state and require protein chaperones and catalysts of protein folding to assist in proper folding. Properly folded proteins traffic from the ER to the Golgi apparatus; misfolded proteins are targeted to degradation. Unfolded protein response (UPR) is a highly regulated intracellular signaling pathway that prevents accumulation of misfolded proteins in the ER lumen. UPR provides an adaptive mechanism by which cells can augment protein folding and processing capacities of the ER. If protein misfolding is not resolved, the UPR triggers apoptotic cascades. Although the molecular mechanisms underlying ER stress-induced apoptosis are not completely understood, increasing evidence suggests that ER and mitochondria cooperate to signal cell death. Mitochondria and ER form structural and functional networks (mitochondria-associated ER membranes [MAMs]) essential to maintain cellular homeostasis and determine cell fate under various pathophysiological conditions. Regulated Ca(2+) transfer from the ER to the mitochondria is important in maintaining control of prosurvival/prodeath pathways. We discuss the signaling/communication between the ER and mitochondria and focus on the role of the mitochondrial permeability transition pore in these complex processes.     

Cellular responses to endoplasmic reticulum stress and apoptosis

The endoplasmic reticulum (ER) is the cell organelle where secretory and membrane proteins are synthesized and folded. Correctly folded proteins exit the ER and are transported to the Golgi and other destinations within the cell, but proteins that fail to fold properly—misfolded proteins—are retained in the ER and their accumulation may constitute a form of stress to the cell—ER stress. Several signaling pathways, collectively known as unfolded protein response (UPR), have evolved to detect the accumulation of misfolded proteins in the ER and activate a cellular response that attempts to maintain homeostasis and a normal flux of proteins in the ER. In certain severe situations of ER stress, however, the protective mechanisms activated by the UPR are not sufficient to restore normal ER function and cells die by apoptosis. Most research on the UPR used yeast or mammalian model systems and only recently Drosophila has emerged as a system to study the molecular and cellular mechanisms of the UPR. Here, we review recent advances in Drosophila UPR research, in the broad context of mammalian and yeast literature.     

内质网相关蛋白的降解及其机制

内质网相关蛋白降解(ER-associated protein degradation,或ER-associated degradation,ERAD)是真核细胞蛋白质质量控制的重要途径,它承担着对错误折叠蛋白的鉴别、分检和降解,清除无功能蛋白在细胞内的积累。ERAD过程包括错误折叠蛋白质的识别、蛋白质从ER向细胞基质逆向转运和蛋白质在细胞基质中的降解三个步骤。ERAD与人类的某些疾病密切相关,有些病毒能巧妙利用ERAD逃遁宿主免疫监控和攻击。     

A versatile selection system for folding competent proteins using genetic complementation in a eukaryotic host

Recombinant expression of native or modified eukaryotic proteins is pivotal for structural and functional studies and for industrial and pharmaceutical production of proteins. However, it is often impeded by the lack of proper folding. Here, we present a stringent and broadly applicable eukaryotic in vivo selection system for folded proteins. It is based on genetic complementation of the Schizosaccharomyces pombe growth marker gene invertase fused C‐terminally to a protein library. The fusion proteins are directed to the secretion system, utilizing the ability of the eukaryotic protein quality‐control systems to retain misfolded proteins in the ER and redirect them for cytosolic degradation, thereby only allowing folded proteins to reach the cell surface. Accordingly, the folding potential of the tested protein determines the ability of autotrophic colony growth. This system was successfully demonstrated using a complex insertion mutant library of TNF‐α, from which different folding competent mutant proteins were uncovered.     

ER signaling in unfolded protein response

Abnormally folded proteins are susceptible to aggregation and accumulation in cells, ultimately leading to cell death. To protect cells against such dangers, expression of various genes including molecular chaperones can be induced and ER-associated protein degradation (ERAD) activated in response to the accumulation of unfolded protein in the endoplasmic reticulum (ER). This is known as the unfolded protein response (UPR). ERAD requires retrograde transport of unfolded proteins from the ER back to the cytosol via the translocon for degradation by the ubiquitin-proteasome system. Hrd1p is a UPR-induced ER membrane protein that acts as a ubiquitin ligase (E3) in the ERAD system. Hrd3p interacts with and stabilizes Hrd1p. We have isolated and identified human homologs (HRD1 and SEL1/HRD3) of Saccharomyces cerevisiae Hrd1p and Hrd3p. Human HRD1 and SEL1 were up-regulated in response to ER stress and overexpression of human IRE1 and ATF6, which are ER stress-sensor molecules in the ER. HEK293T cells overexpressing HRD1 showed resistance to ER stress-induced cell death. These results suggest that HRD1 and SEL1 are up-regulated by the UPR and contribute to protection against the ER stress-induced cell death by degrading unfolded proteins accumulated in the ER.