Genetic disruption of mammalian endoplasmic reticulum-associated protein degradation: Human phenotypes and animal and cellular disease models

Endoplasmic reticulum-associated protein degradation (ERAD) is a stringent quality control mechanism through which misfolded, unassembled and some native proteins are targeted for degradation to maintain appropriate cellular and organelle homeostasis. Several in vitro and in vivo ERAD-related studies have provided mechanistic insights into ERAD pathway activation and its consequent events; however, a majority of these have investigated the effect of ERAD substrates and their consequent diseases affecting the degradation process. In this review, we present all reported human single-gene disorders caused by genetic variation in genes that encode ERAD components rather than their substrates. Additionally, after extensive literature survey, we present various genetically manipulated higher cellular and mammalian animal models that lack specific components involved in various stages of the ERAD pathway.     

GPI锚定蛋白前体C-端附着信号的疏水性决定其内质网相关蛋白质降解途径

目前,已知超过150种糖基磷脂酰肌醇锚定蛋白(glycosylphosphatidylinositol anchored protein, GPI-anchored protein)在哺乳动物细胞中表达,并参与免疫识别、细胞通讯与信号转导等多种生理过程。当蛋白质无法被GPI修饰时,前体蛋白质通过内质网相关蛋白质降解(endoplasmic reticulum associated degradation, ERAD)途径降解。然而,GPI锚定蛋白ERAD的降解机制尚不清楚。为了探究GPI锚定蛋白前体的ERAD途径的具体机制,本研究敲除人胚胎肾细胞293细胞株(HEK293)的GPI转酰胺酶复合物亚基PIGS基因,进而敲除E3泛素连接酶HRD1和GP78基因,之后随机在PIGS-KO,PIGS-HRD1-KO和PIGS-GP78-KO过表达16种GPI锚定蛋白质(以亲本PIGS-KO细胞株作为对照组),Western印迹结果证明,GPI锚定蛋白前体在细胞株PIGS-HRD1-KO中的蛋白质积累量(I_PHK)和PIGS-GP78-KO中的蛋白质积累量(I_PG...     

Export of a cysteine-free misfolded secretory protein from the endoplasmic reticulum for degradation requires interaction with protein disulfide isomerase

Protein disulfide isomerase (PDI) interacts with secretory proteins, irrespective of their thiol content, late during translocation into the ER; thus, PDI may be part of the quality control machinery in the ER. We used yeast pdi1 mutants with deletions in the putative peptide binding region of the molecule to investigate its role in the recognition of misfolded secretory proteins in the ER and their export to the cytosol for degradation. Our pdi1 deletion mutants are deficient in the export of a misfolded cysteine-free secretory protein across the ER membrane to the cytosol for degradation, but ER-to-Golgi complex transport of properly folded secretory proteins is only marginally affected. We demonstrate by chemical cross-linking that PDI specifically interacts with the misfolded secretory protein and that mutant forms of PDI have a lower affinity for this protein. In the ER of the pdi1 mutants, a higher proportion of the misfolded secretory protein remains associated with BiP, and in export-deficient sec61 mutants, the misfolded secretory protein remain bounds to PDI. We conclude that the chaperone PDI is part of the quality control machinery in the ER that recognizes terminally misfolded secretory proteins and targets them to the export channel in the ER membrane.     

Regulation of nicotinic receptor expression by the ubiquitin-proteasome system

Control of ligand-gated ion channel (LGIC) expression is essential for the formation, maintenance and plasticity of synapses. Treatment of mouse myotubes with proteasome inhibitors increased the number of surface nicotinic acetylcholine receptors (AChRs), indicating LGIC expression is regulated by the ubiquitin-proteasome system (UPS). Elevated surface expression resulted from increased AChR delivery to the plasma membrane and not from decreased turnover from the surface. The rise in AChR trafficking was the direct result of increased assembly of subunits in the endoplasmic reticulum (ER). Because proteasome inhibitors also blocked ER-associated degradation (ERAD) of unassembled AChR subunits, the data indicate that the additional AChRs were assembled from subunits normally targeted for ERAD. Our data show that AChR surface expression is regulated by the UPS through ERAD, whose activity determines oligomeric receptor assembly efficiency.     

An in vitro assay for the selective endoplasmic reticulum associated degradation of an unglycosylated secreted protein

The endoplasmic reticulum (ER) represents the first compartment into which nascent secreted proteins traffic, and not coincidentally the ER lumen houses a high concentration of factors that facilitate protein folding, such as molecular chaperones. To off-set the potentially lethal consequences of mis-folded secreted protein accumulation, aberrant proteins may be selected for degradation via a process known as ER associated degradation (ERAD). After their selection ERAD substrates are retro-translocated back to the cytoplasm and then degraded by the 26S proteasome. Key features of the selection, retro-translocation, and degradation steps that constitute the ERAD pathway were elucidated through the development of an in vitro ERAD assay. In this assay the fates of two yeast proteins can be distinguished after their translocation, or import into ER-derived microsomes. Whereas a wild type, glycosylated protein ("Gp(alpha)F") is stable, a non-glycosylated version of the same protein ("p(alpha)F") is rapidly degraded when microsomes containing radiolabeled forms of these substrates are incubated in cytosol and ATP. The purpose of this chapter is first to discuss the experimental findings from the use of the in vitro assay, and then to describe the assay in detail. Finally, future potential uses of the in vitro system are illustrated.     

Quality control system of the endoplasmic reticulum and related diseases

The quality control (QC) system of the endoplasmic reticulum (ER) is an important monitoringmechanism in the protein maturation process,which ensures export of properly folded proteins from the ER.Incorrectly or incompletely folded proteins are retained in the ER for refolding or degradation by the ER-residing proteasome.The calnexin/calreticulin cycle and ER-associated degradation are the key elements inQC.These two mechanisms work together to allow incorrectly folded proteins have additional opportunitiesto achieve their native conformations.The QC dysfunction is involved in many diseases caused by mutantproteins,many of which are causes of neurodegenerative disorders.A better understanding of molecularregulation in the QC system will uncover the molecular pathogenic mechanisms of many diseases caused byprotein misfolding and help discover novel strategies for preventing or treating these diseases.     

Rescuing Trafficking Mutants of the ATP-binding Cassette Protein,ABCA4, with Small Molecule Correctors as a Treatment for Stargardt Eye Disease

Stargardt disease is the most common form of early onset macular degeneration. Mutations in ABCA4, a member of the ATP-binding cassette (ABC) family, are associated with Stargardt disease. Here, we have examined two disease-causing mutations in the NBD1 region of ABCA4, R1108C, and R1129C, which occur within regions of high similarity with CFTR, another ABC transporter gene, which is associated with cystic fibrosis. We show that R1108C and R1129C are both temperature-sensitive processing mutants that engage the cellular quality control mechanism and show a strong interaction with the chaperone Hsp 27. Both mutant proteins also interact with HDCAC6 and are degraded in the aggresome. We also demonstrate that novel corrector compounds that are being tested as treatment for cystic fibrosis, such as VX-809, can rescue the processing of the ABCA4 mutants, particularly their expression at the cell surface, and can reduce their binding to HDAC6. Thus, our data suggest that VX-809 can potentially be developed as a new therapy for Stargardt disease, for which there is currently no treatment.     

Protein dislocation from the endoplasmic reticulum--pulling out the suspect

Proteins that fail to fold properly as well as constitutive or regulated short-lived proteins of the endoplasmic reticulum are subjected to proteolysis by cytosolic 26S proteasomes. This process is known as endoplasmic reticulum-associated protein degradation. In order to become accessible to the proteasome of this system substrates must first be retrogradely transported from the endoplasmic reticulum into the cytosol, in a process termed dislocation. This export step seems to be accompanied by polyubiquitination of such molecules. Surprisingly, protein dislocation from the endoplasmic reticulum seems to require at least some components that mediate import into this compartment. However, protein import and export display differences in the mechanism that provides the driving force and ensures directionality. Of special interest is the cytoplasmic Cdc48p/Npl4p/Ufd1p complex, which is required for the degradation of various endoplasmic reticulum-associated protein degradation substrates and seems to function in a step after polyubiquitination but before proteasomal digestion. In this review, we will summarize our knowledge on protein export during endoplasmic reticulum-associated protein degradation and discuss the possible function of certain components involved in this process.     

Order through destruction: how ER‐associated protein degradation contributes to organelle homeostasis

The endoplasmic reticulum (ER) is a large, dynamic, and multifunctional organelle. ER protein homeostasis is essential for the coordination of its diverse functions and depends on ER‐associated protein degradation (ERAD). The latter process selects target proteins in the lumen and membrane of the ER, promotes their ubiquitination, and facilitates their delivery into the cytosol for degradation by the proteasome. Originally characterized for a role in the degradation of misfolded proteins and rate‐limiting enzymes of sterol biosynthesis, the many branches of ERAD now appear to control the levels of a wider range of substrates and influence more broadly the organization and functions of the ER, as well as its interactions with adjacent organelles. Here, we discuss recent mechanistic advances in our understanding of ERAD and of its consequences for the regulation of ER functions.     

Protein disulfide isomerase, but not binding protein, overexpression enhances secretion of a non-disulfide-bonded protein in yeast

In eukaryotes, secretory proteins are folded and assembled in the endoplasmic reticulum (ER). Many heterologous proteins are retained in the ER due to suboptimal folding conditions. We previously reported that heterologous secretion of Pyrococcus furiosus beta-glucosidase in Saccharomyces cerevisiae resulted in the accumulation of a large fraction of inactive beta-glucosidase in the ER. In this work, we determine the effect of introducing additional genes of ER-resident yeast proteins, Kar2p (binding protein [BiP]) and protein disulfide isomerase (PDI), on relieving this bottleneck. Single-copy expression of BiP and PDI worked synergistically to improve secretion by reverse similar 60%. In an effort to optimize BiP and PDI interactions, we created a library of beta-glucosidase expression strains that incorporated four combinations of constitutively or inducibly-expressed BiP and PDI genes integrated to random gene copynumbers in the yeast chromosome. Approximately 15% of the transformants screened had secretion level improvements higher than that seen with single BiP/PDI gene overexpression, and the highest secreting strain had threefold higher beta-glucosidase levels than the control. Nineteen of the improved strains were re-examined for beta-glucosidase secretion as well as BiP and PDI levels. Within the improved transformants BiP and PDI levels ranged sevenfold and tenfold over the control, respectively. Interestingly, increasing BiP levels decreased beta-glucosidase secretion, whereas increasing PDI levels increased beta-glucosidase secretion. The action of PDI was unexpected because beta-glucosidase is not a disulfide-bonded protein. We suggest that PDI may be acting in a chaperone-like capacity or possibly creating mixed disulfides with the beta-glucosidase's lone cysteine residue during the folding and assembly process.     

Pushing and pulling proteins into the yeast secretory pathway enhances recombinant protein secretion

Yeasts and especially Pichia pastoris (syn Komagataella spp.) are popular microbial expression systems for the production of recombinant proteins. One of the key advantages of yeast host systems is their ability to secrete the recombinant protein into the culture media. However, secretion of some recombinant proteins is less efficient. These proteins include antibody fragments such as Fabs or scFvs. We have recently identified translocation of nascent Fab fragments from the cytosol into the endoplasmic reticulum (ER) as one major bottleneck. Conceptually, this bottleneck requires engineering to increase the flux of recombinant proteins at the translocation step by pushing on the cytosolic side and pulling on the ER side. This engineering strategy is well-known in the field of metabolic engineering. To apply the push-and-pull strategy to recombinant protein secretion, we chose to modulate the cytosolic and ER Hsp70 cycles, which have a key impact on the translocation process. After identifying the relevant candidate factors of the Hsp70 cycles, we combined the push-and-pull factors in a single strain and achieved synergistic effects for antibody fragment secretion. With this concept we were able to successfully engineer strains and improve protein secretion up to 5-fold for different model protein classes. Overall, titers of more than 1.3 g/L Fab and scFv were reached in bioreactor cultivations.     

Protein disulphide isomerase is required for signal peptide peptidase-mediated protein degradation

The human cytomegalovirus glycoprotein US2 induces dislocation of MHC class I heavy chains from the endoplasmic reticulum (ER) into the cytosol and targets them for proteasomal degradation. Signal peptide peptidase (SPP) has been shown to be integral for US2-induced dislocation of MHC class I heavy chains although its mechanism of action remains poorly understood. Here, we show that knockdown of protein disulphide isomerase (PDI) by RNA-mediated interference inhibited the degradation of MHC class I molecules catalysed by US2 but not by its functional homolog US11. Overexpression of the substrate-binding mutant of PDI, but not the catalytically inactive mutant, dominant-negatively inhibited US2-mediated dislocation of MHC class I molecules by preventing their release from US2. Furthermore, PDI associated with SPP independently of US2 and knockdown of PDI inhibited SPP-mediated degradation of CD3δ but not Derlin-1-dependent degradation of CFTR DeltaF508. Together, our data suggest that PDI is a component of the SPP-mediated ER-associated degradation machinery.     

Dissection of Structural and Functional Requirements That Underlie the Interaction of ERdj3 Protein with Substrates in the Endoplasmic Reticulum

ERdj3, a mammalian endoplasmic reticulum (ER) Hsp40/DnaJ family member, binds unfolded proteins, transfers them to BiP, and concomitantly stimulates BiP ATPase activity. However, the requirements for ERdj3 binding to and release from substrates in cells are not well understood. We found that ERdj3 homodimers that cannot stimulate the ATPase activity of BiP (QPD mutants) bound to unfolded ER proteins under steady state conditions in much greater amounts than wild-type ERdj3. This was due to reduced release from these substrates as opposed to enhanced binding, although in both cases dimerization was strictly required for substrate binding. Conversely, heterodimers consisting of one wild-type and one mutant ERdj3 subunit bound substrates at levels comparable with wild-type ERdj3 homodimers, demonstrating that release requires only one protomer to be functional in stimulating BiP ATPase activity. Co-expressing wild-type ERdj3 and a QPD mutant, which each exclusively formed homodimers, revealed that the release rate of wild-type ERdj3 varied according to the relative half-lives of substrates, suggesting that ERdj3 release is an important step in degradation of unfolded client proteins in the ER. Furthermore, pulse-chase experiments revealed that the binding of QPD mutant homodimers remained constant as opposed to increasing, suggesting that ERdj3 does not normally undergo reiterative binding cycles with substrates.     

Search and Destroy: ER Quality Control and ER-Associated Protein Degradation

Abstract

Proteins synthesized in the endoplasmic reticulum (ER) encounter quality control checkpoints that verify their fitness to proceed in the secretory pathway. Molecules undergoing folding and assembly are kept out of the exocytic pathway until maturation is complete. Misfolded side products that inevitably form are removed from the mixture of conformers and returned to the cytosol for degradation. How unfolded proteins are recognized and how irreversibly misfolded proteins are sorted to ER-associated degradation pathways was poorly understood. Recent developments from a combination of genetic and biochemical analyses has revealed new insights into these mechanisms. The emerging view shows distinct pathways working in collaboration to filter the diverse range of unfolded proteins from the transport flow and to divert misfolded molecules for destruction.     

Rkr1/Ltn1 Ubiquitin Ligase-mediated Degradation of Translationally Stalled Endoplasmic Reticulum Proteins

Aberrant nonstop proteins arise from translation of mRNA molecules beyond the coding sequence into the 3′-untranslated region. If a stop codon is not encountered, translation continues into the poly(A) tail, resulting in C-terminal appendage of a polylysine tract and a terminally stalled ribosome. In Saccharomyces cerevisiae, the ubiquitin ligase Rkr1/Ltn1 has been implicated in the proteasomal degradation of soluble cytosolic nonstop and translationally stalled proteins. Rkr1 is essential for cellular fitness under conditions associated with increased prevalence of nonstop proteins. Mutation of the mammalian homolog causes significant neurological pathology, suggesting broad physiological significance of ribosome-associated quality control. It is not known whether and how soluble or transmembrane nonstop and translationally stalled proteins targeted to the endoplasmic reticulum (ER) are detected and degraded. We generated and characterized model soluble and transmembrane ER-targeted nonstop and translationally stalled proteins. We found that these proteins are indeed subject to proteasomal degradation. We tested three candidate ubiquitin ligases (Rkr1 and ER-associated Doa10 and Hrd1) for roles in regulating abundance of these proteins. Our results indicate that Rkr1 plays the primary role in targeting the tested model ER-targeted nonstop and translationally stalled proteins for degradation. These data expand the catalog of Rkr1 substrates and highlight a previously unappreciated role for this ubiquitin ligase at the ER membrane.     

A biosensor of protein foldedness identifies increased “holdase” activity of chaperones in the nucleus following increased cytosolic protein aggregation

Chaperones and other quality control machinery guard proteins from inappropriate aggregation, which is a hallmark of neurodegenerative diseases. However, how the systems that regulate the “foldedness” of the proteome remain buffered under stress conditions and in different cellular compartments remains incompletely understood. In this study, we applied a FRET-based strategy to explore how well quality control machinery protects against the misfolding and aggregation of “bait” biosensor proteins, made from the prokaryotic ribonuclease barnase, in the nucleus and cytosol of human embryonic kidney 293T cells. We found that those barnase biosensors were prone to misfolding, were less engaged by quality control machinery, and more prone to inappropriate aggregation in the nucleus as compared with the cytosol, and that these effects could be regulated by chaperone Hsp70-related machinery. Furthermore, aggregation of mutant huntingtin exon 1 protein (Httex1) in the cytosol appeared to outcompete and thus prevented the engagement of quality control machinery with the biosensor in the cytosol. This effect correlated with reduced levels of DNAJB1 and HSPA1A chaperones in the cell outside those sequestered to the aggregates, particularly in the nucleus. Unexpectedly, we found Httex1 aggregation also increased the apparent engagement of the barnase biosensor with quality control machinery in the nucleus suggesting an independent implementation of “holdase” activity of chaperones other than DNAJB1 and HSPA1A. Collectively, these results suggest that proteostasis stress can trigger a rebalancing of chaperone abundance in different subcellular compartments through a dynamic network involving different chaperone–client interactions.     

Ubiquitin-proteasome system and ER stress in the brain of diabetic rats

The ubiquitin-proteasome system (UPS) has been implicated in the pathogenesis of many neurodegenerative diseases. Endoplasmic reticulum (ER) stress is shown to play a pathological role in the development of diabetes and its complications. Hence, the current study is aimed to investigate the role of UPS and ER stress in the cerebral cortex of diabetic rats and examine the therapeutic effect of 4-phenylbutyric acid (4-PBA), an ER stress inhibitor. Male Sprague-Dawley rats were divided into three groups: control, diabetes, and diabetes plus 4-PBA treatment group. Diabetes was induced by single intraperitoneal streptozotocin injection (37 mg/kg body weight [bw]), and 4-PBA was administered (40 mg/kg bw/d, intraperitoneal) for 2 months, starting from 2 months of diabetes induction. At the end of 4 months, cerebral cortex was collected for analysis. Declined proteasome activity and ubiquitin C-terminal hydrolase (UCH)-L1 expression, increased ubiquitinated proteins, and apoptosis were observed in the diabetic rats. The expression of the ubiquitin-activating enzyme E1, UCHL5, and ER stress markers (ATF6, pPERK, and CHOP) was markedly elevated, whereas the expression of ER-associated protein degradation (ERAD) components was downregulated in the diabetic rats. 4-PBA intervention attenuated ER stress, alterations in UPS, and ERAD components in diabetic rats. Importantly, neuronal apoptosis was lowered in 4-PBA-treated diabetic rats. Our observations demonstrate that altered UPS could be one of the underlying mechanisms of neuronal apoptosis in diabetes and chemical chaperones such as 4-PBA could be potential candidates for preventing these alterations under hyperglycemic conditions.     

OS9 Protein Interacts with Na-K-2Cl Co-transporter (NKCC2) and Targets Its Immature Form for the Endoplasmic Reticulum-associated Degradation Pathway

Mutations in the renal specific Na-K-2Cl co-transporter (NKCC2) lead to type I Bartter syndrome, a life-threatening kidney disease featuring arterial hypotension along with electrolyte abnormalities. We have previously shown that NKCC2 and its disease-causing mutants are subject to regulation by endoplasmic reticulum-associated degradation (ERAD). The aim of the present study was to identify the protein partners specifically involved in ERAD of NKCC2. To this end, we screened a kidney cDNA library through a yeast two-hybrid assay using NKCC2 C terminus as bait. We identified OS9 (amplified in osteosarcomas) as a novel and specific binding partner of NKCC2. Co-immunoprecipitation assays in renal cells revealed that OS9 association involves mainly the immature form of NKCC2. Accordingly, immunocytochemistry analysis showed that NKCC2 and OS9 co-localize at the endoplasmic reticulum. In cells overexpressing OS9, total cellular NKCC2 protein levels were markedly decreased, an effect blocked by the proteasome inhibitor MG132. Pulse-chase and cycloheximide-chase assays demonstrated that the marked reduction in the co-transporter protein levels was essentially due to increased protein degradation of the immature form of NKCC2. Conversely, knockdown of OS9 by small interfering RNA increased NKCC2 expression by increasing the co-transporter stability. Inactivation of the mannose 6-phosphate receptor homology domain of OS9 had no effect on its action on NKCC2. In contrast, mutations of NKCC2 N-glycosylation sites abolished the effects of OS9, indicating that OS9-induced protein degradation is N-glycan-dependent. In summary, our results demonstrate the presence of an OS9-mediated ERAD pathway in renal cells that degrades immature NKCC2 proteins. The identification and selective modulation of ERAD components specific to NKCC2 and its disease-causing mutants might provide novel therapeutic strategies for the treatment of type I Bartter syndrome.     

An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates

The 26S proteasome is the molecular machine at the center of the ubiquitin proteasome system and is responsible for adjusting the concentrations of many cellular proteins. It is a drug target in several human diseases, and assays for the characterization of modulators of its activity are valuable. The 26S proteasome consists of two components: a core particle, which contains the proteolytic sites, and regulatory caps, which contain substrate receptors and substrate processing enzymes, including six ATPases. Current high-throughput assays of proteasome activity use synthetic fluorogenic peptide substrates that report directly on the proteolytic activity of the proteasome, but not on the activities of the proteasome caps that are responsible for protein recognition and unfolding. Here, we describe a simple and robust assay for the activity of the entire 26S proteasome using fluorescence anisotropy to follow the degradation of fluorescently labeled protein substrates. We describe two implementations of the assay in a high-throughput format and show that it meets the expected requirement of ATP hydrolysis and the presence of a canonical degradation signal or degron in the target protein.