内质网压力响应相关的蛋白质降解

内质网相关蛋白质降解途径(ERAD),即蛋白质分泌过程中错误折叠或未折叠的蛋白质在内质网中被识别并逆向运输到细胞质经聚泛素化后由蛋白酶体降解的过程.自从发现该途径后对其机制的阐明一直处于不断探索的阶段.近年来,对ERAD底物识别、逆向运输和泛素化新组分的发现以及新技术的应用,使得该途径的具体分子机制更加清晰.本文全面梳理并综述了内质网应激响应、ERAD降解过程与机理的最新进展,并对模式蛋白底物和最新研究方法进行了总结,以期展示该领域的研究概况.     

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.     

Sec61p is part of the endoplasmic reticulum‐associated degradation machinery

Endoplasmic reticulum‐associated degradation (ERAD) is a cellular pathway for the disposal of misfolded secretory proteins. This process comprises recognition of the misfolded proteins followed by their retro‐translocation across the ER membrane into the cytosol in which polyubiquitination and proteasomal degradation occur. A variety of data imply that the protein import channel Sec61p has a function in the ERAD process. Until now, no physical interactions between Sec61p and other essential components of the ERAD pathway could be found. Here, we establish this link by showing that Hrd3p, which is part of the Hrd‐Der ubiquitin ligase complex, and other core components of the ERAD machinery physically interact with Sec61p. In addition, we study binding of misfolded CPY^* proteins to Sec61p during the process of degradation. We show that interaction with Sec61p is maintained until the misfolded proteins are ubiquitinated on the cytosolic side of the ER. Our observations suggest that Sec61p contacts an ERAD ligase complex for further elimination of ER lumenal misfolded proteins.     

Dfm1 Forms Distinct Complexes with Cdc48 and the ER Ubiquitin Ligases and Is Required for ERAD

Proteins imported into the endoplasmic reticulum (ER) are scanned for their folding status. Those that do not reach their native conformation are degraded via the ubiquitin‐proteasome system. This process is called ER‐associated degradation (ERAD). Der1 is known to be one of the components required for efficient degradation of soluble ERAD substrates like CPY^* (mutated carboxypeptidase yscY). A homologue of Der1 exists, named Dfm1. No function of Dfm1 has been discovered, although a C‐terminally hemagglutinin (HA)₃‐tagged Dfm1 protein has been shown to interact with the ERAD machinery. In our studies, we found Dfm1‐HA₃ to be an ERAD substrate and therefore not suitable for functional studies of Dfm1 in ERAD. We found cellular, non‐tagged Dfm1 to be a stable protein. We identified Dfm1 to be part of complexes which contain the ERAD‐L ligase Hrd1/Der3 and Der1 as well as the ERAD‐C ligase Doa10. In addition, ERAD of Ste6^*‐HA₃ was strongly dependent on Dfm1. Interestingly, Dfm1 forms a complex with the AAA‐ATPase Cdc48 in a strain lacking the Cdc48 membrane‐recruiting component Ubx2. This complex does not contain the ubiquitin ligases Hrd1/Der3 and Doa10. The existence of such a complex might point to an additional function of Dfm1 independent from ERAD.     

The endoplasmic reticulum-associated degradation is necessary for plant salt tolerance

Eukaryotic organisms have quality-control mechanisms that allow misfolded or unassembled proteins to be retained in the endoplasmic reticulum (ER) and subsequently degraded by ER-associated degradation (ERAD). The ERAD pathway is well studied in yeast and mammals; however, the biological functions of plant ERAD have not been reported. Through molecular and cellular biological approaches, we found that ERAD is necessary for plants to overcome salt stress. Upon salt treatment ubiquitinated proteins increased in plant cells, especially unfolded proteins that quickly accumulated in the ER and subsequently induced ER stress responses. Defect in HRD3A of the HRD1/HRD3 complex of the ERAD pathway resulted in alteration of the unfolded protein response (UPR), increased plant sensitivity to salt, and retention of ERAD substrates in plant cells. Furthermore, we demonstrated that Ca(2+) release from the ER is involved in the elevation of UPR and reactive oxygen species (ROS) participates the ERAD-related plant salt response pathway.     

Interaction between Salt-inducible Kinase 2 (SIK2) and p97/Valosin-containing Protein (VCP) Regulates Endoplasmic Reticulum (ER)-associated Protein Degradation in Mammalian Cells

Salt-inducible kinase 2 (SIK2) is an important regulator of cAMP response element-binding protein-mediated gene expression in various cell types and is the only AMP-activated protein kinase family member known to interact with the p97/valosin-containing protein (VCP) ATPase. Previously, we have demonstrated that SIK2 can regulate autophagy when proteasomal function is compromised. Here we report that physical and functional interactions between SIK2 and p97/VCP underlie the regulation of endoplasmic reticulum (ER)-associated protein degradation (ERAD). SIK2 co-localizes with p97/VCP in the ER membrane and stimulates its ATPase activity through direct phosphorylation. Although the expression of wild-type recombinant SIK2 accelerated the degradation and removal of ERAD substrates, the kinase-deficient variant conversely had no effect. Furthermore, down-regulation of endogenous SIK2 or mutation of the SIK2 target site on p97/VCP led to impaired degradation of ERAD substrates and disruption of ER homeostasis. Collectively, these findings highlight a mechanism by which the interplay between SIK2 and p97/VCP contributes to the regulation of ERAD in mammalian cells.     

Selective targeting of proteins within secretory pathway for endoplasmic reticulum-associated degradation

The endoplasmic reticulum-associated degradation (ERAD) is a cellular quality control mechanism to dispose of misfolded proteins of the secretory pathway via proteasomal degradation. SEL1L is an ER-resident protein that participates in identification of misfolded molecules as ERAD substrates, therefore inducing their ER-to-cytosol retrotranslocation and degradation. We have developed a novel class of fusion proteins, termed degradins, composed of a fragment of SEL1L fused to a target-specific binding moiety located on the luminal side of the ER. The target-binding moiety can be a ligand of the target or derived from specific mAbs. Here, we describe the ability of degradins with two different recognition moieties to promote degradation of a model target. Degradins recognize the target protein within the ER both in secretory and membrane-bound forms, inducing their degradation following retrotranslocation to the cytosol. Thus, degradins represent an effective technique to knock-out proteins within the secretory pathway with high specificity.     

The ubiquitin-domain protein HERP forms a complex with components of the endoplasmic reticulum associated degradation pathway

To eliminate misfolded proteins that accumulate in the endoplasmic reticulum (ER) the cell mainly relies on ubiquitin-proteasome dependent ER-associated protein degradation (ERAD). Proteolysis of ERAD substrates by the proteasome requires their ubiquitylation and retro-translocation from the ER to the cytoplasm. Here we describe a high molecular mass protein complex associated with the ER membrane, which facilitates ERAD. It contains the ubiquitin domain protein (UDP) HERP, the ubiquitin protein ligase HRD1, as well as the retro-translocation factors p97, Derlin-1 and VIMP. Our data on the structural arrangement of these ERAD proteins suggest that p97 interacts directly with membrane-resident components of the complex including Derlin-1 and HRD1, while HERP binds directly to HRD1. We propose that ubiquitylation, as well as retro-translocation of proteins from the ER are performed by this modular protein complex, which permits the close coordination of these consecutive steps within ERAD.     

Unconventional roles of nonlipidated LC3 in ERAD tuning and coronavirus infection

Secretory and membrane proteins attain their native structure in the endoplasmic reticulum (ER). Folding-defective polypeptides are selected for degradation by processes collectively defined as ER-associated degradation (ERAD). Enhanced ERAD activity may interfere with protein biogenesis by inappropriately targeting not-yet-native protein folding intermediates for disposal. The regulation of ERAD is therefore crucial to maintain cellular proteostasis. At steady-state, select ERAD regulators are constitutively removed from the ER in a series of processes collectively defined as ERAD tuning. This sets the ERAD activity at levels that do not interfere with completion of ongoing folding programs. Our latest work highlights a crucial, autophagy-independent role of nonlipidated LC3 (LC3-I) as part of a membrane-bound receptor that insures the vesicle-mediated clearance of at least two ERAD regulators from the ER, EDEM1 and OS9. This pathway is hijacked by coronaviruses (CoV), and silencing of LC3 substantially inhibits viral replication.     

Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the process by which aberrant proteins in the ER lumen are exported back to the cytosol and degraded by the proteasome. Although ER molecular chaperones are required for ERAD, their specific role(s) in this process have been ill defined. To understand how one group of interacting lumenal chaperones facilitates ERAD, the fates of pro-alpha-factor and a mutant form of carboxypeptidase Y were examined both in vivo and in vitro. We found that these ERAD substrates are stabilized and aggregate in the ER at elevated temperatures when BiP, the lumenal Hsp70 molecular chaperone, is mutated, or when the genes encoding the J domain-containing proteins Jem1p and Scj1p are deleted. In contrast, deletion of JEM1 and SCJ1 had little effect on the ERAD of a membrane protein. These results suggest that one role of the BiP, Jem1p, and Scj1p chaperones is to maintain lumenal ERAD substrates in a retrotranslocation-competent state.     

Ubiquitin-specific protease 25 functions in Endoplasmic Reticulum-associated degradation

Endoplasmic Reticulum (ER)-associated degradation (ERAD) discards abnormal proteins synthesized in the ER. Through coordinated actions of ERAD components, misfolded/anomalous proteins are recognized, ubiquitinated, extracted from the ER and ultimately delivered to the proteasome for degradation. It is not well understood how ubiquitination of ERAD substrates is regulated. Here, we present evidence that the deubiquitinating enzyme Ubiquitin-Specific Protease 25 (USP25) is involved in ERAD. Our data support a model where USP25 counteracts ubiquitination of ERAD substrates by the ubiquitin ligase HRD1, rescuing them from degradation by the proteasome.     

Misfolded Proteins Induce Aggregation of the Lectin Yos9

A substantial fraction of nascent proteins delivered into the endoplasmic reticulum (ER) never reach their native conformations. Eukaryotes use a series of complementary pathways to efficiently recognize and dispose of these terminally misfolded proteins. In this process, collectively termed ER-associated degradation (ERAD), misfolded proteins are retrotranslocated to the cytosol, polyubiquitinated, and degraded by the proteasome. Although there has been great progress in identifying ERAD components, how these factors accurately identify substrates remains poorly understood. The targeting of misfolded glycoproteins in the ER lumen for ERAD requires the lectin Yos9, which recognizes the glycan species found on terminally misfolded proteins. In a role that remains poorly characterized, Yos9 also binds the protein component of ERAD substrates. Here, we identified a 45-kDa domain of Yos9, consisting of residues 22–421, that is proteolytically stable, highly structured, and able to fully support ERAD in vivo. In vitro binding studies show that Yos9(22–421) exhibits sequence-specific recognition of linear peptides from the ERAD substrate, carboxypeptidase Y G255R (CPY*), and binds a model unfolded peptide ΔEspP and protein Δ131Δ in solution. Binding of Yos9 to these substrates results in their cooperative aggregation. Although the physiological consequences of this substrate-induced aggregation remain to be seen, it has the potential to play a role in the regulation of ERAD.     

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.     

ERAD‐dependent control of the Wnt secretory factor Evi

Active regulation of protein abundance is an essential strategy to modulate cellular signaling pathways. Within the Wnt signaling cascade, regulated degradation of β‐catenin by the ubiquitin‐proteasome system (UPS) affects the outcome of canonical Wnt signaling. Here, we found that abundance of the Wnt cargo receptor Evi (Wls/GPR177), which is required for Wnt protein secretion, is also regulated by the UPS through endoplasmic reticulum (ER)‐associated degradation (ERAD). In the absence of Wnt ligands, Evi is ubiquitinated and targeted for ERAD in a VCP‐dependent manner. Ubiquitination of Evi involves the E2‐conjugating enzyme UBE2J2 and the E3‐ligase CGRRF1. Furthermore, we show that a triaging complex of Porcn and VCP determines whether Evi enters the secretory or the ERAD pathway. In this way, ERAD‐dependent control of Evi availability impacts the scale of Wnt protein secretion by adjusting the amount of Evi to meet the requirement of Wnt protein export. As Wnt and Evi protein levels are often dysregulated in cancer, targeting regulatory ERAD components might be a useful approach for therapeutic interventions.     

SEL1L protein critically determines the stability of the HRD1-SEL1L endoplasmic reticulum-associated degradation (ERAD) complex to optimize the degradation kinetics of ERAD substrates

The mammalian HRD1-SEL1L complex provides a scaffold for endoplasmic reticulum (ER)-associated degradation (ERAD), thereby connecting luminal substrates for ubiquitination at the cytoplasmic surface after their retrotranslocation through the endoplasmic reticulum membrane. In this study the stability of the mammalian HRD1-SEL1L complex was assessed by performing siRNA-mediated knockdown of each of its components. Although endogenous SEL1L is a long-lived protein, the half-life of SEL1L was greatly reduced when HRD1 is silenced. Conversely, transiently expressed SEL1L was rapidly degraded but was stabilized when HRD1 was coexpressed. This was in contrast to the yeast Hrd1p-Hrd3p, where Hrd1p is destabilized by the depletion of Hrd3p, the SEL1L homologue. Endogenous HRD1-SEL1L formed a large ERAD complex (Complex I) associating with numerous ERAD components including ERAD lectin OS-9, membrane-spanning Derlin-1/2, VIMP, and Herp, whereas transiently expressed HRD1-SEL1L formed a smaller complex (Complex II) that was associated with OS-9 but not with Derlin-1/2, VIMP, or Herp. Despite its lack of stable association with the latter components, Complex II supported the retrotranslocation and degradation of model ERAD substrates α1-antitrypsin null Hong-Kong (NHK) and its variant NHK-QQQ lacking the N-glycosylation sites. NHK-QQQ was rapidly degraded when SEL1L was transiently expressed, whereas the simultaneous transfection of HRD1 diminished that effect. SEL1L unassociated with HRD1 was degraded by the ubiquitin-proteasome pathway, which suggests the involvement of a ubiquitin-ligase other than HRD1 in the rapid degradation of both SEL1L and NHK-QQQ. These results indicate that the regulation of the stability and assembly of the HRD1-SEL1L complex is critical to optimize the degradation kinetics of ERAD substrates.     

Mammalian OS-9 Is Upregulated in Response to Endoplasmic Reticulum Stress and Facilitates Ubiquitination of Misfolded Glycoproteins

Proteins that fail to fold or assemble with partner subunits are selectively removed from the endoplasmic reticulum (ER) via the ER-associated degradation (ERAD) pathway. Proteins selected for ERAD are polyubiquitinated and retrotranslocated into the cytosol for degradation by the proteasome. Although it is unclear how proteins are initially identified by the ERAD system in mammalian cells, OS-9 was recently proposed to play a key role in this process. Here we show that OS-9 is upregulated in response to ER stress and is associated both with components of the ERAD machinery and with ERAD substrates. Using RNA interference, we show that OS-9 is required for efficient ubquitination of glycosylated ERAD substrates, suggesting that it helps transfer misfolded proteins to the ubiquitination machinery. We also find that OS-9 binds to a misfolded nonglycosylated protein destined for ERAD, but not to the properly folded wild-type protein. Surprisingly, however, OS-9 is not required for ubiquitination or degradation of this nonglycosylated ERAD substrate. We propose a model in which OS-9 recognises terminally misfolded proteins via polypeptide-based rather than glycan-based signals, but is only required for transferring those bearing N-glycans to the ubiquitination machinery.     

Ubiquitin-Dependent Intramembrane Rhomboid Protease Promotes ERAD of Membrane Proteins

The ER-associated degradation (ERAD) pathway serves as an important cellular safeguard by directing incorrectly folded and unassembled proteins from the ER to the proteasome. Still, however, little is known about the components mediating ERAD of?membrane proteins. Here we show that the evolutionary conserved rhomboid family protein RHBDL4 is a ubiquitin-dependent ER-resident intramembrane protease that is upregulated upon ER stress. RHBDL4 cleaves single-spanning and polytopic membrane proteins with unstable transmembrane helices, leading to their degradation by the canonical ERAD machinery. RHBDL4 specifically binds the AAA+-ATPase p97, suggesting that proteolytic processing and dislocation into the cytosol are functionally linked. The phylogenetic relationship between rhomboids and the ERAD factor derlin suggests that substrates for intramembrane proteolysis and protein dislocation are recruited by?a shared mechanism.     

Traffic-independent function of the Sar1p/COPII machinery in proteasomal sorting of the cystic fibrosis transmembrane conductance regulator

Newly synthesized proteins that do not fold correctly in the ER are targeted for ER-associated protein degradation (ERAD) through distinct sorting mechanisms; soluble ERAD substrates require ER-Golgi transport and retrieval for degradation, whereas transmembrane ERAD substrates are retained in the ER. Retained transmembrane proteins are often sequestered into specialized ER subdomains, but the relevance of such sequestration to proteasomal degradation has not been explored. We used the yeast Saccharomyces cerevisiae and a model ERAD substrate, the cystic fibrosis transmembrane conductance regulator (CFTR), to explore whether CFTR is sequestered before degradation, to identify the molecular machinery regulating sequestration, and to analyze the relationship between sequestration and degradation. We report that CFTR is sequestered into ER subdomains containing the chaperone Kar2p, and that sequestration and CFTR degradation are disrupted in sec12ts strain (mutant in guanine-nucleotide exchange factor for Sar1p), sec13ts strain (mutant in the Sec13p component of COPII), and sec23ts strain (mutant in the Sec23p component of COPII) grown at restrictive temperature. The function of the Sar1p/COPII machinery in CFTR sequestration and degradation is independent of its role in ER-Golgi traffic. We propose that Sar1p/COPII-mediated sorting of CFTR into ER subdomains is essential for its entry into the proteasomal degradation pathway. These findings reveal a new aspect of the degradative mechanism, and suggest functional crosstalk between the secretory and the degradative pathways.     

Jid1 is a J-protein functioning in the mitochondrial matrix, unable to directly participate in endoplasmic reticulum associated protein degradation

J-proteins are a class of molecular chaperones that serve to stimulate the activity of Hsp70s and are often located to recruit Hsp70 to a particular cellular function. Protein degradation associated with the endoplasmic reticulum (ERAD) is one such cellular process that requires Hsp70 on both faces of the endoplasmic reticulum. At least five J-proteins, including Jid1 (DnaJ protein Involved in ER-associated Degradation), have been implicated in controlling ERAD. Here we show that Jid1 is confined within the mitochondrial matrix - Jid1 has the same topology as the J-proteins Pam18 and Mdj2, which stimulate mitochondrial Hsp70 to drive protein import into the mitochondrial matrix. The location of Jid1 within mitochondria makes it unavailable to participate directly in the regulation of ERAD.     

The recognition and retrotranslocation of misfolded proteins from the endoplasmic reticulum

Secretory and membrane proteins that fail to fold in the endoplasmic reticulum (ER) are retained and may be sorted for ER-associated degradation (ERAD). During ERAD, ER-associated components such as molecular chaperones and lectins recognize folding intermediates and specific oligosaccharyl modifications on ERAD substrates. Substrates selected for ERAD are then targeted for ubiquitin- and proteasome-mediated degradation. Because the catalytic steps of the ubiquitin–proteasome system reside in the cytoplasm, soluble ERAD substrates that reside in the ER lumen must be retrotranslocated back to the cytoplasm prior to degradation. In contrast, it has been less clear how polytopic, integral membrane substrates are delivered to enzymes required for ubiquitin conjugation and to the proteasome. In this review, we discuss recent studies addressing how ERAD substrates are recognized, ubiquitinated and delivered to the proteasome and then survey current views of how soluble and integral membrane substrates may be retrotranslocated.