Acts as an E3 ubiquitin-protein ligase which accepts ubiquitin specifically from endoplasmic reticulum-associated UBC7 E2 ligase and transfers it to substrates, promoting their degradation. Component of the endoplasmic reticulum quality control (ERQC) system also called ER-associated degradation (ERAD) involved in ubiquitin-dependent degradation of misfolded endoplasmic reticulum proteins. Also promotes the degradation of normal but naturally short-lived proteins such as SGK. Protects cells from ER stress-induced apoptosis. Protects neurons from apoptosis induced by polyglutamine-expanded huntingtin (HTT) or unfolded GPR37 by promoting their degradation. Sequesters p53/TP53 in the cytoplasm and promotes its degradation, thereby negatively regulating its biological function in transcription, cell cycle regulation and apoptosis.
Synoviolin, also called HRD1, is an E3 ubiquitin ligase and is implicated in endoplasmic reticulum -associated degradation. In mammals, Synoviolin plays crucial roles in various physiological and pathological processes, including embryogenesis and the pathogenesis of arthropathy. However, little is known about the molecular mechanisms of Synoviolin in these actions. To clarify these issues, we analyzed the profile of protein expression in synoviolin-null cells. Here, we report that Synoviolin targets tumor suppressor gene p53 for ubiquitination. Synoviolin sequestrated and metabolized p53 in the cytoplasm and negatively regulated its cellular level and biological functions, including transcription, cell cycle regulation and apoptosis. Furthermore, these p53 regulatory functions of Synoviolin were irrelevant to other E3 ubiquitin ligases for p53, such as MDM2, Pirh2 and Cop1, which form autoregulatory feedback loops. Our results provide novel insights into p53 signaling mediated by Synoviolin.
Stresses that impair the function of the endoplasmic reticulum (ER) lead to an accumulation of unfolded protein in the ER. Under these conditions, the expression of a variety of genes involved in preventing the accumulation of the unfolded proteins is induced. Yeast Hrd1p is an ER stress-inducible ER membrane protein that acts as a ubiquitin ligase (E3) with a RING finger motif and plays a role in the ubiquitination of proteins in the ER. We report here the identification and characterization of a human homolog to yeast Hrd1p. The predicted structures are highly conserved from yeast to humans. Indeed, human HRD1 was localized to the ER and ubiquitinated its substrates. Furthermore, it was found that human HRD1 was up-regulated by ER stress via IRE1 and ATF6, which are ER stress transducers. Interestingly, 293 cells stably expressing wild-type HRD1, but not the C329S mutant, afforded resistance to ER stress-induced apoptosis. These results suggest that the production of HRD1 is up-regulated to protect against ER stress-induced apoptosis by degrading unfolded proteins accumulated in the ER.
Rheumatoid arthritis (RA) is one of the most critical articular diseases with synovial hyperplasia followed by impairment of quality of life. However, the mechanism(s) that regulates synovial cell outgrowth is not fully understood. To clarify its mechanism(s), we carried out immunoscreening by using antirheumatoid synovial cell antibody and identified and cloned "Synoviolin/Hrd1", an E3 ubiquitin ligase. Synoviolin/Hrd1 was highly expressed in the rheumatoid synovium, and mice overexpressing this enzyme developed spontaneous arthropathy. Conversely, synoviolin/hrd1(+/-) mice were resistant to collagen-induced arthritis by enhanced apoptosis of synovial cells. We conclude that Synoviolin/Hrd1 is a novel causative factor for arthropathy by triggering synovial cell outgrowth through its antiapoptotic effects. Our findings provide a new pathogenetic model of RA and suggest that Synoviolin/Hrd1 could be targeted as a therapeutic strategy for RA.
The ubiquitin system plays an important role in endoplasmic reticulum (ER)-associated degradation of proteins that are misfolded, that fail to associate with their oligomerization partners, or whose levels are metabolically regulated. E3 ubiquitin ligases are key enzymes in the ubiquitination process as they recognize the substrate and facilitate coupling of multiple ubiquitin units to the protein that is to be degraded. The Saccharomyces cerevisiae ER-resident E3 ligase Hrd1p/Der3p functions in the metabolically regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and additionally facilitates the degradation of a number of misfolded proteins from the ER. In this study we characterized the structure and function of the putative human orthologue of yeast Hrd1p/Der3p, designated human HRD1. We show that human HRD1 is a non-glycosylated, stable ER protein with a cytosolic RING-H2 finger domain. In the presence of the ubiquitin-conjugating enzyme UBC7, the RING-H2 finger has in vitro ubiquitination activity for Lys(48)-specific polyubiquitin linkage, suggesting that human HRD1 is an E3 ubiquitin ligase involved in protein degradation. Human HRD1 appears to be involved in the basal degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase but not in the degradation that is regulated by sterols. Additionally we show that human HRD1 is involved in the elimination of two model ER-associated degradation substrates, TCR-alpha and CD3-delta.
Serum- and glucocorticoid-induced kinase 1 (Sgk1) regulates many ion channels and transporters in epithelial cells and promotes cell survival under stress conditions. In this study we demonstrate that Sgk1 is a short-lived protein regulated by the endoplasmic reticulum (ER)-associated degradation system and subcellular localization to the ER. We identified a hydrophobic motif (residues 18-30) as the signal for ER localization and rapid degradation by the ubiquitin (Ub)/proteasome pathway in both yeast and mammalian cells. Deletion or reduction of hydrophobicity of the motif redistributes Sgk1 to the cytosol and nucleus and markedly increases its half-life. We determined that the Ub-conjugating UBC6 and UBC7 and the Ub ligase HRD1 are the ER-associated Ub enzymes that mediate degradation of Sgk1; thus, Sgk1 has been identified as a cytosolic substrate for mammalian HRD1. Compartmentalization of Sgk1 controls the functional and spatial specificities of Sgk1-mediated signaling pathways, whereas rapid protein turnover provides a means to rapidly adjust Sgk1 abundance in response to different hormonal and external stimuli that increase Sgk1 gene transcription.
E3 ubiquitin ligases catalyze the conjugation of ubiquitin onto proteins, which acts as a signal for targeting proteins for degradation by the proteasome. Hrd1 is an endoplasmic reticulum (ER) membrane-spanning E3 with its catalytic active RING finger facing the cytosol. We speculated that this topology might allow Hrd1 to ubiquitinate misfolded proteins in the cytosol. We tested this idea by using polyglutamine (polyQ)-containing huntingtin (htt) protein as a model substrate. We found that the protein levels of Hrd1 were increased in cells overexpressing the N-terminal fragment of htt containig an expanded polyQ tract (httN). Forced expression of Hrd1 enhanced the degradation of httN in a RING finger-dependent manner, whereas silencing of endogenous Hrd1 expression by RNA interference stabilized httN. Degradation of httN was found to be p97/VCP-dependent, but independent of Ufd1 and Npl4, all of which are thought to form a complex with Hrd1 during ER-associated degradation. Consistent with its role as an E3 for httN, we demonstrate that Hrd1 interacts with and ubiquitinates httN. Subcellular fractionation and confocal microscopy revealed that Hrd1recruits HttN to the ER and co-localizes with juxtanuclear aggregates of httN in cells. Interaction of Hrd1 with httN was found to be independent of the length of the polyglutamine tract. However, httN with expanded polyglutamine tracts appeared to be a preferred substrate for Hrd1. Functionally, we found that Hrd1 protects cells against the httN-induced cell death. These results suggest that Hrd1 is a novel htt-interacting protein that can target pathogenic httN for degradation and is able to protect cells against httN-induced cell death.
The yeast hHrd1 is a ubiquitin-protein ligase (E3) involved in ER-associated degradation. It was originally identified by genetic methods as an E3 of the yeast cholesterol biosynthetic enzyme HMG-CoA reductase (HMGR). We report the identification and cloning of a human homologue of Hrd1 (hHrd1). Immunofluorescence imaging confirms that the endogenous hHrd1 resides in the ER and in vitro assay demonstrates that it has a ubiquitin-ligase activity. However, the homology between the human and yeast Hrd1 is limited to the N-terminal domain of the proteins, and hHrd1 does not appear to be involved in the degradation of mammalian HMGR.
It has been proposed that in autosomal recessive juvenile parkinsonism (AR-JP), a ubiquitin ligase (E3) Parkin, which is involved in endoplasmic reticulum-associated degradation (ERAD), lacks E3 activity. The resulting accumulation of Parkin-associated endothelin receptor-like receptor (Pael-R), a substrate of Parkin, leads to endoplasmic reticulum stress, causing neuronal death. We previously reported that human E3 HRD1 in the endoplasmic reticulum protects against endoplasmic reticulum stress-induced apoptosis. This study shows that HRD1 was expressed in substantia nigra pars compacta (SNC) dopaminergic neurons and interacted with Pael-R through the HRD1 proline-rich region, promoting the ubiquitylation and degradation of Pael-R. Furthermore, the disruption of endogenous HRD1 by small interfering RNA (siRNA) induced Pael-R accumulation and caspase-3 activation. We also found that ATF6 overexpression, which induced HRD1, accelerated and caused Pael-R degradation; the suppression of HRD1 expression by siRNA partially prevents this degradation. These results suggest that in addition to Parkin, HRD1 is also involved in the degradation of Pael-R.
Nascent secretory proteins are extensively scrutinized at the endoplasmic reticulum (ER). Various signatures of client proteins, including exposure of hydrophobic patches or unpaired sulfhydryls, are coordinately utilized to reduce nonnative proteins in the ER. We report here the cryptic N-glycosylation site as a recognition signal for unfolding of a natively nonglycosylated protein, transthyretin (TTR), involved in familial amyloidosis. Folding and ER-associated degradation (ERAD) perturbation analyses revealed that prolonged TTR unfolding induces externalization of cryptic N-glycosylation site and triggers STT3B-dependent posttranslational N-glycosylation. Inhibition of posttranslational N-glycosylation increases detergent-insoluble TTR aggregates and decreases cell proliferation of mutant TTR-expressing cells. Moreover, this modification provides an alternative pathway for degradation, which is EDEM3-mediated N-glycan-dependent ERAD, distinct from the major pathway of Herp-mediated N-glycan-independent ERAD. Hence we postulate that STT3B-dependent posttranslational N-glycosylation is part of a triage-salvage system recognizing cryptic N-glycosylation sites of secretory proteins to preserve protein homeostasis.
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.
Catalysis of the ligation of an acid to an amino acid via a carbon-nitrogen bond, with the concomitant hydrolysis of the diphosphate bond in ATP or a similar triphosphate.
Stresses that impair the function of the endoplasmic reticulum (ER) lead to an accumulation of unfolded protein in the ER. Under these conditions, the expression of a variety of genes involved in preventing the accumulation of the unfolded proteins is induced. Yeast Hrd1p is an ER stress-inducible ER membrane protein that acts as a ubiquitin ligase (E3) with a RING finger motif and plays a role in the ubiquitination of proteins in the ER. We report here the identification and characterization of a human homolog to yeast Hrd1p. The predicted structures are highly conserved from yeast to humans. Indeed, human HRD1 was localized to the ER and ubiquitinated its substrates. Furthermore, it was found that human HRD1 was up-regulated by ER stress via IRE1 and ATF6, which are ER stress transducers. Interestingly, 293 cells stably expressing wild-type HRD1, but not the C329S mutant, afforded resistance to ER stress-induced apoptosis. These results suggest that the production of HRD1 is up-regulated to protect against ER stress-induced apoptosis by degrading unfolded proteins accumulated in the ER.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionUniProtKB
The recognition of terminally misfolded proteins in the endoplasmic reticulum (ER) and the extraction of these proteins to the cytoplasm for proteasomal degradation are determined by a quality control mechanism in the ER. In yeast, Yos9p, an ER lectin containing a mannose 6-phosphate receptor homology (MRH) domain, enhances ER-associated degradation (ERAD) of glycoproteins. We show here that human XTP3-B (hXTP3-B), an ER lectin containing two MRH domains, has two transcriptional variants, and both isoforms retard ERAD of the human alpha(1)-antitrypsin variant null Hong Kong (NHK), a terminally misfolded glycoprotein. The hXTP3-B long isoform strongly inhibited ERAD of NHK-QQQ, which lacks all of the N-glycosylation sites of NHK, but the short transcriptional variant of hXTP3-B had almost no effect. Examination of complex formation by immunoprecipitation and by fractionation using sucrose density gradient centrifugation revealed that the hXTP3-B long isoform associates with the HRD1-SEL1L membrane-anchored ubiquitin ligase complex and BiP, forming a 27 S ER quality control scaffold complex. The hXTP3-B short isoform, however, is excluded from scaffold formation. Another MRH domain-containing ER lectin, hOS-9, is incorporated into this large complex, but gp78, another mammalian homolog of the yeast ubiquitin ligase Hrd1p, is not. Based on these results, we propose that this large ER quality control scaffold complex, containing ER lectins, a chaperone, and a ubiquitin ligase, provides a platform for the recognition and sorting of misfolded glycoproteins as well as nonglycosylated proteins prior to retrotranslocation into the cytoplasm for degradation.
Evidence
2:
Inferred from Physical InteractionUniProtKB
Terminally misfolded or unassembled proteins in the early secretory pathway are degraded by a ubiquitin- and proteasome-dependent process known as ER-associated degradation (ERAD). How substrates of this pathway are recognized within the ER and delivered to the cytoplasmic ubiquitin-conjugating machinery is unknown. We report here that OS-9 and XTP3-B/Erlectin are ER-resident glycoproteins that bind to ERAD substrates and, through the SEL1L adaptor, to the ER-membrane-embedded ubiquitin ligase Hrd1. Both proteins contain conserved mannose 6-phosphate receptor homology (MRH) domains, which are required for interaction with SEL1L, but not with substrate. OS-9 associates with the ER chaperone GRP94 which, together with Hrd1 and SEL1L, is required for the degradation of an ERAD substrate, mutant alpha(1)-antitrypsin. These data suggest that XTP3-B and OS-9 are components of distinct, partially redundant, quality control surveillance pathways that coordinate protein folding with membrane dislocation and ubiquitin conjugation in mammalian cells.
The chemical reactions and pathways resulting in the breakdown of proteins transported from the endoplasmic reticulum and targeted to cytoplasmic proteasomes for degradation. This process acts on misfolded proteins as well as in the regulated degradation of correctly folded proteins.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
Nascent secretory proteins are extensively scrutinized at the endoplasmic reticulum (ER). Various signatures of client proteins, including exposure of hydrophobic patches or unpaired sulfhydryls, are coordinately utilized to reduce nonnative proteins in the ER. We report here the cryptic N-glycosylation site as a recognition signal for unfolding of a natively nonglycosylated protein, transthyretin (TTR), involved in familial amyloidosis. Folding and ER-associated degradation (ERAD) perturbation analyses revealed that prolonged TTR unfolding induces externalization of cryptic N-glycosylation site and triggers STT3B-dependent posttranslational N-glycosylation. Inhibition of posttranslational N-glycosylation increases detergent-insoluble TTR aggregates and decreases cell proliferation of mutant TTR-expressing cells. Moreover, this modification provides an alternative pathway for degradation, which is EDEM3-mediated N-glycan-dependent ERAD, distinct from the major pathway of Herp-mediated N-glycan-independent ERAD. Hence we postulate that STT3B-dependent posttranslational N-glycosylation is part of a triage-salvage system recognizing cryptic N-glycosylation sites of secretory proteins to preserve protein homeostasis.
Stresses that impair the function of the endoplasmic reticulum (ER) lead to an accumulation of unfolded protein in the ER. Under these conditions, the expression of a variety of genes involved in preventing the accumulation of the unfolded proteins is induced. Yeast Hrd1p is an ER stress-inducible ER membrane protein that acts as a ubiquitin ligase (E3) with a RING finger motif and plays a role in the ubiquitination of proteins in the ER. We report here the identification and characterization of a human homolog to yeast Hrd1p. The predicted structures are highly conserved from yeast to humans. Indeed, human HRD1 was localized to the ER and ubiquitinated its substrates. Furthermore, it was found that human HRD1 was up-regulated by ER stress via IRE1 and ATF6, which are ER stress transducers. Interestingly, 293 cells stably expressing wild-type HRD1, but not the C329S mutant, afforded resistance to ER stress-induced apoptosis. These results suggest that the production of HRD1 is up-regulated to protect against ER stress-induced apoptosis by degrading unfolded proteins accumulated in the ER.
The process whose specific outcome is the progression of the embryo in the uterus over time, from formation of the zygote in the oviduct, to birth. An example of this process is found in Mus musculus.
The glycosylation of protein via the N4 atom of peptidyl-asparagine forming N4-glycosyl-L-asparagine; the most common form is N-acetylglucosaminyl asparagine; N-acetylgalactosaminyl asparagine and N4 glucosyl asparagine also occur. This modification typically occurs in extracellular peptides with an N-X-(ST) motif. Partial modification has been observed to occur with cysteine, rather than serine or threonine, in the third position; secondary structure features are important, and proline in the second or fourth positions inhibits modification.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
Nascent secretory proteins are extensively scrutinized at the endoplasmic reticulum (ER). Various signatures of client proteins, including exposure of hydrophobic patches or unpaired sulfhydryls, are coordinately utilized to reduce nonnative proteins in the ER. We report here the cryptic N-glycosylation site as a recognition signal for unfolding of a natively nonglycosylated protein, transthyretin (TTR), involved in familial amyloidosis. Folding and ER-associated degradation (ERAD) perturbation analyses revealed that prolonged TTR unfolding induces externalization of cryptic N-glycosylation site and triggers STT3B-dependent posttranslational N-glycosylation. Inhibition of posttranslational N-glycosylation increases detergent-insoluble TTR aggregates and decreases cell proliferation of mutant TTR-expressing cells. Moreover, this modification provides an alternative pathway for degradation, which is EDEM3-mediated N-glycan-dependent ERAD, distinct from the major pathway of Herp-mediated N-glycan-independent ERAD. Hence we postulate that STT3B-dependent posttranslational N-glycosylation is part of a triage-salvage system recognizing cryptic N-glycosylation sites of secretory proteins to preserve protein homeostasis.
Protein involved in the response to stress, a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of some stressful conditions. The stress is usually, but not necessarily, exogenous (e.g. temperature, humidity, ionizing radiation, hypertonicity, amino acid deprivation).
Protein involved in ubiquitin-like modifier processing, activation, conjugation or deconjugation such as Ubl-activating enzymes (E1s), Ubl-conjugating enzymes (E2s), Ubl-protein ligases (E3s), some thiol proteases (Ubiquitin carboxyl-terminal hydrolases (UCH), Ubiquitin- specific processing proteases (UBP) and ubiquitin-like proteases) and the ubiquitin-like modifier proteins. Besides signaling proteolysis, ubiquitination for example can be a signal for trafficking, kinase activation and other nonproteolytic fates.
Enzyme that catalyzes the joining of two molecules coupled with the breakdown of a pyrophosphate bond in ATP or a similar triphosphate. Sometimes the terms "synthase", "synthetase" or "carboxylase" are also used for this class of enzymes.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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