Usa1 Protein Facilitates Substrate Ubiquitylation through Two Separate Domains

Background

Defects in protein folding are recognized as the root of many neurodegenerative disorders. In the endoplasmic reticulum (ER), secretory proteins are subjected to a stringent quality control process to eliminate misfolded proteins by the ER-associated degradation (ERAD) pathway. A novel ERAD component Usa1 was recently identified. However, the specific role of Usa1 in ERAD remains obscure.

Methodology/Principal Findings

Here, we demonstrate that Usa1 is important for substrate ubiquitylation. Furthermore, we defined key cis-elements of Usa1 essential for its degradation function. Interestingly, a putative proteasome-binding motif is dispensable for the functioning of Usa1 in ERAD. We identify two separate cytosolic domains critical for Usa1 activity in ERAD, one of which is involved in binding to the Ub-protein ligase Hrd1/Hrd3. Usa1 may have another novel role in substrate ubiquitylation that is separate from the Hrd1 association.

Conclusions/Significance

We conclude that Usa1 has two important roles in ERAD substrate ubiquitylation.     

Misfolded proteins traffic from the endoplasmic reticulum (ER) due to ER export signals

Most misfolded secretory proteins remain in the endoplasmic reticulum (ER) and are degraded by ER-associated degradation (ERAD). However, some misfolded proteins exit the ER and traffic to the Golgi before degradation. Using model misfolded substrates, with or without defined ER exit signals, we found misfolded proteins can depart the ER by continuing to exhibit the functional export signals present in the corresponding correctly folded proteins. Anterograde transport of misfolded proteins utilizes the same machinery responsible for exporting correctly folded proteins. Passive ER retention, in which misfolded proteins fail to exit the ER due to the absence of exit signals or the inability to functionally present them, likely contributes to the retention of nonnative proteins in the ER. Intriguingly, compromising ERAD resulted in increased anterograde trafficking of a misfolded protein with an ER exit signal, suggesting that ERAD and ER exit machinery can compete for binding of misfolded proteins. Disabling ERAD did not result in transport of an ERAD substrate lacking an export signal. This is an important distinction for those seeking possible therapeutic approaches involving inactivating ERAD in anticipation of exporting a partially active protein.     

Probing for membrane domains in the endoplasmic reticulum: retention and degradation of unassembled MHC class I molecules

Quality control of protein biosynthesis requires ER-retention and ER-associated degradation (ERAD) of unassembled/misfolded molecules. Although some evidence exists for the organization of the ER into functionally distinct membrane domains, it is unknown if such domains are involved in the retention and ERAD of unassembled proteins. Here, it is shown that unassembled MHC class I molecules are retained in the ER without accumulating at ER-exit sites or in the ERGIC of beta2m-/- cells. Furthermore, these molecules did not cluster in the ER membrane and appeared to be highly mobile even when ERAD or their association with calnexin were inhibited. However, upon ATP depletion, they were reversibly segregated into an ER membrane domain, distinct from ER exit sites, which included calnexin and COPII, but not the ERGIC marker protein p58. This quality control domain was also observed upon prolonged inhibition of proteasomes. Microtubules were required for its appearance. Segregation of unfolded proteins, ER-resident chaperones, and COPII may be a temporal adaptation to cell stress.     

Proper protein folding in the endoplasmic reticulum is required for attachment of a glycosylphosphatidylinositol anchor in plants

Endoplasmic reticulum (ER) quality control processes recognize and eliminate misfolded proteins to maintain cellular protein homeostasis and prevent the accumulation of defective proteins in the secretory pathway. Glycosylphosphatidylinositol (GPI)-anchored proteins carry a glycolipid modification, which provides an efficient ER export signal and potentially prevents the entry into ER-associated degradation (ERAD), which is one of the major pathways for clearance of terminally misfolded proteins from the ER. Here, we analyzed the degradation routes of different misfolded glycoproteins carrying a C-terminal GPI-attachment signal peptide in Arabidopsis thaliana. We found that a fusion protein consisting of the misfolded extracellular domain from Arabidopsis STRUBBELIG and the GPI-anchor attachment sequence of COBRA1 was efficiently targeted to hydroxymethylglutaryl reductase degradation protein 1 complex-mediated ERAD without the detectable attachment of a GPI anchor. Non-native variants of the GPI-anchored lipid transfer protein 1 (LTPG1) that lack a severely misfolded domain, on the other hand, are modified with a GPI anchor and targeted to the vacuole for degradation. Impaired processing of the GPI-anchoring signal peptide by mutation of the cleavage site or in a GPI-transamidase-compromised mutant caused ER retention and routed the non-native LTPG1 to ERAD. Collectively, these results indicate that for severely misfolded proteins, ER quality control processes are dominant over ER export. For less severely misfolded proteins, the GPI anchor provides an efficient ER export signal resulting in transport to the vacuole.

Severely misfolded proteins carrying a glycosylphosphatidylinositol (GPI)-anchor attachment sequence undergo a stringent quality control process in the endoplasmic reticulum that prevents GPI anchoring.     

TRAPPC9 Mediates the Interaction between p150Glued and COPII Vesicles at the Target Membrane

Background

The transport of endoplasmic reticulum (ER)-derived COPII vesicles toward the ER-Golgi intermediate compartment (ERGIC) requires cytoplasmic dynein and is dependent on microtubules. p150^Glued, a subunit of dynactin, has been implicated in the transport of COPII vesicles via its interaction with COPII coat components Sec23 and Sec24. However, whether and how COPII vesicle tether, TRAPP (Transport protein particle), plays a role in the interaction between COPII vesicles and microtubules is currently unknown.

Principle Findings

We address the functional relationship between COPII tether TRAPP and dynactin. Overexpressed TRAPP subunits interfered with microtubule architecture by competing p150^Glued away from the MTOC. TRAPP subunit TRAPPC9 bound directly to p150^Glued via the same carboxyl terminal domain of p150^Glued that binds Sec23 and Sec24. TRAPPC9 also inhibited the interaction between p150^Glued and Sec23/Sec24 both in vitro and in vivo, suggesting that TRAPPC9 serves to uncouple p150^Glued from the COPII coat, and to relay the vesicle-dynactin interaction at the target membrane.

Conclusions

These findings provide a new perspective on the function of TRAPP as an adaptor between the ERGIC membrane and dynactin. By preserving the connection between dynactin and the tethered and/or fused vesicles, TRAPP allows nascent ERGIC to continue the movement along the microtubules as they mature into the cis-Golgi.     

Protective Unfolded Protein Response in Human Pancreatic Beta Cells Transplanted into Mice

Background

There is great interest about the possible contribution of ER stress to the apoptosis of pancreatic beta cells in the diabetic state and with islet transplantation.

Methods and Findings

Expression of genes involved in ER stress were examined in beta cell enriched tissue obtained with laser capture microdissection (LCM) from frozen sections of pancreases obtained from non-diabetic subjects at surgery and from human islets transplanted into ICR-SCID mice for 4 wk. Because mice have higher glucose levels than humans, the transplanted beta cells were exposed to mild hyperglycemia and the abnormal environment of the transplant site. RNA was extracted from the LCM specimens, amplified and then subjected to microarray analysis. The transplanted beta cells showed an unfolded protein response (UPR). There was activation of many genes of the IRE-1 pathway that provide protection against the deleterious effects of ER stress, increased expression of ER chaperones and ERAD (ER-associated protein degradation) proteins. The other two arms of ER stress, PERK and ATF-6, had many down regulated genes. Downregulation of EIF2A could protect by inhibiting protein synthesis. Two genes known to contribute to apoptosis, CHOP and JNK, were downregulated.

Conclusions

Human beta cells in a transplant site had UPR changes in gene expression that protect against the proapoptotic effects of unfolded proteins.     

Yos9p and Hrd1p mediate ER retention of misfolded proteins for ER-associated degradation

The endoplasmic reticulum (ER) has an elaborate quality control system, which retains misfolded proteins and targets them to ER-associated protein degradation (ERAD). To analyze sorting between ER retention and ER exit to the secretory pathway, we constructed fusion proteins containing both folded carboxypeptidase Y (CPY) and misfolded mutant CPY (CPY*) units. Although the luminal Hsp70 chaperone BiP interacts with the fusion proteins containing CPY* with similar efficiency, a lectin-like ERAD factor Yos9p binds to them with different efficiency. Correlation between efficiency of Yos9p interactions and ERAD of these fusion proteins indicates that Yos9p but not BiP functions in the retention of misfolded proteins for ERAD. Yos9p targets a CPY*-containing ERAD substrate to Hrd1p E3 ligase, thereby causing ER retention of the misfolded protein. This ER retention is independent of the glycan degradation signal on the misfolded protein and operates even when proteasomal degradation is inhibited. These results collectively indicate that Yos9p and Hrd1p mediate ER retention of misfolded proteins in the early stage of ERAD, which constitutes a process separable from the later degradation step.     

Endoplasmic Reticulum Protein Targeting of Phospholamban: A Common Role for an N-Terminal Di-Arginine Motif in ER Retention?

Background

Phospholamban (PLN) is an effective inhibitor of the sarco(endo)plasmic reticulum Ca²⁺-ATPase, which transports Ca²⁺ into the SR lumen, leading to muscle relaxation. A mutation of PLN in which one of the di-arginine residues at positions 13 and 14 was deleted led to a severe, early onset dilated cardiomyopathy. Here we were interested in determining the cellular mechanisms involved in this disease-causing mutation.

Methodology/Principal Finding

Mutations deleting codons for either or both Arg13 or Arg14 resulted in the mislocalization of PLN from the ER. Our data show that PLN is recycled via the retrograde Golgi to ER membrane traffic pathway involving COP-I vesicles, since co-immunoprecipitation assays determined that COP I interactions are dependent on an intact di-arginine motif as PLN RΔ14 did not co-precipitate with COP I containing vesicles. Bioinformatic analysis determined that the di-arginine motif is present in the first 25 residues in a large number of all ER/SR Gene Ontology (GO) annotated proteins. Mutations in the di-arginine motif of the Sigma 1-type opioid receptor, the β-subunit of the signal recognition particle receptor, and Sterol-O-acyltransferase, three proteins identified in our bioinformatic screen also caused mislocalization of these known ER-resident proteins.

Conclusion

We conclude that PLN is enriched in the ER due to COP I-mediated transport that is dependent on its intact di-arginine motif and that the N-terminal di-arginine motif may act as a general ER retrieval sequence.     

Inhibiting endoplasmic reticulum (ER)-associated degradation of misfolded Yor1p does not permit ER export despite the presence of a diacidic sorting signal

Capture of newly synthesized proteins into endoplasmic reticulum (ER)-derived coat protomer type II (COPII) vesicles represents a critical juncture in the quality control of protein biogenesis within the secretory pathway. The yeast ATP-binding cassette transporter Yor1p is a pleiotropic drug pump that shows homology to the human cystic fibrosis transmembrane conductance regulator (CFTR). Deletion of a phenylalanine residue in Yor1p, equivalent to the major disease-causing mutation in CFTR, causes ER retention and degradation via ER-associated degradation. We have examined the relationship between protein folding, ERAD and forward transport during Yor1p biogenesis. Uptake of Yor1p into COPII vesicles is mediated by an N-terminal diacidic signal that likely interacts with the "B-site" cargo-recognition domain on the COPII subunit, Sec24p. Yor1p-DeltaF is subjected to complex ER quality control involving multiple cytoplasmic chaperones and degradative pathways. Stabilization of Yor1p-DeltaF by inhibiting its degradation does not permit access of Yor1p-DeltaF to COPII vesicles. We propose that the ER quality control checkpoint engages misfolded Yor1p even after it has been stabilized by inhibition of the degradative pathway.     

Protein C mutation (A267T) results in ER retention and unfolded protein response activation

Background

Protein C (PC) deficiency is associated with a high risk of venous thrombosis. Recently, we identified the PC-A267T mutation in a patient with PC deficiency and revealed by in vitro studies decreased intracellular and secreted levels of the mutant. The aim of the present study was to characterize the underlying mechanism(s).

Methodology/Principal Findings

CHO-K1 cells stably expressing the wild-type (PC-wt) or the PC mutant were generated. In order to examine whether the PC mutant was subjected to increased intracellular degradation, the cells were treated with several inhibitors of various degradation pathways and pulse-chase experiments were performed. Protein-chaperone complexes were analyzed by treating the cells with a cross-linker followed by Western blotting (WB). Expression levels of the immunoglobulin-binding protein (BiP) and the phosphorylated eukaryotic initiation factor 2α (P-eIF2α), both common ER stress markers, were determined by WB to examine if the mutation induced ER stress and unfolded protein response (UPR) activation. We found no major differences in the intracellular degradation between the PC variants. The PC mutant was retained in the endoplasmic reticulum (ER) and had increased association with the Grp-94 and calreticulin chaperones. Retention of the PC-A267T in ER resulted in UPR activation demonstrated by increased expression levels of the ER stress markers BiP and P-eIF2α and caused also increased apoptotic activity in CHO-K1 cells as evidenced by elevated levels of DNA fragmentation.

Conclusions/Significance

The reduced intracellular level and impaired secretion of the PC mutant were due to retention in ER. In contrast to other PC mutations, retention of the PC-A267T in ER resulted in minor increased proteasomal degradation, rather it induced ER stress, UPR activation and apoptosis.     

Single, context-specific glycans can target misfolded glycoproteins for ER-associated degradation

The endoplasmic reticulum (ER) maintains an environment essential for secretory protein folding. Consequently, the premature transport of polypeptides would be harmful to the cell. To avert this scenario, mechanisms collectively termed "ER quality control" prevent the transport of nascent polypeptides until they properly fold. Irreversibly misfolded molecules are sorted for disposal by the ER-associated degradation (ERAD) pathway. To better understand the relationship between quality control and ERAD, we studied a new misfolded variant of carboxypeptidase Y (CPY). The molecule was recognized and retained by ER quality control but failed to enter the ERAD pathway. Systematic analysis revealed that a single, specific N-linked glycan of CPY was required for sorting into the pathway. The determinant is dependent on the putative lectin-like receptor Htm1/Mnl1p. The discovery of a similar signal in misfolded proteinase A supported the generality of the mechanism. These studies show that specific signals embedded in glycoproteins can direct their degradation if they fail to fold.     

The ERAD inhibitor Eeyarestatin I is a bifunctional compound with a membrane-binding domain and a p97/VCP inhibitory group

Background

Protein homeostasis in the endoplasmic reticulum (ER) has recently emerged as a therapeutic target for cancer treatment. Disruption of ER homeostasis results in ER stress, which is a major cause of cell death in cells exposed to the proteasome inhibitor Bortezomib, an anti-cancer drug approved for treatment of multiple myeloma and Mantle cell lymphoma. We recently reported that the ERAD inhibitor Eeyarestatin I (EerI) also disturbs ER homeostasis and has anti-cancer activities resembling that of Bortezomib.

Methodology and Principal Findings

Here we developed in vitro binding and cell-based functional assays to demonstrate that a nitrofuran-containing (NFC) group in EerI is the functional domain responsible for the cytotoxicity. Using both SPR and pull down assays, we show that EerI directly binds the p97 ATPase, an essential component of the ERAD machinery, via the NFC domain. An aromatic domain in EerI, although not required for p97 interaction, can localize EerI to the ER membrane, which improves its target specificity. Substitution of the aromatic module with another benzene-containing domain that maintains membrane localization generates a structurally distinct compound that nonetheless has similar biologic activities as EerI.

Conclusions and Significance

Our findings reveal a class of bifunctional chemical agents that can preferentially inhibit membrane-bound p97 to disrupt ER homeostasis and to induce tumor cell death. These results also suggest that the AAA ATPase p97 may be a potential drug target for cancer therapeutics.     

Inhibition of endoplasmic reticulum-associated degradation rescues native folding in loss of function protein misfolding diseases

Lysosomal storage disorders are often caused by mutations that destabilize native folding and impair trafficking of secretory proteins. We demonstrate that endoplasmic reticulum (ER)-associated degradation (ERAD) prevents native folding of mutated lysosomal enzymes in patient-derived fibroblasts from two clinically distinct lysosomal storage disorders, namely Gaucher and Tay-Sachs disease. Prolonging ER retention via ERAD inhibition enhanced folding, trafficking, and activity of these unstable enzyme variants. Furthermore, combining ERAD inhibition with enhancement of the cellular folding capacity via proteostasis modulation resulted in synergistic rescue of mutated enzymes. ERAD inhibition was achieved by cell treatment with small molecules that interfere with recognition (kifunensine) or retrotranslocation (eeyarestatin I) of misfolded substrates. These different mechanisms of ERAD inhibition were shown to enhance ER retention of mutated proteins but were associated with dramatically different levels of ER stress, unfolded protein response activation, and unfolded protein response-induced apoptosis.     

Specificity and Regulation of the Endoplasmic Reticulum‐Associated Degradation Machinery

The endoplasmic reticulum‐associated degradation (ERAD) machinery selects native and misfolded polypeptides for dislocation across the ER membrane and proteasomal degradation. Regulated degradation of native proteins is an important aspect of cell physiology. For example, it contributes to the control of lipid biosynthesis, calcium homeostasis and ERAD capacity by setting the turnover rate of crucial regulators of these pathways. In contrast, degradation of native proteins has pathologic relevance when caused by viral or bacterial infections, or when it occurs as a consequence of dysregulated ERAD activity. The efficient disposal of misfolded proteins prevents toxic depositions and persistent sequestration of molecular chaperones that could induce cellular stress and perturb maintenance of cellular proteostasis. In the first section of this review, we survey the available literature on mechanisms of selection of native and non‐native proteins for degradation from the ER and on how pathogens hijack them. In the second section, we highlight the mechanisms of ERAD activity adaptation to changes in the ER environment with a particular emphasis on the post‐translational regulatory mechanisms collectively defined as ERAD tuning.      

The Role of IRE1α in the Degradation of Insulin mRNA in Pancreatic β-Cells

Background

The endoplasmic reticulum (ER) is a cellular compartment for the biosynthesis and folding of newly synthesized secretory proteins such as insulin. Perturbations to ER homeostasis cause ER stress and subsequently activate cell signaling pathways, collectively known as the Unfolded Protein Response (UPR). IRE1α is a central component of the UPR. In pancreatic β-cells, IRE1α also functions in the regulation of insulin biosynthesis.

Principal Findings

Here we report that hyperactivation of IRE1α caused by chronic high glucose treatment or IRE1α overexpression leads to insulin mRNA degradation in pancreatic β-cells. Inhibition of IRE1α signaling using its dominant negative form prevents insulin mRNA degradation. Islets from mice heterozygous for IRE1α retain expression of more insulin mRNA after chronic high glucose treatment than do their wild-type littermates.

Conclusions/Significance

These results reveal a role of IRE1α in insulin mRNA expression under ER stress conditions caused by chronic high glucose. The rapid degradation of insulin mRNA could provide immediate relief for the ER and free up the translocation machinery. Thus, this mechanism would preserve ER homeostasis and help ensure that the insulin already inside the ER can be properly folded and secreted. This adaptation may be crucial for the maintenance of β-cell homeostasis and may explain why the β-cells of type 2 diabetic patients with chronic hyperglycemia stop producing insulin in the absence of apoptosis. This mechanism may also be involved in suppression of the autoimmune type 1 diabetes by reducing the amount of misfolded insulin, which could be a source of “neo-autoantigens.”     

Cytosolic entry of Shiga-like toxin a chain from the yeast endoplasmic reticulum requires catalytically active Hrd1p

Background

Escherichia coli Shiga-like toxin 1 normally traffics to the endoplasmic reticulum (ER) in sensitive mammalian cells from where the catalytic A chain (SLTxA1) dislocates to the cytosol to inactivate ribosomes. Currently, no molecular details of the dislocation process are available. To investigate the mechanism of the dislocation step we expressed SLTxA1 in the ER of Saccharomyces cerevisiae.

Methodology and Principal Findings

Using a combination of growth studies and biochemical tracking in yeast knock-out strains we show that SLTxA1 follows an ER-associated degradation (ERAD) pathway to enter the cytosol in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex. ER-to-cytosol dislocation of the bulk population of SLTxA1 requires Cdc48p and its ubiquitin-handling co-factor Npl4p, and this population of toxin is terminally dispatched by proteasomal degradation. A small sub-population of SLTxA1 uncouples from this classical ERAD pathway and recovers catalytic activity in the cytosol. The pathway that leads to toxicity is also Hrd1p-dependent but, unlike that for the related ricin A chain toxin, SLTxA1 dislocation does require the catalytic cysteine of Hrd1p. However it does not depend on canonical ubiquitylation since toxin variants lacking endogenous lysyl residues also utilize this pathway, and furthermore there is no requirement for a number of Cdc48p co-factors.

Conclusions and Significance

The fraction of SLTxA1 that disengages from the ERAD pathway thus does so upstream of Cdc48p interactions and downstream of Hrd1p interactions, in a step that possibly involves de-ubiquitylation. Mechanistically therefore, the dislocation of this toxin is quite distinct from that of conventional ERAD substrates that are normally degraded, and the toxins partially characterised to date that do not require the catalytic cysteine of the major Hrd1p component of the dislocation apparatus.     

Chemical chaperone 4-phenylbutyrate prevents endoplasmic reticulum stress induced by T17M rhodopsin

Background

Rhodopsin mutations are associated with the autosomal dominant form of retinitis pigmentosa. T17M mutation in rhodopsin predisposes cells to endoplasmic reticulum (ER) stress and induces cell death. This study aimed to examine whether chemical chaperone 4-phenylbutyrate prevents ER stress induced by rhodopsin T17M.

Results

ARPE-19 cells were transfected with myc-tagged wild-type (WT) and T17M rhodopsin constructs. Turnover of WT and T17M rhodopsin was measured by cycloheximide chase analysis. The activity of ubiquitin-proteasome system was evaluated by GFPU reporter. We found that T17M rhodopsin was misfolded, ubiqutinated and eliminated by ER-associated degradation pathway (ERAD) in ARPE-19 cells. Accumulated T17M rhodopsin induced unfolded protein response, but had no effect on the activity of ubiquitin proteasome system. Moreover, chemical chaperone 4-phenylbutyrate facilitated the turnover of T17M rhodopsin and prevented apoptosis and ER stress induced by T17M rhodopsin.

Conclusions

Chemical chaperone could attenuate UPR signaling and ER stress induced by T17M rhodopsin and has potential therapeutic significance for retinitis pigmentosa.

     

Disulfide Bonds within the C2 Domain of RAGE Play Key Roles in Its Dimerization and Biogenesis

Background

The receptor for advanced glycation end products (RAGE) on the cell surface transmits inflammatory signals. A member of the immunoglobulin superfamily, RAGE possesses the V, C1, and C2 ectodomains that collectively constitute the receptor''s extracellular structure. However, the molecular mechanism of RAGE biogenesis remains unclear, impeding efforts to control RAGE signaling through cellular regulation.

Methodology and Result

We used co-immunoprecipitation and crossing-linking to study RAGE oligomerization and found that RAGE forms dimer-based oligomers. Via non-reducing SDS-polyacrylamide gel electrophoresis and mutagenesis, we found that cysteines 259 and 301 within the C2 domain form intermolecular disulfide bonds. Using a modified tripartite split GFP complementation strategy and confocal microscopy, we also found that RAGE dimerization occurs in the endoplasmic reticulum (ER), and that RAGE mutant molecules without the double disulfide bridges are unstable, and are subjected to the ER-associated degradation.

Conclusion

Disulfide bond-mediated RAGE dimerization in the ER is the critical step of RAGE biogenesis. Without formation of intermolecular disulfide bonds in the C2 region, RAGE fails to reach cell surface.

Significance

This is the first report of RAGE intermolecular disulfide bond.