UGGT1 retains proinsulin in the endoplasmic reticulum in an arginine dependent manner.
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ABSTRACT:
SUBMITTER: Cho J
PROVIDER: S-EPMC7863631 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Protein folding in the endoplasmic reticulum (ER) is error prone, and ER quality control (ERQC) processes ensure that only correctly folded proteins are exported from the ER. Glycoproteins can be retained in the ER by ERQC, and this retention contributes to multiple human diseases, termed ER storage diseases. UDP-glucose:glycoprotein glucosyltransferase (UGGT1) acts as a central component of glycoprotein ERQC, monoglucosylating deglucosylated N-glycans of incompletely folded glycoproteins and promoting subsequent reassociation with the lectin-like chaperones calreticulin and calnexin. The extent to which UGGT1 influences glycoprotein folding, however, has only been investigated for a few selected substrates. Using mouse embryonic fibroblasts lacking UGGT1 or those with UGGT1 complementation, we investigated the effect of monoglucosylation on the soluble/insoluble distribution of two misfolded α1-antitrypsin (AAT) variants responsible for AAT deficiency disease: null Hong Kong (NHK) and Z allele. Whereas substrate solubility increases directly with the number of N-linked glycosylation sites, our results indicate that additional solubility is conferred by UGGT1 enzymatic activity. Monoglucosylation-dependent solubility decreases both BiP association with NHK and unfolded protein response activation, and the solubility increase is blocked in cells deficient for calreticulin. These results suggest that UGGT1-dependent monoglucosylation of N-linked glycoproteins promotes substrate solubility in the ER.
| S-EPMC3756913 | biostudies-literature
Project description:Proinsulin folding within the endoplasmic reticulum (ER) remains incompletely understood, but it is clear that in mutant INS gene-induced diabetes of youth (MIDY), progression of the (three) native disulfide bonds of proinsulin becomes derailed, causing insulin deficiency, ?-cell ER stress, and onset of diabetes. Herein, we have undertaken a molecular dissection of proinsulin disulfide bond formation, using bioengineered proinsulins that can form only two (or even only one) of the native proinsulin disulfide bonds. In the absence of preexisting proinsulin disulfide pairing, Cys(B19)-Cys(A20) (a major determinant of ER stress response activation and proinsulin stability) preferentially initiates B-A chain disulfide bond formation, whereas Cys(B7)-Cys(A7) can initiate only under oxidizing conditions beyond that existing within the ER of ?-cells. Interestingly, formation of these two "interchain" disulfide bonds demonstrates cooperativity, and together, they are sufficient to confer intracellular transport competence to proinsulin. The three most common proinsulin disulfide mispairings in the ER appear to involve Cys(A11)-Cys(A20), Cys(A7)-Cys(A20), and Cys(B19)-Cys(A11), each disrupting the critical Cys(B19)-Cys(A20) pairing. MIDY mutations inhibit Cys(B19)-Cys(A20) formation, but treatment to force oxidation of this disulfide bond improves folding and results in a small but detectable increase of proinsulin export. These data suggest possible therapeutic avenues to ameliorate ER stress and diabetes.
| S-EPMC4806660 | biostudies-literature
Project description:The tumor suppressor p53 is a key protein in preventing cell transformation and tumor progression. Activated by a variety of stimuli, p53 regulates cell-cycle arrest and apoptosis. Along with its well-documented transcriptional control over cell-death programs within the nucleus, p53 exerts crucial although still poorly understood functions in the cytoplasm, directly modulating the apoptotic response at the mitochondrial level. Calcium (Ca(2+)) transfer between the endoplasmic reticulum (ER) and mitochondria represents a critical signal in the induction of apoptosis. However, the mechanism controlling this flux in response to stress stimuli remains largely unknown. Here we show that, in the cytoplasm, WT p53 localizes at the ER and at specialized contact domains between the ER and mitochondria (mitochondria-associated membranes). We demonstrate that, upon stress stimuli, WT p53 accumulates at these sites and modulates Ca(2+) homeostasis. Mechanistically, upon activation, WT p53 directly binds to the sarco/ER Ca(2+)-ATPase (SERCA) pump at the ER, changing its oxidative state and thus leading to an increased Ca(2+) load, followed by an enhanced transfer to mitochondria. The consequent mitochondrial Ca(2+) overload causes in turn alterations in the morphology of this organelle and induction of apoptosis. Pharmacological inactivation of WT p53 or naturally occurring p53 missense mutants inhibits SERCA pump activity at the ER, leading to a reduction of the Ca(2+) signaling from the ER to mitochondria. These findings define a critical nonnuclear function of p53 in regulating Ca(2+) signal-dependent apoptosis.
| S-EPMC4330769 | biostudies-literature
Project description:Endoplasmic reticulum (ER) homeostasis is essential for cell function. Increasing evidence indicates that, efficient protein ER export is important for ER homeostasis. However, the consequence of impaired ER export remains largely unknown. Herein, we found that defective ER protein transport caused by either Sar1 mutants or brefeldin A impaired proinsulin oxidative folding in the ER of β-cells. Misfolded proinsulin formed aberrant disulfide-linked dimers and high molecular weight proinsulin complexes, and induced ER stress. Limiting proinsulin load to the ER alleviated ER stress, indicating that misfolded proinsulin is a direct cause of ER stress. This study revealed significance of efficient ER export in maintaining ER protein homeostasis and native folding of proinsulin. Given the fact that proinsulin misfolding plays an important role in diabetes, this study suggests that enhancing ER export may be a potential therapeutic target to prevent/delay β-cell failure caused by proinsulin misfolding and ER stress.
| S-EPMC6613978 | biostudies-literature
Project description:Although endoplasmic reticulum (ER) chaperone binding to mutant proinsulin has been reported, the role of protein chaperones in the handling of wild-type proinsulin is underinvestigated. Here, we have explored the importance of glucose-regulated protein 94 (GRP94), a prominent ER chaperone known to fold insulin-like growth factors, in proinsulin handling within β-cells. We found that GRP94 coimmunoprecipitated with proinsulin and that inhibition of GRP94 function and/or expression reduced glucose-dependent insulin secretion, shortened proinsulin half-life, and lowered intracellular proinsulin and insulin levels. This phenotype was accompanied by post-ER proinsulin misprocessing and higher numbers of enlarged insulin granules that contained amorphic material with reduced immunogold staining for mature insulin. Insulin granule exocytosis was accelerated twofold, but the secreted insulin had diminished bioactivity. Moreover, GRP94 knockdown or knockout in β-cells selectively activated protein kinase R-like endoplasmic reticulum kinase (PERK), without increasing apoptosis levels. Finally, GRP94 mRNA was overexpressed in islets from patients with type 2 diabetes. We conclude that GRP94 is a chaperone crucial for proinsulin handling and insulin secretion.
| S-EPMC6425875 | biostudies-literature
Project description:To maintain secretory pathway fidelity, misfolded proteins are commonly retained in the endoplasmic reticulum (ER) and selected for ER-associated degradation (ERAD). Soluble misfolded proteins use ER chaperones for retention, but the machinery that restricts aberrant membrane proteins to the ER is unclear. In fact, some misfolded membrane proteins escape the ER and traffic to the lysosome/vacuole. To this end, we describe a model substrate, SZ∗, that contains an ER export signal but is also targeted for ERAD. We observe decreased ER retention when chaperone-dependent SZ∗ ubiquitination is compromised. In addition, appending a linear tetra-ubiquitin motif onto SZ∗ overrides ER export. By screening known ubiquitin-binding proteins, we then positively correlate SZ∗ retention with Ubx2 binding. Deletion of Ubx2 also inhibits the retention of another misfolded membrane protein. Our results indicate that polyubiquitination is sufficient to retain misfolded membrane proteins in the ER prior to ERAD.
| S-EPMC8503845 | biostudies-literature
Project description:Insulin is an essential peptide hormone that maintains blood glucose levels. Although the mechanisms underlying insulin exocytosis have been investigated, the mechanism of proinsulin export from the endoplasmic reticulum (ER) remains unclear. Here, we demonstrated that Surf4, a cargo receptor homolog, regulates the ER export of proinsulin via its recruitment to ER exit sites (ERES). Under high-glucose conditions, Surf4 expression was upregulated, and Surf4 proteins mainly localized to the ER at a steady state and accumulated in the ERES, along with proinsulin in rat insulinoma INS-1 cells. Surf4-knockdown resulted in proinsulin retention in the ER and decreased the levels of mature insulin in secretory granules, thereby significantly reducing insulin secretion. Surf4 forms an oligomer and can physically interact with proinsulin and Sec12, essential for COPII vesicle formation. Our findings suggest that Surf4 interacts with proinsulin and delivers it into COPII vesicles for ER export in co-operation with Sec12 and COPII.
| S-EPMC9106718 | biostudies-literature
Project description:The conserved endoplasmic reticulum (ER) membrane protein TRAPα (translocon-associated protein, also known as signal sequence receptor 1, SSR1) has been reported to play a critical but unclear role in insulin biosynthesis. TRAPα/SSR1 is one component of a four-protein complex including TRAPβ/SSR2, TRAPγ/SSR3, and TRAPδ/SSR4. The TRAP complex topologically has a small exposure on the cytosolic side of the ER via its TRAPγ/SSR3 subunit, whereas TRAPβ/SSR2 and TRAPδ/SSR4 function along with TRAPα/SSR1 largely on the luminal side of the ER membrane. Here, we have examined pancreatic β-cells with deficient expression of either TRAPβ/SSR2 or TRAPδ/SSR4, which does not perturb mRNA expression levels of other TRAP subunits, or insulin mRNA. However, deficient protein expression of TRAPβ/SSR2 and, to a lesser degree, TRAPδ/SSR4, diminishes the protein levels of other TRAP subunits, concomitant with deficient steady-state levels of proinsulin and insulin. Deficient TRAPβ/SSR2 or TRAPδ/SSR4 is not associated with any apparent defect of exocytotic mechanism but rather by a decreased abundance of the proinsulin and insulin that accompanies glucose-stimulated secretion. Amino acid pulse labeling directly establishes that much of the steady-state deficiency of intracellular proinsulin can be accounted for by diminished proinsulin biosynthesis, observed in a pulse-labeling as short as 5 minutes. The proinsulin and insulin levels in TRAPβ/SSR2 or TRAPδ/SSR4 null mutant β-cells are notably recovered upon re-expression of the missing TRAP subunit, accompanying a rebound of proinsulin biosynthesis. Remarkably, overexpression of TRAPα/SSR1 can also suppress defects in β-cells with diminished expression of TRAPβ/SSR2, strongly suggesting that TRAPβ/SSR2 is needed to support TRAPα/SSR1 function.
| S-EPMC8106808 | biostudies-literature
Project description:Primary defects in folding of mutant proinsulin can cause dominant-negative proinsulin accumulation in the endoplasmic reticulum (ER), impaired anterograde proinsulin trafficking, perturbed ER homeostasis, diminished insulin production, and β-cell dysfunction. Conversely, if primary impairment of ER-to-Golgi trafficking (which also perturbs ER homeostasis) drives misfolding of nonmutant proinsulin-this might suggest bi-directional entry into a common pathological phenotype (proinsulin misfolding, perturbed ER homeostasis, and deficient ER export of proinsulin) that can culminate in diminished insulin storage and diabetes. Here, we've challenged β-cells with conditions that impair ER-to-Golgi trafficking, and devised an accurate means to assess the relative abundance of distinct folded/misfolded forms of proinsulin using a novel nonreducing SDS-PAGE/immunoblotting protocol. We confirm abundant proinsulin misfolding upon introduction of a diabetogenic INS mutation, or in the islets of db/db mice. Whereas blockade of proinsulin trafficking in Golgi/post-Golgi compartments results in intracellular accumulation of properly-folded proinsulin (bearing native disulfide bonds), impairment of ER-to-Golgi trafficking (regardless whether such impairment is achieved by genetic or pharmacologic means) results in decreased native proinsulin with more misfolded proinsulin. Remarkably, reversible ER-to-Golgi transport defects (such as treatment with brefeldin A or cellular energy depletion) upon reversal quickly restore the ER folding environment, resulting in the disappearance of pre-existing misfolded proinsulin while preserving proinsulin bearing native disulfide bonds. Thus, proper homeostatic balance of ER-to-Golgi trafficking is linked to a more favorable proinsulin folding (as well as trafficking) outcome.
| S-EPMC10955614 | biostudies-literature
Project description:Excessive production of unsaturated aldehydes from oxidized lipoproteins and membrane lipids is a characteristic feature of cardiovascular disease. Our previous studies show that unsaturated lipid peroxidation-derived aldehydes such as 4-hydroxy-trans-2-nonenal (HNE) promote autophagy in rat aortic smooth muscle cells (RASMC). In this study, we examined the mechanism by which HNE induces autophagy. Exposure of RASMC to HNE led to the modification of several proteins, most of which were identified by mass spectrometry and confocal microscopy to be localized to the endoplasmic reticulum (ER). HNE stimulated the phosphorylation of PKR-like ER kinase and eukaryotic initiation factor 2α and increased heme oxygenase-1 (HO-1) abundance. HNE treatment also increased LC3-II formation and the phosphorylation of JNK and p38. Pharmacological inhibition of JNK, but not p38, prevented HNE-induced HO-1 expression and LC3-II formation. Inhibition of JNK increased cell death in HNE-treated cells. Pretreatment with the chemical chaperone phenylbutryic acid prevented LC3-II formation as well as JNK phosphorylation and HO-1 induction. Taken together, these data suggest that autophagic responses triggered by unsaturated aldehydes could be attributed, in part, to ER stress, which stimulates autophagy by a JNK-dependent mechanism and promotes cell survival during oxidative stress.
| S-EPMC3757667 | biostudies-literature