Endoplasmic Reticulum Oxidoreductin-1α (Ero1α) Improves Folding and Secretion of Mutant Proinsulin and Limits Mutant Proinsulin-induced Endoplasmic Reticulum Stress

Upon chronic up-regulation of proinsulin synthesis, misfolded proinsulin can accumulate in the endoplasmic reticulum (ER) of pancreatic β-cells, promoting ER stress and type 2 diabetes mellitus. In Mutant Ins-gene-induced Diabetes of Youth (MIDY), misfolded mutant proinsulin impairs ER exit of co-expressed wild-type proinsulin, limiting insulin production and leading to eventual β-cell death. In this study we have investigated the hypothesis that increased expression of ER oxidoreductin-1α (Ero1α), despite its established role in the generation of H₂O₂, might nevertheless be beneficial in limiting proinsulin misfolding and its adverse downstream consequences. Increased Ero1α expression is effective in promoting wild-type proinsulin export from cells co-expressing misfolded mutant proinsulin. In addition, we find that upon increased Ero1α expression, some of the MIDY mutants themselves are directly rescued from ER retention. Secretory rescue of proinsulin-G(B23)V is correlated with improved oxidative folding of mutant proinsulin. Indeed, using three different variants of Ero1α, we find that expression of either wild-type or an Ero1α variant lacking regulatory disulfides can rescue mutant proinsulin-G(B23)V, in parallel with its ability to provide an oxidizing environment in the ER lumen, whereas beneficial effects were less apparent for a redox-inactive form of Ero1. Increased expression of protein disulfide isomerase antagonizes the rescue provided by oxidatively active Ero1. Importantly, ER stress induced by misfolded proinsulin was limited by increased expression of Ero1α, suggesting that enhancing the oxidative folding of proinsulin may be a viable therapeutic strategy in the treatment of type 2 diabetes.     

The Endoplasmic Reticulum Stress Sensor IRE1α in Intestinal Epithelial Cells Is Essential for Protecting against Colitis

Intestinal epithelial cells (IECs) have critical roles in maintaining homeostasis of intestinal epithelium. Endoplasmic reticulum (ER) stress is implicated in intestinal epithelium homeostasis and inflammatory bowel disease; however, it remains elusive whether IRE1α, a major sensor of ER stress, is directly involved in these processes. We demonstrate here that genetic ablation of Ire1α in IECs leads to spontaneous colitis in mice. Deletion of IRE1α in IECs results in loss of goblet cells and failure of intestinal epithelial barrier function. IRE1α deficiency induces cell apoptosis through induction of CHOP, the pro-apoptotic protein, and sensitizes cells to lipopolysaccharide, an endotoxin from bacteria. IRE1α deficiency confers upon mice higher susceptibility to chemical-induced colitis. These results suggest that IRE1α functions to maintain the intestinal epithelial homeostasis and plays an important role in defending against inflammation bowel diseases.     

3 β-Hydroxysteroid-Δ 24 Reductase (DHCR24) Protects Neuronal Cells from Apoptotic Cell Death Induced by Endoplasmic Reticulum (ER) Stress

3β-Hydroxysteroid-Δ24 reductase (DHCR24) is an endoplasmic reticulum (ER)-localized multifunctional enzyme that possesses anti-apoptotic and cholesterol-synthesizing activities. Accumulating evidence suggests that ER stress is involved in the pathogenesis of neurodegenerative disease. In this study, we investigated whether DHCR24 may function as a neuroprotective protein under ER stress. Neuroblastoma N2A cells were infected with adenovirus expressing myc-tagged DHCR24 (Ad-DHCR24) or lacZ (Ad-lacZ, serving as a control) and subjected to ER-stress, induced with Tunicamycin (TM). Cells infected with Ad-DHCR24-myc were resistant to TM-induced apoptosis, and showed weaker level of caspase-12 activity. These cells also exhibited lower levels of Bip and CHOP proteins than Ad-LacZ-infected cells. Moreover, a stronger and rapid activation of PERK, and a prolonged activation of JNK and p38 were observed in Ad-LacZ–infected cells. The generation of intracellular reactive oxygen species from ER stress was also diminished by the overexpression of DHCR24. Additionally, intracellular cholesterol level was also elevated in the Ad-DHCR24-infected cells, accompanied by a well-organized formation of caveolae (cholesterol-rich microdomain) on the plasma membrane, and improved colocalization of caveolin-1 and insulin-like growth factor 1 receptor. These results demonstrated for the first time that DHCR24 could protect neuronal cells from apoptosis induced by ER stress.     

Inhibition of the Functional Interplay between Endoplasmic Reticulum (ER) Oxidoreduclin-1α (Ero1α) and Protein-disulfide Isomerase (PDI) by the Endocrine Disruptor Bisphenol A

Bisphenol A (BPA) is an endocrine disruptor that may have adverse effects on human health. We recently isolated protein-disulfide isomerase (PDI) as a BPA-binding protein from rat brain homogenates and found that BPA markedly inhibited PDI activity. To elucidate mechanisms of this inhibition, detailed structural, biophysical, and functional analyses of PDI were performed in the presence of BPA. BPA binding to PDI induced significant rearrangement of the N-terminal thioredoxin domain of PDI, resulting in more compact overall structure. This conformational change led to closure of the substrate-binding pocket in b′ domain, preventing PDI from binding to unfolded proteins. The b′ domain also plays an essential role in the interplay between PDI and ER oxidoreduclin 1α (Ero1α), a flavoenzyme responsible for reoxidation of PDI. We show that BPA inhibited Ero1α-catalyzed PDI oxidation presumably by inhibiting the interaction between the b′ domain of PDI and Ero1α; the phenol groups of BPA probably compete with a highly conserved tryptophan residue, located in the protruding β-hairpin of Ero1α, for binding to PDI. Consistently, BPA slowed down the reoxidation of PDI and caused the reduction of PDI in HeLa cells, indicating that BPA has a great impact on the redox homeostasis of PDI within cells. However, BPA had no effect on the interaction between PDI and peroxiredoxin-4 (Prx4), another PDI family oxidase, suggesting that the interaction between Prx4 and PDI is different from that of Ero1α and PDI. These results indicate that BPA, a widely distributed and potentially harmful chemical, inhibits Ero1-PDI-mediated disulfide bond formation.     

ERManI (Endoplasmic Reticulum Class I α-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum-associated Protein Degradation Pathway

Previously, we reported that the mitochondrial translocator protein (TSPO) induces HIV-1 envelope (Env) degradation via the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, but the mechanism was not clear. Here we investigated how the four ER-associated glycoside hydrolase family 47 (GH47) α-mannosidases, ERManI, and ER-degradation enhancing α-mannosidase-like (EDEM) proteins 1, 2, and 3, are involved in the Env degradation process. Ectopic expression of these four α-mannosidases uncovers that only ERManI inhibits HIV-1 Env expression in a dose-dependent manner. In addition, genetic knock-out of the ERManI gene MAN1B1 using CRISPR/Cas9 technology disrupts the TSPO-mediated Env degradation. Biochemical studies show that HIV-1 Env interacts with ERManI, and between the ERManI cytoplasmic, transmembrane, lumenal stem, and lumenal catalytic domains, the catalytic domain plays a critical role in the Env-ERManI interaction. In addition, functional studies show that inactivation of the catalytic sites by site-directed mutagenesis disrupts the ERManI activity. These studies identify ERManI as a critical GH47 α-mannosidase in the ER-associated protein degradation pathway that initiates the Env degradation and suggests that its catalytic domain and enzymatic activity play an important role in this process.     

α-Synuclein Delays Endoplasmic Reticulum (ER)-to-Golgi Transport in Mammalian Cells by Antagonizing ER/Golgi SNAREs

Toxicity of human α-synuclein when expressed in simple organisms can be suppressed by overexpression of endoplasmic reticulum (ER)-to-Golgi transport machinery, suggesting that inhibition of constitutive secretion represents a fundamental cause of the toxicity. Whether similar inhibition in mammals represents a cause of familial Parkinson''s disease has not been established. We tested elements of this hypothesis by expressing human α-synuclein in mammalian kidney and neuroendocrine cells and assessing ER-to-Golgi transport. Overexpression of wild type or the familial disease-associated A53T mutant α-synuclein delayed transport by up to 50%; however, A53T inhibited more potently. The secretory delay occurred at low expression levels and was not accompanied by insoluble α-synuclein aggregates or mistargeting of transport machinery, suggesting a direct action of soluble α-synuclein on trafficking proteins. Co-overexpression of ER/Golgi arginine soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) specifically rescued transport, indicating that α-synuclein antagonizes SNARE function. Ykt6 reversed α-synuclein inhibition much more effectively than sec22b, suggesting a possible neuroprotective role for the enigmatic high expression of ykt6 in neurons. In in vitro reconstitutions, purified α-synuclein A53T protein specifically inhibited COPII vesicle docking and fusion at a pre-Golgi step. Finally, soluble α-synuclein A53T directly bound ER/Golgi SNAREs and inhibited SNARE complex assembly, providing a potential mechanism for toxic effects in the early secretory pathway.