Oxidative protein folding fidelity and redoxtasis in the endoplasmic reticulum.

In eukaryotic cells, oxidative protein folding occurs in the lumen of the endoplasmic reticulum (ER), catalyzed by ER sulfhydryl oxidase 1 (Ero1) and protein disulfide isomerase (PDI). The efficiency and fidelity of oxidative protein folding are vital for the function of secretory cells. Here, we summarize oxidative protein folding in yeast, plants, and mammals, and discuss how the conformation and activity of human Ero1-PDI machinery is regulated through various post-translational modifications (PTMs). We propose that oxidative protein folding fidelity and ER redox homeostasis are maintained by both the precise control of Ero1 oxidase activity and the division of labor between PDI family members. We also discuss how deregulated Ero1-PDI functions contribute to human diseases and can be leveraged for therapeutic interventions.

Reducing oxidative protein folding alleviates senescence by minimizing ER-to-nucleus H2 O2 release.

Oxidative protein folding occurs in the endoplasmic reticulum (ER) to generate disulfide bonds, and the by-product is hydrogen peroxide (H2 O2 ). However, the relationship between oxidative protein folding and senescence remains uncharacterized. Here, we find that the protein disulfide isomerase (PDI), a key oxidoreductase that catalyzes oxidative protein folding, accumulated in aged human mesenchymal stem cells (hMSCs) and deletion of PDI alleviated hMSCs senescence. Mechanistically, knocking out PDI slows the rate of oxidative protein folding and decreases the leakage of ER-derived H2 O2 into the nucleus, thereby decreasing the expression of SERPINE1, which was identified as a key driver of cell senescence. Furthermore, we show that depletion of PDI alleviated senescence in various cell models of aging. Our findings reveal a previously unrecognized role of oxidative protein folding in promoting cell aging, providing a potential target for aging and aging-related disease intervention.

Phosphorylation switches protein disulfide isomerase activity to maintain proteostasis and attenuate ER stress.

Accumulated unfolded proteins in the endoplasmic reticulum (ER) trigger the unfolded protein response (UPR) to increase ER protein folding capacity. ER proteostasis and UPR signaling need to be regulated in a precise and timely manner. Here, we identify phosphorylation of protein disulfide isomerase (PDI), one of the most abundant and critical folding catalysts in the ER, as an early event during ER stress. The secretory pathway kinase Fam20C phosphorylates Ser357 of PDI and responds rapidly to various ER stressors. Phosphorylation of Ser357 induces an open conformation of PDI and turns it from a "foldase" into a "holdase", which is critical for preventing protein misfolding in the ER. Phosphorylated PDI also binds to the lumenal domain of IRE1α, a major UPR signal transducer, and attenuates excessive IRE1α activity. Importantly, PDI-S359A knock-in mice display enhanced IRE1α activation and liver damage under acute ER stress. We conclude that the Fam20C-PDI axis constitutes a post-translational response to maintain ER proteostasis and plays a vital role in protecting against ER stress-induced cell death.

Proteolytic processing of secretory pathway kinase Fam20C by site-1 protease promotes biomineralization.

Family with sequence similarity 20C (Fam20C), the major protein kinase in the secretory pathway, generates the vast majority of the secreted phosphoproteome. However, the regulatory mechanisms of Fam20C transport, secretion, and function remain largely unexplored. Here, we show that Fam20C exists as a type II transmembrane protein within the secretory compartments, with its N-terminal signal peptide-like region serving as a membrane anchor for Golgi retention. The secretion and kinase activity of Fam20C are governed by site-1 protease (S1P), a key regulator of cholesterol homeostasis. We find that only mature Fam20C processed by S1P functions in osteoblast differentiation and mineralization. Together, our findings reveal a unique mechanism for Fam20C secretion and activation via proteolytic regulation, providing a molecular link between biomineralization and lipid metabolism.

Two protein disulfide isomerase subgroups work synergistically in catalyzing oxidative protein folding.

Disulfide bonds play essential roles in the folding of secretory and plasma membrane proteins in the endoplasmic reticulum (ER). In eukaryotes, protein disulfide isomerase (PDI) is an enzyme catalyzing the disulfide bond formation and isomerization in substrates. The Arabidopsis (Arabidopsis thaliana) genome encodes diverse PDIs including structurally distinct subgroups PDI-L and PDI-M/S. It remains unclear how these AtPDIs function to catalyze the correct disulfide formation. We found that one Arabidopsis ER oxidoreductin-1 (Ero1), AtERO1, can interact with multiple PDIs. PDI-L members AtPDI2/5/6 mainly serve as an isomerase, while PDI-M/S members AtPDI9/10/11 are more efficient in accepting oxidizing equivalents from AtERO1 and catalyzing disulfide bond formation. Accordingly, the pdi9/10/11 triple mutant exhibited much stronger inhibition than pdi1/2/5/6 quadruple mutant under dithiothreitol treatment, which caused disruption of disulfide bonds in plant proteins. Furthermore, AtPDI2/5 work synergistically with PDI-M/S members in relaying disulfide bonds from AtERO1 to substrates. Our findings reveal the distinct but overlapping roles played by two structurally different AtPDI subgroups in oxidative protein folding in the ER.

Protein disulfide isomerase is regulated in multiple ways: Consequences for conformation, activities, and pathophysiological functions.

Protein disulfide isomerase (PDI) is one of the most abundant and critical protein folding catalysts in the endoplasmic reticulum of eukaryotic cells. PDI consists of four thioredoxin domains and interacts with a wide range of substrate and partner proteins due to its intrinsic conformational flexibility. PDI plays multifunctional roles in a variety of pathophysiological events, both as an oxidoreductase and a molecular chaperone. Recent studies have revealed that the conformation and activity of PDI can be regulated in multiple ways, including posttranslational modification and substrate/ligand binding. Here, we summarize recent advances in understanding the function and regulation of PDI in different pathological and physiological events. We propose that the multifunctional roles of PDI are regulated by multiple mechanisms. Furthermore, we discuss future directions for the study of PDI, emphasizing how different regulatory modes are linked to the conformational changes and biological functions of PDI in the context of diverse pathophysiologies.

Secretory kinase Fam20C tunes endoplasmic reticulum redox state via phosphorylation of Ero1α.

Family with sequence similarity 20C (Fam20C), the physiological Golgi casein kinase, phosphorylates numerous secreted proteins that are involved in a wide variety of biological processes. However, the role of Fam20C in regulating proteins in the endoplasmic reticulum (ER) lumen is largely unknown. Here, we report that Fam20C interacts with various luminal proteins and that its depletion results in a more reduced ER lumen. We further show that ER oxidoreductin 1α (Ero1α), the pivotal sulfhydryl oxidase that catalyzes disulfide formation in the ER, is phosphorylated by Fam20C in the Golgi apparatus and retrograde-transported to the ER mediated by ERp44. The phosphorylation of Ser145 greatly enhances Ero1α oxidase activity and is critical for maintaining ER redox homeostasis and promoting oxidative protein folding. Notably, phosphorylation of Ero1α is induced under hypoxia, reductive stress, and secretion-demanding conditions such as mammalian lactation. Collectively, our findings open a door to uncover how oxidative protein folding is regulated by phosphorylation in the secretory pathway.

Flavonoids from Lycium barbarum Leaves Exhibit Anti-Aging Effects through the Redox-Modulation.

Lycium barbarum leaves are a kind of vegetable, and modern nutrition studies have found that they have an anti-aging function. Our study aims to investigate the anti-aging effects of Lycium barbarum leaf flavonoid (LBLF) extracts and its underlying molecular mechanism. LBLFs were purified using D101 and polyamide resin, characterized by ultraperformance liquid chromatography coupled with mass spectrometry, and administered to hydrogen peroxide (H2O2)-treated human umbilical vein endothelial cells (HUVECs) and Caenorhabditis elegans. Appropriate enrichment conditions were optimized through dynamic adsorption and desorption experiments, the content of flavonoids reached 909.84 mg/g, rutin and kaempferol being the main ones. LBLFs attenuated H2O2-induced HUVEC apoptosis, decreased reactive oxygen species and malondialdehyde production levels, increased superoxide dismutase, glutathione peroxidase and catalase activities. Furthermore, pre-treatment with LBLF increased mRNA expression of erythropoietin (EPO) and heme oxygenase-1 (HO-1) via the mitogen-activated protein kinase (MAPK) signaling pathway in HUVECs. Compared with 100 µM rutin monomer, LBLF prolonged the lifespan of Caenorhabditis elegans, enhanced their mobility in middle life stages and upregulated expression of sod-2, gcs-1 and skn-1 genes, which indicated that the anti-aging effects of LBLF were due to its redox-modulation.

AtERO1 and AtERO2 Exhibit Differences in Catalyzing Oxidative Protein Folding in the Endoplasmic Reticulum.

Disulfide bonds are essential for the folding of the eukaryotic secretory and membrane proteins in the endoplasmic reticulum (ER), and ER oxidoreductin-1 (Ero1) and its homologs are the major disulfide donors that supply oxidizing equivalents in the ER. Although Ero1 homologs in yeast (Saccharomyces cerevisiae) and mammals have been extensively studied, the mechanisms of plant Ero1 functions are far less understood. Here, we found that both Arabidopsis (Arabidopsis thaliana) ERO1 and its homolog AtERO2 are required for oxidative protein folding in the ER. The outer active site, the inner active site, and a long-range noncatalytic disulfide bond are required for AtERO1's function. Interestingly, AtERO1 and AtERO2 also exhibit significant differences. The ero1 plants are more sensitive to reductive stress than the ero2 plants. In vivo, both AtERO1 and AtERO2 have two distinct oxidized isoforms (Ox1 and Ox2), which are determined by the formation or breakage of the putative regulatory disulfide. AtERO1 is mainly present in the Ox1 redox state, while more AtERO2 exists in the Ox2 state. Furthermore, AtERO1 showed much stronger oxidative protein-folding activity than AtERO2 in vitro. Taken together, both AtERO1 and AtERO2 are required to regulate efficient and faithful oxidative protein folding in the ER, but AtERO1 may serves as the primary sulfhydryl oxidase relative to AtERO2.

Crystal and solution structures of human protein-disulfide isomerase-like protein of the testis (PDILT) provide insight into its chaperone activity.

Protein-disulfide isomerase-like protein of the testis (PDILT), a member of the protein-disulfide isomerase family, is a chaperone essential for the folding of spermatogenesis-specific proteins in male postmeiotic germ cells. However, the structural mechanisms that regulate the chaperone function of PDILTs are unknown. Here, we report the structures of human PDILT (hPDILT) determined by X-ray crystallography to 2.4 Å resolution and small-angle X-ray scattering (SAXS). Distinct from previously reported U-like structures of related PDI family proteins, our structures revealed that hPDILT folds into a compact L-like structure in crystals and into an extended chain-like structure in solution. The hydrophobic regions and the hydrophobic pockets in hPDILT, which are important for substrate recognition, were clearly delineated in the crystal structure. Moreover, our results of the SAXS analysis and of structure-based substitutions and truncations indicated that the C-terminal tail in hPDILT is required for suppression of aggregation of denatured proteins, suggesting that the tail is crucial for the chaperone activity of PDILT. Taken together, our findings have identified the critical regions and conformational changes of PDILT that enable and control its activity. These results advance our understanding of the structural mechanisms involved in the chaperone activity of PDILT.

Novel Roles of the Non-catalytic Elements of Yeast Protein-disulfide Isomerase in Its Interplay with Endoplasmic Reticulum Oxidoreductin 1.

The formation of disulfide bonds in the endoplasmic reticulum (ER) of eukaryotic cells is catalyzed by the sulfhydryl oxidase, ER oxidoreductin 1 (Ero1), and protein-disulfide isomerase (PDI). PDI is oxidized by Ero1 to continuously introduce disulfides into substrates, and feedback regulates Ero1 activity by manipulating the regulatory disulfides of Ero1. In this study we find that yeast Ero1p is enzymatically active even with its regulatory disulfides intact, and further activation of Ero1p by reduction of the regulatory disulfides requires the reduction of non-catalytic Cys(90)-Cys(97)disulfide in Pdi1p. The principal client-binding site in the Pdi1pb' domain is necessary not only for the functional Ero1p-Pdi1p disulfide relay but also for the activation of Ero1p. We also demonstrate by complementary activation assays that the regulatory disulfides in Ero1p are much more stable than those in human Ero1α. These new findings on yeast Ero1p-Pdi1p interplay reveal significant differences from our previously identified mode of human Ero1α-PDI interplay and provide insights into the evolution of the eukaryotic oxidative protein folding pathway.

Different interaction modes for protein-disulfide isomerase (PDI) as an efficient regulator and a specific substrate of endoplasmic reticulum oxidoreductin-1α (Ero1α).

Protein-disulfide isomerase (PDI) and sulfhydryl oxidase endoplasmic reticulum oxidoreductin-1α (Ero1α) constitute the pivotal pathway for oxidative protein folding in the mammalian endoplasmic reticulum (ER). Ero1α oxidizes PDI to introduce disulfides into substrates, and PDI can feedback-regulate Ero1α activity. Here, we show the regulatory disulfide of Ero1α responds to the redox fluctuation in ER very sensitively, relying on the availability of redox active PDI. The regulation of Ero1α is rapidly facilitated by either a or a' catalytic domain of PDI, independent of the substrate binding domain. On the other hand, activated Ero1α specifically binds to PDI via hydrophobic interactions and preferentially catalyzes the oxidation of domain a'. This asymmetry ensures PDI to function simultaneously as an oxidoreductase and an isomerase. In addition, several PDI family members are also characterized to be potent regulators of Ero1α. The novel modes for PDI as a competent regulator and a specific substrate of Ero1α govern efficient and faithful oxidative protein folding and maintain the ER redox homeostasis.

Human protein-disulfide isomerase is a redox-regulated chaperone activated by oxidation of domain a'.

Protein-disulfide isomerase (PDI), with domains arranged as abb'xa'c, is a key enzyme and chaperone localized in the endoplasmic reticulum (ER) catalyzing oxidative folding and preventing misfolding/aggregation of proteins. It has been controversial whether the chaperone activity of PDI is redox-regulated, and the molecular basis is unclear. Here, we show that both the chaperone activity and the overall conformation of human PDI are redox-regulated. We further demonstrate that the conformational changes are triggered by the active site of domain a', and the minimum redox-regulated cassette is located in b'xa'. The structure of the reduced bb'xa' reveals for the first time that domain a' packs tightly with both domain b' and linker x to form one compact structural module. Oxidation of domain a' releases the compact conformation and exposes the shielded hydrophobic areas to facilitate its high chaperone activity. Thus, the study unequivocally provides mechanistic insights into the redox-regulated chaperone activity of human PDI.

Structural insights into the peroxidase activity and inactivation of human peroxiredoxin 4.

Prx4 (peroxiredoxin 4) is the only peroxiredoxin located in the ER (endoplasmic reticulum) and a proposed scavenger for H2O2. In the present study, we solved crystal structures of human Prx4 in three different redox forms and characterized the reaction features of Prx4 with H2O2. Prx4 exhibits a toroid-shaped decamer constructed of five catalytic dimers. Structural analysis revealed conformational changes around helix α2 and the C-terminal reigon with a YF (Tyr-Phe) motif from the partner subunit, which are required for interchain disulfide formation between Cys87 and Cys208, a critical step of the catalysis. The structural explanation for the restricting role of the YF motif on the active site dynamics is provided in detail. Prx4 has a high reactivity with H2O2, but is susceptible to overoxidation and consequent inactivation by H2O2. Either deletion of the YF motif or dissociation into dimers decreased the susceptibility of Prx4 to overoxidation by increasing the flexibility of Cys87.

Growth differentiation factor-15 is independently associated with metabolic syndrome and hyperglycemia in non-elderly subjects.

Metabolic syndrome (MetS) is a major health issue worldwide accompanied by cardiovascular comorbidities. Growth differentiation factor-15 (GDF-15) is a stress-responsive cytokine expressed in cardiomyocytes, adipocytes, macrophages, and endothelial cells. Previous research in elderly subjects revealed that GDF-15 levels were associated with the MetS. However, the association between GDF-15 levels and MetS or its components in the non-elderly subjects remains unclear. In this study, a total of 279 subjects younger than 65-year-old with (n = 84) or without (n = 195) MetS were recruited. MetS was defined according to modified NCEP/ATP III criteria. The GDF-15 levels were measured by an enzyme-linked immunosorbent assay. A multiple linear regression analysis was conducted to identify factors independently associated with GDF-15 levels. Subjects with MetS had higher GDF-15 levels than those without MetS (median (interquartile range), 1.72 ng/mL (1.38, 2.26) vs. 1.63 ng/mL (1.27, 2.07), P = 0.037). With the number of MetS components increased, the GDF-15 levels increased significantly (P for trend = 0.005). Multiple linear regression analysis revealed that the presence of MetS was positively associated with the GDF-15 levels (ß = 0.132, P = 0.037). When substituting MetS with its components, only the presence of hyperglycemia was positively associated with the GDF-15 levels after adjustment for covariates (ß = 0.193, P = 0.003). Taken together, the presence of the MetS in non-elderly was associated with higher GDF-15 levels. Among the MetS components, only hyperglycemia was significantly associated with the GDF-15 levels. Future longitudinal studies will be needed to explore whether GDF-15 has the potential to be a biomarker of gluco-metabolic dysfunction in non-elderly subjects.

Compensatory Increase of Serum Hepassocin Protects Hyperthyroidism-Induced Hepatic Dysfunction.

Hepatic dysfunction is commonly observed in subjects with hyperthyroidism. Hepassocin is a hepatokine playing an important role in metabolic diseases and exhibiting a hepatic protective effect. Nevertheless, the relationship between hepassocin and hyperthyroidism was still unknown. In the present study, a total of 36 subjects with Graves' disease were enrolled, and we found that the alanine aminotransferase (ALT) levels were significantly decreased in parallel with the decrement in serum hepassocin concentrations at 6 months after standard treatment for hyperthyroidism. In addition, HepG2 cell line was used to investigate the role of hepassocin in hyperthyroidism-induced hepatic dysfunction. Treatment of hepassocin recombinant protein in HepG2 cells dose-dependently decreased triiodothyronine (T3)-induced ALT and aspartate aminotransferase (AST) elevation. Moreover, hepassocin significantly increased the expression of phosphoenolpyruvate carboxykinase (PEPCK) in a dose-dependent manner. Deletion of hepassocin in HepG2 cells reversed the effects of T3 on PEPCK expressions. Furthermore, we found that T3 increased the expression of hepassocin through a hepatocyte nuclear factor 1α-dependent pathway. Taken together, these results indicated a compensatory increase in serum hepassocin might have a protective role in hyperthyroidism-induced hepatic dysfunction.

V-ATPase recruitment to ER exit sites switches COPII-mediated transport to lysosomal degradation.

Endoplasmic reticulum (ER)-phagy is crucial to regulate the function and homeostasis of the ER via lysosomal degradation, but how it is initiated is unclear. Here we discover that Z-AAT, a disease-causing mutant of α1-antitrypsin, induces noncanonical ER-phagy at ER exit sites (ERESs). Accumulation of misfolded Z-AAT at the ERESs impairs coat protein complex II (COPII)-mediated ER-to-Golgi transport and retains V0 subunits that further assemble V-ATPase at the arrested ERESs. V-ATPase subsequently recruits ATG16L1 onto ERESs to mediate in situ lipidation of LC3C. FAM134B-II is then recruited by LC3C via its LIR motif and elicits ER-phagy leading to efficient lysosomal degradation of Z-AAT. Activation of this ER-phagy mediated by the V-ATPase-ATG16L1-LC3C axis (EVAC) is also triggered by blocking ER export. Our findings identify a pathway which switches COPII-mediated transport to lysosomal degradation for ER quality control.

The endoplasmic reticulum sulfhydryl oxidase Ero1ß drives efficient oxidative protein folding with loose regulation.

In eukaryotes, disulfide bonds are formed in the endoplasmic reticulum, facilitated by the Ero1 (endoplasmic reticulum oxidoreductin 1) oxidase/PDI (protein disulfide-isomerase) system. Mammals have two ERO1 genes, encoding Ero1α and Ero1ß proteins. Ero1ß is constitutively expressed in professional secretory tissues and induced during the unfolded protein response. In the present work, we show that recombinant human Ero1ß is twice as active as Ero1α in enzymatic assays. Ero1ß oxidizes PDI more efficiently than other PDI family members and drives oxidative protein folding preferentially via the active site in the á domain of PDI. Our results reveal that Ero1ß oxidase activity is regulated by long-range disulfide bonds and that Cys130 plays a critical role in feedback regulation. Compared with Ero1α, however, Ero1ß is loosely regulated, consistent with its role as a more active oxidase when massive oxidative power is required.

The extracellular Ero1α/PDI electron transport system regulates platelet function by increasing glutathione reduction potential.

Protein disulfide isomerase (PDI), an oxidoreductase, possesses two vicinal cysteines in the -Cys-Gly-His-Cys-motif that either form a disulfide bridge (S-S) or exist in a sulfhydryl form (-SH), forming oxidized or reduced PDI, respectively. PDI has been proven to be critical for platelet aggregation, thrombosis, and hemostasis, and PDI inhibition is being evaluated as a novel antithrombotic strategy. The redox states of functional PDI during the regulation of platelet aggregation, however, remain to be elucidated. Endoplasmic reticulum (ER) oxidoreductin-1α (Ero1α) and PDI constitute the pivotal oxidative folding pathway in the ER and play an important role in ER redox homeostasis. Whether Ero1α and PDI constitute an extracellular electron transport pathway to mediate platelet aggregation is an open question. Here, we found that oxidized but not reduced PDI promotes platelet aggregation. On the platelet surface, Ero1α constitutively oxidizes PDI and further regulates platelet aggregation in a glutathione-dependent manner. The Ero1α/PDI system oxidizes reduced glutathione (GSH) and establishes a reduction potential optimal for platelet aggregation. Therefore, platelet aggregation is mediated by the Ero1α-PDI-GSH electron transport system on the platelet surface. We further showed that targeting the functional interplay between PDI and Ero1α by small molecule inhibitors may be a novel strategy for antithrombotic therapy.

SARS-CoV-2 ORF8 reshapes the ER through forming mixed disulfides with ER oxidoreductases.

The replication machinery of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is closely associated with the endoplasmic reticulum (ER) in host cells. Activation of the unfolded protein response (UPR) is a strategy hijacked by coronavirus to facilitate its replication and suppress host innate immunity. Here, we have found that SARS-CoV-2 ORF8 protein accumulates in the ER and escapes the degradation system by forming mixed disulfide complexes with ER oxidoreductases. ORF8 induces the activation of three UPR pathways through targeting key UPR components, remodels ER morphology and accelerates protein trafficking. Moreover, small molecule reducing agents release ORF8 from the mixed disulfide complexes and facilitate its degradation, therefore mitigate ER stress. Our study reveals a unique mechanism by which SARS-CoV-2 ORF8 escapes degradation by host cells and regulates ER reshaping. Targeting ORF8-involved mixed disulfide complexes could be a new strategy to alleviate SARS-CoV-2 induced ER stress and related diseases.