Dual role of the peptide-loading complex as proofreader and limiter of MHC-I presentation.

Antigen presentation via major histocompatibility complex class I (MHC-I) molecules is essential for surveillance by the adaptive immune system. Central to this process is the peptide-loading complex (PLC), which translocates peptides from the cytosol to the endoplasmic reticulum and catalyzes peptide loading and proofreading of peptide-MHC-I (pMHC-I) complexes. Despite its importance, the impact of individual PLC components on the presented pMHC-I complexes is still insufficiently understood. Here, we used stoichiometrically defined antibody-nanobody complexes and engineered soluble T cell receptors (sTCRs) to quantify different MHC-I allomorphs and defined pMHC-I complexes, respectively. Thereby, we uncovered distinct effects of individual PLC components on the pMHC-I surface pool. Knockouts of components of the PLC editing modules, namely tapasin, ERp57, or calreticulin, changed the MHC-I surface composition to a reduced proportion of HLA-A*02:01 presentation compensated by a higher ratio of HLA-B*40:01 molecules. Intriguingly, these knockouts not only increased the presentation of suboptimally loaded HLA-A*02:01 complexes but also elevated the presentation of high-affinity peptides overexpressed in the cytosol. Our findings suggest that the components of the PLC editing module serve a dual role, acting not only as peptide proofreaders but also as limiters for abundant peptides. This dual function ensures the presentation of a broad spectrum of antigenic peptides.

Protein disulfide isomerase-mediated transcriptional upregulation of Nox1 contributes to vascular dysfunction in hypertension.

Nox1 signaling is a causal key element in arterial hypertension. Recently, we identified protein disulfide isomerase A1 (PDI) as a novel regulatory protein that regulates Nox1 signaling in VSMCs. Spontaneously hypertensive rats (SHR) have increased levels of PDI in mesenteric resistance arteries compared with Wistar controls; however, its consequences remain unclear. Herein, we investigated the role of PDI in mediating Nox1 transcriptional upregulation and its effects on vascular dysfunction in hypertension. We demonstrate that PDI contributes to the development of hypertension via enhanced transcriptional upregulation of Nox1 in vascular smooth muscle cells (VSMCs). We show for the first time that PDI sulfenylation by hydrogen peroxide contributes to EGFR activation in hypertension via increased shedding of epidermal growth factor-like ligands. PDI also increases intracellular calcium levels, and contractile responses induced by ANG II. PDI silencing or pharmacological inhibition in VSMCs significantly decreases EGFR activation and Nox1 transcription. Overexpression of PDI in VSMCs enhances ANG II-induced EGFR activation and ATF1 translocation to the nucleus. Mechanistically, PDI increases ATF1-induced Nox1 transcription and enhances the contractile responses to ANG II. Herein we show that ATF1 binding to Nox1 transcription putative regulatory regions is augmented by PDI. Altogether, we provide evidence that HB-EGF in SHR resistance vessels promotes the nuclear translocation of ATF1, under the control of PDI, and thereby induces Nox1 gene expression and increases vascular reactivity. Thus, PDI acts as a thiol redox-dependent enhancer of vascular dysfunction in hypertension and could represent a novel therapeutic target for the treatment of this disease.

Enhancing the expression of the unspecific peroxygenase in Komagataella phaffii through a combination strategy.

The unspecific peroxygenase (UPO) from Cyclocybe aegerita (AaeUPO) can selectively oxidize C-H bonds using hydrogen peroxide as an oxygen donor without cofactors, which has drawn significant industrial attention. Many studies have made efforts to enhance the overall activity of AaeUPO expressed in Komagataella phaffii by employing strategies such as enzyme-directed evolution, utilizing appropriate promoters, and screening secretion peptides. Building upon these previous studies, the objective of this study was to further enhance the expression of a mutant of AaeUPO with improved activity (PaDa-I) by increasing the gene copy number, co-expressing chaperones, and optimizing culture conditions. Our results demonstrated that a strain carrying approximately three copies of expression cassettes and co-expressing the protein disulfide isomerase showed an approximately 10.7-fold increase in volumetric enzyme activity, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. After optimizing the culture conditions, the volumetric enzyme activity of this strain further increased by approximately 48.7%, reaching 117.3 U/mL. Additionally, the purified catalytic domain of PaDa-I displayed regioselective hydroxylation of R-2-phenoxypropionic acid. The results of this study may facilitate the industrial application of UPOs. KEY POINTS: • The secretion of the catalytic domain of PaDa-I can be significantly enhanced through increasing gene copy numbers and co-expressing of protein disulfide isomerase. • After optimizing the culture conditions, the volumetric enzyme activity can reach 117.3 U/mL, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. • The R-2-phenoxypropionic acid can undergo the specific hydroxylation reaction catalyzed by catalytic domain of PaDa-I, resulting in the formation of R-2-(4-hydroxyphenoxy)propionic acid.

Downregulated PDIA3P1 lncRNA Impairs Trophoblast Phenotype by Regulating Snail and SFRP1 in PE.

Preeclampsia (PE) manifests as a pregnancy-specific complication arising from compromised placentation characterized by inadequate trophoblast invasion. A growing body of evidence underscores the pivotal involvement of pseudogenes, a subset of long noncoding RNAs, in the pathological processes of PE. This study presents a novel finding, demonstrating a significant downregulation of the pseudogene PDIA3P1 in PE placental tissues compared to normal tissues. In vitro functional assays revealed that suppressing PDIA3P1 hindered trophoblast proliferation, invasion, and migration, concurrently upregulating the expression of secreted frizzled-related protein 1 (SFRP1). Further exploration of the regulatory role of PDIA3P1 in PE, utilizing human trophoblasts, established that PDIA3P1 exerts its function by binding to HuR, thereby enhancing the stability of Snail expression in trophoblasts. Overall, our findings suggest a crucial role for PDIA3P1 in regulating trophoblast properties and contributing to the pathogenesis of PE, offering potential targets for prognosis and therapeutic intervention.

Efficient production of human interleukin-3 from Escherichia coli using protein disulfide isomerase b'a' domain.

Human interleukin-3 (IL3) is a multifunctional cytokine essential for both clinical and biomedical research endeavors. However, its production in Escherichia coli has historically been challenging due to its aggregation into inclusion bodies, requiring intricate solubilization and refolding procedures. This study introduces an innovative approach employing two chaperone proteins, maltose binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a'), as N-terminal fusion tags. Histidine tag (H) was added at the beginning of each chaperone protein gene for easy purification. This fusion of chaperone proteins significantly improved IL3 solubility across various E. coli strains and temperature conditions, eliminating the need for laborious refolding procedures. Following expression optimization, H-PDIb'a'-IL3 was purified using two chromatographic methods, and the subsequent removal of the H-PDIb'a' tag yielded high-purity IL3. The identity of the purified protein was confirmed through liquid chromatography coupled with tandem mass spectrometry analysis. Biological activity assays using human erythroleukemia TF-1 cells revealed a unique two-step stimulation pattern for both purified IL3 and the H-PDIb'a'-IL3 fusion protein, underscoring the protein's functional integrity and revealing novel insights into its cellular interactions. This study advances the understanding of IL3 expression and activity while introducing novel considerations for protein fusion strategies.

Integrated Transcriptome and Proteome Analysis Reveals the Regulatory Mechanism of Root Growth by Protein Disulfide Isomerase in Arabidopsis.

Protein disulfide isomerase (PDI, EC 5.3.4.1) is a thiol-disulfide oxidoreductase that plays a crucial role in catalyzing the oxidation and rearrangement of disulfides in substrate proteins. In plants, PDI is primarily involved in regulating seed germination and development, facilitating the oxidative folding of storage proteins in the endosperm, and also contributing to the formation of pollen. However, the role of PDI in root growth has not been previously studied. This research investigated the impact of PDI gene deficiency in plants by using 16F16 [2-(2-Chloroacetyl)-2,3,4,9-tetrahydro-1-methyl-1H-pyrido[3,4-b]indole-1-carboxylic acid methyl ester], a small-molecule inhibitor of PDI, to remove functional redundancy. The results showed that the growth of Arabidopsis roots was significantly inhibited when treated with 16F16. To further investigate the effects of 16F16 treatment, we conducted expression profiling of treated roots using RNA sequencing and a Tandem Mass Tag (TMT)-based quantitative proteomics approach at both the transcriptomic and proteomic levels. Our analysis revealed 994 differentially expressed genes (DEGs) at the transcript level, which were predominantly enriched in pathways associated with "phenylpropane biosynthesis", "plant hormone signal transduction", "plant-pathogen interaction" and "starch and sucrose metabolism" pathways. Additionally, we identified 120 differentially expressed proteins (DEPs) at the protein level. These proteins were mainly enriched in pathways such as "phenylpropanoid biosynthesis", "photosynthesis", "biosynthesis of various plant secondary metabolites", and "biosynthesis of secondary metabolites" pathways. The comprehensive transcriptome and proteome analyses revealed a regulatory network for root shortening in Arabidopsis seedlings under 16F16 treatment, mainly involving phenylpropane biosynthesis and plant hormone signal transduction pathways. This study enhances our understanding of the significant role of PDIs in Arabidopsis root growth and provides insights into the regulatory mechanisms of root shortening following 16F16 treatment.

Protein Disulfide Isomerase 4 Is an Essential Regulator of Endothelial Function and Survival.

Endothelial autophagy plays an important role in the regulation of endothelial function. The inhibition of endothelial autophagy is associated with the reduced expression of protein disulfide isomerase 4 (PDIA-4); however, its role in endothelial cells is not known. Here, we report that endothelial cell-specific loss of PDIA-4 leads to impaired autophagic flux accompanied by loss of endothelial function and apoptosis. Endothelial cell-specific loss of PDIA-4 also induced marked changes in endothelial cell architecture, accompanied by the loss of endothelial markers and the gain of mesenchymal markers consistent with endothelial-to-mesenchymal transition (EndMT). The loss of PDIA-4 activated TGFß-signaling, and inhibition of TGFß-signaling suppressed EndMT in PDIA-4-silenced endothelial cells in vitro. Our findings help elucidate the role of PDIA-4 in endothelial autophagy and endothelial function and provide a potential target to modulate endothelial function and/or limit autophagy and EndMT in (patho-)physiological conditions.

PDIA2 has a dual function in promoting androgen deprivation therapy induced venous thrombosis events and castrate resistant prostate cancer progression.

Androgen deprivation therapy (ADT) is the first line of treatment for metastatic prostate cancer (PCa) that effectively delays the tumor progression. However, it also increases the risk of venous thrombosis event (VTE) in patients, a leading cause of mortality. How a pro-thrombotic cascade is induced by ADT remains poorly understood. Here, we report that protein disulfide isomerase A2 (PDIA2) is upregulated in PCa cells to promote VTE formation and enhance PCa cells resistant to ADT. Using various in vitro and in vivo models, we demonstrated a dual function of PDIA2 that enhances tumor-mediated pro-coagulation activity via tumor-derived extracellular vehicles (EVs). It also stimulates PCa cell proliferation, colony formation, and xenograft growth androgen-independently. Mechanistically, PDIA2 activates the tissue factor (TF) on EVs through its isomerase activity, which subsequently triggers a pro-thrombotic cascade in the blood. Additionally, TF-containing EVs can activate the Src kinase inside PCa cells to enhance the AR signaling ligand independently. Androgen deprivation does not alter PDIA2 expression in PCa cells but enhances PDIA2 translocation to the cell membrane and EVs via suppressing the clathrin-dependent endocytic process. Co-recruitment of AR and FOXA1 to the PDIA2 promoter is required for PDIA2 transcription under androgen-deprived conditions. Importantly, blocking PDIA2 isomerase activity suppresses the pro-coagulation activity of patient plasma, PCa cell, and xenograft samples as well as castrate-resistant PCa xenograft growth. These results demonstrate that PDIA2 promotes VTE and tumor progression via activating TF from tumor-derived EVs. They rationalize pharmacological inhibition of PDIA2 to suppress ADT-induced VTE and castrate-resistant tumor progression.

Protein disulfide isomerase cleaves allosteric disulfides in histidine-rich glycoprotein to regulate thrombosis.

The essence of difference between hemostasis and thrombosis is that the clotting reaction is a highly fine-tuned process. Vascular protein disulfide isomerase (PDI) represents a critical mechanism regulating the functions of hemostatic proteins. Herein we show that histidine-rich glycoprotein (HRG) is a substrate of PDI. Reduction of HRG by PDI enhances the procoagulant and anticoagulant activities of HRG by neutralization of endothelial heparan sulfate (HS) and inhibition of factor XII (FXIIa) activity, respectively. Murine HRG deficiency (Hrg-/-) leads to delayed onset but enhanced formation of thrombus compared to WT. However, in the combined FXII deficiency (F12-/-) and HRG deficiency (by siRNA or Hrg-/-), there is further thrombosis reduction compared to F12-/- alone, confirming HRG's procoagulant activity independent of FXIIa. Mutation of target disulfides of PDI leads to a gain-of-function mutant of HRG that promotes its activities during coagulation. Thus, PDI-HRG pathway fine-tunes thrombosis by promoting its rapid initiation via neutralization of HS and preventing excessive propagation via inhibition of FXIIa.

The role and mechanism of TXNDC5 in disease progression.

Thioredoxin domain containing protein-5 (TXNDC5), also known as endothelial protein-disulfide isomerase (Endo-PDI), is confined to the endoplasmic reticulum through the structural endoplasmic reticulum retention signal (KDEL), is a member of the PDI protein family and is highly expressed in the hypoxic state. TXNDC5 can regulate the rate of disulfide bond formation, isomerization and degradation of target proteins through its function as a protein disulfide isomerase (PDI), thereby altering protein conformation, activity and improving protein stability. Several studies have shown that there is a significant correlation between TXNDC5 gene polymorphisms and genetic susceptibility to inflammatory diseases such as rheumatoid, fibrosis and tumors. In this paper, we detail the expression characteristics of TXNDC5 in a variety of diseases, summarize the mechanisms by which TXNDC5 promotes malignant disease progression, and summarize potential therapeutic strategies to target TXNDC5 for disease treatment.

Protein disulfide isomerase 1 is required for RodA assembling-based conidial hydrophobicity of Aspergillus fumigatus.

The hydrophobic layer of Aspergillus conidia, composed of RodA, plays a crucial role in conidia transfer and immune evasion. It self-assembles into hydrophobic rodlets through intramolecular disulfide bonds. However, the secretory process of RodA and its regulatory elements remain unknown. Since protein disulfide isomerase (PDI) is essential for the secretion of many disulfide-bonded proteins, we investigated whether PDI is also involved in RodA secretion and assembly. By gene knockout and phenotypic analysis, we found that Pdi1, one of the four PDI-related proteins of Aspergillus fumigatus, determines the hydrophobicity and integrity of the rodlet layer of the conidia. Preservation of the thioredoxin-active domain of Pdi1 was sufficient to maintain conidial hydrophobicity, suggesting that Pdi1 mediates RodA assembly through its disulfide isomerase activity. In the absence of Pdi1, the disulfide mismatch of RodA in conidia may prevent its delivery from the inner to the outer layer of the cell wall for rodlet assembly. This was demonstrated using a strain expressing a key cysteine-mutated RodA. The dormant conidia of the Pdi1-deficient strain (Δpdi) elicited an immune response, suggesting that the defective conidia surface in the absence of Pdi1 exposes internal immunogenic sources. In conclusion, Pdi1 ensures the correct folding of RodA in the inner layer of conidia, facilitating its secretion into the outer layer of the cell wall and allowing self-assembly of the hydrophobic layer. This study has identified a regulatory element for conidia rodlet assembly.IMPORTANCEAspergillus fumigatus is the major cause of invasive aspergillosis, which is mainly transmitted by the inhalation of conidia. The spread of conidia is largely dependent on their hydrophobicity, which is primarily attributed to the self-assembly of the hydrophobic protein RodA on the cell wall. However, the mechanisms underlying RodA secretion and transport to the outermost layer of the cell wall are still unclear. Our study identified a critical role for Pdi1, a fungal protein disulfide isomerase found in regulating RodA secretion and assembly. Inhibition of Pdi1 prevents the formation of correct S-S bonds in the inner RodA, creating a barrier to RodA delivery and resulting in a defective hydrophobic layer. Our findings provided insight into the formation of the conidial hydrophobic layer and suggested potential drug targets to inhibit A. fumigatus infections by limiting conidial dispersal and altering their immune inertia.

Development of a sensor for disulfide bond formation in diverse bacteria.

In bacteria, disulfide bonds contribute to the folding and stability of proteins important for processes in the cellular envelope. In Escherichia coli, disulfide bond formation is catalyzed by DsbA and DsbB enzymes. DsbA is a periplasmic protein that catalyzes disulfide bond formation in substrate proteins, while DsbB is an inner membrane protein that transfers electrons from DsbA to quinones, thereby regenerating the DsbA active state. Actinobacteria including mycobacteria use an alternative enzyme named VKOR, which performs the same function as DsbB. Disulfide bond formation enzymes, DsbA and DsbB/VKOR, represent novel drug targets because their inhibition could simultaneously affect the folding of several cell envelope proteins including virulence factors, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. We have previously developed a cell-based and target-based assay to identify molecules that inhibit the DsbB and VKOR in pathogenic bacteria, using E. coli cells expressing a periplasmic ß-Galactosidase sensor (ß-Galdbs), which is only active when disulfide bond formation is inhibited. Here, we report the construction of plasmids that allows fine-tuning of the expression of the ß-Galdbs sensor and can be mobilized into other gram-negative organisms. As an example, when expressed in Pseudomonas aeruginosa UCBPP-PA14, which harbors two DsbB homologs, ß-Galdbs behaves similarly as in E. coli, and the biosensor responds to the inhibition of the two DsbB proteins. Thus, these ß-Galdbs reporter plasmids provide a basis to identify novel inhibitors of DsbA and DsbB/VKOR in multidrug-resistant gram-negative pathogens and to further study oxidative protein folding in diverse gram-negative bacteria. IMPORTANCE: Disulfide bonds contribute to the folding and stability of proteins in the bacterial cell envelope. Disulfide bond-forming enzymes represent new drug targets against multidrug-resistant bacteria because inactivation of this process would simultaneously affect several proteins in the cell envelope, including virulence factors, toxins, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. Identifying the enzymes involved in disulfide bond formation in gram-negative pathogens as well as their inhibitors can contribute to the much-needed antibacterial innovation. In this work, we developed sensors of disulfide bond formation for gram-negative bacteria. These tools will enable the study of disulfide bond formation and the identification of inhibitors for this crucial process in diverse gram-negative pathogens.

Hepatitis E virus ORF3 protein hijacking thioredoxin domain-containing protein 5 (TXNDC5) for its stability to promote viral particle release.

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide, responsible for approximately 20 million infections annually. Among the three open reading frames (ORFs) of the HEV genome, the ORF3 protein is involved in virus release. However, the host proteins involved in HEV release need to be clarified. In this study, a host protein, thioredoxin domain-containing protein 5 (TXNDC5), interacted with the non-palmitoylated ORF3 protein by co-immunoprecipitation analysis. We determined that the overexpression or knockdown of TXNDC5 positively regulated HEV release from the host cells. The 17FCL19 mutation of the ORF3 protein lost the ability to interact with TXNDC5. The releasing amounts of HEV with the ORF3 mutation (FCL17-19SSP) were decreased compared with wild-type HEV. The overexpression of TXNDC5 can stabilize and increase ORF3 protein amounts, but not the TXNDC5 mutant with amino acids 1-88 deletion. Meanwhile, we determined that the function of TXNDC5 on the stabilization of ORF3 protein is independent of the Trx-like domains. Knockdown of TXNDC5 could lead to the degradation of ORF3 protein by the endoplasmic reticulum (ER)-associated protein degradation-proteasome system. However, the ORF3 protein cannot be degraded in the knockout-TXNDC5 stable cells, suggesting that it may hijack other proteins for its stabilization. Subsequently, we found that the other members of protein disulfide isomerase (PDI), including PDIA1, PDIA3, PDIA4, and PDIA6, can increase ORF3 protein amounts, and PDIA3 and PDIA6 interact with ORF3 protein. Collectively, our study suggested that HEV ORF3 protein can utilize TXNDC5 for its stability in ER to facilitate viral release. IMPORTANCE: Hepatitis E virus (HEV) infection is the leading cause of acute viral hepatitis worldwide. After the synthesis and modification in the cells, the mature ORF3 protein is essential for HEV release. However, the host protein involved in this process has yet to be determined. Here, we reported a novel host protein, thioredoxin domain-containing protein 5 (TXNDC5), as a chaperone, contributing to HEV release by facilitating ORF3 protein stability in the endoplasmic reticulum through interacting with non-palmitoylated ORF3 protein. However, we also found that in the knockout-TXNDC5 stable cell lines, the HEV ORF3 protein may hijack other proteins for its stabilization. For the first time, our study demonstrated the involvement of TXNDC5 in viral particle release. These findings provide some new insights into the process of the HEV life cycle, the interaction between HEV and host factors, and a new direction for antiviral design.

Cellular responses to silencing of PDIA3 (protein disulphide-isomerase A3): Effects on proliferation, migration, and genes in control of active vitamin D.

The active form of vitamin D, 1,25-dihydroxyvitamin D3, is known to act via VDR (vitamin D receptor), affecting several physiological processes. In addition, PDIA3 (protein disulphide-isomerase A3) has been associated with some of the functions of 1,25-dihydroxyvitamin D3. In the present study we used siRNA-mediated silencing of PDIA3 in osteosarcoma and prostate carcinoma cell lines to examine the role(s) of PDIA3 for 1,25-dihydroxyvitamin D3-dependent responses. PDIA3 silencing affected VDR target genes and significantly altered the 1,25-dihydroxyvitamin D3-dependent induction of CYP24A1, essential for elimination of excess 1,25-dihydroxyvitamin D3. Also, PDIA3 silencing significantly altered migration and proliferation in prostate PC3 cells, independently of 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 increased thermostability of PDIA3 in cellular thermal shift assay, supporting functional interaction between PDIA3 and 1,25-dihydroxyvitamin D3-dependent pathways. In summary, our data link PDIA3 to 1,25-dihydroxyvitamin D3-mediated signalling, underline and extend its role in proliferation and reveal a novel function in maintenance of 1,25-dihydroxyvitamin D3 levels.

Synergistic promotion of transient transgene expression in CHO cells by PDI/XBP-1s co-transfection and mild hypothermia.

Transient gene expression system is an important tool for rapid production of recombinant proteins in Chinese hamster ovary (CHO) cells. However, their low productivity is the main hurdle to overcome. An effective approach through which to obtain high protein yield involves targeting transcriptional, post-transcriptional events (PTEs), and culture conditions. Here, we investigated the effects of protein disulfide isomerase (PDI) and spliced X-box binding protein 1 (XBP-1s) co-overexpression combined with mild hypothermia on the transient yields of recombinant proteins in CHO cells. The results showed that the gene of interest (GOI) and the PDI/XBP-1s helper vector at a co-transfection ratio of 10:1 could obviously increase transient expression level of recombinant protein in CHO cells. However, PDI/XBP-1s overexpression had no significance effect on the mRNA levels of the recombinant protein, suggesting that it targeted PTEs. Moreover, the increased production was due to the enhancing of cell specific productivity, not related to cell growth, viability, and cell cycle. In addition, combined PDI/XBP-1s co-overexpression and mild hypothermia could further improve Adalimumab expression, compared to the control/37 °C and PDI/XBP-1s/37 °C, the Adalimumab volume yield of PDI/XBP-1s/33 °C increased by 203% and 142%, respectively. Mild hypothermia resulted in 3.52- and 2.33-fold increase in the relative mRNA levels of PDI and XBP-1s, respectively. In conclusion, the combination of PDI/XBP-1s overexpression and culture temperature optimization can achieve higher transient expression of recombinant protein, which provides a synergetic strategy to improve transient production of recombinant protein in CHO cells.

Downregulation of PDIA3 inhibits gastric cancer cell growth through cell cycle regulation.

OBJECTIVE: Protein disulfide isomerase A3 (PDIA3) promotes the correct folding of newly synthesized glycoproteins in the endoplasmic reticulum. PDIA3 is overexpressed in most tumors, and it may become a biomarker of cancer prognosis and immunotherapy. Our study aims to detect the expression level of PDIA3 in gastric cancer (GC) and its association with GC development as wells as the underlying mechanisms. METHODS: GC cell lines with PDIA3 knockdown by siRNA, CRISPR-cas9 sgRNAs or a pharmacological inhibitor of LOC14 were prepared and used. PDIA3 knockout GC cells were established by CRISPR-cas9-PDIA3 system. The proliferation, migration, invasion and cell cycle of GC cells were analyzed by cell counting kit-8 assay, wound healing assay, transwell assay and flow cytometry, respectively. Immunodeficient nude mice was used to evaluate the role of PDIA3 in tumor formation. Quantitative PCR and western blot were used for examining gene and protein expressions. RNA sequencing was performed to see the altered gene expression. RESULTS: The expressions of PDIA3 in GC tissues and cells were increased significantly, and its expression was negatively correlated with the three-year survival rate of GC patients. Down-regulation of PDIA3 by siRNA, LOC14 or CRISPR-cas9 significantly inhibited proliferation, invasion and migration of GC cells TMK1 and AGS, with cell cycle arrested at G2/M phase. Meanwhile, decreased PDIA3 significantly inhibited growth of tumor xenograft in vivo. It was found that cyclin G1 (encoded by CCNG1 gene) expression was decreased by downregulation of PDIA3 in GC cells both in vitro and in vivo. In addition, protein levels of other cell cycle related factors including cyclin D1, CDK2, and CDK6 were also significantly decreased. Further study showed that STAT3 was associated with PDIA3-mediated cyclin G1 regulation. CONCLUSION: PDIA3 plays an oncogenic role in GC. Our findings unfolded the functional role of PDIA3 in GC development and highlighted a novel target for cancer therapeutic strategy.

A novel PDIA3/FTO/USP20 positive feedback regulatory loop induces osteogenic differentiation of preosteoblast in osteoporosis.

Osteoporosis is a chronic skeletal disease and the major source of risk for fractures in aged people. It is urgent to investigate the mechanism regulating osteoporosis for developing potential treatment and prevention strategies. Osteogenic differentiation of preosteoblast enhances bone formation, which might be a promising strategy for treatment and prevention of osteoporosis. Protein disulfide isomerase family A, member 3 (PDIA3) could induce bone formation, yet the role of PDIA3 in osteogenic differentiation of preosteoblast remains unknown. In this study, m6 A RNA methylation was detected by methylated RNA immunoprecipitation (MeRIP), while mRNA stability was identified by RNA decay assay. Besides, protein-protein interaction and protein phosphorylation were determined using co-immunoprecipitation (Co-IP). Herein, results revealed that PDIA3 promoted osteogenic differentiation of preosteoblast MC3T3-E1. Besides, PDIA3 mRNA methylation was suppressed by FTO alpha-ketoglutarate dependent dioxygenase (FTO) as RNA methylation reduced PDIA3 mRNA stability during osteogenic differentiation of MC3T3-E1 cells. Moreover, ubiquitin specific peptidase 20 (USP20) improved FTO level through inhibiting FTO degradation while PDIA3 increased FTO level by enhancing USP20 phosphorylation during osteogenic differentiation of MC3T3-E1 cells, suggesting a positive feedback regulatory loop between PDIA3 and FTO. In summary, these findings indicated the mechanism of PDIA3 regulating osteogenic differentiation of preosteoblast and provided potential therapeutic targets for osteoporosis.

CdbC: a disulfide bond isomerase involved in the refolding of mycoloyltransferases in Corynebacterium glutamicum cells exposed to oxidative conditions.

In Corynebacterium glutamicum cells, cdbC, which encodes a protein containing the CysXXCys motif, is regulated by the global redox-responsive regulator OsnR. In this study, we assessed the role of the periplasmic protein CdbC in disulfide bond formation and its involvement in mycomembrane biosynthesis. Purified CdbC efficiently refolded scrambled RNaseA, exhibiting prominent disulfide bond isomerase activity. The transcription of cdbC was decreased in cells grown in the presence of the reductant dithiothreitol (DTT). Moreover, unlike wild-type and cdbC-deleted cells, cdbC-overexpressing (P180-cdbC) cells grown in the presence of DTT exhibited retarded growth, abnormal cell morphology, increased cell surface hydrophobicity and altered mycolic acid composition. P180-cdbC cells cultured in a reducing environment accumulated trehalose monocorynomycolate, indicating mycomembrane deformation. Similarly, a two-hybrid analysis demonstrated the interaction of CdbC with the mycoloyltransferases MytA and MytB. Collectively, our findings suggest that CdbC, a periplasmic disulfide bond isomerase, refolds misfolded MytA and MytB and thereby assists in mycomembrane biosynthesis in cells exposed to oxidative conditions.

Genome-wide analysis and expression divergence of protein disulfide isomerase (PDI) gene family members in chickpea (Cicer arietinum) under salt stress.

Chickpea (Cicer arietinum ) is a grain crop that is an important source of protein, vitamins, carbohydrates and minerals. It is highly sensitive to salt stress, and salt damage to cellular homeostasis and protein folding affects production. Plants have several mechanisms to prevent cellular damages under abiotic stresses, such as proteins in the endoplasmic reticulum (protein isulfide somerases (PDIs) and PDI-like proteins), which help prevent the build-up of mis-folded proteins that are damaged under abiotic stresses. In this study, we completed initial comprehensive genome-wide analysis of the chickpea PDI gene family. We found eight PDI genes are distributed on six out of eight chromosomes. Two pairs of paralogous genes were found to have segmental duplications. The phylogenetic analysis showed that the PDI s have a high degree of homology in C. arietinum, Cicer reticulatum, Lens culinaris, Phaseolus acutifolius, Pisum sativum and Oryza sativa . The gene structure analysis displayed that CaPDI1-CaPDI8 have 9-12 exons except for CaPDI5 , which has 25 exons. Subcellular localisation indicated accumulation of CaPDIs in endoplasmic reticulum. Protein-conserved motifs and domain analysis demonstrated that thioredoxin domains of PDI family is present in all CaPDIs. CaPDI proteins have strong protein-protein interaction. In silico expression analysis showed that four out of eight PDI genes (CPDI2, CaPDI6, CaPDI7 and CaPDI8 ) were expressed under salt stress. Of these, expression of CaPDI2 and CaPDI8 was the highest. This work indicated that PDI genes are involved in salt stress tolerance in chickpea and the CaPDIs may be further studied for their role of inducing salt tolerance.

Protein disulfide isomerase family member 4 promotes triple-negative breast cancer tumorigenesis and radiotherapy resistance through JNK pathway.

BACKGROUND: Despite radiotherapy ability to significantly improve treatment outcomes and survival in triple-negative breast cancer (TNBC) patients, acquired resistance to radiotherapy poses a serious clinical challenge. Protein disulfide isomerase exists in endoplasmic reticulum and plays an important role in promoting protein folding and post-translational modification. However, little is known about the role of protein disulfide isomerase family member 4 (PDIA4) in TNBC, especially in the context of radiotherapy resistance. METHODS: We detected the presence of PDIA4 in TNBC tissues and paracancerous tissues, then examined the proliferation and apoptosis of TNBC cells with/without radiotherapy. As part of the validation process, xenograft tumor mouse model was used. Mass spectrometry and western blot analysis were used to identify PDIA4-mediated molecular signaling pathway. RESULTS: Based on paired clinical specimens of TNBC patients, we found that PDIA4 expression was significantly higher in tumor tissues compared to adjacent normal tissues. In vitro, PDIA4 knockdown not only increased apoptosis of tumor cells with/without radiotherapy, but also decreased the ability of proliferation. In contrast, overexpression of PDIA4 induced the opposite effects on apoptosis and proliferation. According to Co-IP/MS results, PDIA4 prevented Tax1 binding protein 1 (TAX1BP1) degradation by binding to TAX1BP1, which inhibited c-Jun N-terminal kinase (JNK) activation. Moreover, PDIA4 knockdown suppressed tumor growth xenograft model in vivo, which was accompanied by an increase in apoptosis and promoted tumor growth inhibition after radiotherapy. CONCLUSIONS: The results of this study indicate that PDIA4 is an oncoprotein that promotes TNBC progression, and targeted therapy may represent a new and effective anti-tumor strategy, especially for patients with radiotherapy resistance.