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.

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.

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.

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.

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.

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.

Endoplasmic Reticulum Protein TXNDC5 Interacts with PRDX6 and HSPA9 to Regulate Glutathione Metabolism and Lipid Peroxidation in the Hepatic AML12 Cell Line.

Non-alcoholic fatty liver disease or steatosis is an accumulation of fat in the liver. Increased amounts of non-esterified fatty acids, calcium deficiency, or insulin resistance may disturb endoplasmic reticulum (ER) homeostasis, which leads to the abnormal accumulation of misfolded proteins, activating the unfolded protein response. The ER is the primary location site for chaperones like thioredoxin domain-containing 5 (TXNDC5). Glutathione participates in cellular oxidative stress, and its interaction with TXNDC5 in the ER may decrease the disulfide bonds of this protein. In addition, glutathione is utilized by glutathione peroxidases to inactivate oxidized lipids. To characterize proteins interacting with TXNDC5, immunoprecipitation and liquid chromatography-mass spectrometry were used. Lipid peroxidation, reduced glutathione, inducible phospholipase A2 (iPLA2) and hepatic transcriptome were assessed in the AML12 and TXNDC5-deficient AML12 cell lines. The results showed that HSPA9 and PRDX6 interact with TXNDC5 in AML12 cells. In addition, TXNDC5 deficiency reduced the protein levels of PRDX6 and HSPA9 in AML12. Moreover, lipid peroxidation, glutathione and iPLA2 activities were significantly decreased in TXNDC5-deficient cells, and to find the cause of the PRDX6 protein reduction, proteasome suppression revealed no considerable effect on it. Finally, hepatic transcripts connected to PRDX6 and HSPA9 indicated an increase in the Dnaja3, Mfn2 and Prdx5 and a decrease in Npm1, Oplah, Gstp3, Gstm6, Gstt1, Serpina1a, Serpina1b, Serpina3m, Hsp90aa1 and Rps14 mRNA levels in AML12 KO cells. In conclusion, the lipid peroxidation system and glutathione mechanism in AML12 cells may be disrupted by the absence of TXNDC5, a novel protein-protein interacting partner of PRDX6 and HSPA9.

Protein Disulfide Isomerase Family A Member 3 Knockout Abrogate Effects of Vitamin D on Cellular Respiration and Glycolysis in Squamous Cell Carcinoma.

PDIA3 is an endoplasmic reticulum disulfide isomerase, which is involved in the folding and trafficking of newly synthesized proteins. PDIA3 was also described as an alternative receptor for the active form of vitamin D (1,25(OH)2D3). Here, we investigated an impact of PDIA3 in mitochondrial morphology and bioenergetics in squamous cell carcinoma line A431 treated with 1,25(OH)2D3. It was observed that PDIA3 deletion resulted in changes in the morphology of mitochondria including a decrease in the percentage of mitochondrial section area, maximal diameter, and perimeter. The 1,25(OH)2D3 treatment of A431∆PDIA3 cells partially reversed the effect of PDIA3 deletion increasing aforementioned parameters; meanwhile, in A431WT cells, only an increase in mitochondrial section area was observed. Moreover, PDIA3 knockout affected mitochondrial bioenergetics and modulated STAT3 signaling. Oxygen consumption rate (OCR) was significantly increased, with no visible effect of 1,25(OH)2D3 treatment in A431∆PDIA3 cells. In the case of Extracellular Acidification Rate (ECAR), an increase was observed for glycolysis and glycolytic capacity parameters in the case of non-treated A431WT cells versus A431∆PDIA3 cells. The 1,25(OH)2D3 treatment had no significant effect on glycolytic parameters. Taken together, the presented results suggest that PDIA3 is strongly involved in the regulation of mitochondrial bioenergetics in cancerous cells and modulation of its response to 1,25(OH)2D3, possibly through STAT3.

PDIA6 and Maspin in Prostate Cancer.

BACKGROUND/AIM: PDIA6 is a disulphide isomerase of the PDI family, known to mediate disulphide bond formation in the endoplasmic reticulum. However, PDI-related proteins also function in other parts of the cell and PDIA6 has been shown to be involved in many types of cancers. We previously identified PDIA6 as a putative Maspin interactor. Maspin has itself been implicated in prostate cancer progression. Our aim was to further explore the roles of Maspin in prostate cancer and establish whether PDIA6 is also involved in prostate cancer. MATERIALS AND METHODS: RNA levels of PDIA6 and Maspin in prostate cell lines were measured using RT-PCR. Bioinformatics analysis of the TCGA database was used to find RNA levels of PDIA6 and Maspin in prostate cancer. siRNAs were used to knock-down PDIA6, and proliferation and migration assays were conducted on those cells. RESULTS: PDIA6 and Maspin RNA were shown to be expressed at varying levels in prostate cell lines. RNAseq data showed that PDIA6 expression was significantly increased in prostate adenocarcinoma samples, while Maspin RNA expression was decreased. When PDIA6 expression was knocked-down using siRNA in prostate cell lines, proliferation was decreased substantially in the two prostate cancer cell lines (DU145 and PC3) and also decreased in the normal prostate cell line (PNT1a), though less strongly. CONCLUSION: PDIA6 expression is higher in prostate cancer cells compared to normal prostate cells. Decreasing PDIA6 expression decreases proliferation. Thus, PDIA6 is a promising target for prostate cancer therapeutics.

Protein Disulfide Isomerase A2 Is Correlated with Immune Infiltrates and Is a Novel Prognostic Biomarker in Glioma Patients.

OBJECTIVE: Protein disulfide isomerase A2 (PDIA2), a member of the protein disulfide isomerase family, plays a key role in the folding of nascent proteins in the endoplasmic reticulum by forming disulfide bonds, together with enzymes such as thiol isomerase, oxidase, and reductase. This study investigated the clinical significance and potential functions of PDIA2 in glioma. METHODS: The expression of PDIA2 in gliomas was explored using The Cancer Genome Atlas and Gene Expression Omnibus databases. We analyzed the clinical characteristics of glioma patients and the prognostic and diagnostic value of PDIA2 expression. Kaplan-Meier and Cox regression analyses were used to examine the effect of PDIA2 expression on overall survival, progression-free interval, and disease-specific survival. Furthermore, we performed Gene Set Enrichment Analysis and immune infiltration analysis to investigate the functions of PDIA2. PDIA2 mRNA and protein expression was evaluated in cell lines and glioma tissues. RESULTS: PDIA2 was expressed at low levels in glioma patients. Kaplan-Meier survival analysis showed that glioma patients with low PDIA2 levels had a worse prognosis than those with high PDIA2 levels. Receiver operating characteristic curve analysis indicated the diagnostic and prognostic ability of PDIA2 (area under the curve = 0.918). Pathways associated with PD1, PI3K/AKT, cancer immunotherapy via PD1 blockade, Fceri-mediated NF-kB activation, FOXM1, and DNA repair were enriched in glioma patients with low levels of PDIA2. PDIA2 expression levels were negatively correlated with immune cell infiltrate levels. CONCLUSION: PDIA2 levels are significantly downregulated in glioma. PDIA2 expression may be a potential biomarker for the diagnosis and prognosis of glioma patients.

Circular RNA P4HB promotes glycolysis and tumor progression by binding with PKM2 in lung adenocarcinoma.

BACKGROUND: Emerging evidence indicates that circular RNAs (circRNAs) play vital roles in tumor progression, including lung adenocarcinomas (LUAD). However, the mechanisms by which circRNAs promote the progression of LUAD still require further investigation. METHODS: Quantitative real-time PCR was performed to detect the expression of circP4HB in LUAD tissues and cells. Then, Kaplan-Meier analysis was used to determine the prognostic value of circP4HB expression. We employed RNA pull-down, RNA immunoprecipitation, mass spectrometry, cells fraction, glucose consumption, lactate production, pyruvate kinase M2 (PKM2) activity, and macrophage polarization assays to uncover the underlying mechanisms of circP4HB in LUAD. RESULTS: We found that circP4HB is upregulated in LUAD tissues and correlated with advanced TNM stages and lymph node metastasis. LUAD patients with high circP4HB expression had poor prognoses. Functionally, circP4HB promoted LUAD progression in vivo and in vitro. Upregulated circP4HB increased glucose consumption, lactate production and accelerated aerobic glycolysis in LUAD cells. Mechanically, circP4HB mainly accumulated in the cytoplasm of LUAD cells and bound with PKM2 and subsequently upregulating PKM2 enzymatic activity by increasing its tetramer formation. Additionally, circP4HB promoted M2 macrophage phenotype shift via targeting PKM2. Finally, rescue assays further confirmed that circP4HB could promote LUAD cell progression through its interaction with PKM2. CONCLUSION: These results demonstrate that circP4HB could promote LUAD progression, indicating circP4HB might be a potential therapeutic target of LUAD.

Protein Disulfide Isomerase A3 (PDIA3): A Pharmacological Target in Glioblastoma?

The protein disulfide isomerase A3 (PDIA3) is directly or indirectly involved in various physiopathological processes and participates in cancer initiation, progression and chemosensitivity. However, little is known about its involvement in glioblastoma. To obtain specific information, we performed cellular experiments in the T98G and U-87 MG glioblastoma cell lines to evaluate the role of PDIA3. The loss of PDIA3 functions, either through inhibition or silencing, reduced glioblastoma cells spreading by triggering cytotoxic phenomena. PDIA3 inhibition led to a redistribution of PDIA3, resulting in the formation of protein aggregates visualized through immunofluorescence staining. Concurrently, cell cycle progression underwent arrest at the G1/S checkpoint. After PDIA3 inhibition, ROS-independent DNA damage and the activation of the repair system occurred, as evidenced by the phosphorylation of H2A.X and the overexpression of the Ku70 protein. We also demonstrated through a clonogenic assay that PDIA3 inhibition could increase the chemosensitivity of T98G and U-87 MG cells to the approved glioblastoma drug temozolomide (TMZ). Overall, PDIA3 inhibition induced cytotoxic effects in the analyzed glioblastoma cell lines. Although further in vivo studies are needed, the results suggested PDIA3 as a novel therapeutic target that could also be included in already approved therapies.

PDIA3 modulates genomic response to 1,25-dihydroxyvitamin D3 in squamous cell carcinoma of the skin.

An active form of vitamin D3 (1,25-dihydroxyvitamin D3) acts through vitamin D receptor (VDR) initiating genomic response, but several studies described also non-genomic actions of 1,25-dihydroxyvitamin D3, implying the role of PDIA3 in the process. PDIA3 is a membrane-associated disulfide isomerase involved in disulfide bond formation, protein folding, and remodeling. Here, we used a transcriptome-based approach to identify changes in expression profiles in PDIA3-deficient squamous cell carcinoma line A431 after 1,25-dihydroxyvitamin D3 treatment. PDIA3 knockout led to changes in the expression of more than 2000 genes and modulated proliferation, cell cycle, and mobility of cells; suggesting an important regulatory role of PDIA3. PDIA3-deficient cells showed increased sensitivity to 1,25-dihydroxyvitamin D3, which led to decrease migration. 1,25-dihydroxyvitamin D3 treatment altered also genes expression profile of A431ΔPDIA3 in comparison to A431WT cells, indicating the existence of PDIA3-dependent genes. Interestingly, classic targets of VDR, including CAMP (Cathelicidin Antimicrobial Peptide), TRPV6 (Transient Receptor Potential Cation Channel Subfamily V Member 6), were regulated differently by 1,25-dihydroxyvitamin D3, in A431ΔPDIA3. Deletion of PDIA3 impaired 1,25-dihydroxyvitamin D3-response of genes, such as PTGS2, MMP12, and FOCAD, which were identified as PDIA3-dependent. Additionally, response to 1,25-dihydroxyvitamin D3 in cancerous A431 cells differed from immortalized HaCaT keratinocytes, used as non-cancerous control. Finally, silencing of PDIA3 and 1,25-dihydroxyvitamin D3, at least partially reverse the expression of cancer-related genes in A431 cells, thus targeting PDIA3 and use of 1,25-dihydroxyvitamin D3 could be considered in a prevention and therapy of the skin cancer. Taken together, PDIA3 has a strong impact on gene expression and physiology, including genomic response to 1,25-dihydroxyvitamin D3.

Prolyl 4-hydroxylase subunit beta (P4HB) could serve as a prognostic and radiosensitivity biomarker for prostate cancer patients.

BACKGROUND: Prolyl 4-hydroxylase subunit beta (P4HB) has been reported as a suppressor in ferroptosis. However, no known empirical research has focused on exploring relationships between P4HB and prostate cancer (PCa). In this research, we initially examine the function of P4HB in PCa by thorough analysis of numerous databases and proliferation experiment. METHODS: We analyzed the correlations of P4HB expression with prognosis, clinical features, mutation genes, tumor heterogeneity, stemness, tumor immune microenvironment and PCa cells using multiple databases and in vitro experiment with R 3.6.3 software and its suitable packages. RESULTS: P4HB was significantly upregulated in tumor tissues compared to normal tissues and was closely related to biochemical recurrence-free survival. In terms of clinical correlations, we found that higher P4HB expression was significantly related to older age, higher Gleason score, advanced T stage and residual tumor. Surprisingly, P4HB had highly diagnostic accuracy of radiotherapy resistance (AUC 0.938). TGF beta signaling pathway and dorso ventral axis formation were upregulated in the group of low-expression P4HB. For tumor stemness, P4HB expression was positively related to EREG.EXPss and RNAss, but was negatively associated with ENHss and DNAss with statistical significance. For tumor heterogeneity, P4HB expression was positively related to MATH, but was negatively associated with tumor ploidy and microsatellite instability. For the overall assessment of TME, we observed that P4HB expression was negatively associated with all parameters, including B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, dendritic cells, stromal score, immune score and ESTIMATE score. Spearman analysis showed that P4HB expression was negatively related to TIDE score with statistical significance. In vitro experiment, RT-qPCR and western blot showed that three siRNAs of P4HB were effective on the knockdown of P4HB expression. Furthermore, we observed that the downregulation of P4HB had significant influence on the cell proliferation of six PCa cell lines, including LNCap, C4-2, C4-2B, PC3, DU145 and 22RV1 cells. CONCLUSIONS: In this study, we found that P4HB might serve as a prognostic biomarker and predict radiotherapy resistance for PCa patients. Downregulation of P4HB expression could inhibit the cell proliferation of PCa cells.

Protein disulfide-isomerase A4 confers glioblastoma angiogenesis promotion capacity and resistance to anti-angiogenic therapy.

INTRODUCTION: Increasing evidence has revealed the key activity of protein disulfide isomerase A4 (PDIA4) in the endoplasmic reticulum stress (ERS) response. However, the role of PDIA4 in regulating glioblastoma (GBM)-specific pro-angiogenesis is still unknown. METHODS: The expression and prognostic role of PDIA4 were analyzed using a bioinformatics approach and were validated in 32 clinical samples and follow-up data. RNA-sequencing was used to search for PDIA4-associated biological processes in GBM cells, and proteomic mass spectrum (MS) analysis was used to screen for potential PDIA4 substrates. Western blotting, real-time quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assays (ELISA) were used to measure the levels of the involved factors. Cell migration and tube formation assays determined the pro-angiogenesis activity of PDIA4 in vitro. An intracranial U87 xenograft GBM animal model was constructed to evaluate the pro-angiogenesis role of PDIA4 in vivo. RESULTS: Aberrant overexpression of PDIA4 was associated with a poor prognosis in patients with GBM, although PDIA4 could also functionally regulate intrinsic GBM secretion of vascular endothelial growth factor-A (VEGF-A) through its active domains of Cys-X-X-Cys (CXXC) oxidoreductase. Functionally, PDIA4 exhibits pro-angiogenesis activity both in vitro and in vivo, and can be upregulated by ERS through transcriptional regulation of X-box binding protein 1 (XBP1). The XBP1/PDIA4/VEGFA axis partially supports the mechanism underlying GBM cell survival under ER stress. Further, GBM cells with higher expression of PDIA4 showed resistance to antiangiogenic therapy in vivo. CONCLUSIONS: Our findings revealed the pro-angiogenesis role of PDIA4 in GBM progression and its potential impact on GBM survival under a harsh microenvironment. Targeting PDIA4 might help to improve the efficacy of antiangiogenic therapy in patients with GBM.

Targeting unfolded protein response using albumin-encapsulated nanoparticles attenuates temozolomide resistance in glioblastoma.

BACKGROUND: Chemoresistant cancer cells frequently exhibit a state of chronically activated endoplasmic reticulum (ER) stress. Engaged with ER stress, the unfolded protein response (UPR) is an adaptive reaction initiated by the accumulation of misfolded proteins. Protein disulfide isomerase (PDI) is a molecular chaperone known to be highly expressed in glioblastomas with acquired resistance to temozolomide (TMZ). We investigate whether therapeutic targeting of PDI provides a rationale to overcome chemoresistance. METHODS: The activity of PDI was suppressed in glioblastoma cells using a small molecule inhibitor CCF642. Either single or combination treatment with TMZ was used. We prepared nanoformulation of CCF642 loaded in albumin as a drug carrier for orthotopic tumour model. RESULTS: Inhibition of PDI significantly enhances the cytotoxic effect of TMZ on glioblastoma cells. More importantly, inhibition of PDI is able to sensitise glioblastoma cells that are initially resistant to TMZ treatment. Nanoformulation of CCF642 is well-tolerated and effective in suppressing tumour growth. It activates cell death-triggering UPR beyond repair and induces ER perturbations through the downregulation of PERK signalling. Combination treatment of TMZ with CCF642 significantly reduces tumour growth compared with either modality alone. CONCLUSION: Our study demonstrates modulation of ER stress by targeting PDI as a promising therapeutic rationale to overcome chemoresistance.

Periostin Protects Against Alcohol-related Liver Disease by Activating Autophagy by Interacting With Protein Disulfide Isomerase.

BACKGROUND & AIMS: The matricellular protein periostin plays a critical role in liver inflammation, fibrosis, and even carcinoma. Here, the biological function of periostin in alcohol-related liver disease (ALD) was investigated. METHODS: We used wild-type (WT), Postn-null (Postn-/-) mice and Postn-/- mice with periostin recovery to investigate the biological function of periostin in ALD. Proximity-dependent biotin identification analysis identified the protein that interacted with periostin, and coimmunoprecipitation analysis validated the interaction between protein disulfide isomerase (PDI) and periostin. Pharmacological intervention and genetic knockdown of PDI were used to investigate the functional correlation between periostin and PDI in ALD development. RESULTS: Periostin was markedly upregulated in the livers of mice that were fed ethanol. Interestingly, periostin deficiency severely aggravated ALD in mice, whereas the recovery of periostin in the livers of Postn-/- mice significantly ameliorated ALD. Mechanistic studies showed that the upregulation of periostin alleviated ALD by activating autophagy through inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) pathway, which was verified in murine models treated with the mTOR inhibitor rapamycin and the autophagy inhibitor MHY1485. Furthermore, a protein interaction map of periostin was generated by proximity-dependent biotin identification analysis. Interaction profile analysis identified PDI as a key protein that interacted with periostin. Intriguingly, periostin-mediated enhancement of autophagy by inhibiting the mTORC1 pathway in ALD depended on its interaction with PDI. Moreover, alcohol-induced periostin overexpression was regulated by transcription factor EB. CONCLUSIONS: Collectively, these findings clarify a novel biological function and mechanism of periostin in ALD and the periostin-PDI-mTORC1 axis is a critical determinant of ALD.

Splice variants of protein disulfide isomerase - identification, distribution and functional characterization in the rat.

BACKGROUND: Protein Disulfide Isomerase (PDI) enzyme is an emerging therapeutic target in oncology and hematology. Although PDI reductase activity has been studied with isolated fragments of the protein, natural structural variations affecting reductase activity have not been addressed. METHODS: In this study, we discovered four coding splice variants of the Pdi pre-mRNA in rats. In vitro Michaelis constants and apparent maximum steady-state rate constants after purification and distribution in different rat tissues were determined. RESULTS: The consensus sequence was found to be the most expressed splice variant while the second most expressed variant represents 15 to 35% of total Pdi mRNA. The third variant shows a quasi-null expression profile and the fourth was not quantifiable. The consensus sequence splice variant and the second splice variant are widely expressed (transcription level) in the liver and even more present in males. Measurements of the reductase activity of recombinant PDI indicate that the consensus sequence and third splice variant are fully active variants. The second most expressed variant, differing by a lack of signal peptide, was found active but less than the consensus sequence. GENERAL SIGNIFICANCE: Our work emphasizes the importance of taking splice variants into account when studying PDI-like proteins to understand the full biological functionalities of PDI.

A conserved protein disulfide isomerase enhances plant resistance against herbivores.

Herbivore-associated molecular patterns (HAMPs) enable plants to recognize herbivores and may help plants adjust their defense responses. Here, we report on herbivore-induced changes in a protein disulfide isomerase (PDI) widely distributed across arthropods. PDI from the spider mite Tetranychus evansi (TePDI), a mesophyll-feeding agricultural pest worldwide, triggered immunity in multiple Solanaceae plants. TePDI-mediated cell death in Nicotiana benthamiana required the plant signaling proteins SGT1 (suppressor of the G2 allele of skp1) and HSP90 (heat shock protein 90), but was suppressed by spider mite effectors Te28 and Te84. Moreover, PDIs from phylogenetically distinct herbivorous and nonherbivorous arthropods triggered plant immunity. Finally, although PDI-induced plant defenses impaired the performance of spider mites on plants, RNAi experiments revealed that PDI genes are essential for the survival of mites and whiteflies. Our findings indicate that plants recognize evolutionarily conserved HAMPs to activate plant defense and resist pest damage, pointing to opportunities for broad-spectrum pest management.