Protein Disulfide Isomerase A3 Research Articles

Investigation of endoplasmic reticulum stress-regulated chaperones as biomarkers in idiopathic nonobstructive azoospermia.

Azoospermia is a condition in which sperm cells are completely absent in a male's ejaculate. Typically, sperm production occurs in the testes and is regulated by a complex series of cellular and molecular interactions. Endoplasmic reticulum (ER) stress arises when there is a deviation from or damage to the normal functions of the ER within cells. In response to this stress, a cascade of response mechanisms is activated to regulate ER stress within cells. This study aims to investigate the role of ERstress-regulated chaperones as potential biomarkers in male infertility. ER stress associated with azoospermia can manifest in cells such as spermatogonia in the testes and can impact sperm production. As a result of ER stress, the expression and activity of a variety of proteins within cells can be altered. Among these proteins are chaperone proteins that regulate the ER stress response. The sample size was calculated to be a minimum of 36 patients ineach group. In this preliminary study, we measured and compared serum levels of protein disulfide-isomerase A1, protein disulfide-isomerase A3 (PDIA3), mesencephalic astrocyte-derived neurotrophic factor (MANF), glucose regulatory protein 78 (GRP78), clusterin (CLU), calreticulin (CRT), and calnexin (CNX) between male subjects with idiopathic nonobstructive azoospermia and a control group of noninfertile males. Serum PDIA1 (P=0.0004), MANF (P=0.018), PDIA3 (P<0.0001), GRP78 (P=0.0027), andCRT (P=0.0009) levels were higher in the infertile group compared to the control. In summary, this study presents novel findings in a cohort of male infertile patients, emphasizing the significance of incorporating diverse biomarkers. It underscores the promising role of ER stress-regulated proteins as potential serum indicators for male infertility. By elucidating the impact of ER stress on spermatogenic cells, the research illuminates the maintenance or disruption of cellular health. A deeper understanding of these results could open the door to novel treatment approaches for reproductive conditions, including azoospermia.

Protein disulfide isomerase A4 binds to Brucella BtpB and mediates intracellular NAD+/NADH metabolism in RAW264.7 cells

The Toll/interleukin-1 receptor (TIR) signaling domain is distributed widely in mammalian Toll-like receptors and adaptors, plant nucleotide-binding leucine-rich repeat receptors, and specific bacterial virulence proteins. Proteins that possess TIR domain exhibit NADase activity which is distinct from the canonical signaling function of these domains. However, the effects of bacterial TIR domain proteins on host metabolic switches and the underlying mechanism of NADase activity in these proteins remain unclear. Here, we utilized Brucella TIR domain-containing type IV secretion system effector protein, BtpB, to explore the mechanism of NADase activity in host cells. We showed that using ectopic expression BtpB not only generates depletion of NAD+ but also loss of NADH and ATP in RAW264.7 macrophage cells. Moreover, immunoprecipitation-mass spectrometry, co-immunoprecipitation, and confocal microscope assays revealed that BtpB interacted with host protein disulfide isomerase A4 (PDIA4). The Brucella mutant strain deleted the gene for BtpB, significantly decreased PDIA4 expression. Furthermore, our data revealed that PDIA4 played an important role in regulating intracellular NAD+/NADH levels in macrophages, and PDIA4 overexpression restored the decline of intracellular NAD+ and NADH levels induced by Brucella BtpB. The results provide new insights into the metabolic regulatory activity of TIR domain proteins in the critical human and animal pathogen Brucella.

The PDIA3-STAT3 protein complex regulates IBS formation and development via CTSS/MHC-II pathway-mediated intestinal inflammation

Irritable bowel syndrome (IBS) is a persistent functional gastrointestinal disorder characterised by abdominal pain and altered patterns of defecation. This study aims to clarify an increase in the expression and interaction of protein disulfide-isomerase A3 (PDIA3) and Signal Transducer and Activator of Transcription 3 (STAT3) within the membrane of dendritic cells (DCs) from individuals with IBS. Mechanistically, the heightened interaction between PDIA3 and STAT3 at the DC membrane results in reduced translocation of phosphorylated STAT3 (p-STAT3) into the nucleus. The reduction of p-STAT3 to nuclear transport subsequently increased the levels of cathepsin S (CTSS) and major histocompatibility complex class II (MHC-II). Consequently, activated DCs promote CD4+ T cell proliferation and cytokine secretion, including interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-9 (IL-9), and tumour necrosis factor-alpha (TNF-α), thereby contributing to the development of IBS. Importantly, the downregulation of PDIA3 and the administration of punicalagin (Pun), a crucial active compound found in pomegranate peel, alleviate IBS symptoms in rats, such as increased visceral hypersensitivity and abnormal stool characteristics. Collectively, these findings highlight the involvement of the PDIA3-STAT3 protein complex in IBS, providing a novel perspective on the modulation of immune and inflammatory responses. Additionally, this research advances our understanding of the role and mechanisms of PDIA3 inhibitors, presenting new therapeutic possibilities for managing IBS.

Therapeutic Options of Crystallin Mu and Protein Disulfide Isomerase A3 for Cuprizone-Induced Demyelination in Mouse Hippocampus

This study investigates the changes in hippocampal proteomic profiles during demyelination and remyelination using the cuprizone model. Employing two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry for protein profiling, we observed significant alterations in the expression of ketimine reductase mu-crystallin (CRYM) and protein disulfide isomerase A3 precursor (PDIA3) following exposure to and subsequent withdrawal from cuprizone. Immunohistochemical staining validated these protein expression patterns in the hippocampus, revealing that both PDIA3 and CRYM were downregulated in the hippocampal CA1 region during demyelination and upregulated during remyelination. Additionally, we explored the potential protective effects of CRYM and PDIA3 against cuprizone-induced demyelination by synthesizing cell-permeable Tat peptide-fusion proteins (Tat-CRYM and Tat-PDIA3) to facilitate their crossing through the blood–brain barrier. Our results indicated that administering Tat-CRYM and Tat-PDIA3 mitigated the reduction in proliferating cell and differentiated neuroblast counts compared to the group receiving cuprizone alone. Notably, Tat-PDIA3 demonstrated significant effects in enhancing myelin basic protein expression alongside phosphorylation of CREB in the hippocampus, suggesting its potential therapeutic role in the prevention or treatment of demyelination, and by extension, in conditions such as multiple sclerosis.

The protein disulfide isomerase A3 and osteopontin axis promotes influenza-induced lung remodelling.

Fibrotic lung remodelling after a respiratory viral infection represents a debilitating clinical sequela. Studying or managing viral-fibrotic sequela remains challenging, due to limited therapeutic options and lack of understanding of mechanisms. This study determined whether protein disulfide isomerase A3 (PDIA3) and secreted phosphoprotein 1 (SPP1), which are associated with pulmonary fibrosis, can promote influenza-induced lung fibrotic remodelling and whether inhibition of PDIA3 or SPP1 can resolve viral-mediated fibrotic remodelling. A retrospective analysis of TriNetX data sets was conducted. Serum from healthy controls and influenza A virus (IAV)-infected patients was analysed. An inhibitor of PDIA3, punicalagin, and a neutralizing antibody for SPP1 were administered in mice. Macrophage cells treated with macrophage colony-stimulating factor (M-CSF) were used as a cell culture model. The TriNetX data set showed an increase in lung fibrosis and decline in lung function in flu-infected acute respiratory distress syndrome (ARDS) patients compared with non-ARDS patients. Serum samples revealed a significant increase in SPP1 and PDIA3 in influenza-infected patients. Lung PDIA3 and SPP1 expression increased following viral infection in mouse models. Punicalagin administration 2 weeks after IAV infection in mice caused a significant decrease in lung fibrosis and improved oxygen saturation. Administration of neutralizing SPP1 antibody decreased lung fibrosis. Inhibition of PDIA3 decreased SPP1secretion from macrophages, in association with diminished disulfide bonds in SPP1. The PDIA3-SPP1 axis promotes post-influenza lung fibrosis in mice and that pharmacological inhibition of PDIA3 or SPP1 can treat virus-induced lung fibrotic sequela.

Single-cell analysis identified PDIA3 as regulator of malignant characteristics and macrophage function in human cancers.

Protein disulfide isomerase A3 (PDIA3) is an endoplasmic reticulum (ER) protein. It has different functions including glycoprotein folding in the ER. The unfavorable prognosis of cancer patients was related to the abnormal PDIA3 expression level. However, it is unclear how PDIA3 correlates with the malignant characteristics of different tumors and its impact on tumor immunity. Pan-cancer data were downloaded from several databases for large-scale bioinformatics analysis. The immunological functions of PDIA3 were systematically explored at the single-cell sequencing level, including cell communication, cell metabolism, cell evolution and epigenetic modification. We performed immunofluorescence staining to visualize PDIA3 expression and infiltration of macrophages in pan-cancer samples. Further, we performed a loss-of-function assay of PDIA3 in vitro. The CCK8 assay, clone formation assay, and transwell assay were performed. M2 macrophages were co-cultured with different cell lines before the transwell assay was performed. The immunofluorescence staining of pan-cancer samples presented a higher expression of PDIA3 than those of the paired normal tissues. According to single-cell sequencing analysis, expression of PDIA3 was closely associated with cell communication, cell metabolism, cell evolution and epigenetic modification. The knockdown of PDIA3 in tumor cells inhibited cell proliferation and invasion, and restrained cocultured M2 macrophage migration. Furthermore, PDIA3 displayed predictive value in immunotherapy response in human cancer cohorts, indicating a potential therapeutic target. Our study showed that PDIA3 was associated with tumor malignant characteristics and could mediate the migration of M2 macrophages in various tumor types. PDIA3 could be a promising target to achieve tumor control and improve the immune response on a pan-cancer scale.

Suppressing ERp57 diminishes osteoclast activity and ameliorates ovariectomy-induced bone loss via the intervention in calcium oscillation and the calmodulin/calcineurin/Nfatc1 pathway

BackgroundIncreased osteoclast activity constitutes the primary etiology of excessive bone erosion in postmenopausal osteoporosis. ERp57, otherwise referred to as protein disulfide isomerase A3 (PDIA3), plays a crucial role in the regulation of intracellular calcium signaling. This is documented to exert a profound impact on osteoclast differentiation and functionality. MethodsTo ascertain the potential role of ERp57 in disease progression, prevention, and treatment, network pharmacology and bioinformatics analyses were conducted in relation to postmenopausal osteoporosis and ERp57 inhibitor (Loc14). Then, subsequent experimental verifications were employed in vitro on osteoclast and osteoblast, and in vivo on ovariectomy (OVX) mice models. ResultsMultiple enrichment analyses suggested that the “calcium signaling pathway” may constitute a potential avenue for therapeutic intervention by Loc14 in the treatment of postmenopausal osteoporosis. In vitro experiments demonstrated inhibition of ERp57 could block osteoclast differentiation and function by interfering with the expression of osteoclast marker genes (Traf6, Nfatc1, and Ctsk). Further mechanisms studies based on calcium imaging, qPCR, and WB established that ERp57 inhibitor (Loc14) could obstruct calcium oscillation in osteoclast precursor cells (OPCs) by limiting the entry sources of cytosolic Ca2+ and interfering with calmodulin/calcineurin/Nfatc1 pathway. Evidence from Micro-CT scanning and double calcein labeling confirmed that the application of Loc14 in vivo could alleviate bone loss and partially reversed the osteogenic impairment caused by OVX in mice. ConclusionsOur findings proved the suppressive effects of Loc14 on osteoclastogenesis via attenuating calcium oscillation and associated singling pathways, providing ERp57 as a potential therapeutic target for postmenopausal osteoporosis.

Redox Enzymes P4HB and PDIA3 Interact with STIM1 to Fine-Tune Its Calcium Sensitivity and Activation.

Sensing the lowering of endoplasmic reticulum (ER) calcium (Ca2+), STIM1 mediates a ubiquitous Ca2+ influx process called the store-operated Ca2+ entry (SOCE). Dysregulated STIM1 function or abnormal SOCE is strongly associated with autoimmune disorders, atherosclerosis, and various forms of cancers. Therefore, uncovering the molecular intricacies of post-translational modifications, such as oxidation, on STIM1 function is of paramount importance. In a recent proteomic screening, we identified three protein disulfide isomerases (PDIs)-Prolyl 4-hydroxylase subunit beta (P4HB), protein disulfide-isomerase A3 (PDIA3), and thioredoxin domain-containing protein 5 (TXNDC5)-as the ER-luminal interactors of STIM1. Here, we demonstrated that these PDIs dynamically associate with STIM1 and STIM2. The mutation of the two conserved cysteine residues of STIM1 (STIM1-2CA) decreased its Ca2+ affinity both in cellulo and in situ. Knockdown of PDIA3 or P4HB increased the Ca2+ affinity of wild-type STIM1 while showing no impact on the STIM1-2CA mutant, indicating that PDIA3 and P4HB regulate STIM1's Ca2+ affinity by acting on ER-luminal cysteine residues. This modulation of STIM1's Ca2+ sensitivity was further confirmed by Ca2+ imaging experiments, which showed that knockdown of these two PDIs does not affect STIM1-mediated SOCE upon full store depletion but leads to enhanced SOCE amplitudes upon partial store depletion. Thus, P4HB and PDIA3 dynamically modulate STIM1 activation by fine-tuning its Ca2+ binding affinity, adjusting the level of activated STIM1 in response to physiological cues. The coordination between STIM1-mediated Ca2+ signaling and redox responses reported herein may have implications for cell physiology and pathology.

PDIA3 orchestrates effector T cell program by serving as a chaperone to facilitate the non-canonical nuclear import of STAT1 and PKM2

Dysregulated T cell activation underpins the immunopathology of rheumatoid arthritis (RA), yet the machineries that orchestrate T cell effector program remain incompletely understood. Herein, we leveraged bulk and single-cell RNA sequencing data from RA patients and validated protein disulfide-isomerase A3 (PDIA3) as a potential therapeutic target. PDIA3 is remarkably upregulated in pathogenic CD4 T cells derived from RA patients and positively correlates with C-reactive protein (CRP) level and disease activity score 28 (DAS28). Pharmacological inhibition or genetic ablation of PDIA3 alleviates RA-associated articular pathology and autoimmune responses. Mechanistically, T cell receptor (TCR) signaling triggers intracellular calcium flux to activate NFAT1, a process that is further potentiated by Wnt5a under RA settings. Activated NFAT1 then directly binds to the Pdia3 promoter to enhance the expression of PDIA3, which complexes with STAT1 or PKM2 to facilitate their nuclear import for transcribing Th1 and Th17 lineage-related genes, respectively. This non-canonical regulatory mechanism likely occurs under pathological conditions as PDIA3 could only be highly induced following aberrant external stimuli. Together, our data support that targeting PDIA3 is a vital strategy to mitigate autoimmune diseases, such as RA, in clinical settings.

Integrating Phenotypic and Chemoproteomic Approaches to Identify Covalent Targets of Dietary Electrophiles in Platelets.

A large variety of dietary phytochemicals has been shown to improve thrombosis and stroke outcomes in preclinical studies. Many of these compounds feature electrophilic functionalities that potentially undergo covalent addition to the sulfhydryl side chain of cysteine residues within proteins. However, the impact of such covalent modifications on the platelet activity and function remains unclear. This study explores the irreversible engagement of 23 electrophilic phytochemicals with platelets, unveiling the unique antiplatelet selectivity of sulforaphane (SFN). SFN impairs platelet responses to adenosine diphosphate (ADP) and a thromboxane A2 receptor agonist while not affecting thrombin and collagen-related peptide activation. It also substantially reduces platelet thrombus formation under arterial flow conditions. Using an alkyne-integrated probe, protein disulfide isomerase A6 (PDIA6) was identified as a rapid kinetic responder to SFN. Mechanistic profiling studies revealed SFN's nuanced modulation of PDIA6 activity and substrate specificity. In an electrolytic injury model of thrombosis, SFN enhanced the thrombolytic activity of recombinant tissue plasminogen activator (rtPA) without increasing blood loss. Our results serve as a catalyst for further investigations into the preventive and therapeutic mechanisms of dietary antiplatelets, aiming to enhance the clot-busting power of rtPA, currently the only approved therapeutic for stroke recanalization that has significant limitations.

Differential protein repertoires related to sperm function identified in extracellular vesicles (EVs) in seminal plasma of distinct fertility buffalo (Bubalus bubalis) bulls.

Buffalo bulls are backbone of Indian dairy industry, and the quality of semen donating bulls determine the overall production efficiency of dairy farms. Seminal plasma harbor millions of lipid bilayer nanovesicles known as extracellular vesicles (EVs). These EVs carry a heterogenous cargo of essential biomolecules including fertility-associated proteins which contribute to fertilizing potential of spermatozoa. In this study, we explored size, concentration, and complete proteome profiles of SP EVs from two distinct fertility groups to uncover proteins influencing bull fertility. Through Dynamic Light Scattering (DLS) it was found that purified EVs were present in 7-14 size exclusion chromatographic (SEC) fractions with sizes ranging from 146.5 to 258.7nm in high fertile (HF) and low fertile (LF) bulls. Nanoparticle Tracking Analysis (NTA) confirmed the size of seminal EVs up to 200nm, and concentrations varying from 2.84 to 6.82 × 1011 and 3.57 to 7.74 × 1011 particles per ml in HF and LF bulls, respectively. No significant difference was observed in size and concentration of seminal EVs between two groups. We identified a total of 1,862 and 1,807 proteins in seminal EVs of HF and LF bulls, respectively using high throughput LC-MS/MS approach. Out of these total proteins, 1,754 proteins were common in both groups and about 87 proteins were highly abundant in HF group while 1,292 were less abundant as compared to LF bulls. Gene ontology (GO) analysis, revealed that highly abundant proteins in HF group were mainly part of the nucleus and involved in nucleosome assembly along with DNA binding. Additionally, highly abundant proteins in EVs of HF group were found to be involved in spermatogenesis, motility, acrosome reaction, capacitation, gamete fusion, and cryotolerance. Two highly abundant proteins, protein disulfide-isomerase A4 and gelsolin, are associated with sperm-oocyte fusion and acrosome reaction, respectively, and their immunolocalization on spermatozoa may indicate that these proteins are transferred through EVs. Our evidences support that proteins in EVs and subsequently their presence on sperm, are strongly associated with sperm functions. Altogether, our investigation indicates that SPEVs possess crucial protein repertoires that are essential for enhancing sperm fertilizing capacity.

PDIA4 Is a Host Factor Important for Lymphocytic Choriomeningitis Virus Infection.

Mammalian arenaviruses are rodent-borne zoonotic viruses, some of which can cause fatal hemorrhagic diseases in humans. The first discovered arenavirus, lymphocytic choriomeningitis virus (LCMV), has a worldwide distribution and can be fatal for transplant recipients. However, no FDA-approved drugs or vaccines are currently available. In this study, using a quantitative proteomic analysis, we identified a variety of host factors that could be needed for LCMV infection, among which we found that protein disulfide isomerase A4 (PDIA4), a downstream factor of endoplasmic reticulum stress (ERS), is important for LCMV infection. Biochemical analysis revealed that LCMV glycoprotein was the main viral component accounting for PDIA4 upregulation. The inhibition of ATF6-mediated ERS could prevent the upregulation of PDIA4 that was stimulated by LCMV infection. We further found that PDIA4 can affect the LCMV viral RNA synthesis processes and release. In summary, we conclude that PDIA4 could be a new target for antiviral drugs against LCMV.

The role of PDIA3 in oral squamous cell carcinoma and its value as A diagnostic and prognostic biomarker

BackgroundThis study aimed to investigate the role of protein disulfide isomerase A3 (PDIA3) in oral squamous cell carcinoma (OSCC) and evaluate its significance as a diagnostic and prognostic biomarker. MethodsComprehensive bioinformatics analysis of the OSCC dataset from The Cancer Genome Atlas (TCGA) was performed. PDIA3 was depleted in CAL27 and SCC25 OSCC cells by transfection with PDIA3-specific siRNA oligos. The effects of PDIA3 downregulation on cell viability, apoptosis, and cell migration were evaluated using CCK8, ELISA, and wound healing assays, respectively. ResultsThe mRNA and protein expression of PDIA3 was significantly up-regulated in OSCC tissues compared to adjacent normal tissues. Knockdown of PDIA3 led to significantly decreased cell viability, increased apoptosis, and suppressed migratory ability in OSCC cells. The Kaplan-Meier survival curve showed that patients with higher PDIA3 expression levels had shorter survival than those with low PDIA3 levels. The receiver operating characteristic (ROC) curve indicated that PDIA3 had high sensitivity and accuracy for detecting OSCC (area under the curve (AUC): 0.917, CI: 0.879–0.955). Univariate and multivariate Cox regression analyses identified PDIA3 as an independent prognostic factor of OSCC. Furthermore, the depletion of PDIA3 inhibited AKT activity in OSCC cells. Gene set enrichment analysis (GSEA) indicated that PDIA3 is involved in various important biological functions and signaling pathways closely related to cancer development. ConclusionPDIA3 plays an oncogenic role in OSCC and represents a good candidate as a diagnostic and prognostic biomarker for OSCC.

Generation of Genetically Modified Mouse Models to Study Protein Disulfide Isomerase A3 Involvement in Neurological Disorders

Generation of Genetically Modified Mouse Models to Study Protein Disulfide Isomerase A3 Involvement in Neurological Disorders

Protein disulfide isomerase A3 as novel biomarker for endometrial cancer.

This study aims to investigate the potential of PDIA3 as a novel prognostic biomarker and therapeutic target for Endometrial Cancer (EC) with the ultimate goal of improving survival rates in EC patients. This study employed a combination of public database analysis and clinical tissue sample assays. The analysis included comparing the gene expression of PDIA3 between EC and adjacent paracancerous tissues, investigating this expression status using qPCR and immunohistochemistry (IHC) assays, studying the correlation of expression with different parameters using Chi-square test, Cox Regression, and log-rank test, as well as exploring the PDIA3-related immune infiltration and metabolic pathway using TIMER and GSEA. The analysis of public datasets revealed that PDIA3 mRNA and protein expression was significantly higher in EC tissues compared to adjacent tissues (P = 4.1e-03, P = 1.95e-14, and P = 1.6e-27, respectively). The qPCR analysis supported this finding (P = 0.029). IHC analysis revealed a significant increase in PDIA3 expression in endometrial cancer (EC) tissues compared to adjacent normal tissues (P = 0.01). Furthermore, PDIA3 expression showed significant correlations with cancer stage and tumor grade. Multivariate Cox regression analysis suggested that the PDIA3 gene holds promise as a prognostic factor for EC patients (HR = 0.47, 95% CI [0.27, 0.82], P = 0.008). The results from TIMER demonstrated a positive correlation between PDIA3 and tumor-infiltrating CD8 T cells and macrophages, and a negative correlation with tumor-infiltrating CD4 T cells. Additionally, the GSEA results indicated that PDIA3 overexpression was associated with various metabolic processes in EC patients. PDIA3 has been validated as a potential biomarker for EC, and its expression is further associated with pathological staging and prognosis.

Vitamin D, a Secosteroid Hormone and Its Multifunctional Receptor, Vitamin D Receptor, in Alzheimer's Type Neurodegeneration.

Vitamin D is a secosteroid hormone exerting neurosteroid-like properties. Its well-known nuclear hormone receptor, and recently proposed as a mitochondrial transcription factor, vitamin D receptor, acts for its primary functions. The second receptor is an endoplasmic reticulum protein, protein disulfide isomerase A3 (PDIA3), suggested to act as a rapid response. Vitamin D has effects on various systems, particularly through calcium metabolism. Among them, the nervous system has an important place in the context of our subject. Recent studies have shown that vitamin D and its receptors have numerous effects on the nervous system. Neurodegeneration is a long-term process. Throughout a human life span, so is vitamin D deficiency. Our previous studies and others have suggested that the out-come of long-term vitamin D deficiency (hypovitaminosis D or inefficient utilization of vitamin D), may lead neurons to be vulnerable to aging and neurodegeneration. We suggest that keeping vitamin D levels at adequate levels at all stages of life, considering new approaches such as agonists that can activate vitamin D receptors, and utilizing other derivatives produced in the synthesis process with UVB are crucial when considering vitamin D-based intervention studies. Given most aspects of vitamin D, this review outlines how vitamin D and its receptors work and are involved in neurodegeneration, emphasizing Alzheimer's disease.

Chemical Hypoxic Preconditioning Improves Survival and Proliferation of Mesenchymal Stem Cells.

Increasing evidence has demonstrated that mesenchymal stem cells (MSCs) have been linked to tissue regeneration both in vitro and in vivo. However, poor engraftment and low survival rate of transplanted MSCs are still a major concern. It has been found that the proliferation, survival, and migration of MSCs are all increased by hypoxic preconditioning. However, the molecular mechanism through which hypoxic preconditioning enhances these beneficial properties of MSCs remains to be fully investigated. Therefore, the present study is aimed to investigate the mechanism by which hypoxic preconditioning enhances the survival of MSCs. We used proteomic analysis to explore the molecules that may contribute to the survival and proliferation of hypoxic preconditioned (HP) MSCs. The analysis revealed a higher expression of prelamin A/C (Lmna), glutamate dehydrogenase 1(Glud1), Actin, cytoplasmic 1(Actb), Alpha-enolase (Eno1), Glucose-6-phosphate 1-dehydrogenase (G6pd), Protein disulfide-isomerase A3 (Pdia3), Malate dehydrogenase (Mdh1), Peroxiredoxin-6 (Prdx6), Superoxide dismutase (Sod1), and Annexin A2 (Anxa2) in HP-MSCs. These proteins are possibly involved in cellular survival and proliferation through various cellular pathways. This research could aid in understanding the processes involved in hypoxic preconditioning of MSCs and designing of cell-based therapeutic strategies for tissue regeneration.

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.

A Proteomic Analysis of Detergent-Resistant Membranes in HIV Virological Synapse: The Involvement of Vimentin in CD4 Polarization.

The cell-cell contact between HIV-1-infected and uninfected cells forms a virological synapse (VS) to allow for efficient HIV-1 transmission. Not only are HIV-1 components polarized and accumulate at cell-cell interfaces, but viral receptors and lipid raft markers are also. To better understand the nature of the HIV-1 VS, detergent-resistant membrane (DRM) fractions were isolated from an infected-uninfected cell coculture and compared to those from non-coculture samples using 2D fluorescence difference gel electrophoresis. Mass spectrometry revealed that ATP-related enzymes (ATP synthase subunit and vacuolar-type proton ATPase), protein translation factors (eukaryotic initiation factor 4A and mitochondrial elongation factor Tu), protein quality-control-related factors (protein disulfide isomerase A3 and 26S protease regulatory subunit), charged multivesicular body protein 4B, and vimentin were recruited to the VS. Membrane flotation centrifugation of the DRM fractions and confocal microscopy confirmed these findings. We further explored how vimentin contributes to the HIV-1 VS and found that vimentin supports HIV-1 transmission through the recruitment of CD4 to the cell-cell interface. Since many of the molecules identified in this study have previously been suggested to be involved in HIV-1 infection, we suggest that a 2D difference gel analysis of DRM-associated proteins may reveal the molecules that play crucial roles in HIV-1 cell-cell transmission.

Interaction with ERp57 is required for progranulin protection against Type 2 Gaucher disease.

Gaucher disease (GD), one of the most common lysosomal storage diseases, is caused by GBA1 mutations resulting in defective glucocerebrosidase (GCase) and consequent accumulation of its substrates β-glucosylceramide (β-GlcCer). We reported progranulin (PGRN), a secretary growth factor-like molecule and an intracellular lysosomal protein was a crucial co-factor of GCase. PGRN binds to GCase and recruits Heat Shock Protein 70 (Hsp70) to GCase through its C-terminal Granulin (Grn) E domain, termed as ND7. In addition, both PGRN and ND7 are therapeutic against GD. Herein we found that both PGRN and its derived ND7 still displayed significant protective effects against GD in Hsp70 deficient cells. To delineate the molecular mechanisms underlying PGRN's Hsp70-independent regulation of GD, we performed a biochemical co-purification and mass spectrometry with His-tagged PGRN and His-tagged ND7 in Hsp70 deficient cells, which led to the identification of ERp57, also referred to as protein disulfide isomerase A3 (PDIA3), as a protein that binds to both PGRN and ND7. Within type 2 neuropathic GD patient fibroblasts L444P, bearing GBA1 L444P mutation, deletion of ERp57 largely abolished the therapeutic effects of PGRN and ND7, as manifested by loss of effects on lysosomal storage, GCase activity, and β-GlcCer accumulation. Additionally, recombinant ERp57 effectively restored the therapeutic effects of PGRN and ND7 in ERp57 knockout L444P fibroblasts. Collectively, this study reports ERp57 as a previously unrecognized binding partner of PGRN that contributes to PGRN regulation of GD.