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The interplay between metal ion binding and the activity of thiol proteins, particularly within the protein disulfide isomerase family, remains an area of active investigation due to the critical role that these proteins play in many vital processes. This research investigates the interaction between recombinant human PDIA1 and zinc ions, focusing on the subsequent implications for PDIA1's conformational stability and enzymatic activity. Employing isothermal titration calorimetry and differential scanning calorimetry, we systematically compared the zinc binding capabilities of both oxidized and reduced forms of PDIA1 and assessed the structural consequences of this interaction. Our results demonstrate that PDIA1 can bind zinc both in reduced and oxidized states, but with significantly different stoichiometry and more pronounced conformational effects in the reduced form of PDIA1. Furthermore, zinc binding was observed to inhibit the catalytic activity of reduced-PDIA1, likely due to induced alterations in its conformation. These findings unveil a potential regulatory mechanism in PDIA1, wherein metal ion binding under reductive conditions modulates its activity. Our study highlights the potential role of zinc in regulating the catalytic function of PDIA1 through conformational modulation, suggesting a nuanced interplay between metal binding and protein stability in the broader context of cellular redox regulation.

Background: The endoplasmic reticulum (ER) transmembrane chaperones DNAJB12(B12) and DNAJB14(B14) are cofactors that cooperate with cytosolic Heat Shock-70 protein (HSC70) facilitating folding/degradation of nascent membrane proteins and supporting the ER-membrane penetration of viral particles. Here, we assessed structural/functional features of B12/B14 with respect to their regulation by ER stress and their involvement in ER stress-mediated protein reflux., Methods: We investigated the effect of Unfolded Protein Response(UPR)-eliciting drugs on the expression/regulation of B12-B14 and their roles in ER-to-cytosol translocation of Protein Disulfide Isomerase-A1(PDI)., Results: We show that B12 and B14 are similar but do not seem redundant. They share predicted structural features and show high homology of their cytosolic J-domains, while their ER-lumen DUF1977 domains are quite dissimilar. Interactome analysis suggested that B12/B14 associate with different biological processes. UPR activation did not significantly impact on B12 gene expression, while B14 transcripts were up-regulated. Meanwhile, B12 and B14 (33.4 kDa isoform) protein levels were degraded by the proteasome upon acute reductive challenge. Also, B12 degradation was impaired upon sulfenic-acid trapping by dimedone. We originally report that knockdown of B12/B14 and their cytosolic partner SGTA in ER-stressed cells significantly impaired the amount of the ER redox-chaperone PDI in a cytosolic-enriched fraction. Additionally, B12 but not B14 overexpression increased PDI relocalization in non-stressed cells., Conclusions and General Significance: Our findings reveal that B12/B14 regulation involves thiol redox processes that may impact on their stability and possibly on physiological effects. Furthermore, we provide novel evidence that these proteins are involved in UPR-induced ER protein reflux., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Francisco Rafael Martins Laurindo reports financial support was provided by State of Sao Paulo Research Foundation., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background and Aims: Redox signaling is involved in the pathophysiology of aortic aneurysm/dissection. Protein Disulfide Isomerases and its prototype PDIA1 are thiol redox chaperones mainly from endoplasmic reticulum (ER), while PDIA1 cell surface pool redox-regulates thrombosis, cytoskeleton remodeling and integrin activation, which are mechanisms involved in aortic disease. Here we investigate the roles of PDIA1 in aortic dissection., Methods: Initially, we assessed the outcome of aortic aneurysm/dissection in transgenic PDIA1-overexpressing FVB mice using a model of 28-day exposure to lysyl oxidase inhibitor BAPN plus angiotensin-II infusion. In a second protocol, we assessed the effects of PDIA1 inhibitor isoquercetin (IQ) against aortic dissection in C57BL/6 mice exposed to BAPN for 28 days., Results: Transgenic PDIA1 overexpression associated with ca. 50% (p = 0.022) decrease (vs.wild-type) in mortality due to abdominal aortic rupture and protected against elastic fiber breaks in thoracic aorta. Conversely, exposure of mice to IQ increased thoracic aorta dissection-related mortality rates, from ca. 18%-50% within 28-days (p = 0.019); elastic fiber disruption and collagen deposition were also enhanced. The structurally-related compound diosmetin, which does not inhibit PDI, had negligible effects. In parallel, stretch-tension curves indicated that IQ amplified a ductile-type of biomechanical failure vs. control or BAPN-exposed mice aortas. IQ-induced effects seemed unassociated with nonspecific antioxidant effects or ER stress. In both models, echocardiographic analysis of surviving mice suggested that aortic rupture was dissociated from progressive dilatation., Conclusions: Our data indicate a protective role of PDIA1 against aortic dissection/rupture and potentially uncovers a novel integrative mechanism coupling redox and biomechanical homeostasis in vascular remodeling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The Microtubule-Associated Protein Tau is expressed in several cancers, including low-grade gliomas and glioblastomas. We have previously shown that Tau is crucial for the 2D motility of several glioblastoma cell lines, including U87-MG cells. Using an RNA interference (shRNA), we tested if Tau contributed to glioblastoma in vivo tumorigenicity and analyzed its function in a 3D model of multicellular spheroids (MCS). Tau depletion significantly increased median mouse survival in an orthotopic glioblastoma xenograft model. This was accompanied by the inhibition of MCS growth and cell evasion, as well as decreased MCS compactness, implying N-cadherin mislocalization. Intracellular Signaling Array analysis revealed a defective activation of the PI3K/AKT pathway in Tau-depleted cells. Such a defect in PI3K/AKT signaling was responsible for reduced MCS growth and cell evasion, as demonstrated by the inhibition of the pathway in control MCS using LY294002 or Perifosine, which did not significantly affect Tau-depleted MCS. Finally, analysis of the glioblastoma TCGA dataset showed a positive correlation between the amount of phosphorylated Akt-Ser473 and the expression of MAPT RNA encoding Tau, underlining the relevance of our findings in glioblastoma disease. We suggest a role for Tau in glioblastoma by controlling 3D cell organization and functions via the PI3K/AKT signaling axis.

Mechanisms whereby fibrillin-1 mutations determine thoracic aorta aneurysms/dissections (TAAD) in Marfan Syndrome (MFS) are unclear. Most aortic aneurysms evolve from mechanosignaling deregulation, converging to impaired vascular smooth muscle cell (VSMC) force-generating capacity accompanied by synthetic phenotype switch. However, little is known on VSMC mechanoresponses in MFS pathophysiology. Here, we investigated traction force-generating capacity in aortic VSMC cultured from 3-month old mg∆ lpn MFS mice, together with morpho-functional and proteomic data. Cultured MFS-VSMC depicted marked phenotype changes vs. wild-type (WT) VSMC, with overexpressed cell proliferation markers but either lower (calponin-1) or higher (SM alpha-actin and SM22) differentiation marker expression. In parallel, the increased cell area and its complex non-fusiform shape suggested possible transition towards a mesenchymal-like phenotype, confirmed through several markers (e.g. N-cadherin, Slug). MFS-VSMC proteomic profile diverged from that of WT-VSMC particularly regarding lower expression of actin cytoskeleton-regulatory proteins. Accordingly, MFS-VSMC displayed lower traction force-generating capacity and impaired contractile moment at physiological substrate stiffness, and markedly attenuated traction force responses to enhanced substrate rigidity. Such impaired mechanoresponses correlated with decreased number, altered morphology and delocalization of focal adhesions, as well as disorganized actin stress fiber network vs. WT-VSMC. In VSMC cultured from 6-month-old mice, phenotype changes were attenuated and both WT-VSMC and MFS-VSMC generated less traction force, presumably involving VSMC aging, but without evident senescence. In summary, MFS-VSMC display impaired force-generating capacity accompanying a mesenchymal-like phenotype switch connected to impaired cytoskeleton/focal adhesion organization. Thus, MFS-associated TAAD involves mechanoresponse impairment common to other TAAD types, but through distinct mechanisms., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Redox-related plasma proteins are candidate reporters of protein signatures associated with endothelial structure/function. Thiol-proteins from protein disulfide isomerase (PDI) family are unexplored in this context. Here, we investigate the occurrence and physiological significance of a circulating pool of PDI in healthy humans. We validated an assay for detecting PDI in plasma of healthy individuals. Our results indicate high inter-individual (median = 330 pg/mL) but low intra-individual variability over time and repeated measurements. Remarkably, plasma PDI levels could discriminate between distinct plasma proteome signatures, with PDI-rich (>median) plasma differentially expressing proteins related to cell differentiation, protein processing, housekeeping functions and others, while PDI-poor plasma differentially displayed proteins associated with coagulation, inflammatory responses and immunoactivation. Platelet function was similar among individuals with PDI-rich vs. PDI-poor plasma. Remarkably, such protein signatures closely correlated with endothelial function and phenotype, since cultured endothelial cells incubated with PDI-poor or PDI-rich plasma recapitulated gene expression and secretome patterns in line with their corresponding plasma signatures. Furthermore, such signatures translated into functional responses, with PDI-poor plasma promoting impairment of endothelial adhesion to fibronectin and a disturbed pattern of wound-associated migration and recovery area. Patients with cardiovascular events had lower PDI levels vs. healthy individuals. This is the first study describing PDI levels as reporters of specific plasma proteome signatures directly promoting contrasting endothelial phenotypes and functional responses., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Protein disulfide isomerases including PDIA1 are implicated in cancer progression, but underlying mechanisms are unclear. PDIA1 is known to support vascular Nox1 NADPH oxidase expression/activation. Since deregulated reactive oxygen species (ROS) production underlies tumor growth, we proposed that PDIA1 is an upstream regulator of tumor-associated ROS. We focused on colorectal cancer (CRC) with distinct KRas activation levels. Analysis of RNAseq databanks and direct validation indicated enhanced PDIA1 expression in CRC with constitutive high (HCT116) vs. moderate (HKE3) and basal (Caco2) Ras activity. PDIA1 supported Nox1-dependent superoxide production in CRC; however, we first reported a dual effect correlated with Ras-level activity: in Caco2 and HKE3 cells, loss-of-function experiments indicate that PDIA1 sustains Nox1-dependent superoxide production, while in HCT116 cells PDIA1 restricted superoxide production, a behavior associated with increased Rac1 expression/activity. Transfection of Rac1 G12V active mutant into HKE3 cells induced PDIA1 to become restrictive of Nox1-dependent superoxide, while in HCT116 cells treated with Rac1 inhibitor, PDIA1 became supportive of superoxide. PDIA1 silencing promoted diminished cell proliferation and migration in HKE3, not detectable in HCT116 cells. Screening of cell signaling routes affected by PDIA1 silencing highlighted GSK3β and Stat3. Also, E-cadherin expression after PDIA1 silencing was decreased in HCT116, consistent with PDIA1 support of epithelial-mesenchymal transition. Thus, Ras overactivation switches the pattern of PDIA1-dependent Rac1/Nox1 regulation, so that Ras-induced PDIA1 bypass can directly activate Rac1. PDIA1 may be a crucial regulator of redox-dependent adaptive processes related to cancer progression.