Protein disulfide isomerase A1 regulates fenestration dynamics in primary mouse liver sinusoidal endothelial cells (LSECs).

Protein disulfide isomerases (PDIs) are involved in many intracellular and extracellular processes, including cell adhesion and cytoskeletal reorganisation, but their contribution to the regulation of fenestrations in liver sinusoidal endothelial cells (LSECs) remains unknown. Given that fenestrations are supported on a cytoskeleton scaffold, this study aimed to investigate whether endothelial PDIs regulate fenestration dynamics in primary mouse LSECs. PDIA3 and PDIA1 were found to be the most abundant among PDI isoforms in LSECs. Taking advantage of atomic force microscopy, the effects of PDIA1 or PDIA3 inhibition on the fenestrations in LSECs were investigated using a classic PDIA1 inhibitor (bepristat) and novel aromatic N-sulfonamides of aziridine-2-carboxylic acid derivatives as PDIA1 (C-3389) or PDIA3 (C-3399) inhibitors. The effect of PDIA1 inhibition on liver perfusion was studied in vivo using dynamic contrast-enhanced magnetic resonance imaging. Additionally, PDIA1 inhibitors were examined in vitro in LSECs for effects on adhesion, cytoskeleton organisation, bioenergetics, and viability. Inhibition of PDIA1 with bepristat or C-3389 significantly reduced the number of fenestrations in LSECs, while inhibition of PDIA3 with C-3399 had no effect. Moreover, the blocking of free thiols by the cell-penetrating N-ethylmaleimide, but not by the non-cell-penetrating 4-chloromercuribenzenesulfonate, resulted in LSEC defenestration. Inhibition of PDIA1 did not affect LSEC adhesion, viability, and bioenergetics, nor did it induce a clear-cut rearrangement of the cytoskeleton. However, PDIA1-dependent defenestration was reversed by cytochalasin B, a known fenestration stimulator, pointing to the preserved ability of LSECs to form new pores. Importantly, systemic inhibition of PDIA1 in vivo affected intra-parenchymal uptake of contrast agent in mice consistent with LSEC defenestration. These results revealed the role of intracellular PDIA1 in the regulation of fenestration dynamics in LSECs, and in maintaining hepatic sinusoid homeostasis.

Comparison of anti-cancer effects of novel protein disulphide isomerase (PDI) inhibitors in breast cancer cells characterized by high and low PDIA17 expression.

BACKGROUND: Protein disulphide isomerases (PDIs) play an important role in cancer progression. However, the relative contribution of the various isoforms of PDI in tumorigenesis is not clear. METHODS: The content of PDI isoforms in 22 cancer cells lines was investigated using LC-MS/MS-based proteomic analysis. The effects of PDIA1, PDIA3 and PDIA17 inhibition on the proliferation, migration and adhesion of MCF-7 and MDA-MB-231 cells, identified as high and low PDIA17 expressing cells, respectively, were assessed using novel aromatic N-sulphonamides of aziridine-2-carboxylic acid derivatives as PDI inhibitors. RESULTS: PDIA1 and PDIA3 were the most abundant in cancer cell lysates and were also detected extracellularly in breast cancer cells (MDA-MB-231 and MCF-7). Some cancer cell lines (e.g., MCF-7, HT-29) showed upregulated expression of PDIA17, whereas in others (e.g., MDA-MB-231, 67NR), PDIA17 was not detected. The simultaneous inhibition of PDIA1 and PDIA3 showed similar anti-proliferative effects in MCF-7 and MDA-MB-231 breast cancer cells. However, the inhibition of PDIA1 and PDIA17 in the MCF-7 cell line resulted in more effective anti-adhesive and anti-proliferative effects. CONCLUSIONS: PDIA1 and PDIA3 represent major isoforms of multiple cancer cells, and their non-selective inhibition displays significant anti-proliferative effects irrespective of whether or not PDIA17 is present. The more pronounced anti-adhesive effects of PDI inhibition in hormone-sensitive MCF-7 cells featured by higher levels of PDIs when compared to triple-negative MDA-MB-231 cells suggests that targeting extracellular PDIA1 and PDIA3 with or without additional PDIA17 inhibition may represent a strategy for personalized anti-adhesive, anti-metastatic therapy in cancers with high PDI expression.

Protein disulfide isomerase-A1 regulates intraplatelet reactive oxygen species-thromboxane A2 -dependent pathway in human platelets.

BACKGROUND: Platelet-derived protein disulfide isomerase 1 (PDIA1) regulates thrombus formation, but its role in the regulation of platelet function is not fully understood. AIMS: The aim of this study was to characterize the role of PDIA1 in human platelets. METHODS: Proteomic analysis of PDI isoforms in platelets was performed using liquid chromatography tandem mass spectometry, and the expression of PDIs on platelets in response to collagen, TRAP-14, or ADP was measured with flow cytometry. The effects of bepristat, a selective PDIA1 inhibitor, on platelet aggregation, expression of platelet surface activation markers, thromboxane A2 (TxA2 ), and reactive oxygen species (ROS) generation were evaluated by optical aggregometry, flow cytometry, ELISA, and dihydrodichlorofluorescein diacetate-based fluorescent assay, respectively. RESULTS: PDIA1 was less abundant compared with PDIA3 in resting platelets and platelets stimulated with TRAP-14, collagen, or ADP. Collagen, but not ADP, induced a significant increase in PDIA1 expression. Bepristat potently inhibited the aggregation of washed platelets induced by collagen or convulxin, but only weakly inhibited platelet aggregation induced by TRAP-14 or thrombin, and had the negligible effect on platelet aggregation induced by arachidonic acid. Inhibition of PDIA1 by bepristat resulted in the reduction of TxA2 and ROS production in collagen- or thrombin-stimulated platelets. Furthermore, bepristat reduced the activation of αIIbß3 integrin and expression of P-selectin. CONCLUSIONS: PDIA1 acts as an intraplatelet regulator of the ROS-TxA2 pathway in collagen-GP VI receptor-mediated platelet activation that is a mechanistically distinct pathway from extracellular regulation of αIIbß3 integrin by PDIA3.

Protein Disulphide Isomerase A1 Is Involved in the Regulation of Breast Cancer Cell Adhesion and Transmigration via Lung Microvascular Endothelial Cells.

Cancer cell cross-talk with the host endothelium plays a crucial role in metastasis, but the underlying mechanisms are still not fully understood. We studied the involvement of protein disulphide isomerase A1 (PDIA1) in human breast cancer cell (MCF-7 and MDA-MB-231) adhesion and transendothelial migration. For comparison, the role of PDIA1 in proliferation, migration, cell cycle and apoptosis was also assessed. Pharmacological inhibitor, bepristat 2a and PDIA1 silencing were used to inhibit PDIA1. Inhibition of PDIA1 by bepristat 2a markedly decreased the adhesion of breast cancer cells to collagen type I, fibronectin and human lung microvascular endothelial cells. Transendothelial migration of breast cancer cells across the endothelial monolayer was also inhibited by bepristat 2a, an effect not associated with changes in ICAM-1 expression or changes in cellular bioenergetics. The silencing of PDIA1 produced less pronounced anti-adhesive effects. However, inhibiting extracellular free thiols by non-penetrating blocker p-chloromercuribenzene sulphonate substantially inhibited adhesion. Using a proteomic approach, we identified that ß1 and α2 integrins were the most abundant among all integrins in breast cancer cells as well as in lung microvascular endothelial cells, suggesting that integrins could represent a target for PDIA1. In conclusion, extracellular PDIA1 plays a major role in regulating the adhesion of cancer cells and their transendothelial migration, in addition to regulating cell cycle and caspase 3/7 activation by intracellular PDIA1. PDIA1-dependent regulation of cancer-endothelial cell interactions involves disulphide exchange and most likely integrin activation but is not mediated by the regulation of ICAM-1 expression or changes in cellular bioenergetics in breast cancer or endothelial cells.

Rat serum carboxylesterase partly hydrolyses gamma-butyrobetaine esters.

Although described some time ago, gamma-butyrobetaine esters and related compounds have not gained much attention from researchers, and their physiological function remains obscure. Formerly we detected GBB-esterase enzymatic activity in rat blood serum using phenylated gamma-butyrobetaine as an artificial substrate of the enzyme and HPLC. The aim of the present work was to develop an assay that would enable spectrophotometric or colorimetric determination of the reaction products of GBB-esterase activity and to reveal individual proteins performing GBB-esterase activity in rat blood serum. For this purpose gamma-butyrobetaine 1-naphthyl ester was synthesised. Hydrolysis of this ester releases 1-naphthol, which increases the optical absorbance at 322 nm. We have shown that the enzymatic hydrolysis of GBB 1-naphthyl ester to 1-naphthol in rat blood serum is due to GBB-esterase activity. An attempt was done to purify the enzyme from rat blood serum. By combining DEAE Sepharose at pH 4.2 and affinity chromatography with procainamide we achieved a 68-fold enrichment of GBB-esterase activity in our preparations. Separation of fraction proteins in 2D protein electrophoresis with following mass-spectrometry indicated that GBB esterase activity in rat blood serum is performed in part by carboxylesterase.