Is protein disulfide isomerase a redox-dependent molecular chaperone?
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Protein disulfide isomerase (PDI) is a multifunctional protein catalysing the formation of disulfide bonds, acting as a molecular chaperone and being a component of the enzymes prolyl 4-hydroxylase (P4H) and microsomal triglyceride transfer protein. The role of PDI as a molecular chaperone or polypeptide-binding protein is mediated primarily through an interaction of substrates with its b' domain. It has been suggested that this binding is regulated by the redox state of PDI, with association requiring the presence of glutathione, and dissociation the presence of glutathione disulfide. To determine whether this is the case, we investigated the ability of PDI to bind to a folding polypeptide chain within a functionally intact endoplasmic reticulum and to be dissociated from the alpha-subunit of P4H in vitro in the presence of reducing or oxidizing agents. Our results clearly demonstrate that binding of PDI to these polypeptides is not regulated by its redox state. We also demonstrate that the dissociation of PDI from substrates observed in the presence of glutathione disulfide can be explained by competition for the peptide-binding site on PDI. (+info)
Inhibition of p38 mitogen-activated protein kinase blocks activation of rat pancreatic stellate cells.
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Activated pancreatic stellate cells (PSCs) have recently been implicated in the pathogenesis of pancreatic fibrosis and inflammation. However, the signal transduction pathways in PSCs remain largely unknown. We examined the role of p38 mitogen-activated protein (MAP) kinase in the activation of PSCs. PSCs were isolated from rat pancreas tissue and used in their culture-activated, myofibroblast-like phenotype. Activation of p38 MAP kinase was determined by Western blotting using anti-phosphospecific antibody. The effects of two p38 MAP kinase inhibitors, 4-(4-flurophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole (SB203580) and 4-(4-flurophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole (SB202190), on the parameters of PSC activation, including proliferation, expression of alpha-smooth muscle actin, alpha1(I) procollagen, and prolyl 4-hydroxylase (alpha) genes, and monocyte chemoattractant protein-1 production were evaluated. Interleukin-1beta and platelet-derived growth factor-BB activated p38 MAP kinase. Platelet-derived growth factor-induced PSC proliferation was inhibited by SB203580 and SB202190. These reagents decreased alpha-smooth muscle actin protein expression, and alpha1(I) procollagen and prolyl 4-hydroxylase (alpha) mRNA levels. Treatment with these p38 MAP kinase inhibitors also resulted in inhibition of monocyte chemoattractant protein-1 expression. In addition, SB203580 inhibited spontaneous activation of freshly isolated PSCs in culture on plastic. Thus, inhibition of p38 MAP kinase modulated profibrogenic and proinflammatory actions in PSCs, implying a potential application of p38 MAP kinase inhibitors for the treatment of pancreatic fibrosis and inflammation. (+info)
Cyclosporin A prevents the hypoxic adaptation by activating hypoxia-inducible factor-1alpha Pro-564 hydroxylation.
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The mechanism by which hypoxia induces gene transcription involves the inhibition of hypoxia-inducible factor (HIF)-1alpha prolyl hydroxylase activity, which prevents von Hippel-Lindau (vHL)-dependent targeting of HIF-1alpha to the ubiquitin-proteasome pathway. HIF-1alpha is stabilized, translocates to the nucleus, interacts with hypoxia-responsive elements, and promotes the activation of target genes. This report shows that cyclosporin A (CsA) interferes with the hypoxic signaling cascade in C6 glioma cells. CsA inhibits hypoxia-dependent gene transcription in a reporter gene assay and prevents the hypoxic accumulation of HIF-1alpha. Addition of the 530-603 C-terminal oxygen-dependent degradation (ODD) domain of HIF-1alpha to the green fluorescent protein (GFP) destabilized the protein in an oxygen-dependent manner. CsA prevented the hypoxic stabilization of an ODD.GFP fusion protein. An assay for 2-oxoglutarate-dependent dioxygenases was developed using a light mitochondrial kidney fraction as a source of enzyme. It uses the capacity of specific peptides to stimulate the degradation of [(14)C]2-oxoglutarate. CsA stimulated the enzymatic activity in the presence of a peptide that mimicked the 557-576 sequence of HIF-1alpha. The enzyme promoted [(35)S]vHL binding to glutathione S-transferase (GST).ODD fusion protein. This association increased in the presence of CsA. CsA effects were not observed when the proline residue corresponding to Pro-564 in the HIF-1alpha sequence was replaced by a hydroxyproline or an alanine residue. Finally, CsA increased vHL-ODD interaction during hypoxia. We conclude that CsA destabilizes HIF-1alpha by promoting hydroxylation of Pro-564 in the ODD domain. Such a mechanism may prevent hypoxic adaptation during CsA-induced nephrotoxicity and contribute to the adverse effects of this drug. (+info)
Intracellular localisation of human HIF-1 alpha hydroxylases: implications for oxygen sensing.
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Hypoxia-inducible factor1 (HIF-1) is an essential transcription factor for cellular adaptation to decreased oxygen availability. In normoxia the oxygen-sensitive alpha-subunit of HIF-1 is hydroxylated on Pro564 and Pro402 and thus targeted for proteasomal degradation. Three human oxygen-dependent HIF-1 alpha prolyl hydroxylases (PHD1, PHD2, and PHD3) function as oxygen sensors in vivo. Furthermore, the asparagine hydroxylase FIH-1 (factor inhibiting HIF) has been found to hydroxylate Asp803 of the HIF-1 C-terminal transactivation domain, which results in the decreased ability of HIF-1 to bind to the transcriptional coactivator p300/CBP. We have fused these enzymes to the N-terminus of fluorescent proteins and transiently transfected the fusion proteins into human osteosarcoma cells (U2OS). Three-dimensional 2-photon confocal fluorescence microscopy showed that PHD1 was exclusively present in the nucleus, PHD2 and FIH-1 were mainly located in the cytoplasm and PHD3 was homogeneously distributed in cytoplasm and nucleus. Hypoxia did not influence the localisation of any enzyme under investigation. In contrast to FIH-1, each PHD inhibited nuclear HIF-1 alpha accumulation in hypoxia. All hydroxylases suppressed activation of a cotransfected hypoxia-responsive luciferase reporter gene. Endogenous PHD2mRNA and PHD3mRNA were hypoxia-inducible, whereas expression of PHD1mRNA and FIH-1mRNA was oxygen independent. We propose that PHDs and FIH-1 form an oxygen sensor cascade of distinct subcellular localisation. (+info)
Activation of the prolyl hydroxylase oxygen-sensor results in induction of GLUT1, heme oxygenase-1, and nitric-oxide synthase proteins and confers protection from metabolic inhibition to cardiomyocytes.
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Recently an oxygen-sensing/transducing mechanism has been identified as a family of O2-dependent prolyl hydroxylase domain-containing enzymes (PHD). In normoxia, PHD hydroxylates a specific proline residue that directs the degradation of constitutively synthesized hypoxia-inducible factor-1alpha. During hypoxia, the cessation of hydroxylation of this proline results in less degradation and thus increases hypoxia-inducible factor-1alpha protein levels. In this study we have examined the consequences of activating the PHD oxygen-sensing pathway in cultured neonatal myocytes using ethyl-3,4 dihydroxybenzoate and dimethyloxalylglycine, inhibitors that, similar to hypoxia, inhibit this family of O2-dependent PHD enzymes. Increased glucose uptake and enhanced glycolytic metabolism are classical cellular responses to hypoxia. Ethyl-3,4 dihydroxybenzoate treatment of cardiomyocyte cultures for 24 h increased [3H]deoxy-4-glucose uptake concurrent with an induction of GLUT1 protein. In addition, ethyl-3,4 dihydroxybenzoate, dimethyloxalylglycine, and hypoxia treatments were found to induce protein levels of nitricoxide synthase-2 and heme oxygenase-1, two important cardioregulatory proteins whose expression in response to hypoxic conditions is poorly understood. In conjunction with these changes in gene expression, activation of the PHD oxygen-sensing mechanism was found to preserve myocyte viability in the face of metabolic inhibition with cyanide and 2-deoxyglucose. These results point to a key role for the PHD pathway in the phenotypic changes that are observed in a hypoxic myocyte and may suggest a strategy to pharmacologically induce protection in heart. (+info)
Inhibition of prolyl-4-hydroxylase ameliorates chronic rejection of mouse kidney allografts.
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Interstitial fibrosis, glomerulosclerosis and arteriosclerosis are the major components of chronic allograft nephropathy (CAN), the leading cause of late graft failure after transplantation. To investigate the mechanism of collagen deposition in CAN, we studied the effects of prolyl-hydroxylase inhibitor (PHI), an enzyme essential for collagen formation, using a mouse model of kidney transplantation. Kidneys from H-2b mice were transplanted into MHC-incompatible H-2d recipients (allografts) and at 3 weeks post-transplant, received either PHI or vehicle treatment daily for 3 weeks. At 6 weeks post-transplant, GFR was significantly improved in the allografts receiving PHI (3.3 +/- 0.5 mL/min/kg) compared with those receiving vehicle (1.8 +/- 0.5 mL/min/kg, p < 0.05), while renal function was relatively unimpaired in the nonrejecting isografts (6.45 +/- 0.53 mL/min/kg). Allografts had histologic changes of CAN but the severity was significantly reduced with PHI treatment compared with vehicle, with reductions in interstitial inflammation and fibrosis. Furthermore, TGFa and connective tissue growth factor mRNA expression was enhanced in both allograft groups compared with the isografts. In conclusion, PHI-treated allografts had improved renal function and reduced the severity of renal injury as a result of CAN. Inhibition of matrix synthesis may be a useful adjunct in ameliorating the development of CAN in humans. (+info)
Effect of ascorbate on the activity of hypoxia-inducible factor in cancer cells.
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Hypoxia-inducible factor (HIF) plays an important role in determining patterns of gene expression in cancer. HIF is down-regulated in oxygenated cells by a series of Fe (II) and 2-oxoglutarate dependent dioxygenases that hydroxylate specific residues in the regulatory HIF-alpha subunits. Because these enzymes require ascorbate for activity in vitro we analyzed the effects of ascorbate on HIF in human cancer cell lines. Ascorbate at physiological concentrations (25 micro M) strikingly suppressed HIF-1alpha protein levels and HIF transcriptional targets, particularly when the system was oncogenically activated in normoxic cells. Similar results were obtained with iron supplementation. These results indicate that both ascorbate and iron availability have major effects on HIF, and imply that the system is commonly regulated by limiting hydroxylase activity under normoxic tissue culture conditions. (+info)
Activation of the hypoxia-inducible factor-pathway and stimulation of angiogenesis by application of prolyl hydroxylase inhibitors.
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Hypoxia-inducible transcription factors (HIF) mediate complex adaptations to reduced oxygen supply, including neoangiogenesis. Regulation of HIF occurs mainly through oxygen-dependent destruction of its alpha subunit. In the presence of oxygen, two HIFalpha prolyl residues undergo enzymatic hydroxylation, which is required for its proteasomal degradation. We therefore tested whether pharmacological activation of HIFalpha by hydroxylase inhibitors may provide a novel therapeutic strategy for the treatment of ischemic diseases. Three distinct prolyl 4-hydroxylase inhibitors-l-mimosine (L-Mim), ethyl 3,4-dihydroxybenzoate (3,4-DHB), and 6-chlor-3-hydroxychinolin-2-carbonic acid-N-carboxymethylamid (S956711)-demonstrated similar effects to hypoxia (0.5% O2) by inducing HIFalpha protein in human and rodent cells. L-Mim, S956711, and, less effectively, 3,4-DHB also induced HIF target genes in cultured cells, including glucose transporter 1 and vascular endothelial growth factor, as well as HIF-dependent reporter gene expression. Systemic administration of L-Mim and S956711 in rats led to HIFalpha induction in the kidney. In a sponge model for angiogenesis, repeated local injection of the inhibitors strongly increased invasion of highly vascularized tissue into the sponge centers. In conclusion, structurally distinct inhibitors of prolyl hydroxylation are capable of inducing HIFalpha and HIF target genes in vitro and in vivo and induce adaptive responses to hypoxia, including angiogenesis. (+info)