Dataset Information

Substrate preference of the HIF-prolyl hydroxylase-2 (PHD2) and substrate-induced conformational change.

ABSTRACT: HIF prolyl-4-hydroxylase 2 (PHD2) is a non-heme Fe, 2-oxoglutarate (2OG) dependent dioxygenase that regulates the hypoxia inducible transcription factor (HIF) by hydroxylating two conserved prolyl residues in N-terminal oxygen degradation domain (NODD) and C-terminal oxygen degradation domain (CODD) of HIF-1?. Prior studies have suggested that the substrate preference of PHD2 arises from binding contacts with the ?2?3 loop of PHD2. In this study we tested the substrate selectivity of PHD2 by kinetic competition assays, varied ionic strength, and global protein flexibility using amide H/D exchange (HDX). Our results revealed that PHD2 preferred CODD by 20-fold over NODD and that electrostatics influenced this effect. Global HDX monitored by mass spectrometry indicated that binding of Fe(II) and 2OG stabilized the overall protein structure but the saturating concentrations of either NODD or CODD caused an identical change in protein flexibility. These observations imply that both substrates stabilize the ?2?3 loop to the same extent. Under unsaturated substrate conditions NODD led to a higher HDX rate than CODD due to its lower binding affinity to PHD2. Our results suggest that loop closure is the dominant contributor to substrate selectivity in PHD2.

SUBMITTER: Pektas S

PROVIDER: S-EPMC4046702 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Substrate preference of the HIF-prolyl hydroxylase-2 (PHD2) and substrate-induced conformational change.

Pektas Serap S Knapp Michael J MJ

Journal of inorganic biochemistry 20130521

HIF prolyl-4-hydroxylase 2 (PHD2) is a non-heme Fe, 2-oxoglutarate (2OG) dependent dioxygenase that regulates the hypoxia inducible transcription factor (HIF) by hydroxylating two conserved prolyl residues in N-terminal oxygen degradation domain (NODD) and C-terminal oxygen degradation domain (CODD) of HIF-1α. Prior studies have suggested that the substrate preference of PHD2 arises from binding contacts with the β2β3 loop of PHD2. In this study we tested the substrate selectivity of PHD2 by kin ...[more]

PMID: 23787140

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Project description:BACKGROUND:In muscular dystrophy and old age, skeletal muscle repair is compromised leading to fibrosis and fatty tissue accumulation. Therefore, therapies that protect skeletal muscle or enhance repair would be valuable medical treatments. Hypoxia-inducible factors (HIFs) regulate gene transcription under conditions of low oxygen, and HIF target genes EPO and VEGF have been associated with muscle protection and repair. We tested the importance of HIF activation following skeletal muscle injury, in both a murine model and human volunteers, using prolyl hydroxylase inhibitors that stabilize and activate HIF. METHODS:Using a mouse eccentric limb injury model, we characterized the protective effects of prolyl hydroxylase inhibitor, GSK1120360A. We then extended these studies to examine the impact of EPO modulation and infiltrating immune cell populations on muscle protection. Finally, we extended this study with an experimental medicine approach using eccentric arm exercise in untrained volunteers to measure the muscle-protective effects of a clinical prolyl hydroxylase inhibitor, daprodustat. RESULTS:GSK1120360A dramatically prevented functional deficits and histological damage, while accelerating recovery after eccentric limb injury in mice. Surprisingly, this effect was independent of EPO, but required myeloid HIF1?-mediated iNOS activity. Treatment of healthy human volunteers with high-dose daprodustat reduced accumulation of circulating damage markers following eccentric arm exercise, although we did not observe any diminution of functional deficits with compound treatment. CONCLUSION:The results of these experiments highlight a novel skeletal muscle protective effect of prolyl hydroxylase inhibition via HIF-mediated expression of iNOS in macrophages. Partial recapitulation of these findings in healthy volunteers suggests elements of consistent pharmacology compared to responses in mice although there are clear differences between these two systems.

Project description:Vascular calcification is a life-threatening clinical condition in chronic kidney disease (CKD) and is associated with reduced zinc serum levels. Anemia is another frequent complication of CKD. Hypoxia-inducible factor (HIF) stabilizers, also known as HIF prolyl hydroxylase inhibitors (PHI), are promising candidates to treat CKD-associated anemia by increasing erythropoietin synthesis. Recent evidence suggests that HIFs play a pivotal role in vascular calcification. Our study explored feasible impacts of HIF PHI on phosphate (Pi)-induced calcification of vascular smooth muscle cells (VSMCs) and tested whether zinc might inhibit this mineralization process. Treatment of VSMCs with PHI aggravated Pi-induced calcium deposition and Pi uptake. PHI promoted Pi-induced loss of smooth muscle cell markers (ACTA-2, MYH11, SM22?) and enhanced osteochondrogenic gene expression (Msx-2, BMP-2, Sp7) triggering osteochondrogenic phenotypic switch of VSMCs. These effects of PHI paralleled with increased pyruvate dehydrogenase kinase 4 (PDK4) expression, decreased Runx2 Ser451 phosphorylation, and reduced cell viability. Zinc inhibited Pi-induced mineralization of VSMCs in a dose-dependent manner and also attenuated the pro-calcification effect of PHI in Pi-induced mineralization. Zinc inhibited osteochondrogenic phenotypic switch of VSMCs reflected by lowering Pi uptake, decreasing the expressions of Msx-2, BMP-2, and Sp7 as well as the loss of smooth muscle cell-specific markers. Zinc preserved phosphorylation state of Runx2 Ser451, decreased PDK4 level, and restored cell viability. PHI alone reduced the expression of smooth muscle markers without inducing mineralization, which was also inhibited by zinc. In addition, we observed a significantly lower serum zinc level in CKD as well as in patients undergoing carotid endarterectomy compared to healthy individuals. Conclusion - PHI promoted the loss of smooth muscle markers and augmented Pi-induced osteochondrogenic phenotypic switch leading to VSMCs calcification. This mineralization process was attenuated by zinc. Enhanced vascular calcification is a potential risk factor during PHI therapy in CKD which necessitates the strict follow up of vascular calcification and zinc supplementation.

Dataset Information

Substrate preference of the HIF-prolyl hydroxylase-2 (PHD2) and substrate-induced conformational change.

Publications

Substrate preference of the HIF-prolyl hydroxylase-2 (PHD2) and substrate-induced conformational change.

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