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Dynamic regulation of NADPH oxidase 5 by intracellular heme levels and cellular chaperones.

ABSTRACT: NADPH oxidase 5 (NOX5) is a transmembrane signaling enzyme that produces superoxide in response to elevated cytosolic calcium. In addition to its association with numerous human diseases, NOX5 has recently been discovered to play crucial roles in the immune response and cardiovascular system. Details of NOX5 maturation, and specifically its response to changes in intracellular heme levels have remained unclear. Here we establish an experimental system in mammalian cells that allows us to probe the influence of heme availability on ROS production by NOX5. We identified a mode of dynamic regulatory control over NOX5 activity through modulation of its heme saturation and oligomeric state by intracellular heme levels and Hsp90 binding. This regulatory mechanism allows for fine-tuning and reversible modulation of NOX5 activity in response to stimuli.

SUBMITTER: Sweeny EA

PROVIDER: S-EPMC7394750 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Dynamic regulation of NADPH oxidase 5 by intracellular heme levels and cellular chaperones.

Sweeny Elizabeth A EA Schlanger Simon S Stuehr Dennis J DJ

Redox biology 20200726

NADPH oxidase 5 (NOX5) is a transmembrane signaling enzyme that produces superoxide in response to elevated cytosolic calcium. In addition to its association with numerous human diseases, NOX5 has recently been discovered to play crucial roles in the immune response and cardiovascular system. Details of NOX5 maturation, and specifically its response to changes in intracellular heme levels have remained unclear. Here we establish an experimental system in mammalian cells that allows us to probe t ...[more]

PMID: 32738790

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Project description:NADPH oxidase5 (Nox5) is a novel Nox isoform which has recently been recognized as having important roles in the pathogenesis of coronary artery disease, acute myocardial infarction, fetal ventricular septal defect and cancer. The activity of Nox5 and production of reactive oxygen species is regulated by intracellular calcium levels and phosphorylation. However, the kinases that phosphorylate Nox5 remain poorly understood. Previous studies have shown that the phosphorylation of Nox5 is PKC dependent, but this contention was based on the use of pharmacological inhibitors and the isoforms of PKC involved remain unknown. Thus, the major goals of this study were to determine whether PKC can directly regulate Nox5 phosphorylation and activity, to identify which isoforms are involved in the process, and to understand the functional significance of this pathway in disease. We found that a relatively specific PKCα inhibitor, Ro-32-0432, dose-dependently inhibited PMA-induced superoxide production from Nox5. PMA-stimulated Nox5 activity was significantly reduced in cells with genetic silencing of PKCα and PKCε, enhanced by loss of PKCδ and the silencing of PKCθ expression was without effect. A constitutively active form of PKCα robustly increased basal and PMA-stimulated Nox5 activity and promoted the phosphorylation of Nox5 on Ser490, Thr494, and Ser498. In contrast, constitutively active PKCε potently inhibited both basal and PMA-dependent Nox5 activity. Co-IP and in vitro kinase assay experiments demonstrated that PKCα directly binds to Nox5 and modifies Nox5 phosphorylation and activity. Exposure of endothelial cells to high glucose significantly increased PKCα activation, and enhanced Nox5 derived superoxide in a manner that was in prevented by a PKCα inhibitor, Go 6976. In summary, our study reveals that PKCα is the primary isoform mediating the activation of Nox5 and this maybe of significance in our understanding of the vascular complications of diabetes and other diseases with increased ROS production.

Dataset Information

Dynamic regulation of NADPH oxidase 5 by intracellular heme levels and cellular chaperones.

Publications

Dynamic regulation of NADPH oxidase 5 by intracellular heme levels and cellular chaperones.

Similar Datasets

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