Dissertation

Post-translational regulation of NADPH oxidase 5 (Nox5) mediated via phosphorylation and SUMOylation

Pandey, Deepesh

2011

Overview

Increased levels of reactive oxygen species (ROS) are a hallmark of cardiovascular disease and are most prominently observed in blood vessels from humans and animals with diabetes, atherosclerosis and hypertension [1]. The NADPH oxidase (Nox) family of enzymes is comprised of seven members, Nox1-5 and Duox 1 and 2 [2] has been shown to be a major source of ROS including superoxide (O 2·-) and hydrogen peroxide (H2O 2) in vascular cells [3]. Nox5 was the most recent of the conventional Nox enzymes to be identified and because it has been lost from rodent genomes (mice and rats) which have become our primary models for experimentation, very little is known about the molecular regulation and functional significance of Nox5. Our first goal was to determine whether Nox5 and its splice variants α, β, δ, γ and Nox5 Short (ϵ) are expressed in human blood vessels. We detected Nox5 mRNA and protein expression in human blood vessels, smooth muscle cells and endothelial cells, but not in fibroblasts. The primary splice variants of Nox5 detected were α and β whereas δ and γ were undetected. We also found that Nox5 α and β were active and produced extracellular superoxide and H2O2, while Nox5, δ, γ and ϵ did not produce measurable ROS. As much as we lack knowledge about functional significance of Nox5, we are not so far ahead in understanding its molecular regulation. The mechanisms controlling the activity of NADPH oxidase 5 (Nox5) are unique in that they appear to be independent of the protein: protein interactions that coordinate the activation of other Nox isoforms [4]. Instead, the primary driving force for Nox5 activity is calcium [5]. While calcium is absolutely required for Nox5 activity, discrepancies between the amount of calcium needed to initiate ROS production versus that measured inside cells has led to the discovery by our laboratory and others that the calcium sensitivity of Nox5 can be modified by the specific phosphorylation of serine/threonine residues in response to the protein kinase C (PKC)-agonist, PMA resulting in a sustained activation of Nox5 at resting levels of calcium [6, 7]. However, the specific kinase(s) mediating the phosphorylation and activation of Nox5 are not known and their identification was the goal of our study. Using pharmacological inhibitors, dominant negative mutants and knockdown of endogenous genes (MEK1, MEK2 and CAMKIIα) using siRNA approach, we demonstrated that MEK1/2-ERK1/2 and CAMKIIα signaling pathways can positively regulate Nox5 activity by inducing the specific phosphorylation of S498 and S475, respectively. While much attention has been given to the mechanisms that positively regulate Nox activity, little is known about mechanisms that suppress Nox function. Cellular stress arising from changes in osmotic pressure, heat, cold etc are potent stimuli for protein SUMOylation. Importantly, oxidative stress arising from increased ROS is one of the best recognized stimuli for regulating protein SUMOylation [8, 9]. Hence, we investigated whether SUMO could influence the activity of Nox and thus limit the damaging effects of these molecules. We found that SUMO-1 and the SUMO-specific conjugating enzyme, UBC-9 potently suppressed the activity of Nox5 as well as other Nox isoforms (Nox1, 2, 3 and 4). We also found that co-expression of SUMO-1 does not result in the SUMOylation of Nox5 and that mutation of predicted sites of SUMOylation and conserved lysines on Nox5 failed to prevent the SUMO-1 driven inhibition of ROS production. In summary, we have identified the expression of Nox5 and more specifically the α and β splice variants in human blood vessels and tissues. Our data suggest that Nox5 α and β are the only variants capable of producing ROS in human blood vessels, but also that the inactive variants can function as dominant negatives. Additionally, we have shown that MAPK and CAMKIIα signaling pathways positively regulate Nox5 activity via changes in phosphorylation whereas SUMO-1 negatively regulates activity through a yet to be defined mechanism. INDEX WORDS: Nox5, siRNA, SUMO, MAPK, ERK, MEK and CAMKIIα

• Publisher: ProQuest Dissertations & Theses
• Subject: Cellular biology / Pharmacology / Physiology
• ISBN: 9781124625492, 1124625496