Protein disulfide isomerase is a central regulator of NADPH oxidase activity

Abstract

Redox state is a critical factor determining enzyme activity. The protein disulfide isomerase (PDI) belongs to a class of enzymes that maintains proteins in the adequate redox state by isomerising thiols. Vascular NADPH oxidases are important sources of reactive oxygen species (ROS). As NADPH oxidases are likely to be thiol oxidized by the radicals generated by themselves, we hypothesize that the PDI promotes oxidative stress in vascular cells by maintaining NADPH oxidase activity. Overexpression of fluorescence-tagged fusion constructs revealed colocalization of the NADPH oxidase proteins Nox1, Nox2 and Nox4 with endogenous PDI. Moreover, immuno-precipitation of PDI resulted in co-precipitation of Nox 1, 2 and 4 as well as of the p22phox subunit of the NAPDH oxidase. ROS formation in NADPH oxidase-over-expressing HEK cells was reduced by siRNA directed against PDI. Analysis of NADPH oxidase homologues revealed several highly conserved cysteines. Exchange of some of these amino acids to valine or serine resulted in a substantial loss of function. In cultured vascular smooth muscle cells (VSMC) inhibition of PDI using bacitracin or DNA antisense oligonucleotides inhibited NADPH oxidase activity and significantly attenuated angiotensin II-induced radical-mediated activation of AKT. Therefore, PDI via its action on redox-sensitive cysteines is a novel regulator of NADPH oxidases.