O14	Protein disulfide isomerase is a central regulator of NADPH oxidase activity.
1M.Janiszewski, 2F.Laurindo, 1R.Busse, 1R.P.Brandes
1J.W. Goethe-Universität, Frankfurt/Main, DE; 2Incor, Sao Paulo, BR.

The oxidative state is a critical factor determining enzyme activity. The protein disulfide isomerase (PDI) belongs to a class of redox active enzymes that maintain enzymes in the adequate redox state by transfer of thiols. The vascular NADPH oxidase is the most important source of reactive oxygen species in vascular cells. As NADPH oxidase however may also be thiol oxidized by the radicals generated by it self, we hypothesize that PDI promotes oxidative stress in vascular cells by maintaining NADPH oxidase activity. Overexpression of fluorescence-tagged fusion constructs of NADPH oxidases revealed perfect colocalization of the NADPH oxidase proteins Nox1, Nox2 and No4 with endogenous PDI. Moreover, overexpression of Nox subunits increased the endogenous PDI protein level and immuno-precipitation of PDI resulted in co-precipitation of the Nox 1, 2 and 4 as well as of the p22phox subunit of the NAPDH oxidase. Disruption of heme-binding of the Nox subunits and thus inactivation of the oxidase significantly attenuated the interaction of PDI with the Nox proteins. Radical generation in NADPH oxidase expressing HEK cells was rapidly reduced by bacitracin, a pharmacological inhibitor of PDI. More importantly, transfection of siRNA directed against PDI resulted in a substantial inhibition of Nox1- as well as of Nox4-dependent radical generation. 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 is a novel regulator of NADPH oxidase activity. Given that oxidative stress is particularly associated with vascular disease, inhibition of PDI might provide a specific tool to reduce oxidative burden and to restore normal vascular homeostasis.

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