Abstract
101 - Redox-Dependent Interaction Between Protein Disulfide Isomerase and P47phox activates Nox1 NADPH Oxidase in Vascular Disease
Free radical biology & medicine, Vol.87(Suppl 1), pp.S54-S54
10/2015
DOI: 10.1016/j.freeradbiomed.2015.10.141
Abstract
We previously showed that Protein disulfide isomerase (PDI) expression and NADPH oxidase-dependent ROS generation are increased in resistance arteries in hypertension. Here, we sought to investigate the molecular mechanisms through which PDI regulates Nox1 activation. Recombinant proteins were expressed in Escherichia coli transformed with plasmids encoding p47phox, PDI wild type (PDIwt) and PDI mutated (PDImut) to serine in all four cysteines of the catalytic motifs (C36, 39, 383, 386S). Incubation of PDIwt with p47phox demonstrated intermolecular disulfide bonding of the two proteins, as detected by non-reducing western blot, only after addition of arachidonic acid, confirming this interaction is dependent on p47phox activation and unfolding. This bonding was not detected when p47phox was incubated with PDImut. The overexpression of PDTwt, but not PDImut, increased superoxide generation in vascular smooth muscle cells and ERK phosphorylation in thrombin and TNF-a-stimulated HEK293 cells. Next, Cos phox cells constitutively expressing p22phox were transfected with Nox1, p47phox, p67phox, and PDIwt or PDImut. PDIwt, but not PDImut, increased superoxide generation and recruitment of p47phox to the membrane in resting conditions and after thrombin stimulation. Furthermore, recruitment of PDI to the oxidase complex was impaired with the PDImut. These data implicate a role for the redox cysteines of PDI in Nox1 complex assembly and activation. Mass spectrometry analysis identified PDI/p47phox homo and hetero-dipeptides and revealed several disulfide bonds between PDI and p47phox. Mutation of p47phox cysteine 196 to alanine decreased superoxide generation and Nox1 complex assembly by PDI in resting and stimulated cells. These findings suggest that PDI facilitates redox-mediated recruitment of Nox1 regulatory subunits to the plasma membrane and contributes to Nox1 signaling in vascular disease.
Details
- Title: Subtitle
- 101 - Redox-Dependent Interaction Between Protein Disulfide Isomerase and P47phox activates Nox1 NADPH Oxidase in Vascular Disease
- Creators
- Marcela Gimenez - University of IowaBrandon Schickling - University of IowaIlka Wittig - Goethe University FrankfurtFrancis Miller - University of IowaLucia Rossetti Lopes - Universidade de São Paulo
- Resource Type
- Abstract
- Publication Details
- Free radical biology & medicine, Vol.87(Suppl 1), pp.S54-S54
- DOI
- 10.1016/j.freeradbiomed.2015.10.141
- ISSN
- 0891-5849
- eISSN
- 1873-4596
- Publisher
- Elsevier Inc
- Language
- English
- Date published
- 10/2015
- Academic Unit
- Obstetrics and Gynecology
- Record Identifier
- 9984966823302771
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