P-153 - Nuclear translocation and alternative conformation of cytochrome c after peroxynitrite-mediated challenge to cells

Abstract

Cytochrome c (cyt c) is a mitochondrial heme-containing protein that functions as an electron carrier in the mitochondrial respiratory chain and as a pro-apoptotic mediator in the cytosol. A monoclonal antibody (mAb 1D3) was generated that recognizes a region in cyt c surrounding residue Pro44 in an alternative conformation of the protein (such as that present after cyt c nitration Tyr74). Previous studies shown that in a peroxynitrite-treated Hela cell model, cyt c translocate to the cellular nuclei as detected by the 1D3 suggesting its oxidative modification. The molecular interactions of cyt c in this unusual location are not known. The aim of this study was to gain insights into the identification of the possible partners of oxidatively-modified and/or nitrated cyt c with nuclear proteins. Herein, we employed four cell lines (Hela, Vero, BAECs and B16F1), exposed the cultures to peroxynitrite, and evaluated general redox status by flow cytometry, and the presence of nitrated proteins by SILAC technology 24 hours after treatment. In all cases, cyt c was shown in the cellular nuclei. The identification of the cyt c interacting proteins following oxidative modification is under way using cellular fractionation, 2D-gel electrophoresis and mass spectrometry.