Proteomic analysis of peroxynitrite-induced protein nitration in isolated beef heart mitochondria

0489848 - FGÚ 2019 RIV CZ eng J - Journal Article
Kohutiar, Matej - Eckhardt, Adam - Mikšík, Ivan - Šantorová, J. - Wilhelm, Jiří
Proteomic analysis of peroxynitrite-induced protein nitration in isolated beef heart mitochondria.
Physiological Research. Roč. 67, č. 2 (2018), s. 239-250. ISSN 0862-8408. E-ISSN 1802-9973
R&D Projects: GA ČR(CZ) GAP303/11/0298; GA ČR(CZ) GA15-01948S
Institutional support: RVO:67985823
Keywords : mitochondrial proteome * peroxynitrite * 3-nitrotyrosine * 2D electrophoresis * mass spectrometry
OECD category: Biochemistry and molecular biology
Impact factor: 1.701, year: 2018

Mitochondria are exposed to reactive nitrogen species under physiological conditions and even more under several pathologic states. In order to reveal the mechanism of these processes we studied the effects of peroxynitrite on isolated beef heart mitochondria in vitro. Peroxynitrite has the potential to nitrate protein tyrosine moieties, break the peptide bond, and eventually release the membrane proteins into the solution. All these effects were found in our experiments. Mitochondrial proteins were resolved by 2D electrophoresis and the protein nitration was detected by immunochemical methods and by nano LC-MS/MS. Mass spectrometry confirmed nitration of ATP synthase subunit beta, pyruvate dehydrogenase E1 component subunit beta, citrate synthase and acetyl-CoA acetyltransferase. Immunoblot detection using chemiluminiscence showed possible nitration of other proteins such as cytochrome b-c1 complex subunit 1, NADH dehydrogenase [ubiquinone] iron-sulfur protein 2, elongation factor Tu, NADH dehydrogenase [ubiquinone] flavoprotein 2, heat shock protein beta-1 and NADH dehydrogenase [ubiquinone] iron-sulfur protein 8. ATP synthase beta subunit was nitrated both in membrane and in fraction prepared by osmotic lysis. The high sensitivity of proteins to nitration by peroxynitrite is of potential biological importance, as these enzymes are involved in various pathways associated with energy production in the heart.
Permanent Link: http://hdl.handle.net/11104/0284181