Analysis of peroxynitrite-mediated post-translational modifications of caveolin-1

Abstract

Caveolin-1 is an important protein in caveolae, which plays a role in cholesterol transport, signal transduction, and transcytosis and tumor suppression. Caveolin-1 is found in endothelial cells, smooth muscle cells and adipocytes. The main focus of this study is to investigate the peroxynitrite-mediated in vitro post-translational modifications (PTMs) of caveolin-1.

Bovine brain was used to isolate caveolin-1 as an initial step for isolation method development. Density gradient centrifugation was used to isolate caveolin-1 from bovine brain. From the isolate, caveolin-1α, caveolin-1β isomers and caveolin dimer were identified by western blotting with anti-caveolin monoclonal antibody. Glutathione S-transferase (GST)-caveolin fusion protein was used to isolate caveolin-1 and used for in vitro experiments in this study.

During normal and pathological conditions, endothelial cells are subjected to locally generated reactive oxygen species such as peroxynitrite. Peroxynitrite is capable of modifying amino acids such as tyrosine, cysteine, tryptophan and methionine.

Peroxynitrite mediated tyrosine nitration of caveolin-1 was detected by SDS-PAGE followed by western blotting with anti-nitrotyrosine monoclonal antibody. The approach used to identify potentially modified peptide sequences of caveolin-1 was ESI-MS/MS. Fluorometry was used to detect formation of dityrosine.

Caveolin-1 was treated with different concentrations of peroxynitrite in caveolin- under the physiological conditions and found that caveolin-1 form dimer and oligomer under the physiological conditions. The stability of caveolin-1 dimer and oligomer suggests that the coupling mechanism could most likely be occurred via a covalent bond. Western blotting with anti-nitrotyrosine monoclonal antibody revealed the formation of nitrotyrosine upon the exposure to peroxynitrite.

In this study, we report the nitration of specific tyrosine residues of caveolin-1 for the first time. ESI-MS/MS analysis revealed that peroxynitrite can selectively nitrate Tyr6 and Tyr14 located in the tryptic peptide YVDSEGHLYTVPIR under physiological conditions. Caveolin-1 can form dityrosine upon exposure to peroxynitrite as shown by fluorometry. Oxidative and nitrative modifications due to the reaction of peroxynitrite with caveolin-1 may lead to several pathological conditions. Our study can provide authentic standards of modified proteins, which will be used to determine post-translational modifications of caveolin-1 in vivo.