INVESTIGADORES
CORVI Maria Martha
libros
Título:
Characterization of carbamoyl phosphate synthetase 1 palmitoylation and development of a chemical biology approach to detect and identify new palmitoylated proteins
Autor/es:
CORVI, MM
Editorial:
University of Alberta
Referencias:
Lugar: Edmonton; Año: 2006 p. 161
ISSN:
9780494230091
Resumen:
S-acylation is the modification of proteins by a variety of fatty acids on cysteine residues via thioester bond. Since palmitate (C16:0) is the most abundant fatty acid in cells and as such the most frequently found attached to proteins, S-acylation is preferentially referred to as palmitoylation. In mitochondria, palmitoylation occurs mainly in the matrix where metabolic processes take place, suggesting that palmitate plays a role in the regulation of metabolism. Rat liver carbamoyl phosphate synthetase 1 (CPS 1) is a prominent palmitoylated 165 kDa mitochondrial protein. Palmitoylation inhibits CPS 1 activity at physiological concentrations of palmitoyl-CoA. This inhibition corresponds to an irreversible inactivation of CPS 1 and occurred in a timeand concentration-dependent manner. Palmitoylation of CPS 1 was prevented by pre-incubation with N-ethylmaleimide and 5'-p-fluorosulfonylbenzoyladenosine, an ATP analog that reacts with CPS 1 active site cysteine residues, suggesting that palmitoylation of CPS 1 very likely occurs on at least one of its essential active site cysteine residues. Site-directed mutagenesis analysis of these cysteines failed to confirm the palmitoylation site on CPS 1 due to a lack of an appropriate recombinant expression system. In order to facilitate and promote palmitoylation research, we developed a non-radioactive method allowing for the rapid detection and identification of mitochondrial palmitoylated proteins. This method is based on the modified Staudinger reaction in which an azido-palmitate moiety is incorporated into proteins and specifically reacted with a tagged-triaryl-phosphine capture reagent. The resulting modified proteins can be detected using western blot/ECL. Our results show that mitochondrial proteins are azido-fatty acylated on cysteine residues, suggesting that the azido moiety does not affect the specificity of the modification. This methodology allowed the identification of pyruvate carboxylase (PC) and aspartate aminotransferase (AAT) as putative mitochondrial palmitoylated proteins.