Regulation of Mitochondrial Carbamoyl-phosphate Synthetase 1 Activity by Active Site Fatty Acylation

Abstract

In addition to its role in reversible membrane localization of signal-transducing proteins, protein fatty acylation could play a role in the regulation of mitochondrial metabolism. Previous studies have shown that several acylated proteins exist in mitochondria isolated from COS-7 cells and rat liver. Here, a prominent fatty-acylated 165-kDa protein from rat liver mitochondria was identified as carbamoyl-phosphate synthetase 1 (CPS 1). Covalently attached palmitate was linked to CPS 1 via a thioester bond resulting in an inhibition of CPS 1 activity at physiological concentrations of palmitoyl-CoA. This inhibition corresponds to irreversible inactivation of CPS 1 and occurred in a time-and concentration-dependent manner. Fatty acylation of CPS 1 was prevented by preincubation with N-ethylmaleimide and 5-p-fluorosulfonylbenzoyladenosine, an ATP analog that reacts with CPS 1 active site cysteine residues. Our results suggest that fatty acylation of CPS 1 is specific for long-chain fatty acyl-CoA and very likely occurs on at least one of the essential cysteine residues inhibiting the catalytic activity of CPS 1. Inhibition of CPS 1 by long-chain fatty acylCoAs could reduce amino acid degradation and urea secretion, thereby contributing to nitrogen sparing during starvation.The covalent modification of proteins by lipids alters their physical and functional properties. Several types of lipids are covalently bound to proteins as follows: isoprenoids, glycosylphosphatidylinositols, cholesterol, and fatty acids (1-4). Protein fatty acylation is the modification of proteins by fatty acids. It is divided into two categories, myristoylation and palmitoylation. In myristoylation, the 14-carbon myristate is co-translationally attached to the N-terminal glycine residue of a protein via a stable amide bond. Palmitoylation is characterized by the post-translational attachment of the 16-carbon fatty acid palmitate to cysteine residues of a protein via a thioester bond. Due to its reversible nature, palmitoylation has been shown recently to regulate the subcellular localization of several proteins involved in signal transduction processes (1,5,6). For protein palmitoylation to occur, palmitate needs to be activated in the form of its coenzyme A derivative, palmitoyl-CoA.Interestingly, palmitoyl-CoA, the acyl donor for protein palmitoylation, inhibits several enzymes including rat adipocyte pyruvate dehydrogenase (7), rat liver ADP/ATP translocase (8), bovine liver glutamate dehydrogenase (9), and bovine liver methylmalonyl semialdehyde dehydrogenase (MMSDH)

Rapid and selective detection of fatty acylated proteins using ω-alkynyl-fatty acids and click chemistry

“…This resulted in an approximate 100 M circulating concentration of -alkynyl-palmitate. After 1 or 4 h postinjection, livers were excised and a homogenate was made as described in Corvi et al ( 23 ). Samples (5 mg) of postnuclear supernatant (1,000 g ) were solubilized in RIPA buffer and used in immunoprecipitation experiments with rat anti-pan-Ras 259 antibody as described above.…”

“…Crude mitochondrial fractions were obtained using differential centrifugation as described in Corvi et al ( 23 ). All buffers contained freshly added EDTA-free Complete protease inhibitor cocktail.…”

“…Using the palmitoyl-CoA analog -azido-tetradecanoyl-CoA as label, we identifi ed 21 palmitoylated proteins in rat liver mitochondria, including 3-hydroxyl-3-methylglutaryl-CoA synthase (HMGCS), the rate-limiting enzyme in ketogenesis ( 21 ). Palmitoylated mitochondrial proteins are surprisingly numerous and the characterization of the role of their acylation is still pending in the vast majority of cases (21)(22)(23)(24). In two characterized cases, the acylation of methylmalonyl semialdehyde dehydrogenase and carbamoyl phosphate synthetase 1 was shown to occur on the active site cysteine residues, thereby inhibiting these catabolic enzymes ( 22,23 ).…”

“…Palmitoylated mitochondrial proteins are surprisingly numerous and the characterization of the role of their acylation is still pending in the vast majority of cases (21)(22)(23)(24). In two characterized cases, the acylation of methylmalonyl semialdehyde dehydrogenase and carbamoyl phosphate synthetase 1 was shown to occur on the active site cysteine residues, thereby inhibiting these catabolic enzymes ( 22,23 ). In addition, Gross et al ( 25 ) showed that the glycolytic metabolic enzyme GAPDH is also a palmitoylated protein.…”

Role of Cys-1327 and Cys-1337 in Redox Sensitivity and Allosteric Monitoring in Human Carbamoyl Phosphate Synthetase

“…First, studies on palmitoylation in mitochondria isolated from COS-7 cells and rat liver found that the majority of fatty-acylated protein in the mitochondrial matrix was urea-specific CPS (26). Although this study did not determine the site of palmitoylation, the findings that reaction with palmitoyl-CoA inactivated CPS and that the inactivation was prevented by ATP inclusion or prior FSBA treatment are consistent with Cys-1327 and/or Cys-1337 as the site(s) of palmitoylation.…”