Protein tyrosine nitration of mitochondrial carbamoyl phosphate synthetase 1 and its functional consequences

Abstract

Mitochondria are the primary locus for the generation of reactive nitrogen species including peroxynitrite and subsequent protein tyrosine nitration. Protein tyrosine nitration may have important functional and biological consequences such as alteration of enzyme catalytic activity. In the present study, mouse liver mitochondria were incubated with peroxynitrite, and the mitochondrial proteins were separated by 1D and 2D gel electrophoresis. Nitrotyrosinylated proteins were detected with an anti-nitrotyrosine antibody. One of the major proteins nitrated by peroxynitrite was carbamoyl phosphate synthetase 1 (CPS1) as identified by LC-MS protein analysis and Western blotting. The band intensity of nitration normalized to CPS1 was increased in a peroxynitrite concentration-dependent manner. In addition, CPS1 activity was decreased by treatment with peroxynitrite in a peroxynitrite concentration- and time-dependent manner. The decreased CPS1 activity was not recovered by treatment with reduced glutathione, suggesting that the decrease of the CPS1 activity is due to tyrosine nitration rather than cysteine oxidation. LC-MS analysis of in-gel digested samples, and a Popitam-based modification search located 5 out of 36 tyrosine residues in CPS1 that were nitrated. Taken together with previous findings regarding CPS1 structure and function, homology modeling of mouse CPS1 suggested that nitration at Y1450 in an α-helix of allosteric domain prevents activation of CPS1 by its activator, N-acetyl-l-glutamate. In conclusion, this study demonstrated the tyrosine nitration of CPS1 by peroxynitrite and its functional consequence. Since CPS1 is responsible for ammonia removal in the urea cycle, nitration of CPS1 with attenuated function might be involved in some diseases and drug-induced toxicities associated with mitochondrial dysfunction.

Figure 1

Western blot analysis of mouse mitochondrial proteins after treatment with peroxynitrite. (A) The mitochondrial fractions collected from three mice were individually incubated with each concentration of peroxynitrite (0, 0.1 and 1.0 mM) at 37°C for 10 min and subjected to 1D SDS gel electrophoresis. The blots were probed with antibodies to nitrotyrosine. (B) The blots were probed with antibodies to CPS1. (C) Densitometry analysis of nitration to CPS1 was performed using the Odyssey Infrared Imaging System. (D) The mitochondrial proteins pooled from three mice were incubated with 1.0 mM peroxynitrite at 37°C for 10 min and subjected to 2D gel electrophoresis. The blots of nitrated proteins (green) were superimposed with those of CPS1 (red). The overlap is represented by yellow.

Figure 2

The effects of peroxynitrite treatment on CPS1 activity in mouse mitochondrial fractions. (A) The mitochondrial fractions were incubated with 1.0 mM peroxynitrite at 37°C for different time periods and subjected to the CPS1 activity assay. (B) The mitochondrial fractions were incubated with different concentrations of peroxynitrite at 37°C for 10 min and subjected to the CPS1 activity assay with or without 10 mM NAG. (C) The mitochondrial fractions were incubated with 1.0 mM peroxynitrite at 37°C for 10 min and subsequently incubated with or without 5.0 mM GSH at 37°C for 10 min, followed by the CPS1 activity assay with 10 mM NAG.

Figure 3

Representative LC-MS/MS spectra and assignments of the sequence-specific fragment ions of the nitrated peptides. The sequence-specific fragment ions are labeled as y and b ions on the spectra. For the peptide 2, the mass difference of 208 detected between b₄ and b₅ and between y₅ and y₆ corresponds to nitrated tyrosine residue (163 (Tyr) + 45 (nitration)). For the peptide 5, the mass difference of 208 detected between b₅ and b₆ and between y₃ and y₄ corresponds to nitrated tyrosine residue.

Figure 4

Homology modeling of mouse CPS1. (A) The superimposed image of the created model of mouse CPS1 (yellow) with the template structure for the E. Coli CPS (white, PDB file: 1JDB). Three active sites are shown in blue (1: glutamine binding site, 2: bicarbonate binding site, 3: carbamoyl phosphate binding site) and the molecular tunnel is shown in red. (B) The superimposed image of the created model of the mouse NAG binding domain (pink) with the template structure for the human NAG binding domain (white, PBD file: 2YVQ). (C) The sequences (residues 1442 - 1459) in NAG binding domain of CPS1s from mouse (Mus musculus), human (Homo sapiens), rat (Rattus norvegicus) and monkey (Macaca mulatta), were obtained from the NCBI protein database.