Nitration and Inactivation of Tyrosine Hydroxylase by Peroxynitrite

Abstract

Tyrosine hydroxylase (TH) is modified by nitration after exposure of mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydrophenylpyridine. The temporal association of tyrosine nitration with inactivation of TH activity in vitro suggests that this covalent post-translational modification is responsible for the in vivo loss of TH function (Ara, J., Przedborski, S., Naini, A. B., Jackson-Lewis, V., Trifiletti, R. R., Horwitz, J., and Ischiropoulos, H. Tyrosine hydroxylase (TH) 1 (EC 1.14.16.2) is a non-heme iron, tetrahydrobiopterin-dependent protein that catalyzes the conversion of tyrosine to L-dihydroxyphenylalanine (L-DOPA) and represents the rate-limiting step in the biosynthesis of catecholamines (1). Loss of ability to synthesize catecholamines is an important step in the development of Parkinson's disease (PD) and other neurodegenerative diseases (2-6). Early loss of TH activity followed by a decline in TH protein is thought to contribute to the dopamine deficiency and phenotypic expression in PD and the MPTP mouse model (4). Tyrosine hydroxylase is a selective target for nitration following administration of the parkinsonian toxin MPTP to mice and following exposure of PC12 cells to either peroxynitrite or 1-methyl-4-phenylpyridiniun ion (7). Nitration of one or more tyrosine residues of TH was temporally associated with loss of enzymatic activity. The magnitude of inactivation was proportional to the number of TH molecules that were nitrated in PC12 cells. In the mouse striatum, the tyrosine nitration-mediated loss in TH activity parallels the decline in dopamine levels whereas the levels of TH protein remain unchanged for the first 6 h post-MPTP injection (7).However, a recent report indicated that exposure of recombinant purified TH to peroxynitrite in vitro results not only in nitration of tyrosine residues but also in the formation of covalently linked dimers and oxidation of cysteine residues (8). The same report also indicated that cysteine oxidation rather than tyrosine nitration is responsible for the loss of TH enzymatic activity (8). Cysteine, methionine, tryptophan, and tyrosine appear to be the principal amino acids in proteins modified by peroxynitrite in vitro (9 -14). To resolve the apparent differences, the reaction of peroxynitrite with recombinant purified rat TH in vitro was re-examined, and no evidence of cysteine oxidation was found. Oxidation of one cysteine residue per molecule of TH was observed only at high peroxynitrite concentrations, and three cysteine residues were oxidized in partially unfolded protein. Amino acid analysis failed to show any * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Nitric oxide mediates neurodegeneration and breakdown of the blood-brain barrier in tPA-dependent excitotoxic injury in mice

“…In the whole animal, ONOOmight not have an opportunity to interact with the BBB. ONOO -has a short half-life (Beckman et al, 1990), reacts quickly with lipids (Radi et al, 1991;Rubbo et al, 1994) and might influence protein function by nitrating tyrosine (Blanchard-Fillion et al, 2001;MacMillan-Crow et al, 1998;Nielsen et al, 2004). If the ONOO -is formed inside the cells, it might cause DNA damage (Estevez et al, 1995), ultimately leading to apoptosis.…”

Peroxynitrite-induced Nitration of Tyrosine Hydroxylase

“…DISCUSSION TH is the initial and rate-limiting enzyme in dopamine biosynthesis, and, as such, alterations in its activity will have corresponding effects on the availability of dopamine for release into synaptic activity. TH is inhibited by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in vivo through a process that is thought to involve ONOO Ϫ -induced nitration of tyrosine residues in the enzyme (1,5). This finding could offer insight into early biochemical processes that contribute to neuronal dopamine deficiencies.…”

“…A more recent paper from the same group purports that Tyr 423 , not Tyr 225 , is the actual site mediating enzyme inactivation after nitration by ONOO Ϫ (5). A Y423F mutant of TH was extensively nitrated by high concentrations of ONOO Ϫ , but its catalytic activity was not inhibited (5). ONOO Ϫ is a powerful oxidant that can modify cysteine, tryptophan, methionine, and tyrosine residues in proteins.…”

“…Based on the importance of TH to dopamine neuronal function, and considering the possibility that ONOO Ϫ causes the inhibition of TH as an early event in the process of dopamine neuronal degeneration (1,5), we sought to determine the sites at which TH is modified by ONOO Ϫ . MALDI-TOF mass spectrometry and tyrosine-scanning mutagenesis have identified tyrosines 423, 428, and 432 as the sites of ONOO Ϫ -induced nitration.…”

Redox Sensitivity of Tyrosine Hydroxylase Activity and Expression in Dopaminergic Dysfunction

“…ONOO -has been implicated as an etiological factor in the DA neuronal degeneration associated with PD and in other neurodegenerative and neurotoxic conditions [32,33]. Exposure of TH to ONOO -inhibits its catalytic activity and results in extensive tyrosine nitration of the protein [34,35]. However, ONOO -is also a powerful oxidant.…”