Superoxide Dismutase Catalyzes Nitration of Tyrosines by Peroxynitrite in the Rod and Head Domains of Neurofilament‐L

Abstract

Superoxide dismutase (SOD) catalyzes the nitration of specific tyrosine residues in proteins by peroxynitrite (ONOO), which may be the damaging gain-offunction resulting from mutations to SOD associated with familial amyotrophic lateral sclerosis (ALS). We found that disassembled neurofilament-L (light subunit) was more susceptible to tyrosine nitration catalyzed by SOD in vitro. Neurofilament-L was selectively nitrated compared with the majority of other proteins present in brain homogenates. Assembled neurofilament-L was more resistant to nitration, suggesting that the susceptible tyrosine residues were protected by intersubunit contacts in assembled neurofilaments. Electrospray mass spectrometry of trypsin-digested neurofilament-L showed that tyrosine 17 in the head region and tyrosines 138, 177, and 265 in a-helical coil regions of the rod domain of neurofilament-L were particularly susceptible to SOD-catalyzed nitration. Nitrated neurofilament-L inhibited the assembly of unmodified neurofilament subunits, suggesting that the affected tyrosines are located in regions important for intersubunit contacts. Neurofilaments are major structural proteins expressed in motor neurons and known to be important for their survival in vivo. We suggest that SODcatalyzed nitration of neurofilament-L may have a significant role in the pathogenesis of ALS.

Effects of oxidative and nitrosative stress in brain on p53 proapoptotic protein in amnestic mild cognitive impairment and Alzheimer disease

“…Because eight of the nine tyrosines in the p53 molecule are located in the critical DNA binding region, it is possible that covalent modification of these residues by p53 could result in a mutant p53 conformation or directly interfere with DNA binding residues. Further, it is been suggested that tyrosine residues next to glutamate in an amino acid sequence could be easily nitrated in neurofilaments [45]. In human p53 protein, there are three tyrosines with match these characteristics, i.e., Y 205, Y 220 and Y 327.…”

Inducible nitric oxide synthase (NOS2) expressed in septic patients is nitrated on selected tyrosine residues: implications for enzymic activity

“…The mechanism(s) explaining how the negative charge directs nitration to a neighbouring tyrosine residue is (are) still a matter of controversy. On one hand, Crow and associates [20] suggested that the carboxy group of acidic residues facilitates the nitration of nearby tyrosine residues by hydrogen-bonding with one of the two equivalent Figure 3 For legend see facing page.…”

Proteomic identification of brain proteins in the canine model of human aging following a long-term treatment with antioxidants and a program of behavioral enrichment: Relevance to Alzheimer's disease

“…The protein levels of NF-L in brains of AD, Down syndrome, and ALS patients is signsificantly decreased [8,9], suggesting that normal NF-L expression could be critical to central nervous system (CNS) function. Oxidation or nitration of neurofilament (NF) proteins transform the α-helix secondary structure to β-sheet and random coil conformations, destabilizing the interactions between the NF proteins and resulting in axonal damage [41] and CNS dysfunction. We have previously shown that NF68 was significantly oxidized in the brain of the gracile axonal dystrophy (gad) mouse [35].…”