Characterization of nitroproteome in neuron‐like PC12 cells differentiated with nerve growth factor: Identification of two nitration sites in α‐tubulin

Abstract

Nitric oxide (NO) is a precursor of reactive nitrating species, peroxynitrite and nitrogen dioxide, which modify proteins to generate oxidized species such as 3‐nitrotyrosine that has been used as a hallmark of peroxynitrite‐mediated oxidative stress on proteins. In the last few years however, a growing body of evidence indicates that NO also regulates a myriad of physiologic responses by modifying tyrosine residues. Looking for the molecular event triggered by NO in nerve growth factor (NGF)‐induced neuronal differentiation, we recently reported that in differentiating PC12 cells, the cytoskeleton becomes the main cellular fraction containing nitrotyrosinated proteins, and α‐tubulin is the major target. In the present work, we focus on the investigation of the sites of tyrosine nitration in α‐tubulin purified by two‐dimensional gel electrophoresis following anti‐α‐tubulin immunoprecipitation of protein extract from NGF‐treated PC12 cells. Using Western blotting and matrix‐assisted laser desorption/ionization‐time of flight analysis, we show for the first time, both in vivo and in vitro, that nitration can occur on α‐tubulin at sites other than the C‐terminus and we positively identify Tyr 161 and Tyr 357 as two specific amino acids endogenously nitrated.

In Vitro and in Vivo Protein-bound Tyrosine Nitration Characterized by Diagonal Chromatography

“…Both tyrosines were found by Tedeschi et al (36) to play an important role during nerve growth factorinduced neuronal differentiation. In addition, nitration of Tyr 224 found here was previously described by Fiore et al (37), specifically during grade IV human glioma.…”

Nitrosative protein tyrosine modifications: biochemistry and functional significance

“…For the mass spectrometric analysis of nitrated target proteins, proteins of interest were digested into peptides by site-specific proteases, such as trypsin, GluC, and chymotrypsin, similar to general proteomic techniques, followed by mass spectrometry. In the case of MS analysis of tyrosine-nitrated peptides, a characteristic mass increase of 45 Da of the nitrated peptides was observed by both electrospray ionization (ESI) (63) and matrix-assisted laser desorption ionization (MALDI) methods (61). During MALDI analysis of the nitrated peptides, two additional modified peaks with mass shifts of +13 Da and +29 Da were observed (65)(66)(67)(68).…”

Redox Regulation of the NPR1-TGA1 System of Arabidopsis thaliana by Nitric Oxide

“…In this way, NO can act as activator or inhibitor of enzymes, ion channels, or transcription factors and regulate specific processes during abiotic or biotic stress situations in plants (Beltran et al, 2000;Kim et al, 2002;Zhang et al, 2005;Sayed et al, 2007;Asada et al, 2009). In addition to the formation of protein Tyr nitrates (Tedeschi et al, 2005) and metallonitrosyls (Brandish et al, 1998;Russwurm and Koesling, 2004), NO can also form S-nitrosothiols (via S-nitrosylation) (Stamler, 1994;Stamler et al, 2001;Gaston et al, 2003). The majority of all NO-affected proteins seem to be regulated by S-nitrosylation, which occurs either by oxygen-dependent chemical reactions or by the transfer of NO from a nitrosothiol to a protein sulfhydryl group (transnitrosylation).…”