Functional proteomics approaches for the identification of transnitrosylase and denitrosylase targets.

Protein S-nitrosylation is a dynamic post-translational modification (PTM) of specific cysteines within a target protein. Both proteins and small molecules are known to regulate the attachment and removal of this PTM, and proteins exhibiting such a function are transnitrosylase or denitrosylase candidates. With the advent of the biotin switch technique coupled to high-throughput proteomics workflows, the identification and quantification of large numbers of S-nitrosylated proteins and peptides is now possible. Proper analysis and interpretation of high throughout and quantitative proteomics data will help identify specific transnitrosylase and denitrosylase target peptide sequences and contribute to an understanding of the function and regulation of specific S-nitrosylation events. Here we describe the application of a quantitative proteomics approach using isotope-coded affinity tags (ICAT) in the biotin switch approach for the identification of transnitrosylation and denitrosylation targets of thioredoxin 1, an enigmatic protein with both reported transnitrosylase and denitrosylase activities.

P2X7 Receptor Augments LPS-Induced Nitrosative Stress by Regulating Nrf2 and GSH Levels in the Mouse Hippocampus.

P2X7 receptor (P2X7R) regulates inducible nitric oxide synthase (iNOS) expression/activity in response to various harmful insults. Since P2X7R deletion paradoxically decreases the basal glutathione (GSH) level in the mouse hippocampus, it is likely that P2X7R may increase the demand for GSH for the maintenance of the intracellular redox state or affect other antioxidant defense systems. Therefore, the present study was designed to elucidate whether P2X7R affects nuclear factor-erythroid 2-related factor 2 (Nrf2) activity/expression and GSH synthesis under nitrosative stress in response to lipopolysaccharide (LPS)-induced neuroinflammation. In the present study, P2X7R deletion attenuated iNOS upregulation and Nrf2 degradation induced by LPS. Compatible with iNOS induction, P2X7R deletion decreased S-nitrosylated (SNO)-cysteine production under physiological and post-LPS treated conditions. P2X7R deletion also ameliorated the decreases in GSH, glutathione synthetase, GS and ASCT2 levels concomitant with the reduced S-nitrosylations of GS and ASCT2 following LPS treatment. Furthermore, LPS upregulated cystine:glutamate transporter (xCT) and glutaminase in P2X7R+/+ mice, which were abrogated by P2X7R deletion. LPS did not affect GCLC level in both P2X7R+/+ and P2X7R-/- mice. Therefore, our findings indicate that P2X7R may augment LPS-induced neuroinflammation by leading to Nrf2 degradation, aberrant glutamate-glutamine cycle and impaired cystine/cysteine uptake, which would inhibit GSH biosynthesis. Therefore, we suggest that the targeting of P2X7R, which would exert nitrosative stress with iNOS in a positive feedback manner, may be one of the important therapeutic strategies of nitrosative stress under pathophysiological conditions.

Identification of endogenously S-nitrosylated proteins in Arabidopsis plantlets: effect of cold stress on cysteine nitrosylation level.

S-nitrosylation is a nitric oxide (NO)-based post-translational modification regulating protein function and signalling. We used a combination between the biotin switch method and labelling with isotope-coded affinity tag to identify endogenously S-nitrosylated peptides in Arabidopsis thaliana proteins extracted from plantlets. The relative level of S-nitrosylation in the identified peptides was compared between unstressed and cold-stress seedlings. We thereby detected 62 endogenously nitrosylated peptides out of which 20 are over-nitrosylated following cold exposure. Taken together these data provide a new repertoire of endogenously S-nitrosylated proteins in Arabidopsis with cysteine S-nitrosylation site. Furthermore they highlight the quantitative modification of the S-nitrosylation status of specific cysteine following cold stress.