Differential S-nitrosylation of proteins in Alzheimer's disease

Abstract

Numerous studies have provided evidence regarding the involvement of protein S-nitrosylation in the progression of Alzheimer's disease (AD) pathology and its implication in the formation and accumulation of misfolded protein aggregates. The identification of S-nitrosylated proteins can be a major step toward the understanding of mechanisms leading to neuronal degeneration. The present study targeted S-nitrosylated proteins in AD hippocampus, substantia nigra and cortex using the following work-flow that combines S-nitrosothiol-specific antibody detection, classical biotin switch method labeled with fluorescence dye followed by electrospray ionization quadrupole time of flight tandem MS (ESI-QTOF MS/MS) identification. Endogenous nitrosocysteines were identified in 45 proteins, mainly involved in metabolism, signaling pathways, apoptosis and redox regulation as assigned by REACTOME and KEGG pathway database analysis. Superoxide dismutase (SOD2) [Mn], fructose-bisphosphate aldolase C (ALDOC) and voltage-dependent anion-selective channel protein 2 (VDAC2) showed differential S-nitrosylation signal, not previously reported in AD regions. Extensive neuronal atrophy with increased protein S-nitrosylation in AD regions is also evident from immunofluorescence studies using S-nitrosocysteine antibody. A number of plausible cysteine modification sites were predicted via Group-based Prediction System-S-nitrosothiols (GPS-SNO) 1.0 while STRING 8.3 analysis revealed functional annotations in the modified proteins. The findings are helpful in characterization of functional abnormalities and may facilitate the understanding of molecular mechanisms and biological function of S-nitrosylation in AD pathology.

Keywords: 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; 2DE; ACT; AD; ALDO; Alzheimer’s disease; BSM; ENO; ESI-QTOF-MS/MS; GAPDH; GPS; HEPES; IP; IPG; LDH; N-ethylmaleimide; NADP; NDDs; NEM; NO; PEBP; PTMs; RNS; S-nitrosocysteine; S-nitrosothiols; SNO; SNO-Cys; SOD2; TBST; TPI; TUBA; TUBB; Tris-buffered saline and Tween 20; VDAC2; WEB-based Gene SeT AnaLysis Toolkit; WEBGESTALT; actin; aldolase; biotin switch method; cortex; electrospray ionization quadrupole time of flight tandem MS; enolase; glyceraldehyde-3-phosphate dehydrogenase; group-based prediction system; hippocampus; immobilized pH gradients; immunoprecipitation; lactate dehydrogenase; neurodegenerative diseases; nicotinamide adenine dinucleotide phosphate; nitric oxide; phosphatidylethanolamine-binding protein; post-translational modifications; protein S-nitrosylation; proteomics; reactive nitrogen species; substantia nigra; superoxide dismutase; triose phosphate isomerase; tubulin alpha; tubulin beta; two-dimensional electrophoresis; voltage-dependent anion-selective channel protein 2.