S -nitrosation of proteins relevant to Alzheimer’s disease during early stages of neurodegeneration

Abstract

Protein S-nitrosation (SNO-protein), the nitric oxide-mediated posttranslational modification of cysteine thiols, is an important regulatory mechanism of protein function in both physiological and pathological pathways. A key first step toward elucidating the mechanism by which S-nitrosation modulates a protein's function is identification of the targeted cysteine residues. Here, we present a strategy for the simultaneous identification of SNO-cysteine sites and their cognate proteins to profile the brain of the CK-p25-inducible mouse model of Alzheimer's disease-like neurodegeneration. The approach-SNOTRAP (SNO trapping by triaryl phosphine)-is a direct tagging strategy that uses phosphine-based chemical probes, allowing enrichment of SNO-peptides and their identification by liquid chromatography tandem mass spectrometry. SNOTRAP identified 313 endogenous SNO-sites in 251 proteins in the mouse brain, of which 135 SNO-proteins were detected only during neurodegeneration. S-nitrosation in the brain shows regional differences and becomes elevated during early stages of neurodegeneration in the CK-p25 mouse. The SNO-proteome during early neurodegeneration identified increased S-nitrosation of proteins important for synapse function, metabolism, and Alzheimer's disease pathology. In the latter case, proteins related to amyloid precursor protein processing and secretion are S-nitrosated, correlating with increased amyloid formation. Sequence analysis of SNO-cysteine sites identified potential linear motifs that are altered under pathological conditions. Collectively, SNOTRAP is a direct tagging tool for global elucidation of the SNO-proteome, providing functional insights of endogenous SNO proteins in the brain and its dysregulation during neurodegeneration.S-nitrosation | Alzheimer's disease | secretase pathway | presenilin pathway | neurodegeneration P rotein S-nitrosation (SNO-protein), in which a cysteine (Cys) thiol is converted to a nitrosothiol (RSNO), is an important posttranslational modification (PTM). Cys residues targeted for S-nitrosation often impact enzyme activity, protein localization, and protein-protein interactions (1). SNO begins with the production of nitric oxide radicals (NO • ) via conversion of L-arginine to L-citrulline by nitric oxide synthase 1 (NOS1) (neuronal), NOS2 (inducible), and NOS3 (endothelial). NO-mediated SNO PTMs are thought to occur in vivo predominantly through radical recombination between NO • and a thiyl radical, transnitrosation by low-molecular weight NO carriers such as S-nitrosoglutathione (GSNO), or protein-assisted transnitrosation (2-8). In the healthy brain, low levels of NO and normal SNO PTMs play important roles in regulating synaptic plasticity, gene expression, and neuronal survival. In contrast, elevated NO levels associated with aging and environmental stress have been linked to neurological pathologies, including Alzheimer's (AD), Parkinson's, and Huntington's disease (9). AD is the most prevalent form of human dementia, with a frequency that progressiv...

Quantitative proteomics of synaptosome S‐nitrosylation in Alzheimer’s disease

“…In general, the protocol applied in this study yields good comparability when compared to other studies on the synaptic proteome. Nevertheless, only a small overlap was found between the identified proteins and other studies on the SNO-proteome (Seneviratne et al 2016;Amal et al 2019). Yet, these studies analyzed whole-brain regions utilizing different models and enrichment strategies for nitrosylated peptides.…”

“…Cytokine‐induced NOS2 expression causes post‐translational protein modifications through the sustained generation of NO, which can be taken as a fingerprint of neuroinflammatory activity in the brain (Heneka and Feinstein ; Colton et al ). Increased levels of nitrated proteins have been reported in the brain and cerebrospinal fluid (CSF) of patients with AD, indicating a sustained activity of NOS2 in the central nervous system (Nakamura et al ; Seneviratne et al ).…”

“…Studies carried out in NOS2 knockout mice have shown a correlation between NOS2, amyloid beta (Ab) deposition, and cognitive dysfunction in AD mouse models (Colton et al 2008;Kummer et al 2011). The SNO-proteome in cortex and hippocampus tissues is altered in both the CK-p25 mouse model of AD and the P301S mouse model of tauopathy (Seneviratne et al 2016;Amal et al 2019).…”

Dual Maleimide Tagging for Relative and Absolute Quantitation of Cysteine‐Containing Peptides by MALDI‐TOF MS

“…Cysteine thiols are often subjected to a variety of covalent post‐translational modifications (PTMs), which subsequently function as important mediators of redox signaling and regulation . The biomedical significance of several kinds of reversible cysteine PTMs has increasingly been recognized, including disulfide formation, S‐nitrosylation (SNO), S‐glutathionylation (SSG), S‐sulfenylation (SOH), and S‐acylation (alternatively called palmitoylation) . These modifications have been discovered to be linked to many diseases.…”

Chemoproteomic Strategy to Quantitatively Monitor Transnitrosation Uncovers Functionally Relevant S -Nitrosation Sites on Cathepsin D and HADH2

“…These include the SNO-RAC and d-Switch methodologies, which introduce resin-assisted capture and isotopic labeling, to identify and quantify S -nitrosated species from proteomes (Fares et al, 2014; Forrester et al, 2009b; Sinha et al, 2010). Furthermore, protein S -nitrosothiols can be enriched with mercury resin (Doulias et al, 2013; Gould et al, 2015), phosphine reagents that undergo a Staudinger-type reaction (Bechtold et al, 2010; Seneviratne et al, 2013; Seneviratne et al, 2016; Wang and Xian, 2008), and biotinylated sulfinic acids that form stable thiosulfonates (Majmudar et al, 2016). …”