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.

Keywords: Alzheimer’s disease; S-nitrosation; neurodegeneration; presenilin pathway; secretase pathway.

Fig. 1.

Site-specific identification of SNO-protein. (A) Schematic for selective labeling and analysis of SNO-proteins. Unmodified Cys-thiols were blocked, and SNO-Cys sites were labeled with the b-SNOTRAP probe. For approach A, b-SNOTRAP–tagged proteins were enriched using streptavidin beads, washed, eluted with denaturing conditions, trypsin-digested, and analyzed by LC–MS/MS. For approach B, the proteome was trypsin-digested, and b-SNOTRAP–tagged peptides were enriched using streptavidin beads, followed by release of the biotin linker by TCEP; alkylated with NEM; and analyzed by LC–MS/MS. (B) Generation of DFIs from NEM-modified peptides.

Fig. S1.

Site-specific identification of SNO-protein. (A) Schematic for selective tagging of protein S-nitrosothiols using the biotin-SNOTRAP (b-SNOTRAP) probe. The SNOTRAP reagent directly reacts with the –SNO group to yield an azaylide intermediate, which rearranges to form the disulfide–iminophosphorane compound. (B) Representative MS/MS spectra for SNO-Cys150 of GAPDH and SNO-Cys284 of GPHN (SNO-Cys is highlighted in red). (C) Representative MS1 spectrums of GAPDH (monoisotopic m/z 944.4688, MH⁺error 0.1 ppm) and GPHN (monoisotopic m/z 565.2814, MH⁺ error 0.2 ppm) identified in cortex with low ppm mass error. (D) Representative total ion chromatograms (TICs) after b-SNOTRAP capture showing ion intensity of untreated (black), TCEP-treated (red), UV-treated (green), and ascorbate-Cu–treated (blue) samples. Chromatogram corresponding to TCEP treatment was plotted with a y axis offset of 1E4. (E) Representative Western blot of total b-SNOTRAP–captured proteins of untreated (cortex control) and TCEP-treated samples. (F) Current methodologies used for detection of SNO-peptides and SNO-Cys sites (, –32).

Fig. 2.

Altered levels of SNO are detected in CK-p25 mice during early stages of neurodegeneration. (A) Timeline of the pathological progression of the CK-p25 mouse model of neurodegeneration. (B) GSNO concentration (μM) in the cortex, hippocampus, and cerebellum of control or CK-p25 mice following induction of p25 expression for 2 wk or 6 wk. Two-way ANOVA; Dunnett’s multiple comparisons; ****P < 0.0001, ***P < 0.001, *P < 0.05; n = 4 for 2-wk samples and n = 3 for 6-wk samples; mean ± SEM. (C) Number of SNO-proteins identified in the cortex, hippocampus, and cerebellum of control and CK-p25 mice following 2-wk induction. (D) Gene ontology analysis of total SNO-proteins identified in the cortex of control mice (dark blue, KEGG pathways; light blue, BPs). (E) Gene ontology analysis of SNO-proteins exclusively identified in the cortex of CK-p25 mice following 2-wk induction (dark red, KEGG pathways; light red, BPs).

Fig. S2.

Altered SNO-proteins during early neurodegeneration. (A) SNO-proteins identified by MS were validated by SNOTRAP Western blot analysis. Representative Western blot of SNO-Synaptophysin, SNO-GAPDH, and SNO-GSK3β in control and CK-p25 mice following 2-wk induction (n = 2). (B) Neuronal proteins previously identified as S-nitrosated that were not in our MS datasets were examined by SNOTRAP Western blot. Representative Western blot of SNO-PTEN, SNO-Cdk5, SNO-Stargazin, SNO-HDAC2, and SNO-PSD95 in control and CK-p25 mice following 2-wk induction (n = 2). (C) Linear motif generated by pLOGO using published sequences that flank SNO-Cys sites detected in C57BL/6 wild-type mice (47).

Fig. 3.

SNO-site linear motif analysis indicates differential preference for SNO-Cys sites during early neurodegeneration. (A) Linear motif generated by pLOGO using sequences flanking SNO-Cys sites detected in control mice. (B) Linear motif generated by pLOGO using sequences flanking SNO-Cys sites exclusively detected in 2-wk–induced CK-p25 mice.

Fig. 4.

Pathways affected by S-nitrosation during neurodegeneration. (A) SNO-LRP1 and SNO-PKC isoforms were identified in CK-p25 mice after 2-wk induction and have been implicated in secretase processing of APP and amyloid clearance. (B) SNO-GSK3β and SNO-TAU were identified in CK-p25 mice after 2-wk induction and have been implicated in neurofibrillary tangle formation and AD pathology. The functional implications of previously unidentified SNO-proteins are represented with a question mark.