Stress-induced Changes in the S-palmitoylation and S-nitrosylation of Synaptic Proteins

Abstract

The precise regulation of synaptic integrity is critical for neuronal network connectivity and proper brain function. Essential aspects of the activity and localization of synaptic proteins are regulated by posttranslational modifications. S-palmitoylation is a reversible covalent modification of the cysteine with palmitate. It modulates affinity of the protein for cell membranes and membranous compartments. Intracellular palmitoylation dynamics are regulated by crosstalk with other posttranslational modifications, such as S-nitrosylation. S-nitrosylation is a covalent modification of cysteine thiol by nitric oxide and can modulate protein functions. Therefore, simultaneous identification of endogenous site-specific proteomes of both cysteine modifications under certain biological conditions offers new insights into the regulation of functional pathways. Still unclear, however, are the ways in which this crosstalk is affected in brain pathology, such as stress-related disorders. Using a newly developed mass spectrometry-based approach Palmitoylation And Nitrosylation Interplay Monitoring (PANIMoni), we analyzed the endogenous S-palmitoylation and S-nitrosylation of postsynaptic density proteins at the level of specific single cysteine in a mouse model of chronic stress. Among a total of 813 S-PALM and 620 S-NO cysteine sites that were characterized on 465 and 360 proteins, respectively, we sought to identify those that were differentially affected by stress. Our data show involvement of S-palmitoylation and S-nitrosylation crosstalk in the regulation of 122 proteins including receptors, scaffolding proteins, regulatory proteins and cytoskeletal components. Our results suggest that atypical crosstalk between the S-palmitoylation and S-nitrosylation interplay of proteins involved in synaptic transmission, protein localization and regulation of synaptic plasticity might be one of the main events associated with chronic stress disorder, leading to destabilization in synaptic networks.

Keywords: Animal models; Imaging Visualization Tools; Label-free quantification; Neurobiology; PLA; Post-translational modifications; Psychiatric disease; S-nitrosylation; S-palmitoylation; postsynaptic density; synapse.

Graphical abstract

Fig. 1.

Schematic representation of PANIMoni proteomic approach for large-scale, site-specific S-palmitoylation and S-nitrosylation monitoring. Protein modifications were selectively replaced with biotin groups. After trypsin digestion and enrichment on avidin resin, the S-PALM and S-NO peptides were analyzed using MS in two independent runs (LC-MS and LC-MS/MS). MSparky software was used for site-specific data analysis (21).

Fig. 2.

Crosstalk between protein S-palmitoylation and S-nitrosylation. A, Imaging of total proteome palmitoylation in neuronal cultures subjected to click chemistry reaction with Oregon Green 488 dye. Scale bar represent 50 μm. B, Quantitation of average fluorescence intensity of Oregon Green under the same treatment conditions as in (A). C, Western blot analysis of ABE used to detect total S-palmitoylome in primary cortical neurons treated with corticosterone, 2Br, SNAP, and l-NAME. Results are presented as the mean ± S.E.

Fig. 3.

S-palmitoylome of mouse PSD proteins. A, Schematic representation of S-palmitoylation analysis. B, Western blot analysis of S-palmitoylation in the PSD protein fraction. C, Mass spectrometry chromatograms LC-MS/MS showing base peak chromatograms of eluted peptides ions (MS1 mode). Results depict PANIMoni analysis of PSDs proteins in negative control (- hydroxylamine) and positive sample (+ hydroxylamine). Note the detection of peptide ions at low levels in negative control and abundant levels in positive sample. D, Mass spectrometry-based analysis of S-palmitoylation that shows a high correlation between two biological replicates (CV < 1%).

Fig. 4.

Schematic representation of the mouse model of chronic restraint stress and behavioral tests. A, Schematic illustration of mouse treatment. B, Tail suspension test. Results are represented as mean ± S.E. with N_mice = 12 in each group. C, Corticosterone levels. Results are represented as mean ± S.E. with N_mice = 15 in each group. D, Changes in body mass recorded every other day throughout the experiment. Results are represented as mean ± S.E. with N_mice = 15 in each group. Values are significant at P * <0.05, ***<0.01 as compared with control group. Statistical significance was determined by the heteroscedastic two-tailed t test.

Fig. 5.

Analysis of changes in protein expression in mouse brains after chronic restraint stress. A, Principal Component Analysis of identified proteins in control and chronically stressed mice. Black points represent proteins; the color-coded points represent samples, either from control (blue) or stressed (red). The 2-dimensional graph represents the first two main components of the samples. As expected, the control/stressed status is the principal component responsible for the differences among samples. (n_samples = 3, N_mice = 3) B, Volcano plot showing proteins differentially expressed between control and chronic stress PSDs (n_samples = 3, N_mice = 3). Proteins with statistically significant differential expression (≥0.5-fold change <0.5, p < 0.05) are located in the right upper and left quadrants. C, Distributions of the log-ratios (vertical axis) and q values (horizontal axis) of proteins from control and chronically stressed mice (data analyzed using Diffprot). The numbers of peptides per protein are color-coded as shown in the inserts. In the Diffprot analysis, 6 protein clusters out of 1293 were assigned q-values below 0.05 (i.e. they are significantly regulated). D, Bar-graphs show detailed differential analysis of down and up-regulated proteins after chronic stress.

Fig. 6.

Analysis of changes in protein S-palmitoylation in mouse brains after chronic restraint stress. A, Western blot analysis of S-palmitoylation pattern in control mice and chronically stressed mice. S-palmitoylation sites were selectively labeled with S-S-biotin (ABE). The visualization of biotinylated proteins was achieved using anti-biotin antibodies. (n_samples = 3, N_mice = 6; pulled PSDs fractions from two mice per one sample). Negatives - negative controls without specific reduction of S-acyl bonds with hydroxylamine (pulled 3 negative controls into 1 control (negatives 1) and 1 chronic stress (negatives 2). B, Venn diagram comparisons of the numbers of S-PALM proteins and sites that were identified in synaptic protein fractions from control mice and chronically stressed mice. (n_samples = 3, N_mice = 6; pulled PSDs fractions from two mice per one sample). C, Schematically depicted results from mass spectrometry for the newly discovered targets of S-PALM, Striatin4 Shank3 and a well-known PSD-95. Differential analysis of S-PALM in control and chronic stress PSDs using the Acyl biotin-PEG exchange method (AbPE) reveals site-specific S-PALM of protein targets, Striatin-4 and Shank3. S-palmitoylation of PSD protein was used as an internal control (n_samples = 3, N_mice = 6).

Fig. 7.

Differential protein S-nitrosylation after chronic stress. A, Schematic representation of S-nitrosylation analysis. B, Western blot analysis of S-nitrosylation pattern in control mice and chronically stressed mice. S-nitrosylation sites were selectively labeled with S-S-biotin (BSM). The visualization of biotinylated proteins was achieved using anti-biotin antibodies. (n_samples = 3, N_mice = 6; pulled PSDs fractions from two mice per one sample) Negatives - negative controls without specific reduction of S-NO bonds with ascorbate (pulled 3 negative controls into one (negatives 1) and chronic stress (negatives 2). C, Venn diagram comparisons of the numbers of S-NO proteins and sites that were identified in synaptic protein fractions from control mice and chronically stressed mice.

Fig. 8.

Aberrant S-palmitoylation/S-nitrosylation crosstalk caused by chronic stress. A, Venn diagram comparisons of the numbers of S-PALM and S-NO proteins and sites that were identified in synaptic protein fractions from control mice and chronically stressed mice. B, 4-Tailed Venn diagram comparison of all identified S-PALM and S-NO proteins. C, S-PALM pattern in control mice and chronically stressed mice that shows differential S-PALM for important synaptic proteins.

Fig. 9.

Bioinformatics tools for the analysis of proteins with aberrant S-PALM/S-NO crosstalk. A–C, Panther software analysis in terms of (A) biological processes (GO_BP), (B) REACTOME pathway analysis, (C) KEGG pathway analysis (D) STRING analysis of protein interactome. E, Bingo analysis of enriched biological classes. The analysis was done using the 'hyper geometric test', and all GO terms that were significant with p < 0.05 (after correcting multiple term testing by Benjamini and Hochberg false discovery rate corrections) were selected as overrepresented.