SwissPalm: Protein Palmitoylation database

Abstract

Protein S-palmitoylation is a reversible post-translational modification that regulates many key biological processes, although the full extent and functions of protein S-palmitoylation remain largely unexplored. Recent developments of new chemical methods have allowed the establishment of palmitoyl-proteomes of a variety of cell lines and tissues from different species. As the amount of information generated by these high-throughput studies is increasing, the field requires centralization and comparison of this information. Here we present SwissPalm ( http://swisspalm.epfl.ch), our open, comprehensive, manually curated resource to study protein S-palmitoylation. It currently encompasses more than 5000 S-palmitoylated protein hits from seven species, and contains more than 500 specific sites of S-palmitoylation. SwissPalm also provides curated information and filters that increase the confidence in true positive hits, and integrates predictions of S-palmitoylated cysteine scores, orthologs and isoform multiple alignments. Systems analysis of the palmitoyl-proteome screens indicate that 10% or more of the human proteome is susceptible to S-palmitoylation. Moreover, ontology and pathway analyses of the human palmitoyl-proteome reveal that key biological functions involve this reversible lipid modification. Comparative analysis finally shows a strong crosstalk between S-palmitoylation and other post-translational modifications. Through the compilation of data and continuous updates, SwissPalm will provide a powerful tool to unravel the global importance of protein S-palmitoylation.

Keywords: Acyl-RAC; Acyl-biotin exchange; S-palmitoylation; database; palmitoyl-proteomes; proteomics.

Figure 1.. Palmitoyl-proteomes integrated in SwissPalm.

A: Database content: Primary data on S-palmitoylation of proteins are extracted from MS large scale experiments on different species. Curated data on S-palmitoylation are obtained from the literature and input together with the MS information in the same data structure. In order to perform complex query related to S-palmitoylation, we have integrated in the database various external sources like orthology databases (Ortho_DB and OMA), UniProt features and subcellular localization information and Interpro domains. We ran also programs to have additional data, like multiple alignments of orthologous proteins or protein isoforms, and results from existing S-palmitoylation site predictors (CSS-Palm 4.0 and PalmPred). Web interface: The web interface presents the knowledge on S-palmitoylation in protein-centric pages. These pages are accessible through queries on a search engine. Some tools to analyse S-palmitoylation datasets are available online, like the orthologs comparison tool, aiming to perform cross-species S-palmitoylation comparison. B: 19 palmitoyl-proteomes from 7 species and various cell types and tissues were selected from published literature and integrated to SwissPalm. In total the dataset includes 5199 proteins.

Figure 2.. Improved confidence in S-palmitoylation protein hits.

A: Analysis of the 1372 human hits contained in 8 palmitoyl proteomes: 672 S-palmitoylation hits are present in at least 2 human palmitoyl proteomes or are annotated as “high confident hits” (HC). 204 are only found in at least 2 human palmitoyl proteomes, 136 are only classified as HC and 332 S-palmitoylation hits are both found in more than one palmitoyl-proteome and classified as HC. Out of the 672 hits, 345 are identified with 2 independent techniques. 63 out of the 672 S-palmitoylation hits have been validated in targeted studies, while 24 out of 700 hits only found in 1 human palmitoyl proteome have been validated. B: Analysis of the 1747 mouse hits contained in 6 palmitoyl proteomes as described in A. C: Analysis of the 2541 human orthologous hits contained in 19 palmitoyl proteomes as described in A. D: Number of the S-palmitoylation hits by the occurrence of palmitoyl-proteomes in which they have been identified.

Figure 3.. Search and result page.

A: SwissPalm search page: Example of query for “calnexin” shows that it has been found in palmitoyl-proteomes from several species: human (7 out of 8 screens), mouse (6 out of 6), rat (1 out of 1) and Arabidopsis thaliana (1 out of 1). For human, mouse and rat calnexin was classified as a high confident hit and for human and mouse identified by two independent techniques (metabolic labeling and chemical capture). Finally, calnexin S-palmitoylation was also subject to targeted studies and 2 cysteine residues (502 and 503 in human calnexin) were identified. B: Results Page from human calnexin display summary boxes containing the main information related to S-palmitoylation: number of occurrences in palmitoyl-proteome screens and targeted studies, sites information, cysteine prediction.

Figure 4.. Additional information.

A: (upper) Global alignment of isoform sequences highlighting all cysteine residues. (lower) When available, information on protein topology, disulfide bond involvement and prediction scores from CSS-Palm 4.0 and PalmPred are provided for each cysteine residue in the different isoform sequences. B: Global alignment of orthologs sequences show conserved cysteine residues (502 and 503 in human calnexin) across species.

Figure 5.. Abundance and distribution of S-palmitoylated proteins in the mammalian proteome.

A: Percentage of S-palmitoylation hits combined from 8 human or 6 palmitoyl proteomes in the human and mouse proteomes. The analysis was extended to human orthologs of mouse and all S-palmitoylation hits present in the dataset. B: Percentage of targeted studies present in palmitoyl-proteomes. C: Topology of human and mouse S-palmitoylation hits. D and E: Distribution of human and mouse S-palmitoylation hits in cellular compartments. F: Enrichment of amino acid nearby validated S-palmitoylated cysteines in all, only cytoplasmic or only membrane proteins.

Figure 6.. Ontology and network analysis of S-palmitoylated proteins.

A: GO term analysis of 470 human S-palmitoylation hits found by 2 independent techniques or by targeted studies. GO terms sharing the same biological functions enriched in S-palmitoylation Hit proteins are clustered. The distance between GO terms corresponds to the inverse numbers of proteins common to the two terms and the size of the circle to the number of proteins associated with the GO term. B: Protein-protein interactions networks analysis of 470 human S-palmitoylation hits found by 2 independent techniques or by targeted studies using STRING software. The interactions (high confidence score > 0.9) are shown in evidence view (pink: experimental evidences and blue database evidences).

Figure 7.. S-palmitoylation of mammalian protein complexes.

A and B: Human and mouse protein complexes enriched in S-palmitoylation hits using the CORUM database. Proteins complexes containing more than 6 proteins and enriched by at least 50% of S-palmitoylation hits were selected. C: Representation of the TRiC complex subunits. Color circles represent the species in which each CCT subunit was identified as S-palmitoylated. The star indicated proteins identified by 2 independent techniques. D: Palmitoylation of the subunits was validated by Acyl-RAC on CCT1, CCT2, CCT3, CCT4 and CCT5 subunits and by ³H-palmitate labelling on CCT1 and CCT2 subunits. (TCE: Total cell extract, NH ₂OH: Hydroxylamine treatment, ³H-palm: radioactive palmitate signal, WB: Western blot signal).

Figure 8.. S-palmitoylation and other posttranslational modifications.

A: Venn diagram displaying the strong co-occurrence of S-palmitoylation, S-nitrosylation and S-glutathionylation modifications within the same proteins. For this analysis human orthologs of mouse proteins were included. B and C: Percentage of phosphorylation and ubiquitination sites in human and mouse S-palmitoylation hits compared to the total protein population. C: Ubiquitination pattern in WT and DHHC2, 5 and 6 HAP1 cells. Total protein extracts from HAP1 cells were subject to Western blot and probe using anti-ubiquitin antibody and actin as loading control.

Supplementary figure S1.. Percentage of human and mouse S-palmitoylation hits in cellular compartments.

Supplementary figure S2.. Strategy to obtain a high confidence list of 470 human and 443 mouse S-palmitoylation hits to perform ontology and network analysis of S-palmitoylation hits.

Supplementary figure S3.. GO term analysis of mouse palmitoyl-proteome hits.

GO term analysis of 443 mouse S-palmitoylation hits found by 2 independent techniques or by targeted studies. GO terms sharing the same biological functions enriched in S-palmitoylation Hit proteins are clustered. The distance between GO terms corresponds to the inverse numbers of proteins common to the two terms and the size of the circle to the number of proteins associated with the GO term.

Supplementary figure S4.. Protein-protein interaction networks within mouse S-palmitoylated proteins.

Protein-protein interactions networks analysis of 443 human S-palmitoylation hits found by 2 independent techniques or by targeted studies using STRING software. The interactions (high confidence score > 0.9) are shown in evidence view (pink: experimental evidences and blue database evidences).