ac839d1bd4ac19e38fcf4e3d47031492 pmc_9867282.pdf 8f7b2608947aa70356166ba1bea8881f5328d311 pmc_9867282.pdf b2aa3125907dace1348d17925c235b2119fca5e01912edaa5a58438fe44ae801 pmc_9867282.pdf Title: Proximity Mapping of CCP6 Reveals Its Association with Centrosome Organization and Cilium Assembly Subject: The cytosolic carboxypeptidase 6 (CCP6) catalyzes the deglutamylation of polyglutamate side chains, a post-translational modification that affects proteins such as tubulins or nucleosome assembly proteins. CCP6 is involved in several cell processes, such as spermatogenesis, antiviral activity, embryonic development, and pathologies like renal adenocarcinoma. In the present work, the cellular role of CCP6 has been assessed by BioID, a proximity labeling approach for mapping physiologically relevant protein–protein interactions (PPIs) and bait proximal proteins by mass spectrometry. We used HEK 293 cells stably expressing CCP6-BirA* to identify 37 putative interactors of this enzyme. This list of CCP6 proximal proteins displayed enrichment of proteins associated with the centrosome and centriolar satellites, indicating that CCP6 could be present in the pericentriolar material. In addition, we identified cilium assembly-related proteins as putative interactors of CCP6. In addition, the CCP6 proximal partner list included five proteins associated with the Joubert syndrome, a ciliopathy linked to defects in polyglutamylation. Using the proximity ligation assay (PLA), we show that PCM1, PIBF1, and NudC are true CCP6 physical interactors. Therefore, the BioID methodology confirms the location and possible functional role of CCP6 in centrosomes and centrioles, as well as in the formation and maintenance of primary cilia. Keywords: cytosolic carboxypeptidases; CCP6; polyglutamylation; proteomics; biotin-dependent proximity labeling (BioID); BirA*; centrioles; cilia; Joubert syndrome; cilium assembly Author: Sergi Rodriguez-Calado, Petra Van Damme, Francesc Xavier Avilés, Ana Paula Candiota, Sebastian Tanco and Julia Lorenzo Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Jan 9 08:35:43 2023 CET ModDate: Tue Jan 17 01:09:08 2023 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 21 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2680654 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- BTFGED+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 XQKSOB+VnURWPalladioL Type 1 Custom yes yes no 15 0 XFJLWT+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 XSVZTY+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 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147 0 FENMCD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 150 0 FENMBP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 164 0 FENMCB+PalatinoLinotype TrueType WinAnsi yes yes no 167 0 FENMCD+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 170 0 NJWNDJ+VnURWPalladioL-Italic Type 1 Custom yes yes no 208 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-10-06 02:24:31 CEST RepresentationInformation: pmc_9867282.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-01-26 02:11:19 CET Size: 2680654 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 576 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Children: Item: Title: Identification of CCP6 Proximal Interactors Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: CCP6 Interacts with PCM1 and PIBF1 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: CCP6 Interacts with NudC Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Children: Item: Title: Cell Lines and Cell Culture Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Interactomic Studies Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Children: Item: Title: Molecular Cloning and Generation of Stable Cell Lines Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: BioID Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Mass Spectrometry Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Gene Ontology Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Human Cell Map Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Co-Immunoprecipitation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Western Blotting Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Confocal Microscopy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Children: Item: Title: Proximity Ligation Assay (PLA) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Immunocytochemistry and Image Acquisition Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Info: Title: Proximity Mapping of CCP6 Reveals Its Association with Centrosome Organization and Cilium Assembly Author: Sergi Rodriguez-Calado, Petra Van Damme, Francesc Xavier Avilés, Ana Paula Candiota, Sebastian Tanco and Julia Lorenzo Subject: The cytosolic carboxypeptidase 6 (CCP6) catalyzes the deglutamylation of polyglutamate side chains, a post-translational modification that affects proteins such as tubulins or nucleosome assembly proteins. CCP6 is involved in several cell processes, such as spermatogenesis, antiviral activity, embryonic development, and pathologies like renal adenocarcinoma. In the present work, the cellular role of CCP6 has been assessed by BioID, a proximity labeling approach for mapping physiologically relevant protein protein interactions (PPIs) and bait proximal proteins by mass spectrometry. We used HEK 293 cells stably expressing CCP6-BirA* to identify 37 putative interactors of this enzyme. This list of CCP6 proximal proteins displayed enrichment of proteins associated with the centrosome and centriolar satellites, indicating that CCP6 could be present in the pericentriolar material. In addition, we identified cilium assembly-related proteins as putative interactors of CCP6. In addition, the CCP6 proximal partner list included five proteins associated with the Joubert syndrome, a ciliopathy linked to defects in polyglutamylation. Using the proximity ligation assay (PLA), we show that PCM1, PIBF1, and NudC are true CCP6 physical interactors. Therefore, the BioID methodology confirms the location and possible functional role of CCP6 in centrosomes and centrioles, as well as in the formation and maintenance of primary cilia. Keywords: cytosolic carboxypeptidases; CCP6; polyglutamylation; proteomics; biotin-dependent proximity labeling (BioID); BirA*; centrioles; cilia; Joubert syndrome; cilium assembly Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Jan 09 08:35:43 CET 2023 ModDate: Tue Jan 17 01:09:08 CET 2023 ID: 0xef88ce7fdd37c77ddf590847821fd6c4, 0xef88ce7fdd37c77ddf590847821fd6c4 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: LEVXPH+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: LEVXPH+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: NJWNDJ+VnURWPalladioL-Italic FontSubset: true FirstChar: 189 LastChar: 226 FontDescriptor: FontName: NJWNDJ+VnURWPalladioL-Italic Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: WBVKDJ+URWPalladioL-BoldItal FontSubset: true FirstChar: 112 LastChar: 112 FontDescriptor: FontName: 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