title: Proteomic Analysis of Connexin 43 Reveals Novel Interactors Related to Osteoarthritis.
abstract: We have previously reported that articular chondrocytes in tissue contain long cytoplasmic arms that physically connect two distant cells. Cell-to-cell communication occurs through connexin channels termed Gap Junction (GJ) channels, which achieve direct cellular communication by allowing the intercellular exchange of ions, small RNAs, nutrients, and second messengers. The Cx43 protein is overexpressed in several human diseases and inflammation processes and in articular cartilage from patients with osteoarthritis (OA). An increase in the level of Cx43 is known to alter gene expression, cell signaling, growth, and cell proliferation. The interaction of proteins with the C-terminal tail of connexin 43 (Cx43) directly modulates GJ-dependent and -independent functions. Here, we describe the isolation of Cx43 complexes using mild extraction conditions and immunoaffinity purification. Cx43 complexes were extracted from human primary articular chondrocytes isolated from healthy donors and patients with OA. The proteomic content of the native complexes was determined using LC-MS/MS, and protein associations with Cx43 were validated using Western blot and immunolocalization experiments. We identified >100 Cx43-associated proteins including previously uncharacterized proteins related to nucleolar functions, RNA transport, and translation. We also identified several proteins involved in human diseases, cartilage structure, and OA as novel functional Cx43 interactors, which emphasized the importance of Cx43 in the normal physiology and structural and functional integrity of chondrocytes and articular cartilage. Gene Ontology (GO) terms of the proteins identified in the OA samples showed an enrichment of Cx43-interactors related to cell adhesion, calmodulin binding, the nucleolus, and the cytoskeleton in OA samples compared with healthy samples. However, the mitochondrial proteins SOD2 and ATP5J2 were identified only in samples from healthy donors. The identification of Cx43 interactors will provide clues to the functions of Cx43 in human cells and its roles in the development of several diseases, including OA.
authors.affiliation: From the ‡CellCOM Research Group. Instituto de Investigación Biomédica A Coruña (INIBIC), XXIAC, University of A Coruña. Xubias de Arriba 84, 15006 A Coruña, Spain;
authors.forename: Raquel
authors.initials: R
authors.lastname: Gago-Fuentes
authors.affiliation: §Rheumatology Division, ProteoRed/ISCIII, Proteomics Group, Instituto de Investigación Biomédica A Coruña (INIBIC), XXIAC. University of A Coruña. Xubias de Arriba 84, 15006 A Coruña, Spain; ¶Rheumatology Division, CIBER-BBN/ISCIII, Instituto de Investigación Biomédica A Coruña (INIBIC), XXIAC. University of A Coruña. Xubias de Arriba 84, 15006 A Coruña, Spain;
authors.forename: Patricia
authors.initials: P
authors.lastname: Fernández-Puente
authors.affiliation: ‖Confocal Microscopy Core Unit. Centro Nacional de Investigaciones Oncológicas (CNIO), 28029, Madrid, Spain.
authors.forename: Diego
authors.initials: D
authors.lastname: Megias
authors.affiliation: From the ‡CellCOM Research Group. Instituto de Investigación Biomédica A Coruña (INIBIC), XXIAC, University of A Coruña. Xubias de Arriba 84, 15006 A Coruña, Spain;
authors.forename: Paula
authors.initials: P
authors.lastname: Carpintero-Fernández
authors.affiliation: §Rheumatology Division, ProteoRed/ISCIII, Proteomics Group, Instituto de Investigación Biomédica A Coruña (INIBIC), XXIAC. University of A Coruña. Xubias de Arriba 84, 15006 A Coruña, Spain; ¶Rheumatology Division, CIBER-BBN/ISCIII, Instituto de Investigación Biomédica A Coruña (INIBIC), XXIAC. University of A Coruña. Xubias de Arriba 84, 15006 A Coruña, Spain;
authors.forename: Jesus
authors.initials: J
authors.lastname: Mateos
authors.affiliation: From the ‡CellCOM Research Group. Instituto de Investigación Biomédica A Coruña (INIBIC), XXIAC, University of A Coruña. Xubias de Arriba 84, 15006 A Coruña, Spain;
authors.forename: Benigno
authors.initials: B
authors.lastname: Acea
authors.affiliation: From the ‡CellCOM Research Group. Instituto de Investigación Biomédica A Coruña (INIBIC), XXIAC, University of A Coruña. Xubias de Arriba 84, 15006 A Coruña, Spain;
authors.forename: Eduardo
authors.initials: E
authors.lastname: Fonseca
authors.affiliation: §Rheumatology Division, ProteoRed/ISCIII, Proteomics Group, Instituto de Investigación Biomédica A Coruña (INIBIC), XXIAC. University of A Coruña. Xubias de Arriba 84, 15006 A Coruña, Spain; ¶Rheumatology Division, CIBER-BBN/ISCIII, Instituto de Investigación Biomédica A Coruña (INIBIC), XXIAC. University of A Coruña. Xubias de Arriba 84, 15006 A Coruña, Spain;
authors.forename: Francisco Javier
authors.initials: FJ
authors.lastname: Blanco
authors.affiliation: From the ‡CellCOM Research Group. Instituto de Investigación Biomédica A Coruña (INIBIC), XXIAC, University of A Coruña. Xubias de Arriba 84, 15006 A Coruña, Spain; Ma.Dolores.Mayan.Santos@sergas.es fblagar@sergas.es.
authors.forename: Maria Dolores
authors.initials: MD
authors.lastname: Mayan
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D002358:Cartilage, Articular / Q000473:pathology; D002467:Cell Nucleus / Q000378:metabolism; D019902:Chondrocytes / Q000378:metabolism; D018031:Connexin 43 / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008875:Middle Aged; D010003:Osteoarthritis / Q000378:metabolism* / Q000473:pathology; D011485:Protein Binding; D025941:Protein Interaction Mapping; D021381:Protein Transport; D040901:Proteomics / Q000379:methods*; D014746:Vimentin / Q000378:metabolism
chemical_list: D018031:Connexin 43; C500216:GJA1 protein, human; D018841:HSP90 Heat-Shock Proteins; D014746:Vimentin