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.
Highlights
IntroductionXubias de Arriba 84, 15006 A Coruna, Spain; §Rheumatology Division, ProteoRed/ISCIII, Proteomics Group, Instituto de Investigacion Biomedica A Coruna (INIBIC), XXIAC
One hundred and eighteen proteins were specific to the connexin 43 (Cx43) immunoprecipitation (IP) and not identified in the control IP, which was performed without antibody (See supplemental Tables S1 and S2)
The meta-analysis to determine the degree of overlap between the identified interactors in this study and the identified proteins in previous studies revealed a set of proteins (n ϭ 108 for healthy and n ϭ 129 for chondrocytes from OA patients) that were exclusive to the present study and 15 and 28 common interactors
Summary
Xubias de Arriba 84, 15006 A Coruna, Spain; §Rheumatology Division, ProteoRed/ISCIII, Proteomics Group, Instituto de Investigacion Biomedica A Coruna (INIBIC), XXIAC. Many aspects of Cx43 function, for example cellular transport, plaque assembly and stability, and channel conductivity are most likely governed by interactions with regulatory and structural proteins that bind to the cytoplasmic domains of Cx43 These proteins include the tight junction protein zonula occludens-1 (ZO-1) [12,13,14], 14 –3-3 [15], Drebrin [16], -tubulin [17], c-Src, v-Src [10], and other potential Cx43-interacting proteins that target Cx43 to points of cell– cell contact and regulate gap junctional intercellular communication (GJIC) [10]. These and other studies raise the possibility that the C-terminal tail of Cx43 can control cell-cycle, gene expression, or different signaling pathways via binding proteins independently of its channel function [18]
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