Study On Identification And Degradation Pathway Of Ligand Linker 31.1 Interaction Protein

Gap junctions are channels that link the cytoplasm of adjacent cells. These channels allows for the passage of small molecules of<1,000Da, such as ions and secondary messenger molecules. Gap junctions play important roles in various physiologic functions such as regulation of cell proliferation, cell differentiation, tissue development, and cell apoptosis.Gap junctions are made of two hemi-channels, called connexons, which are composed of six molecules of the membrane spanning connexin protein. There are more than21connexin (Cx) genes in the human genome.Connexin31.1, also known as gap junction protein B5(GJB5), is a273amino acid peptide with a molecular mass of31.088kD. It is predominantly expressed in the testes and the skin epidermis. It plays a critical role in early mouse placental development. We separated possible interacting protein with Cx31.1using co-immunoprecipitaion. After in-gel digested and analyzed by HPLC-MS/MS, the possible proteins were identified by peptide sequence tags and database searching in Swiss-prot. Five reproduction-related proteins were identified. The expression levels of gene family of GAGE were tested and the expression levels of GAGE family were reduced when Cx31.1expression level increased.Connexins can be rapidly modulated by their degradation because they have relative short half-lives. Connexin-31.1(Cx31.1) was newly reported to be down-regulated in non-small cell lung cancer (NSCLC) cell lines, and its tumor-suppressive properties include reducing cell proliferation and suppressing cell migration and invasion. However, no reports have tried to describe how a cell regulates Cx31.1level. In this study, Cx31.1was suggested to be degraded through both ubiquitin proteasome system (UPS) and autophagy. Blockage of UPS with MG-132increased Cx31.1level in cells but could not inhibit Cx31.1degradation completely. Autophagy is a degradation pathway that can implicate several diseases and can be induced by cellular stress such as starvation. In HI299cells stably express Cx31.1, level of Cx31.1reduced when autophagy was induced through starvation or Brefeldin A treatment; the reduction was blocked by treatment with autophagy inhibitor as well as by knockdown of the autophagy-related protein Atg5. We also observed Cx31.1was enclosed by double membrane structures-autophagosome. Moreover, Cx31.1degradation through UPS could interact with autophagy system through linkers such as p62.As a conclusion, our study suggested that Cx31.1is a protein involved in reproduction function. Cx31.1has a short half-life of only1-2hours, both autophagy pathway and proteasomal pathway work in harmony in Cx31.1degradation. In this system, Cx31.1ubiquitination and p62may work as a linker between the two degradation pathways.