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Study of connexin-43 protein-protein interactions involved in glioma migration, A
Brandon University, Faculty of Science
xi, 78 pages : graphs, charts
Includes bibliographical references (pages 59-65).
"In partial fulfillment of the requirements for the degree of Master of Science, Evironmental and Life Sciences."
Glioblastoma Multiforme (GBM) are aggressive tumors of the central nervous system (CNS) that are characterized by proliferation and a strong tendency of cancer cells to infiltrate the brain. The mechanisms underlying GBM migration involves the degradation and remodeling of the extracellular matrix (ECM). Gap junctions are intercellular channels that permit the exchange of small metabolites ([less than] 1 kDa) between adjacent cells. Gap junction proteins (connexins, Cxs) have been shown to promote the in vivo and in vitro invasiveness of GBM. We have demonstrated previously connexin43 (Cx43) increases glioma motility and increases the expression of promalignancy factors such as MMP3 and osteopontin. The cellular mechanisms of connexin-induced migration are poorly understood. As glioma migration and invasiveness are hallmarks of GBM malignancy, we have sought to identify protein-protein interactions and signaling networks downstream of Cx43. In this study, we have identified the interaction of Cx43 with annexinA2, a calcium-dependent binding protein linked to the increase of cell migration and MMP/protease activation. The interaction of Cx43/annexinA2 was identified by Cx43 co-IPs (LC-MS/MS), and the proteomic analysis gap junction-enriched fractions (differential centrifugation, and highthroughput, stable isotope labeling, HPLC-MS/MS) and reciprocal Co-IPs (SDS-PAGE/Western blot). As network bioinformatic analysis of Cx43 pathways in glioma predicts increases cell motility, we further demonstrate peptide inhibitors down-stream of Cx43/annexinA2 downregulates MMP-3 activity (by gelatin zymographic assays) and expression (by Western blot). Our characterization of interactions Cx43/annexinA2 identifies potentially druggable targets for GBM.
Glioblastoma multiforme--Genetic aspectsCancer invasivenessExtracellular matrix proteinsConnexinsMetalloproteinasesProtein bindingGenetic regulation
Brandon University.Faculty of Science