Ubiquitin Mediated Regulation of NF-kappa B Signaling




Pineda, Gabriel

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NF-κB signaling is involved in many vital cellular functions such as immunity, cell proliferation, inflammation, and apoptosis. The activation of NF-κB signaling requires the process of ubiquitination. K63-and K48-linked ubiquitin chains have been shown to have distinct roles and biological function in NF-κB signaling. K63-linked ubiquitin chains are required for the activation of TAK1, which leads to the activation of IKK. Activation of IKK leads to K48-linked ubiquitination, and the subsequent proteasomal degradation of IκBalpha . Two important areas of research focusing on ubiquitin regulation of NF-κB signaling are addressed in this dissertation. The areas addressed include understanding how ubiquitinated substrates are targeted for proteasomal degradation and how CYLD negatively regulates NF-κB signaling. In these studies, I investigated the molecular mechanisms involved in the regulation of IκBalpha degradation. Using a siRNA approach, NPL4 was shown to be required for IκBalpha degradation. In vitro proteasomal degradation assays demonstrated that the NPL4 complex is required for IκBalpha degradation. Evidence from both in vitro and in vivo studies suggest NPL4 is required for IκBalpha degradation, but not for IKK activation. These results suggest NPL4 is working at a step after ubiquitination of IκBalpha , but before proteasomal degradation. I propose that ubiquitinated IκBalpha is targeted to the proteasome by an interaction between the NPL4 complex that is mediated through the zinc finger domain of NPL4. The cylindromatosis tumor suppressor gene (CYLD) encodes a 110 kDa deubiquitination enzyme that negatively regulates NF-κB signaling. Loss-of-function mutations in CYLD lead to the disease Familial Cylindromatosis, which is characterized by the formation of benign skin tumors that originate from the head and neck of individuals afflicted with the disease. Here I present in vitro evidence that CYLD inhibits both TAK1 and IKK activation by TRAF6 in a cell free system. I also demonstrate, using a highly purified in vitro system, that CYLD specifically cleaves K63 linked ubiquitin chains and harbors endoproteolytic activity. Furthermore, the third CAPGLY domain of CYLD was shown to be a novel ubiquitin binding domain. My results provide biochemical evidence that CYLD functions as a K63 deubiquitinase to attenuate NF-κB signaling.

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