Ubiquitin Mediated Regulation of NF-KB Signaling

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Title: Ubiquitin Mediated Regulation of NF-KB Signaling
Author: Pineda, Gabriel
Abstract: 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 .
URI: http : / /hdl .handle .net /2152 .5 /391
Date: 2008-05-13


Ubiquitin Mediated Regulation of NF-KB Signaling. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /391 .

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