Regulation of apoptosis by ubiquitination : the functional consequences after Livin associates with SMAC/DIABLO; ARF complexes with COMMD1

• Main Author: Huang, Yafen
• Other Authors: Li Hoi Yeung
• Format: Theses and Dissertations
• Language: English
• Published: 2009
• Subjects: DRNTU::Science::Biological sciences::Biochemistry
• Online Access: https://hdl.handle.net/10356/14963
• Institution: Nanyang Technological University

Ubiquitination is a posttranslational modification that the substrate proteins are covalently conjugated with an ubiquitin or a polyubiquitin chain. Ubiquitination regulates a wide range of cellular progresses including cell cycle progression, transcription and antigen processing in both proteasome dependent and independent manner. Recent findings also substantiate many essential roles of ubiquitination in the regulation of apoptosis. In this thesis, I report my studies on how ubiquitination controls the stability and functions of proteins involved in apoptosis. Livin is a member of the IAP (Inhibitor of apoptosis protein) family. It has been reported that Livin inhibits caspase-3 and -7 in vitro and caspase-9 in vivo and is negatively regulated by Smac/DIABLO (Second mitochondria derived activator of caspases/Direct IAP binding protein with low PI). However the detailed mechanism underlying its anti-apoptotic function has not yet been fully characterized. In the first part of my thesis, the anti-apoptotic function of Livin through its ability to ubiquitinate and degrade death inducer Smac is presented. In summary, it was found that Livin can act as an E3 ubiquitin ligase to target Smac or itself for degradation by the proteasome. RING domain mediated E3 ligase activity is essential for the autoubiquitination and rapid degradation of Livin under normal state. However upon apoptotic stimulation, Livin promotes degradation of its antagonist Smac by accelerating the ubiquitination process. Both BIR domain and RING finger domain of Livin are required for the ubiquitination of Smac in vivo and in vitro. Mutation in BIR domain nullifies the association of Livin and Smac and thus results in a reduced anti-apoptotic function of Livin. Together, these findings provide a novel function of Livin: it exhibits E3 ubiquitin ligase activity to degrade the pivotal apoptotic regulator Smac through the ubiquitin–proteasome pathway. The second part of this thesis is mainly focused on the studies of the functional and physical interaction between tumor suppressor ARF (Alternate reading frame) and COMMD1 (Copper metabolism gene MURR1 domain containing protein 1). In summary, it was found that ARF associates with COMMD1 and promotes K63 mediated polyubiquitination of COMMD1 in a p53 independent way. I found that ARF interacts with COMMD1 in vivo. Deletion analysis of ARF suggests that N-terminal amino acids 15-45 are important for its interaction with COMMD1. In addition, endogenous ARF redistributes from nucleolus to nucleoplasm and interacts with COMMD1 when DNA is damaged by actinomycin D. Interestingly, ARF promotes polyubiquitination of nuclear COMMD1 through the K63 of ubiquitin but not K48, which does not target COMMD1 for proteasomal proteolysis. ARF induced K63 ubiquitination stabilizes COMMD1 in a proteasome independent manner.