Mechanistic studies of mitochondrial outer membrane permeabilization (MOMP)

• Main Author: Pang, Xiaming
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2013
• Subjects: Biochemistry; Cellular biology; Oncology
• ISBN: 1303722968; 9781303722967

The mechanism of Bax/Bak-dependent mitochondrial outer membrane permeabilization (MOMP), a central apoptotic event primarily controlled by the Bcl-2 family proteins, remains not well understood. The major work of this study is to investigate MOMP regulation via phage/bacteria system. We express active Bax/Bak in bacteria, the putative origin of mitochondria, and examine their functional similarities to the λ bacteriophage (λ) holin. As critical effectors for bacterial lysis, holin oligomers form membrane lesions, through which endolysin, a muralytic enzyme, escapes the cytoplasm to attack the cell wall at the end of the infection cycle. We found that active Bax/Bak, but not any other Bcl-2 family protein, displays holin behavior, causing bacterial lysis by releasing endolysin in an oligomerization-dependent manner. Strikingly, replacing the holin gene with active alleles of Bax/Bak results in plaque-forming phages. Furthermore, we provide evidence that active Bax produces large membrane holes, the size of which is controlled by structural elements of Bax. Notably, lysis by active Bax is inhibited by Bcl-xL, and the lysis activity of the wild-type Bax is stimulated by a BH3-only protein, tBid or Bim. These results mechanistically link MOMP to holin-mediated hole formation in the bacterial plasma membrane. In this study, we also aim to understand how MOMP is regulated by protein stability control. As a critical mediator for MOMP, the BH3-only protein Noxa was found to be degraded by the proteasome in a ubiquitin-independent manner. The mechanism by which Noxa is degraded remains unclear. By mutagenesis analysis, we show that proteasomal degradation of Noxa in Hela cells is dependent on a novel degron sequence, which is able to destabilize GFP and Bcl-xL. Interestingly, the Noxa degron sequence is also involved in the regulation of Mcl-1 stability. These results provide important clues to the regulation of Noxa stability and how Noxa targets Mcl-1 for proteasomal degradation. Overall, this work contributes to our understanding of the mechanisms of Bax/Bak-mediated MOMP, and provides critical insights into the design of therapeutics against cancer and/or other apoptosis-related diseases.