Rapid turnover of McL-1 couples translation to cell survival and apoptosis
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Abstract
Global inhibition of translation contributes to the induction of apoptosis by various cell stimuli including inhibition of PI 3-kinase/Akt survival signaling, viral infection, lipopolysaccharide, and tumor necrosis factor alpha. However, the mechanism by which translation inhibition promotes apoptosis has not been established. This dissertation investigates the mechanism by which inhibition of translation induces apoptosis using the drug cycloheximide as a tool to isolate and study the direct effects of translation inhibition. Cycloheximide-induced cell death was detected within 2 - 4 hours of treatment in various cell types. Pre-treatment with several proteasome inhibitors blocked cell death, indicating that the loss of short-lived protein(s) via proteasome-mediated degradation was responsible for cell death. Multiple observations demonstrated that cycloheximide induces cell death by activation of the intrinsic/mitochondrial pathway of apoptosis. Cycloheximide-induced cell death was blocked by inhibition of caspases or overexpression of Bcl-2 family proteins Bak and Bax were activated concomitantly with detection of cell death following cycloheximide treatment. Moreover, inhibition of the proteasome abrogated Bak/Bax activation, indicating that the loss of short-lived protein(s) induces mitochondrial apoptosis via Bak/Bax activation. Further studies investigated candidate proteins whose loss may be directly responsible for apoptosis upon translation inhibition. These proteins include mitochondria hexokinases I/II, the caspase-8 inhibitor c-FLIP (FLICE inhibitory protein), and the IAP protein survivin. Most prominently, these studies showed a critical role for the anti-apoptotic Bcl-2 family protein Mcl-1, which is rapidly lost following translation inhibition with a half-life of approximately 30 minutes, preceding Bak/Bax activation and detection of cell death. Overexpression of Mcl-1 abrogated cycloheximide-induced apoptosis, while knockdown of Mcl-1 by RNA interference was sufficient to induce apoptosis. Furthermore, knockdown of the pro-apoptotic Bcl-2 family proteins Bim and Bak, which are normally sequestered by Mcl-1, inhibited cycloheximide-induced apoptosis, as did knockdown of Bax. Apoptosis resulting from inhibition of translation thus involves the rapid degradation of Mcl-1, leading to activation of Bim, Bak and Bax. Therefore, due to its rapid turnover, Mcl-1 may serve as a convergence point for signals that affect global translation, coupling translation to cell survival and the apoptotic machinery.
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Thesis (Ph.D.)--Boston University
PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.