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Ubiquitin-mediated degradation of the proapoptotic active form of bid. A functional consequence on apoptosis
K Breitschopf1, A M Zeiher, S Dimmeler
1Division of Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.
Abstract:
Under basal conditions, the proapoptotic protein Bid is a long-lived protein. Pro-apoptotic stimuli such as tumor necrosis factor-alpha (TNFalpha) or Fas induce its caspase-8-mediated cleavage into two fragments. The COOH-terminal cleavage fragment of Bid (tBid) becomes localized to mitochondrial membranes and triggers the release of cytochrome c. Here we show that tBid is ubiquitinated and subsequently degraded by the 26 S proteasome. Degradation of tBid is significantly inhibited by the proteasome inhibitors MG-132 and lactacystin. In contrast, caspase-specific or lysosomal inhibitors do not affect tBid stability. Furthermore, mutation of the putative ubiquitin acceptor sites within tBid results in a stabilized protein as assessed by pulse-chase analysis. To address whether tBid degradation might be regulated by interaction with other Bcl-2-like proteins, cotransfection studies were performed. However, neither the presence of proapoptotic Bax nor antiapoptotic Bcl-2 or Bcl-XL affected tBid degradation. Finally, we determined the functional role of tBid degradation. Overexpression of stabilized tBid proteins significantly enhanced cytochrome c release and subsequent apoptosis induction approximately 2-fold compared with wild type tBid. Similarly, tBid-induced apoptosis was considerably amplified by inhibition of tBid degradation using the proteasome-specific inhibitor MG-132. Thus, proteasomal degradation of tBid limits the extent of apoptosis in living cells.
Insights
The proapoptotic protein Bid (tBid) is degraded by the proteasome, limiting apoptosis. Inhibiting this degradation enhances tBid
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The proapoptotic protein Bid is cleaved by caspase-8 into tBid upon apoptotic stimuli.
- tBid translocates to mitochondria, initiating cytochrome c release and apoptosis.
- The regulation of tBid stability and its role in apoptosis extent are not fully understood.
Purpose of the Study:
- To investigate the post-translational modifications and degradation pathways of tBid.
- To determine the functional consequences of tBid degradation on apoptosis induction.
- To elucidate the role of proteasomal degradation in regulating apoptosis.
Main Methods:
- Pulse-chase analysis to assess protein stability.
- Treatment with proteasome inhibitors (MG-132, lactacystin) and other inhibitors.
- Site-directed mutagenesis of putative ubiquitination sites.
- Cotransfection studies with Bcl-2 family proteins.
- Assessment of cytochrome c release and apoptosis induction.
Main Results:
- tBid is ubiquitinated and degraded by the 26 S proteasome.
- Proteasome inhibitors and mutation of ubiquitination sites stabilize tBid.
- Caspase or lysosomal inhibitors do not affect tBid stability.
- Bcl-2 family proteins do not influence tBid degradation.
- Stabilized tBid enhances cytochrome c release and apoptosis induction.
Conclusions:
- Proteasomal degradation of tBid serves as a negative feedback mechanism to limit apoptosis.
- Targeting tBid degradation could be a strategy to enhance apoptosis induction in certain contexts.
- tBid stability is tightly regulated by the ubiquitin-proteasome system.