Programmed cell death by apoptosis is required for normal development and tissue homeostasis. Perturbations of the critical signaling pathways that regulate apoptosis drive a number of pathologic diseases; therefore, a deep understanding of the apoptotic regulatory networks and methods for therapeutically modulating them is highly warranted. As constituents of the intrinsic pathway of apoptosis, pro-apoptotic BCL-2 family proteins respond to internal signals of cell stress to activate cell death through permeabilization of the outer mitochondrial membrane to release cytochrome c and other apoptogenic factors. Anti-apoptotic BCL-2 proteins block apoptosis by forming stable heterodimers with pro-apoptotic BAX and BAK. Specifically, the C-terminal binding groove of BCL-2 sequesters the BCL-2 homology 3 (BH3) death domain of BAX to prevent BAX oligomerization. The N-terminal BH4 domain of BCL-2 is also believed to confer anti-apoptotic activity but the mechanism by which this occurs remains unknown. A direct, inhibitory interaction between the BCL-2 BH4 domain and BAX was established through application of a peptide stapling technology to preserve the α-helical character of the BH4 domain outside the context of full-length BCL-2. Photoaffinity labeling identified a new mode of BH4 domain interaction at the C-terminal face of BAX, revealing an additional layer of apoptotic regulation. Examination of the conformational activation of full-length BAX in a lipid membrane by hydrogen-deuterium exchange mass spectrometry revealed that the BCL-2 BH4 helix blocks the BAX conformational changes triggered by an activating BH3 peptide to preserve the inactive BAX fold. Thus, the mechanistic paradigm for BCL-2 inhibition of BAX has been expanded to allow future opportunities for modulation of apoptosis by mimicking or inhibiting the BH4 motif.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/13070041 |
| Date | January 2014 |
| Creators | Barclay, Lauren Anne |
| Contributors | Walensky, Loren David |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | closed access |
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