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A double point mutation at residues Ile14 and Val15 of Bcl-2 uncovers a role for BH4 domain in both protein stability and function

Journal Contribution - Journal Article

B-cell lymphoma 2 (Bcl-2) protein is the archetype apoptosis suppressor protein. The N-terminal Bcl-2-homology 4 (BH4) domain of Bcl-2 is required for the anti-apoptotic function of this protein at the mitochondria and endoplasmic reticulum (ER). The involvement of the BH4 domain in Bcl-2's anti-apoptotic functions has been proposed based on Gly-based substitutions of the Ile14/Val15 amino acids, two hydrophobic residues located in the centre of Bcl-2's BH4 domain. Following this strategy, we recently showed that a BH4-domain-derived peptide in which Ile14 and Val15 have been replaced by Gly residues, was unable to dampen pro-apoptotic Ca(2+) -release events from the ER. Here, we investigated the impact of these mutations on the overall structure, stability and function of full-length Bcl-2 as a regulator of Ca(2+) signalling and cell death. Our results indicate that full-length Bcl-2 I14G/V15G, in contrast to wild-type Bcl-2, (i) displayed severely reduced structural stability and a shortened protein half-life; (ii) failed to interact with BAX, to inhibit the inositol 1,4,5-trisphosphate receptor (IP3 R) and to protect against Ca(2+) -mediated apoptosis. We conclude that the hydrophobic face of Bcl-2's BH4 domain (Ile14, Val15) is an important structural regulatory element by affecting protein stability and turnover, thereby likely reducing Bcl-2's ability to modulate the function of its targets, like IP3 R and BAX. Therefore, Bcl-2 structure/function studies require pre-emptive and reliable determination of protein stability upon introduction of point mutations at the level of the BH4 domain. This article is protected by copyright. All rights reserved.
Journal: FEBS JOURNAL
ISSN: 1742-464X
Issue: 1
Volume: 285
Pages: 127 - 145
Publication year:2018
BOF-keylabel:yes
IOF-keylabel:yes
BOF-publication weight:1
CSS-citation score:1
Authors:International
Authors from:Higher Education
Accessibility:Open