Abstract
BackgroundBcl-2 family proteins are key regulators of mitochondrial integrity and comprise both pro- and anti-apoptotic proteins. Bax a pro-apoptotic member localizes as monomers in the cytosol of healthy cells and accumulates as oligomers in mitochondria of apoptotic cells. The Bcl-2 homology-3 (BH3) domain regulates interactions within the family, but regions other than BH3 are also critical for Bax function. Thus, the N-terminus has been variously implicated in targeting to mitochondria, interactions with BH3-only proteins as well as conformational changes linked to Bax activation. The transmembrane (TM) domains (α5-α6 helices in the core and α9 helix in the C-terminus) in Bax are implicated in localization to mitochondria and triggering cytotoxicity. Here we have investigated N-terminus modulation of TM function in the context of regulation by the anti-apoptotic protein Bcl-xL.ResultsDeletion of 29 amino acids in the Bax N-terminus (Bax 30–192) caused constitutive accumulation at mitochondria and triggered high levels of cytotoxicity, not inhibited by Bcl-xL. Removal of the TM domains (Bax 30–105) abrogated mitochondrial localization but resulted in Bcl-xL regulated activation of endogenous Bax and Bax-Bak dependent apoptosis. Inclusion of the α5-α6 helices/TMI domain (Bax 30–146) phenocopied Bax 30–192 as it restored mitochondrial localization, Bcl-xL independent cytotoxicity and was not dependent on endogenous Bax-Bak. Inhibition of function and localization by Bcl-xL was restored in Bax 1–146, which included the TM1 domain. Regardless of regulation by Bcl-xL, all N-terminal deleted constructs immunoprecipitated Bcl-xLand converged on caspase-9 dependent apoptosis consistent with mitochondrial involvement in the apoptotic cascade. Sub-optimal sequence alignments of Bax and Bcl-xL indicated a sequence similarity between the α5–α6 helices of Bax and Bcl-xL. Alanine substitutions of three residues (T14A-S15A-S16A) in the N-terminus (Bax-Ala3) attenuated regulation by the serine-threonine kinase Akt/PKB but not by Bcl-xL indicative of distinct regulatory mechanisms.ConclusionCollectively, the analysis of Bax deletion constructs indicates that the N-terminus drives conformational changes facilitating inhibition of cytotoxicity by Bcl-xL. We speculate that the TM1 helices may serve as 'structural antagonists' for BH3-Bcl-xL interactions, with this function being regulated by the N-terminus in the intact protein.
Highlights
Bcl-2 family proteins are key regulators of mitochondrial integrity and comprise both proand anti-apoptotic proteins
Bcl-2 family proteins are central to the regulation of mitochondrial integrity and cell death pathways that converge on this organelle [1,2]
Apoptosis triggered by a Bax N-terminal deletion mutant (Bax 30–192) is poorly inhibited by the anti apoptotic proteins, Bcl-2 and Bcl-xL Apoptosis triggered by a deletion mutant that lacks the first 29 amino acids (Bax 30–192) was poorly inhibited by Bcl-xL (Figure 1A) or Bcl-2 (Figure 1B) in the Jurkat T-cell line
Summary
Bcl-2 family proteins are key regulators of mitochondrial integrity and comprise both proand anti-apoptotic proteins. The N-terminus has been variously implicated in targeting to mitochondria, interactions with BH3-only proteins as well as conformational changes linked to Bax activation. Bcl-2 family proteins are central to the regulation of mitochondrial integrity and cell death pathways that converge on this organelle [1,2]. This family comprises both prosurvival (Bcl-2, Bcl-xL, Mcl-1, Bcl-w) and pro-apoptotic (Bax, Bak, Bim, Bid, Bad) proteins. All members of this family are characterized by the presence of BH (Bcl-2 homology) domain(s) [3,4]. Selectivity in interactions between pro- and anti-apoptotic members of the Bcl-2 family has been reported [10]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
