The N-terminus and alpha-5, alpha-6 helices of the pro-apoptotic protein Bax, modulate functional interactions with the anti-apoptotic protein Bcl-xL.
BACKGROUND
Bcl-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 (alpha5-alpha6 helices in the core and alpha9 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.


RESULTS
Deletion 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 alpha5-alpha6 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 alpha5-alpha6 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.


CONCLUSIONS
Collectively, 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.
Parikh
Neha
N
National Centre for Biological Sciences, Bangalore, Karnataka, India. parikh@ncbs.res.in <parikh@ncbs.res.in>
Koshy
Caroline
C
Dhayabaran
Vaigundan
V
Perumalsamy
Lakshmi R
LR
Sowdhamini
R
R
Sarin
Apurva
A
D020816:Amino Acid Motifs; D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D051028:bcl-2-Associated X Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051020:bcl-X Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C494811:BCL2L1 protein, human; C494827:Bax protein, mouse; D021122:Protein Subunits; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein