Contribution of BH3-domain and Transmembrane-domain to the Activity and Interaction of the Pore-forming Bcl-2 Proteins Bok, Bak, and Bax

Daniel Stehle,Stephanie Einsele-Scholz,Friederike Ladwig,Janina Johänning,Frank Essmann,Klaus Schulze-Osthoff,Bernhard Gillissen,Gerd Fischer,Melanie Grimm

doi:10.1038/s41598-018-30603-6

Abstract

Central to intrinsic apoptosis signaling is the release of cytochrome c from mitochondria, which depends on the pro-apoptotic effector proteins Bax, Bak or Bok. These pore-forming effector proteins share four Bcl-2 homology (BH) domains, a functionally essential and conserved sequence of hydrophobic amino acids in their BH3-domain and a C-terminal transmembrane-domain whose specific function remains rather unknown. To elucidate the molecular basis of Bok-mediated apoptosis we analyzed apoptosis induction by transmembrane-domain deficient BokΔTM compared to the respective Bax and Bak proteins and proteins in which the first leucine in the BH3-stretch was mutated to glutamic acid. We show that deletion of the C-terminal transmembrane-domain reduces the pro-apoptotic function of each protein. Mutation of the first leucine in the BH3-domain (L78E) blocks activity of Bak, while mutation of the homologue residues in Bax or Bok (L63E and L70E respectively) does not affect apoptosis induction. Unexpectedly, combined mutation of the BH3-domain and deletion of the transmembrane-domain enhances the pro-apoptotic activity of Bok(L70E)ΔTM by abolishing the interaction with anti-apoptotic proteins, especially the primary Bok-inhibitory protein Mcl-1. These results therefore suggest a specific contribution of the transmembrane-domain to the pro-apoptotic function and interaction of Bok.

Highlights

In their inactive conformation the N-terminus of Bax and Bak folds back onto the globular protein[8]
The proteasomal degradation proposed for the regulation of Bok activity significantly differs from the mechanism of Bax and Bak activation that is controlled by interaction with anti-apoptotic Bcl-2 and/or BH3-only proteins that regulate induction of a conformational change
To gain a systematic picture of the molecular basis for MDP mediated apoptosis we investigated apoptosis induction by transmembrane-domain deficient variants (ΔTM) of the MDPs and mutant variants in which the first conserved amino acid residue of the hydrophobic BH3-stretch was substituted by a glutamic acid (L70E)

Summary

Introduction

In their inactive conformation the N-terminus of Bax and Bak folds back onto the globular protein[8]. The yet-to-be verified Bok antagonist Mcl-1 itself is quickly degraded by the proteasome with a half-life of 0.5–3 h16,17 – the same regulatory mechanism Llambi et al propose for Bok[15] These findings raise questions about the function of Bok as a genuine MDP, the regulation of Bok-induced apoptosis, if Bok interacts with Mcl-1 and whether Bok acts independently of Bax and Bak or not[6,14,15]. To gain a systematic picture of the molecular basis for MDP mediated apoptosis we investigated apoptosis induction by transmembrane-domain deficient variants (ΔTM) of the MDPs and mutant variants in which the first conserved amino acid residue (leucine) of the hydrophobic BH3-stretch was substituted by a glutamic acid (L70E) These analyses indicate that the regulation of Bok activity differs from that of Bak and presumably Bax because mutation of the BH3-domain does not influence apoptosis induction by Bok. Unexpectedly the L70E mutation of the BH3-domain restores apoptosis induction by Bok lacking the TM-domain. The transmembrane-domain of Bok is dispensable for apoptosis induction but essential for efficient inhibition of Bok-induced apoptosis by Mcl-1

Methods

Results

Conclusion