title: Bid chimeras indicate that most BH3-only proteins can directly activate Bak and Bax, and show no preference for Bak versus Bax.
abstract: The mitochondrial pathway of apoptosis is initiated by Bcl-2 homology region 3 (BH3)-only members of the Bcl-2 protein family. On upregulation or activation, certain BH3-only proteins can directly bind and activate Bak and Bax to induce conformation change, oligomerization and pore formation in mitochondria. BH3-only proteins, with the exception of Bid, are intrinsically disordered and therefore, functional studies often utilize peptides based on just their BH3 domains. However, these reagents do not possess the hydrophobic membrane targeting domains found on the native BH3-only molecule. To generate each BH3-only protein as a recombinant protein that could efficiently target mitochondria, we developed recombinant Bid chimeras in which the BH3 domain was replaced with that of other BH3-only proteins (Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk). The chimeras were stable following purification, and each immunoprecipitated with full-length Bcl-xL according to the specificity reported for the related BH3 peptide. When tested for activation of Bak and Bax in mitochondrial permeabilization assays, Bid chimeras were ~1000-fold more effective than the related BH3 peptides. BH3 sequences from Bid and Bim were the strongest activators, followed by Puma, Hrk, Bmf and Bik, while Bad and Noxa were not activators. Notably, chimeras and peptides showed no apparent preference for activating Bak or Bax. In addition, within the BH3 domain, the h0 position recently found to be important for Bax activation, was important also for Bak activation. Together, our data with full-length proteins indicate that most BH3-only proteins can directly activate both Bak and Bax.
authors.affiliation: 1] The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia [2] Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: C
authors.initials: C
authors.lastname: Hockings
authors.affiliation: 1] The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia [2] Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: K
authors.initials: K
authors.lastname: Anwari
authors.affiliation: 1] The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia [2] Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: R L
authors.initials: RL
authors.lastname: Ninnis
authors.affiliation: 1] The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia [2] Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: J
authors.initials: J
authors.lastname: Brouwer
authors.affiliation: 1] The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia [2] Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: M
authors.initials: M
authors.lastname: O'Hely
authors.affiliation: 1] The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia [2] Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: M
authors.initials: M
authors.lastname: Evangelista
authors.affiliation: School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: M G
authors.initials: MG
authors.lastname: Hinds
authors.affiliation: 1] The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia [2] Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: P E
authors.initials: PE
authors.lastname: Czabotar
authors.affiliation: 1] The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia [2] Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: E F
authors.initials: EF
authors.lastname: Lee
authors.affiliation: 1] The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia [2] Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: W D
authors.initials: WD
authors.lastname: Fairlie
authors.affiliation: 1] The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia [2] Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: G
authors.initials: G
authors.lastname: Dewson
authors.affiliation: 1] The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia [2] Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: R M
authors.initials: RM
authors.lastname: Kluck
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D051023:BH3 Interacting Domain Death Agonist Protein / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D013379:Substrate Specificity; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000378:metabolism*; D051020:bcl-X Protein / Q000378:metabolism
chemical_list: C494772:BAK1 protein, human; C494826:BAX protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; C474007:Bax protein (53-86); D033681:Mitochondrial Membrane Transport Proteins; D015514:Oncogene Proteins, Fusion; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; C581062:RTL10 protein, human; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein