RESUMEN
To elicit apoptosis, BAX metamorphoses from an inert cytosolic monomer into homo-oligomers that permeabilize the mitochondrial outer membrane (MOM). A long-standing puzzle is that BH3 domains apparently activate BAX by not only its canonical groove but also a proposed site involving helices α1 and α6. Our mutagenesis studies reveal that late steps like oligomerization require activation through the groove but probably not earlier steps like MOM association. Conversely, α1 or α6 obstruction and alanine mutagenesis scanning implicate these helices early in BAX activation. The α1 and α6 mutations lowered BH3 binding, altered the BAX conformation, and reduced its MOM translocation and integration; their exposure of the BAX α1-α2 loop allosterically sequestered its α9 membrane anchor in the groove. The crystal structure of an α6 mutant revealed additional allosteric effects. The results suggest that the α1 and α6 region drives MOM association and integration, whereas groove binding favors subsequent steps toward oligomerization.
Asunto(s)
Mitocondrias Hepáticas/metabolismo , Membranas Mitocondriales/metabolismo , Mutación , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Fragmentos de Péptidos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Alineación de SecuenciaRESUMEN
Intrinsic apoptosis is critical to prevent tumor formation and is engaged by many anti-cancer agents to eliminate tumor cells. BAX and BAK, the two essential mediators of apoptosis, are thought to be regulated through similar mechanisms and act redundantly to drive apoptotic cell death. From an unbiased genome-wide CRISPR/Cas9 screen, we identified VDAC2 (voltage-dependent anion channel 2) as important for BAX, but not BAK, to function. Genetic deletion of VDAC2 abrogated the association of BAX and BAK with mitochondrial complexes containing VDAC1, VDAC2, and VDAC3, but only inhibited BAX apoptotic function. Deleting VDAC2 phenocopied the loss of BAX in impairing both the killing of tumor cells by anti-cancer agents and the ability to suppress tumor formation. Together, our studies show that efficient BAX-mediated apoptosis depends on VDAC2, and reveal a striking difference in how BAX and BAK are functionally impacted by their interactions with VDAC2.
