Abstract
BNIP3 is a proapoptotic protein that induces cell death through a mitochondria-mediated pathway. We reported previously that mitochondrial localization of BNIP3 and translocation of EndoG from mitochondria to the nucleus are critical steps of the BNIP3 pathway. It is not clear, however, that how BNIP3 interacts with mitochondria. Here we show that expression of BNIP3 resulted in mitochondrial release and nuclear translocation of EndoG. Incubation of a recombinant GST-BNIP3 protein with freshly isolated mitochondria led to the integration of BNIP3 into mitochondria, reduction in the levels of EndoG in mitochondria and the presence of EndoG in the supernatant that was able to cleave chromatin DNA. Co-immunoprecipitation and mass spectrometry analysis reveals that BNIP3 interacted with the voltage-dependent anion channel (VDAC) to increase opening probabilities of mitochondrial permeability transition (PT) pores and induce mitochondrial release of EndoG. Blocking VDAC with a VDAC antibody largely abolished mitochondrial localization of BNIP3 and prevented EndoG release. Together, the data identify VDAC as an interacting partner of BNIP3 and support endonuclease G as a mediator of the BNIP3 pathway.
Highlights
BNIP3 (Bcl-2/adenovirus E1B 19 kDa protein-interacting protein 3, known as NIP3) is a member of a Bcl-2 subfamily of death-inducing mitochondrial proteins [1]
endonuclease G (EndoG) is encoded in the nucleus, synthesized as a propeptide in the cytoplasm and imported into mitochondria through a process mediated by its amino-terminal mitochondrion-targeting sequence
We previously observed the involvement of EndoG in BNIP3-induced cell death in models of hypoxia and cerebral ischemia [8, 9, 26]
Summary
BNIP3 (Bcl-2/adenovirus E1B 19 kDa protein-interacting protein 3, known as NIP3) is a member of a Bcl-2 subfamily of death-inducing mitochondrial proteins [1]. Loss of BNIP3 expression contributes to chemoresistance of cancer cells [4] This 194-amino acid protein has 4 domains: a PEST domain that targets BNIP3 for degradation, a putative Bcl-2 homology 3 (BH3) domain that is homologous to other members of the Bcl-2 family, a conserved CD domain and a C-terminal transmembrane domain (TM). We previously reported that the BNIP3 pathway involves mitochondrial release and nuclear translocation of the endonuclease G (EndoG) [8, 9]. It is not clear, that how BNIP3 interacts with mitochondria. We show that BNIP3 interacts with the voltage-dependent anion channel (VDAC) to directly induce mitochondrial release and nuclear translocation of EndoG. Our data identify VDAC as an interacting partner of BNIP3 and provide direct evidence to support that EndoG is a mediator of the BNIP3 cell death pathway
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