title: Bnip3 functions as a mitochondrial sensor of oxidative stress during myocardial ischemia and reperfusion.
abstract: Bcl-2/adenovirus E1B 19-kDa protein-interacting protein 3 (Bnip3) is a member of the Bcl-2 homology domain 3-only subfamily of proapoptotic Bcl-2 proteins and is associated with cell death in the myocardium. In this study, we investigated the potential mechanism(s) by which Bnip3 activity is regulated. We found that Bnip3 forms a DTT-sensitive homodimer that increased after myocardial ischemia-reperfusion (I/R). The presence of the antioxidant N-acetylcysteine reduced I/R-induced homodimerization of Bnip3. Overexpression of Bnip3 in cells revealed that most of exogenous Bnip3 exists as a DTT-sensitive homodimer that correlated with increased cell death. In contrast, endogenous Bnip3 existed mainly as a monomer under normal conditions in the heart. Screening of the Bnip3 protein sequence revealed a single conserved cysteine residue at position 64. Mutation of this cysteine to alanine (Bnip3C64A) or deletion of the NH2-terminus (amino acids 1-64) resulted in reduced cell death activity of Bnip3. Moreover, mutation of a histidine residue in the COOH-terminal transmembrane domain to alanine (Bnip3H173A) almost completely inhibited the cell death activity of Bnip3. Bnip3C64A had a reduced ability to interact with Bnip3, whereas Bnip3H173A was completely unable to interact with Bnip3, suggesting that homodimerization is important for Bnip3 function. A consequence of I/R is the production of reactive oxygen species and oxidation of proteins, which promotes the formation of disulfide bonds between proteins. Thus, these experiments suggest that Bnip3 functions as a redox sensor where increased oxidative stress induces homodimerization and activation of Bnip3 via cooperation of the NH2-terminal cysteine residue and the COOH-terminal transmembrane domain.
authors.affiliation: BioScience Center, San Diego State University, 5500 Campanile Dr., San Diego, CA 92182-4650, USA.
authors.forename: Dieter A
authors.initials: DA
authors.lastname: Kubli
authors.forename: Melissa N
authors.initials: MN
authors.lastname: Quinsay
authors.forename: Chengqun
authors.initials: C
authors.lastname: Huang
authors.forename: Youngil
authors.initials: Y
authors.lastname: Lee
authors.forename: Asa B
authors.initials: AB
authors.lastname: Gustafsson
mesh_terms: D000818:Animals; D017209:Apoptosis; D003545:Cysteine; D004195:Disease Models, Animal; D006367:HeLa Cells; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008929:Mitochondria, Heart / Q000378:metabolism* / Q000473:pathology; D024101:Mitochondrial Proteins; D009154:Mutation; D015428:Myocardial Reperfusion Injury / Q000378:metabolism* / Q000473:pathology; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000473:pathology; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010477:Perfusion; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
chemical_list: C505650:BNIP3 protein, rat; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011518:Proto-Oncogene Proteins; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D003545:Cysteine
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