Bnip3 functions as a mitochondrial sensor of oxidative stress during myocardial ischemia and reperfusion

Am J Physiol Heart Circ Physiol. 2008 Nov;295(5):H2025-31. doi: 10.1152/ajpheart.00552.2008. Epub 2008 Sep 12.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis
  • Cysteine
  • Disease Models, Animal
  • HeLa Cells
  • Humans
  • Male
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mice
  • Mitochondria, Heart / metabolism*
  • Mitochondria, Heart / pathology
  • Mitochondrial Proteins
  • Mutation
  • Myocardial Reperfusion Injury / metabolism*
  • Myocardial Reperfusion Injury / pathology
  • Myocytes, Cardiac / metabolism*
  • Myocytes, Cardiac / pathology
  • Oxidation-Reduction
  • Oxidative Stress*
  • Perfusion
  • Protein Multimerization
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins / chemistry
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism*
  • Rats
  • Rats, Sprague-Dawley
  • Reactive Oxygen Species / metabolism
  • Recombinant Fusion Proteins / metabolism
  • Transfection

Substances

  • BNIP3 protein, rat
  • Membrane Proteins
  • Mitochondrial Proteins
  • Proto-Oncogene Proteins
  • Reactive Oxygen Species
  • Recombinant Fusion Proteins
  • Cysteine