title: Acidosis regulates the stability, hydrophobicity, and activity of the BH3-only protein Bnip3.
abstract: Bnip3 is a prodeath member of the so-called BH3-only subfamily of Bcl-2 proteins. A major function of this class of proteins is to regulate the permeability state of the outer mitochondrial membrane by forming homoand hetero-oligomers inside the membrane. We reported previously that Bnip3 accumulates in cardiac myocytes during exposure to hypoxia, but coincident acidosis is required to activate the death program. Acidosis increased the rate of intracellular accumulation of Bnip3 and promoted a tighter association with mitochondria. Here we report that acidic pH mediates increased half-lives of Bnip3 dimers and monomers (>3-) as well as that of a faster-migrating fragment (>10-) and confers protection against degradation by protease. Hydrophobic partitioning experiments revealed that Bnip3 monomers and oligomers from hypoxia-acidic cell fractions associated significantly with the detergent layer, whereas protein from hypoxia-neutral myocytes did not. Acidosis promoted homodimerization of Bcl-xL but did not increase its association with detergent. Neutralization of the extracellular medium of cardiac myocyte cultures under hypoxia-acidosis resulted in rapid degradation of accumulated Bnip3 (half life, <2 h), coincident with cessation of the death program. Bnip3 monomers appear to be the active species because substitution of alanine for histidine at position 173 within the transmembrane (TM) domain prevented homodimerization but did not inhibit the death function. These results demonstrate a pH-sensitive shift in the stability and apparent hydrophobicity of Bnip3 monomers that correlates closely with membrane binding and function.
authors.affiliation: Department of Pharmacology and the Vascular Biology Institute, University of Miami Medical Center, Florida 33136, USA.
authors.forename: Donna P
authors.initials: DP
authors.lastname: Frazier
authors.forename: Amber
authors.initials: A
authors.lastname: Wilson
authors.forename: Regina M
authors.initials: RM
authors.lastname: Graham
authors.forename: John W
authors.initials: JW
authors.lastname: Thompson
authors.forename: Nanette H
authors.initials: NH
authors.lastname: Bishopric
authors.forename: Keith A
authors.initials: KA
authors.lastname: Webster
mesh_terms: D000138:Acidosis / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D015687:Cell Hypoxia; D002460:Cell Line; D002478:Cells, Cultured; D019281:Dimerization; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins; D009154:Mutation / Q000235:genetics; D032383:Myocytes, Cardiac / Q000378:metabolism; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D014162:Transfection; D051020:bcl-X Protein / Q000378:metabolism
chemical_list: C505650:BNIP3 protein, rat; C494813:Bcl2l1 protein, rat; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011518:Proto-Oncogene Proteins; D051020:bcl-X Protein