Frazier Donna P, Wilson Amber, Graham Regina M, Thompson John W, Bishopric Nanette H, Webster Keith A
Department of Pharmacology and the Vascular Biology Institute, University of Miami Medical Center, Florida 33136, USA.
Antioxid Redox Signal. 2006 Sep-Oct;8(9-10):1625-34. doi: 10.1089/ars.2006.8.1625.
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.
Bnip3是Bcl-2蛋白中所谓仅含BH3结构域亚家族的促死亡成员。这类蛋白的一个主要功能是通过在膜内形成同型和异型寡聚体来调节线粒体外膜的通透性状态。我们之前报道过，在缺氧暴露期间Bnip3在心肌细胞中积累，但同时需要酸中毒来激活死亡程序。酸中毒增加了Bnip3在细胞内的积累速率，并促进其与线粒体更紧密的结合。在此我们报道，酸性pH介导了Bnip3二聚体和单体（>3倍）以及一个迁移速度更快的片段（>10倍）半衰期的延长，并赋予其对蛋白酶降解的保护作用。疏水分配实验表明，来自缺氧-酸性细胞组分的Bnip3单体和寡聚体与去污剂层显著相关，而来自缺氧-中性心肌细胞的蛋白则不然。酸中毒促进了Bcl-xL的同源二聚化，但没有增加其与去污剂的结合。在缺氧-酸中毒条件下对心肌细胞培养物的细胞外培养基进行中和，导致积累的Bnip3迅速降解（半衰期，<2小时），同时死亡程序停止。Bnip3单体似乎是活性形式，因为在跨膜（TM）结构域内第173位的组氨酸被丙氨酸取代可防止同源二聚化，但不抑制死亡功能。这些结果表明Bnip3单体的稳定性和表观疏水性发生了pH敏感的变化，这与膜结合和功能密切相关。
