Hendgen-Cotta Ulrike B, Esfeld Sonja, Rudi Katharina, Miinalainen Ilkka, Klare Johann P, Rassaf Tienush
Department of Cardiology and Vascular Medicine, West German Heart and Vascular Center, Medical Faculty, University Hospital Essen, Hufelandstr. 55, 45147 Essen, Germany.
Department of Physics, University of Osnabrück, Barbarastr. 7, 49069 Osnabrück, Germany.
Int J Mol Sci. 2017 Mar 23;18(4):687. doi: 10.3390/ijms18040687.
The primary function of mitochondria is energy production, a task of particular importance especially for cells with a high energy demand like cardiomyocytes. The B-cell lymphoma (BCL-2) family member BCL-2 adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is linked to mitochondrial targeting after homodimerization, where it functions in inner membrane depolarization and permeabilization of the mitochondrial outer membrane (MOM) mediating cell death. We investigated the basal distribution of cardiac BNIP3 in vivo and its physical interaction with the pro-death protein BCL2 associated X, apoptosis regulator (BAX) and with mitochondria using immunoblot analysis, co-immunoprecipitation, and continuous wave and pulsed electron paramagnetic resonance spectroscopy techniques. We found that BNIP3 is present as a dimer in the cytosol and in the outer membrane of cardiac mitochondria under basal conditions. It forms disulfide-bridged, but mainly non-covalent dimers in the cytosol. Heterodimers with BAX are formed exclusively in the MOM. Furthermore, our results suggest that BNIP3 interacts with the MOM directly via mitochondrial BAX. However, the physical interactions with BAX and the MOM did not affect the membrane potential and cell viability. These findings suggest that another stimulus other than the mere existence of the BNIP3/BAX dimer in the MOM is required to promote BNIP3 cell-death activity; this could be a potential disturbance of the BNIP3 distribution homeostasis, namely in the direction of the mitochondria.
线粒体的主要功能是产生能量，这一任务尤为重要，特别是对于像心肌细胞这种有高能量需求的细胞。B细胞淋巴瘤（BCL-2）家族成员BCL-2腺病毒E1B 19 kDa相互作用蛋白3（BNIP3）在同二聚化后与线粒体靶向相关，它在内膜去极化和线粒体外膜（MOM）通透性改变中发挥作用，介导细胞死亡。我们使用免疫印迹分析、免疫共沉淀以及连续波和脉冲电子顺磁共振波谱技术，研究了体内心脏BNIP3的基础分布及其与促死亡蛋白B细胞淋巴瘤-2相关X蛋白（凋亡调节因子，BAX）以及线粒体的物理相互作用。我们发现，在基础条件下，BNIP3以二聚体形式存在于心肌线粒体的细胞质和外膜中。它在细胞质中形成二硫键连接但主要是非共价的二聚体。与BAX的异二聚体仅在MOM中形成。此外，我们的结果表明，BNIP3通过线粒体BAX直接与MOM相互作用。然而，与BAX和MOM的物理相互作用并未影响膜电位和细胞活力。这些发现表明，除了MOM中单纯存在BNIP3/BAX二聚体之外，还需要其他刺激来促进BNIP3的细胞死亡活性；这可能是BNIP3分布稳态的潜在干扰，即朝着线粒体方向的干扰。
