Department of Pharmacology, SUNY Upstate Medical University, 750 E Adams Street, Syracuse, NY, 13210, USA.
Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA.
Cell Death Differ. 2019 Dec;26(12):2682-2694. doi: 10.1038/s41418-019-0327-4. Epub 2019 Apr 11.
Bok (Bcl-2-related ovarian killer) is a member of the Bcl-2 protein family that governs the intrinsic apoptosis pathway, but the cellular role that Bok plays is controversial. Remarkably, endogenous Bok is constitutively bound to inositol 1,4,5-trisphosphate receptors (IPRs) and is stabilized by this interaction. Here we report that despite the strong association with IPRs, deletion of Bok expression by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease)-mediated gene editing does not alter calcium mobilization via IPRs or calcium influx into the mitochondria. Rather, Bok deletion significantly reduces mitochondrial fusion rate, resulting in mitochondrial fragmentation. This phenotype is reversed by exogenous wild-type Bok and by an IPR binding-deficient Bok mutant, and may result from a decrease in mitochondrial motility. Bok deletion also enhances mitochondrial spare respiratory capacity and membrane potential. Finally, Bok does not play a major role in apoptotic signaling, since Bok deletion does not alter responsiveness to various apoptotic stimuli. Overall, despite binding to IPRs, Bok does not alter IPR-mediated Ca signaling, but is required to maintain normal mitochondrial fusion, morphology, and bioenergetics.
Bok（Bcl-2 相关卵巢杀手）是 Bcl-2 蛋白家族的一员，它控制着内在的细胞凋亡途径，但 Bok 在细胞中的作用存在争议。值得注意的是，内源性 Bok 与肌醇 1,4,5-三磷酸受体（IPRs）持续结合，并通过这种相互作用得到稳定。在这里，我们报告尽管与 IPRs 强烈相关，但通过 CRISPR/Cas9（成簇的规则间隔短回文重复序列/CRISPR 相关蛋白-9 核酸酶）介导的基因编辑删除 Bok 表达并不会改变 IPR 介导的钙动员或钙流入线粒体。相反，Bok 缺失会显著降低线粒体融合率，导致线粒体碎片化。这种表型可以通过外源性野生型 Bok 和 IPR 结合缺陷的 Bok 突变体逆转，可能是由于线粒体运动性降低所致。Bok 缺失还增强了线粒体备用呼吸能力和膜电位。最后，Bok 在凋亡信号中不起主要作用，因为 Bok 缺失不会改变对各种凋亡刺激的反应性。总的来说，尽管与 IPRs 结合，Bok 不会改变 IPR 介导的 Ca 信号，但 Bok 缺失会导致线粒体融合、形态和生物能量学的正常维持。
