The Cyto-Protective Role of Hexokinase II During Metabolic Stress

描述（由申请人提供）：肾缺血以近端小管上皮细胞为靶点，是导致发病和死亡的重要原因。大量证据表明，肾缺血激活Bax，一种促凋亡的BCL2蛋白，其增加线粒体膜通透性并导致细胞凋亡，是器官衰竭的重要因素。代谢应激通过引起“线粒体裂变”破坏细胞器形态，这是一个促进细胞凋亡的过程。初步研究表明，代谢应激是一种已建立的肾缺血模型，其目标是己糖激酶II (HKII)，它是糖酵解的限速酶，也是bax介导的细胞死亡途径的潜在调节因子。代谢应激导致HKII与肾上皮细胞线粒体分离并经历快速降解。此外，HKII与分子伴侣Hsp70相互作用，表明应激破坏了HKII的构象和功能。最后，应激导致动力蛋白相关蛋白1 （DRP1）在分离的线粒体中积累，DRP1是细胞器裂变的主要调节因子。选择性表达HKII可增加线粒体HKII含量，防止线粒体Bax积累，减少膜损伤，显著提高应激后细胞存活率。应激时HKII的保护机制目前尚不清楚。我们假设HKII通过以下途径促进应激后细胞活力：(A)拮抗bax介导的线粒体膜损伤和/或(B)阻止线粒体裂变。此外，我们认为通过与HKII相互作用，Hsp70抑制HKII降解和/或促进线粒体HKII易位，这些事件促进细胞存活。通过测量代谢应激细胞的AIF渗漏、caspase 3活化、膜联蛋白V染色，研究HKII或drp1表达升高或降低对线粒体损伤和凋亡的影响。线粒体相关Bax和drp1的积累以及drp1磷酸化将在完整细胞和分离线粒体中进行研究。蛋白-蛋白相互作用和HKII泛素化将通过免疫沉淀研究。线粒体分裂将通过共聚焦显微镜使用线粒体染料和线粒体靶向，光激活的绿色荧光蛋白进行评估。最后，应激细胞中HKII泛素化和半衰期与HK含量有关。肾缺血损伤肾小管细胞，引起上皮细胞死亡，是导致发病和死亡的重要原因。这些建议的研究将为肾细胞死亡的机制提供分子见解，并可能促进治疗急性缺血性肾功能衰竭的治疗干预措施的发展。

DESCRIPTION (provided by applicant): Renal ischemia targets the proximal tubule epithelial cell and is an important cause of morbidity and mortality. Substantial evidence shows that renal ischemia activates Bax,a pro-apoptotic BCL2 protein that increases mitochondrial membrane permeability and causes apoptosis, an important contributor to organ failure. Metabolic stress disrupts organelle morphology by causing "mitochondrial fission", a process that promotes apoptosis. Preliminary studies show that metabolic stress, an established model of renal ischemia, targets hexokinase II (HKII), the rate-limiting enzyme in glycolysis and a potential regulator of the Bax-mediated cell death pathway. Metabolic stress causes HKII to dissociate from renal epithelial cell mitochondria and to undergo rapid degradation. Furthermore, HKII interacts with Hsp70, a molecular chaperone, suggesting that stress disrupts HKII conformation and function. Finally, stress causes dynamin-related protein 1 (DRP1), the primary regulator of organelle fission, to accumulate in isolated mitochondria. Selective HKII expression increases mitochondrial HKII content, prevents mitochondrial Bax accumulation, reduces membrane injury and dramatically improves cell survival after stress. The protective mechanism of HKII during stress is presently unknown. We hypothesize that HKII promotes cell viability after stress by: (A) antagonizing Bax-mediated mitochondrial membrane injury and/or (B) preventing mitochondrial fission. Furthermore, we suggest that by interacting with HKII, Hsp70 inhibits HKII degradation and/or facilitates mitochondrial HKII translocation, events that promote cell survival. The effect of increased or decreased HKII or DRP1expression on mitochondrial injury and apoptosis will be studied by measuring AIF leakage, caspase 3 activation, annexin V staining in cells subjected to metabolic stress. Accumulation of mito- chondrial-associated Bax and DRP1and DRP1phosphorylaton will be studied using intact cells and isolated mitochondria. Protein-protein interactions and HKII ubiquitination will be studied by immuno-precipitation. Mitchondrial fission will be evaluated by confocal microscopy using a mitochondrial dye and mitochondrial targeted, photoactivatable GFP. Lastly, HKII ubiquitination and half-life will be correlated with HK content in cells subjected to stress. Renal ischemia, an important cause of morbidity and mortality, damages kidney tubular cells and causes epithelial cell death. The proposed studies will provide molecular insights into the mechanism of renal cell death and may promote the development of therapeutic interventions for treating acute, ischemic renal failure.