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目标，是发病率和死亡率的重要原因。大量证据表明，肾缺血激活Bax，一种促凋亡的bcl2蛋白，增加线粒体膜通透性，导致细胞凋亡，这是导致器官衰竭的重要因素。代谢应激通过引起“线粒体分裂”来破坏细胞器的形态，线粒体分裂是一种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脏缺血模型，其靶标是糖酵解中的限速酶己糖激酶II(HKII)，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从肾上皮细胞线粒体解离，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，压力会导致细胞器分裂的主要调节因子--动力蛋白相关蛋白1(Drp1)在分离的线粒体中积累。选择性的HKII表达增加了线粒体HKII的含量，防止了线粒体Bax的积累，减少了膜损伤，显著提高了应激后的细胞存活率。HKII在应激过程中的保护机制目前尚不清楚。我们推测，HKII通过以下途径促进应激后的细胞活性：(A)拮抗Bax介导的线粒体膜损伤和/或(B)阻止线粒体分裂。此外，我们认为，通过与HKII的相互作用，Hsp70抑制HKII的降解和/或促进线粒体HKII的易位，从而促进细胞存活。通过检测代谢应激下细胞的AIF漏出、caspase3活性、Annexin V染色，研究HKII或DRP1表达的增加或减少对线粒体损伤和细胞凋亡的影响。我们将使用完整的细胞和分离的线粒体来研究线粒体相关的Bax和DRP1以及DRP1磷酸化的积累。蛋白质之间的相互作用和HKII泛素化将用免疫沉淀法进行研究。线粒体分裂将通过使用线粒体染料和线粒体靶向的、可光激活的绿色荧光蛋白的共聚焦显微镜进行评估。最后，HKII泛素化和半衰期将与应激状态下细胞中的HK含量相关。肾缺血损伤肾小管上皮细胞，导致肾小管上皮细胞死亡，是导致发病和死亡的重要原因。建议的研究将提供对肾细胞死亡机制的分子洞察力，并可能促进治疗急性缺血性肾功能衰竭的治疗措施的发展。

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.