CYTOPROTECTIVE ROLE OF HSP72 IN RENAL CELL INJURY

HSP72 在肾细胞损伤中的细胞保护作用

• 批准号: 7086854
• 负责人: STEVEN C. BORKAN
• 金额: $ 34.51万
• 依托单位: BOSTON MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7086854
• 关键词: BCL2 gene /protein; Bax gene /protein; adenosine triphosphate; antisense nucleic acid; apoptosis; cysteine endopeptidases; cytochrome c; cytoprotection; endonuclease; enzyme activity; gene deletion mutation; heat shock proteins; mitochondria; mitochondrial membrane; nucleic acid metabolism; phosphorylation; protein binding; protein folding; protein structure function; renal ischemia /hypoxia

DESCRIPTION (provided by applicant): Renal ischemia in vivo and ATP depletion in vitro cause renal epithelial cells to die by apoptosis. Apoptosis is caused by mitochondrial membrane injury that is regulated by well-characterized members of the BCL2 protein family. We have established that ATP depletion in renal cells increases bax (pro-apoptotic), decreases bcl2 (anti-apoptotic) and causes apoptosis. Concomitant with this pro-apoptotic change in the bcl2:bax ratio, cytochrome c and apoptosis inducing factor (AIF) translocate from mitochondria into the cytosol and state III mitochondrial respiration is reduced. Cytosolic cytochrome c activates caspases, whereas AIF enters the nucleus, activates endonucleases and causes DNA degradation. Prior heat stress, sufficient to induce hsp72, a molecular chaperone, stabilizes the bcl2:bax ratio, decreases cytochrome c and AIF leak, reduces caspase activation and DNA degradation, completely preserves organeile function and enhances long term survival after ATP depletion. The selective over-expression of hsp72 mimics the protective effects of prior heat stress on apoptosis and renal cell survival. The fact that hsp72 binds bcl2 (but not bax), cytochrome c and AIF suggests that hsp72 itself is an anti-apoptotic protein. We hypothesize that hsp72 decreases primary injury to the mitochondrial membrane caused by bax and inhibits the secondary, pro-apoptotic effects of leaked cytochrome c and AIF on caspase activation and DNA degradation, respectively. In isolated organelles and intact ceils, this study will: (1) determine whether cytoprotection by hsp72 against bax-mediated mitochondrial membrane injury requires bcl2; (2) evaluate the ability of hsp72 to rescue bcl2 function, prevent caspase activation and/or to inhibit AIF-mediated DNA degradation and (3) identify the specific hsp72 domain(s) responsible for its anti-apoptotic actions. To achieve these AIMS, hsp72, bax and bcl2 content will be manipulated in renal epithelial cells using adenoviral infection with established vectors including wild type human hsp72, antisense and known hsp72 deletion mutants with specific functional defects. These studies will identify the anti-apoptotic mechanism(s) of hsp72. These insights can prompt the development of preemptive strategies for preventing renal cell injury during ischemic insults as well as other forms of renal injury in which apoptosis contributes to organ failure.

描述（由申请人提供）：体内肾脏缺血和ATP体外耗竭会导致肾上皮细胞因凋亡而死亡。细胞凋亡是由线粒体膜损伤引起的，该膜受到BCL2蛋白家族的良好特征成员的调节。我们已经确定，肾细胞中的ATP耗竭会增加BAX（促凋亡），减少BCL2（抗凋亡）并引起凋亡。与Bcl2：BAX比，细胞色素C和凋亡诱导因子（AIF）迁移因子（AIF）从线粒体转化为细胞质和状态III线粒体呼吸的促凋亡变化相关。胞质细胞色素C激活胱天蛋白酶，而AIF进入核，激活核酸内切酶并导致DNA降解。足以诱导HSP72的先前热应激，即分子伴侣，可以稳定BCL2：BAX比率，降低细胞色素C和AIF泄漏，减少caspase的激活和DNA降解，完全保留了ATP DEPETION后长期存活的器官函数。 HSP72的选择性过表达模仿了先前热应激对凋亡和肾细胞存活的保护作用。 HSP72结合Bcl2（而不是BAX），细胞色素C和AIF的事实表明，HSP72本身是一种抗凋亡蛋白。我们假设HSP72降低了由BAX引起的线粒体膜的一级损伤，并抑制了泄漏的细胞色素C和AIF对胱天蛋白酶激活和DNA降解的继发性，促凋亡作用。在孤立的细胞器和完整的天花板中，本研究将：（1）确定HSP72对BAX介导的线粒体膜损伤是否需要BCL2的细胞保护作用； （2）评估HSP72挽救BCl2功能，防止caspase激活和/或抑制AIF介导的DNA降解的能力，并且（3）确定负责其抗凋亡作用的特定HSP72域。为了实现这些目标，使用腺病毒感染具有既定的载体，包括野生型人类HSP72，反义和已知的HSP72缺失突变体，具有特定功能缺陷。这些研究将确定HSP72的抗凋亡机制。这些见解可以促使人们开发预先性策略，以防止缺血性损伤期间肾细胞损伤以及其他形式的肾脏损伤，其中细胞凋亡会导致器官衰竭。