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（申请人提供）：体内肾缺血和体外ATP耗竭导致肾上皮细胞凋亡。细胞凋亡是由线粒体膜损伤引起的，线粒体膜损伤由 BCL2 蛋白家族的明确成员调节。我们已经确定，肾细胞中 ATP 消耗会增加 bax（促凋亡）、减少 bcl2（抗凋亡）并导致细胞凋亡。伴随着 bcl2:bax 比例的这种促凋亡变化，细胞色素 c 和凋亡诱导因子 (AIF) 从线粒体转移到细胞质中，并且状态 III 线粒体呼吸减少。胞质细胞色素 c 激活半胱天冬酶，而 AIF 进入细胞核，激活核酸内切酶并导致 DNA 降解。先前的热应激足以诱导 hsp72（一种分子伴侣）稳定 bcl2:bax 比例，减少细胞色素 c 和 AIF 泄漏，减少 caspase 激活和 DNA 降解，完全保留细胞器功能并增强 ATP 耗尽后的长期存活率。 hsp72 的选择性过度表达模拟了先前热应激对细胞凋亡和肾细胞存活的保护作用。 hsp72 结合 bcl2（但不结合 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 减少了 bax 引起的对线粒体膜的初级损伤，并分别抑制泄漏的细胞色素 c 和 AIF 对 caspase 激活和 DNA 降解的继发性促凋亡作用。在分离的细胞器和完整细胞中，本研究将：（1）确定hsp72针对bax介导的线粒体膜损伤的细胞保护是否需要bcl2； (2) 评估 hsp72 拯救 bcl2 功能、防止 caspase 激活和/或抑制 AIF 介导的 DNA 降解的能力，以及 (3) 鉴定负责其抗凋亡作用的特定 hsp72 结构域。为了实现这些目标，将使用已建立的载体（包括野生型人 hsp72、反义和具有特定功能缺陷的已知 hsp72 缺失突变体）进行腺病毒感染来操纵肾上皮细胞中的 hsp72、bax 和 bcl2 含量。这些研究将确定 hsp72 的抗凋亡机制。这些见解可以促进预防性策略的发展，以预防缺血性损伤期间的肾细胞损伤以及细胞凋亡导致器官衰竭的其他形式的肾损伤。