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: 6922030
• 负责人: STEVEN C. BORKAN
• 金额: $ 35.34万
• 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/6922030
• 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（由申请方提供）：体内肾缺血和体外ATP耗竭导致肾上皮细胞通过凋亡死亡。细胞凋亡是由线粒体膜损伤引起的，其由BCL 2蛋白家族的充分表征的成员调节。我们已经确定，肾细胞中的ATP耗竭增加bax（促凋亡），减少bcl 2（抗凋亡），并导致细胞凋亡。伴随着bcl 2：bax比率的这种促凋亡变化，细胞色素c和凋亡诱导因子（AIF）从线粒体易位到胞质溶胶中，并且状态III线粒体呼吸减少。细胞溶质细胞色素c激活半胱天冬酶，而AIF进入细胞核，激活核酸内切酶并引起DNA降解。先前的热应激，足以诱导热休克蛋白72，分子伴侣，稳定bcl 2：bax的比例，减少细胞色素c和AIF泄漏，减少半胱天冬酶的激活和DNA降解，完全保留细胞器功能，并提高ATP耗竭后的长期生存。热休克蛋白72的选择性过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。hsp 72结合bcl 2（但不结合bax）、细胞色素c和AIF的事实表明hsp 72本身是抗凋亡蛋白。我们推测，热休克蛋白72减少Bax引起的线粒体膜的原发性损伤，并抑制继发性的，促凋亡的影响泄漏的细胞色素c和AIF的caspase激活和DNA降解，分别。在分离的细胞器和完整细胞中，本研究将：（1）确定hsp 72针对bax介导的线粒体膜损伤的细胞保护是否需要bcl 2;（2）评估hsp 72拯救bcl 2功能、防止半胱天冬酶活化和/或抑制AIF介导的DNA降解的能力;和（3）鉴定负责其抗凋亡作用的特定hsp 72结构域。为了实现这些目标，将使用腺病毒感染在肾上皮细胞中操纵hsp 72、bax和bcl 2含量，所述腺病毒感染具有已建立的载体，包括野生型人hsp 72、反义和具有特定功能缺陷的已知hsp 72缺失突变体。这些研究将确定hsp 72的抗凋亡机制。这些见解可以促进预防缺血性损伤期间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的先发制人策略的发展。