Protein ubiquitination based neuroprotection for stroke

基于蛋白质泛素化的中风神经保护

• 批准号: 6862336
• 负责人: ROBERT MELLER
• 金额: $ 17.92万
• 依托单位: LEGACY EMANUEL HOSPITAL AND HEALTH CENTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-15 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6862336
• 关键词: cell death; cerebral ischemia /hypoxia; immunoprecipitation; ischemic preconditioning; laboratory rat; mass spectrometry; neuroprotectants; newborn animals; posttranslational modifications; tissue /cell culture; ubiquitin

Tolerance, the cellular response to mild stress, which protects against a toxic stress, is a conserved feature of many organisms. Classic tolerance is gene-mediated, protein synthesis-dependent and requires at least 24 hours to occur. In contrast, rapid ischemic tolerance occurs within 1 hour of the preconditioning ischemia and is not dependent on the synthesis of new proteins. As such understanding the endogenous mechanism of neuroprotection which are involved in rapid ischemic tolerance, may identify novel fast acting therapeutic strategies to treat acute stroke. Our preliminary data suggests that cell death promoting proteins are rapidly down regulated, following preconditioning, which may account for the neuroprotective effects observed in rapid ischemic tolerance. Our central hypothesis is that brief ischemia stimulates the generation of rapid ischemic tolerance by acute alterations in the levels of cell death promoting proteins by the process of ubiquitination, which targets the proteins for degradation by the proteasome. The specific aims are:-1. We will test our hypothesis using a candidate cell death promoting protein, Bim. Experiments are designed to investigate Bim ubiquitination and degradation in the 1 hour period following ischemic preconditioning. 2. We will further test our hypothesis broadly with immunoprecipitation and mass spectrographic analysis to identify novel proteins that are ubiquitinated following preconditioning ischemia. Experiments will then further validate this data, and identify potential new targets for regulating cell death following ischemia. These studies will support our hypothesis that protein down regulation is a powerful endogenous neuroprotective mechanism in the brain. We will identify the molecular mechanisms that favor the degradation of pro-apoptotic proteins, which may offer new acute therapeutic targets for the treatment of stroke and other neurological insults, which would not be available via gene modulation strategies based on classic tolerance, requiring over 24h to exert a neuroprotective effect.

耐受性是细胞对温和压力的反应，它可以抵御有毒压力，是许多生物体的保守特征。经典的耐受性是由基因介导的，依赖蛋白质合成，至少需要24小时才能发生。相反，快速缺血耐受发生在预适应缺血后1小时内，并且不依赖于新蛋白质的合成。因此，了解参与快速缺血耐受的神经保护的内源性机制，可能会发现治疗急性卒中的新的快速有效的治疗策略。我们的初步数据表明，细胞死亡促进蛋白在预适应后迅速下调，这可能解释了在快速缺血耐受中观察到的神经保护作用。我们的中心假设是，短暂的缺血通过泛素化过程中促进细胞死亡的蛋白水平的急剧变化来刺激快速缺血耐受的产生，泛素化是蛋白酶体降解蛋白质的靶点。具体目标是：-1.我们将使用候选细胞死亡促进蛋白Bim来验证我们的假设。实验旨在研究缺血后1小时内Bim的泛素化和降解 预适应。2.我们将通过免疫沉淀和质谱分析进一步广泛地检验我们的假设，以确定在预适应缺血后泛素化的新蛋白。然后，实验将进一步验证这一数据，并确定调节缺血后细胞死亡的潜在新靶点。这些研究将支持我们的假设，即蛋白质下调是大脑中一种强大的内源性神经保护机制。我们将确定有利于促凋亡蛋白降解的分子机制，这可能为治疗肺癌提供新的急性治疗靶点。 中风和其他神经侮辱，这些都不能通过基于经典耐受性的基因调制策略来实现，需要超过24小时才能发挥神经保护作用。