Protein ubiquitination based neuroprotection for stroke

耐受性是细胞对轻度胁迫的反应，它可以保护细胞免受毒性胁迫，是许多生物体的保守特征。经典耐受性是基因介导的，依赖于蛋白质合成，需要至少24小时才能发生。相反，快速缺血耐受发生在预处理缺血后1小时内，并且不依赖于新蛋白质的合成。因此，了解与快速缺血耐受相关的神经保护的内源性机制，可以找到治疗急性中风的新型速效治疗策略。我们的初步数据表明，细胞死亡促进蛋白迅速下调，预处理后，这可能是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，短暂的缺血刺激快速缺血耐受的产生，通过急性改变细胞死亡促进蛋白的水平，通过泛素化的过程，其目标是蛋白质的蛋白酶体降解。具体目标是：1。我们将使用候选细胞死亡促进蛋白Bim来测试我们的假设。实验旨在研究缺血后1小时内Bim泛素化和降解， 预处理2.我们将进一步测试我们的假设广泛的免疫沉淀和质谱分析，以确定新的蛋白质，泛素化后预处理缺血。然后，实验将进一步验证这些数据，并确定潜在的新靶点来调节缺血后的细胞死亡。这些研究将支持我们的假设，蛋白质下调是一个强大的内源性神经保护机制在大脑中。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 中风和其他神经损伤，这将无法通过基于经典耐受性的基因调节策略获得，需要超过24小时才能发挥神经保护作用。

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.