ElF4g Cleavage and Persistent Protein Synthesis Inhibition Following Ischemia

描述(由申请人提供)： 脑缺血事件(即中风)后的神经系统并发症会导致严重的临床疾病。因此，我们的长期目标是阐明缺血后导致临床疾病的机制，以潜在地确定新的治疗策略。缺血和再灌流导致所有受缺血影响的神经元的蛋白质合成立即和全面停止。最初的蛋白质合成抑制(PSI)之后蛋白质合成几乎完全恢复，或者PSI持续存在。正是这种持续性PSI与神经退行性变和最终的神经元死亡高度相关。因此，我们的工作假设是持续性PSI介导了脑缺血后的细胞死亡。我们的具体假设是，真核细胞起始因子4G(ElF4G)被Calain等酶降解可能介导持续性PSI，阻止elF4G切割可能阻断持续性PSI，从而提供神经保护。这一假说基于以下数据：1)elF4G在体外和体内的其他蛋白质合成起始因子减少之前开始切割，2)体外切割的时间与体外持续的PSI相关，3)体外缺血导致一种新的大小特异性切割产物的产生，以及4)通过过表达其内源性抑制物calastatin来抑制Calain的活性，部分恢复体外缺血后的蛋白质合成。根据这些数据，我们的重点是确定calain在elF4G裂解中的作用，并确定elF4G裂解在持续性PSI和细胞死亡中的作用。我们的具体目标是：1)验证体外缺血后elF4G裂解和持续性PSI是由Calain激活介导的假说。我们将：i)确定在体外缺血后是否可以防止elF4G的切割(I)通过过表达内源性钙蛋白酶抑制剂calastatin和(Ii)使用来自calain基因敲除小鼠的神经元。我们还将(Iii)确定通过抑制calain来防止elF4G裂解是否可以挽救体外缺血后的蛋白质合成。2)验证防止elF4G裂解可减轻持续性PSI并对体外缺血提供神经保护的假说。我们将(I)过表达elF4G以尝试恢复基线elF4G水平，(Ii)确定过表达elF4G是否会在体外缺血后提供神经保护，以及(Iii)在体外利用重组蛋白确定calain对elF4G的切割位点。 公共卫生相关性：最终，描述持续PSI的机制可能会导致一种新的脑缺血治疗策略。

DESCRIPTION (provided by applicant): The neurological complications following cerebral ischemic events (i.e. stroke) produce severe clinical disease. Our long term goal is thus to elucidate mechanisms following ischemia that produce clinical disease to potentially identify novel therapeutic strategies. Ischemia and reperfusion cause immediate and global arrest of protein synthesis in all ischemia-affected neurons. Initial protein synthesis inhibition (PSI) is either followed by near complete restoration of protein synthesis or there is a persistence of PSI. It is this persistent PSI that is highly correlated with neurodegeneration and eventual neuronal death. Thus our working hypothesis is that persistent PSI mediates cell death following cerebral ischemia. Our specific hypothesis is that proteolytic cleavage of eukaryotic initiation factor 4G (elF4G) by proteases such as calpain may mediate persistent PSI, and prevention of elF4G cleavage may block persistent PSI and thus provide neuroprotection against ischemic injury. This hypothesis is based on the following data: 1) elF4G cleavage begins prior to decreases in other protein synthesis initiation factors in vitro and in vivo, 2) the time course of cleavage is correlated with that of persistent PSI in vitro, 3) in vitro ischemia results in the production of a novel size-specific cleavage product, and 4) inhibition of calpain activity by overexpression of its endogenous inhibitor calpastatin partially restores protein synthesis following in vitro ischemia. As a result of these data, our specific focus is to determine the role of calpain in elF4G cleavage and to identify the role of elF4G cleavage in persistent PSI and cell death. Our specific aims are: 1) to test the hypothesis that elF4G cleavage and persistent PSI after in vitro ischemia is mediated by calpain activation. We will i) determine if elF4G cleavage is prevented following in vitro ischemia (i) by overexpression the endogenous calpain inhibitor calpastatin and (ii) using neurons derived from calpain knock out mice. We will also (iii) determine if prevention of elF4G cleavage via calpain inhibition can rescue protein synthesis following in vitro ischemia. 2) To test the hypothesis that prevention of elF4G cleavage alleviates persistent PSI and confers neuroprotection against in vitro ischemia. We will (i) overexpress elF4G to attempt to restore baseline elF4G levels, (ii) determine if overexpression of elF4G will confer neuroprotection following in vitro ischemia and (iii) identify the cleavage sites of elF4G by calpain using recombinant proteins in vitro. PUBLIC HEALTH RELEVANCE: Ultimately, the delineation of the mechanism governing persistent PSI may lead to a novel therapeutic strategy for cerebral ischemia.