ElF4g Cleavage and Persistent Protein Synthesis Inhibition Following Ischemia

缺血后 ElF4g 裂解和持续的蛋白质合成抑制

• 批准号: 7545042
• 负责人: PETER Stephen VOSLER
• 金额: $ 4.45万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7545042
• 关键词: Affect; Antibodies; Binding; Brain Ischemia; C-terminal; Calpain; Calpain I; Caspase; Cell Death; Cells; Cellular Stress Response; Cerebral Ischemia; Cerebrum; Cessation of life; Clinical; Complex; Cysteine Protease; Data; Disease; Endopeptidases; Eukaryotic Initiation Factor-4G; Eukaryotic Initiation Factors; Event; Genetic Translation; Glucose; Goals; Hour; Impairment; In Vitro; Injury; Investigation; Ischemia; Knockout Mice; Lead; Length; Mediating; Messenger RNA; Modeling; Molecular; Nerve Degeneration; Neurologic; Neurons; Oxygen; Peptide Hydrolases; Peptide Initiation Factors; Phase; Physiological reperfusion; Poly(A)-Binding Proteins; Prevention; Production; Protein Binding; Protein Biosynthesis; Protein Overexpression; Protein Synthesis Inhibition; Proteins; Public Health; RNA Cap-Binding Proteins; RNA Helicase; Rattus; Recombinant Proteins; Recombinants; Recovery; Reperfusion Therapy; Ribosomes; Role; Scaffolding Protein; Site; Stroke; Testing; Thinking; Time; Work; base; calpain inhibitor; calpastatin; deprivation; experience; in vitro Model; in vivo; inhibitor/antagonist; interest; knockout gene; loss of function; neuron loss; neuroprotection; novel; novel therapeutics; prevent; protein degradation; research study; restoration; size; small hairpin RNA

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.

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产生严重的临床疾病。因此，我们的长期目标是阐明缺血后产生临床疾病的机制，以潜在地确定新的治疗策略。缺血和再灌注会导致所有受缺血影响的神经元中蛋白质合成立即全面停止。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。正是这种持续的 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的 PSI 介导脑缺血后的细胞死亡。我们的具体假设是，钙蛋白酶等蛋白酶对真核起始因子 4G (elF4G) 的蛋白水解裂解可能介导持续性 PSI，而预防 eF4G 裂解可能会阻止持续性 PSI，从而提供针对缺血性损伤的神经保护。该假设基于以下数据：1) eF4G 裂解开始于体外和体内其他蛋白质合成起始因子减少之前，2) 裂解的时间进程与体外持续 PSI 的时间进程相关，3) 体外缺血导致新的大小特异性裂解产物的产生，4) 通过其过表达来抑制钙蛋白酶活性 内源性抑制剂 Calpastatin 可部分恢复体外缺血后的蛋白质合成。根据这些数据，我们的具体重点是确定钙蛋白酶在 eF4G 裂解中的作用，并确定 eF4G 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）检验体外缺血后 eF4G 裂解和持续 PSI 是由钙蛋白酶激活介导的假设。我们将 i) 确定在体外缺血后是否可以阻止 eF4G 裂解 (i) 通过过表达内源性钙蛋白酶抑制剂 calpastatin 和 (ii) 使用源自钙蛋白酶敲除小鼠的神经元。我们还将 (iii) 确定通过抑制钙蛋白酶来预防 eF4G 裂解是否可以挽救体外缺血后的蛋白质合成。 2) 检验以下假设：预防 eF4G 裂解可减轻持续性 PSI 并赋予神经保护作用以对抗体外缺血。我们将（i）过表达elF4G以尝试恢复基线elF4G水平，（ii）确定elF4G的过表达是否会在体外缺血后赋予神经保护作用，以及（iii）使用体外重组蛋白鉴定钙蛋白酶对elF4G的切割位点。 公共卫生相关性：最终，对持续性 PSI 控制机制的描述可能会带来一种新的脑缺血治疗策略。