The Unfolded Protein Response After Brain Ischemia

脑缺血后未折叠的蛋白质反应

• 批准号: 7072615
• 负责人: DONALD J DEGRACIA
• 金额: $ 31.52万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7072615
• 关键词: apoptosis; cAMP response element binding protein; cerebral ischemia /hypoxia; endoribonucleases; genetic translation; immunocytochemistry; immunoprecipitation; laboratory rat; matrix assisted laser desorption ionization; nervous system regeneration; neurons; phosphorylation; protein biosynthesis; protein folding; protein transport; reperfusion; translation factor; western blottings

DESCRIPTION (provided by applicant): Brain ischemia and reperfusion injury prevents greater than 90% of the 70,000 patients per year resuscitated from cardiac arrest from resuming their normal lives. Our long-term goal is sufficient understanding of the injury mechanisms to formulate clinically effective therapy. Inhibition of protein synthesis during brain reperfusion correlates with regional selective vulnerability and neuronal death, and is due to modification of two translation initiation factors: the phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha), and the proteolytic fragmentation of eukaryotic initiation factor 4G (eIF4G). eIF2 phosphorylation and eIF4G fragmentation affect not only the overall protein synthesis rate, but also which peptides are synthesized from the available mRNAs. Moreover, the kinase that phosphorylates eIF2alpha immediately after brain ischemia and reperfusion, PERK, is known to be activated only by the endoplasmic reticulum stress signaling system termed the unfolded protein response (UPR). The UPR can signal either an adaptive pro-survival response, or it can trigger cell death. Thus suppression of protein translation is likely to be part of a more comprehensive cellular response that determines the ultimate fate of reperfused neurons. We hypothesize: (1) the UPR is activated during early brain reperfusion, (2) vulnerable, but not resistant, neurons fail to resolve the UPR, and (3) there is synthesis of only a limited number of proteins during early reperfusion, as a consequence of eIF2alpha phosphorylation and eIF4G fragmentation, that may determine the outcome of neuronal recovery or death. Our Specific Aims are the following. Aim I will compare in ischemia and reperfusion vulnerable and resistant brain regions the activation of the UPR by characterizing activation of its three effectors ATF6, IRE1alpha, and PERK. Aim 2 will examine in ischemia and reperfusion vulnerable and resistant brain regions whether the UPR is resolved (by determining if synthesis of the pro-survival proteins GRP78, XBP-1, GADD34 and SERCA2b occurs), or if the UPR fails to resolve (by determining if synthesis of the pro-cell death proteins ATF4 and CHOP occurs). Aim 3 will identify those proteins being synthesized by residual translation during the early hours of reperfusion and compare them between ischemia and reperfusion vulnerable and resistant brain regions. This approach provides an integrated examination during brain ischemia and reperfusion of: (1) the occurrence and the consequences of UPR activation, (2) the consequences of translation initiation factor alterations on residual protein synthesis, and (3) the relationship of these two events to the selective vulnerability of the brain to ischemia and reperfusion injury.

描述(由申请人提供)：脑缺血和再灌注损伤会阻止每年从心脏骤停中复苏的70,000名患者中超过90%的人恢复正常生活。我们的长期目标是充分了解损伤机制，以制定临床有效的治疗方法。脑再灌注时蛋白质合成的抑制与局部选择性易损性和神经元死亡有关，这是由于两种翻译启动因子的修饰：真核启动因子2的α亚基(eIF2α)的磷酸化和真核启动因子4G(EIF4G)的蛋白降解碎片。EIF2的磷酸化和eIF4G的断裂不仅影响总的蛋白质合成速度，而且还影响从现有的mRNAs合成哪些肽。此外，在脑缺血和再灌流后立即磷酸化eIF2α的激酶PERK已知只被内质网应激信号系统激活，称为未折叠蛋白反应(UPR)。UPR既可以发出适应性促进生存反应的信号，也可以触发细胞死亡。因此，抑制蛋白质翻译很可能是更全面的细胞反应的一部分，这决定了再灌流神经元的最终命运。我们假设：(1)UPR在脑再灌注早期被激活，(2)脆弱但不耐受的神经元不能分解UPR，(3)由于eIF2α磷酸化和eIF4G断裂，在再灌流早期只有有限数量的蛋白质合成，这可能决定神经元恢复或死亡的结果。我们的具体目标如下。目的通过研究UPR的三个效应因子ATF6、IRE1α和PERK的激活特征，比较缺血再灌注易损和耐受脑区UPR的激活情况。AIM 2将检查在缺血和再灌注脆弱和抵抗的脑区UPR是否被分解(通过确定是否合成有利于生存的蛋白质GRP78、XBP-1、Gadd34和SERCA2b)，或者是否UPR无法分解(通过确定是否合成亲细胞死亡蛋白ATF4和CHOP)。目的3将鉴定那些在再灌流早期通过残存翻译合成的蛋白质，并将它们在缺血和再灌流的脆弱和抵抗脑区进行比较。这一方法在脑缺血和再灌注过程中提供了一个综合的检测：(1)UPR激活的发生和后果，(2)翻译起始因子改变对残余蛋白质合成的影响，以及(3)这两个事件与脑对缺血和再灌注损伤的选择性易感性的关系。