The Unfolded Protein Response After Brain Ischemia

脑缺血后未折叠的蛋白质反应

基本信息

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
项目类别：
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

项目摘要

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）：每年有 70,000 名心脏骤停复苏的患者中，超过 90% 的患者因脑缺血和再灌注损伤而无法恢复正常生活。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。脑再灌注期间蛋白质合成的抑制与区域选择性脆弱性和神经元死亡相关，这是由于两个翻译起始因子的修饰所致：真核起始因子 2 (eIF2α) 的 α 亚基的磷酸化和真核起始的蛋白水解片段化因子 4G (eIF4G)。 eIF2 磷酸化和 eIF4G 片段化不仅影响总体蛋白质合成速率，还影响从可用 mRNA 合成的肽。此外，已知在脑缺血和再灌注后立即磷酸化 eIF2α 的激酶 PERK 只能被称为未折叠蛋白反应 (UPR) 的内质网应激信号系统激活。 UPR 可以发出适应性促生存反应的信号，也可以触发细胞死亡。因此，蛋白质翻译的抑制可能是决定再灌注神经元最终命运的更全面细胞反应的一部分。我们假设：(1) UPR 在早期脑再灌注期间被激活，(2) 脆弱但不具有抵抗力的神经元无法解析 UPR，以及 (3) 在早期再灌注期间仅合成有限数量的蛋白质，如eIF2α 磷酸化和 eIF4G 片段化的结果，可能决定神经元恢复或死亡的结果。我们的具体目标如下。目的我将通过表征其三个效应器 ATF6、IRE1alpha 和 PERK 的激活来比较缺血和再灌注易损和抵抗脑区域中 UPR 的激活。目标 2 将检查缺血和再灌注脆弱和抵抗的大脑区域中 UPR 是否得到解决（通过确定促生存蛋白 GRP78、XBP-1、GADD34 和 SERCA2b 的合成是否发生），或者 UPR 是否未能解决（通过确定是否发生促细胞死亡蛋白 ATF4 和 CHOP 的合成）。目标 3 将识别再灌注早期通过残余翻译合成的蛋白质，并在缺血和再灌注易受影响和抵抗的大脑区域之间比较它们。这种方法提供了脑缺血和再灌注期间的综合检查：（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后果，以及（3）这两个事件的关系大脑对缺血和再灌注损伤的选择性脆弱性。