Regulation of the Unfolded Protein Response after Acute Brain Injury

急性脑损伤后未折叠蛋白反应的调节

基本信息

批准号：
8739331
负责人：
JAMES D LECHLEITER
金额：
$ 16.45万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-29 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8739331
关键词：
Acute Acute Brain Injuries Area Astrocytes Attenuated Binding Biochemical Biological Assay Brain Injuries Calcineurin Calmodulin Carbodiimides Cell Culture Techniques Cell Death Cell Survival Cell physiology Cells Cerebral Ischemia Cerebrum Co-Immunoprecipitations Confocal Microscopy Data Dependence Development Diazomethane Dimerization Endoplasmic Reticulum Glucose Goals Image In Vitro Individual Injury Intervention Ischemia Ischemic Stroke Label Lead Maps Mass Spectrum Analysis Measures Membrane Molecular Molecular Target Oxidative Stress Oxygen Peptide Fragments Peptides Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Physiological Positioning Attribute Process Prolactin Protein Inhibition Proteins Recombinant Proteins Recombinants Regulation Reporting Research Research Proposals Role Solvents Source Stress Surface Testing Therapeutic Time Translations Traumatic Brain Injury Work autophosphorylation-dependent multifunctional protein kinase base biophysical techniques calcineurin phosphatase combat deprivation efficacy testing endoplasmic reticulum stress in vitro Model in vitro testing in vivo innovation knock-down mouse model mutant neuron loss novel novel therapeutics overexpression protein complex public health relevance research study response sensor sulfo-N-hydroxysuccinimide-biotin tool

项目摘要

DESCRIPTION (provided by applicant): The proposed research investigates novel molecular targets and processes that promise to minimize brain damage after cerebral ischemic stroke (CIS) and traumatic brain injury (TBI). Current therapeutic strategies to combat acute brain injuries have been largely unsuccessful. We discovered that the Ca2+ dependent phosphatase calcineurin (CN) can bind to PERK, a stress sensor in the endoplasmic reticulum (ER), increases its auto-phosphorylation and enhance a cellular process called the Unfolded Protein Response (UPR). The UPR attenuates protein translation during stress and gives the cell more time to recover. Significantly, our preliminary data suggest that this new protective role for CN increases cell viability after ischemic conditions in cell culture. The goal of this R21 proposal i to develop molecular interventions that can be used to specifically regulate PERK auto-phosphorylation in vivo. Ultimately, data generated from this proposal will be used to delineate the therapeutic potential of regulating the UPR during CIS and TBI. Our overall hypothesis is that, under ischemic conditions, CN directly interacts with PERK with Ca2+ as a co-factor. The formation of this protein complex promotes PERK dimerization/oligomerization and auto-phosphorylation. This, in turn, enhances inhibition of protein translation and cell viability, whic reduces brain damage after injury. We have two Specific Aims: 1) Develop molecular interventions that promote, disrupt or mimic CN binding to PERK. 2) Delineate the Ca2+ dependence of CN binding to PERK in vivo. Biochemical assays and biophysical techniques will be used to map the binding interaction of CN and PERK and to generate the peptide fragments. Primary cultures of astrocytes will be used to test the efficacy of these molecular interventions i vivo. Confocal microscopy will be used to image changes in microdomains of Ca2+ near the ER. Oxygen Glucose Deprivation, an in vitro model of ischemia, will be used to determine the physiological impact of PERK auto-phosphorylation as well as our molecular interventions. If successful, the development of these peptides will serve as attractive therapeutic tools for the treatment of brain injuries.

描述（由申请人提供）：拟议的研究调查了新的分子靶点和过程，有望最大限度地减少脑缺血性中风（CIS）和创伤性脑损伤（TBI）后的脑损伤。目前对抗急性脑损伤的治疗策略基本上不成功。我们发现 Ca2+ 依赖性磷酸酶钙调神经磷酸酶 (CN) 可以与内质网 (ER) 中的应激传感器 PERK 结合，增加其自身磷酸化并增强称为未折叠蛋白反应 (UPR) 的细胞过程。 UPR 会减弱应激期间的蛋白质翻译，并为细胞提供更多时间恢复。值得注意的是，我们的初步数据表明，CN 的这种新的保护作用可提高细胞培养中缺血条件后的细胞活力。 R21 提案的目标是开发可用于特异性调节体内 PERK 自磷酸化的分子干预措施。最终，该提案生成的数据将用于描述 CIS 和 TBI 期间调节 UPR 的治疗潜力。我们的总体假设是，在缺血条件下，CN 直接与 PERK 相互作用，Ca2+ 作为辅助因子。该蛋白质复合物的形成促进 PERK 二聚化/寡聚化和自磷酸化。这反过来又增强了对蛋白质翻译和细胞活力的抑制，从而减少了受伤后的脑损伤。我们有两个具体目标：1) 开发促进、破坏或模拟 CN 与 PERK 结合的分子干预措施。 2) 描述体内 CN 与 PERK 结合的 Ca2+ 依赖性。生化测定和生物物理技术将用于绘制 CN 和 PERK 的结合相互作用图并生成肽片段。星形胶质细胞的原代培养物将用于测试这些分子干预措施的体内功效。共聚焦显微镜将用于对 ER 附近 Ca2+ 微区的变化进行成像。缺氧葡萄糖剥夺是一种体外缺血模型，将用于确定 PERK 自磷酸化的生理影响以及我们的分子干预措施。如果成功，这些肽的开发将成为治疗脑损伤的有吸引力的治疗工具。