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：
8623859
负责人：
JAMES D LECHLEITER
金额：
$ 20.5万
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 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8623859
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 Techniques Testing Therapeutic Time Translations Traumatic Brain Injury Work autophosphorylation-dependent multifunctional protein kinase base 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

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 is 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, which 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 in 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.

拟议的研究调查了新的分子靶标和过程，这些靶标有望最大程度地减少大脑脑缺血性中风（CIS）和创伤性脑损伤（TBI）后的损害。当前的治疗策略抗击急性脑损伤基本上没有成功。我们发现CA2+依赖磷酸酶钙调蛋白（CN）可以与内质网（ER）中的应力传感器（ER）中的应力传感器结合增加其自磷酸化并增强称为展开蛋白质反应的细胞过程（UPR）。 UPR在压力期间减弱蛋白质的翻译，并使细胞更多的时间恢复。值得注意的是，我们的初步数据表明，CN的新保护作用可提高细胞的活力细胞培养中的缺血条件。该R21提案的目的是开发分子干预措施可用于特异性调节体内PERK自磷酸化。最终，从中生成的数据建议将用于描述在顺式和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与Ca2+作为副因素直接与PERK相互作用。这种蛋白质复合物的形成促进了PERK二聚/寡聚和自磷酸化。反过来，这增强了蛋白质翻译和细胞活力的抑制受伤。我们有两个具体的目的：1）开发促进，破坏或模仿CN的分子干预措施与PERK结合。 2）描述CN与体内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的结合相互作用并生成肽片段。星形胶质细胞的主要培养物将用于测试这些分子干预在体内。共聚焦显微镜将用于图像微区域的变化 Ca2+在ER附近。氧气剥夺是一种缺血的体外模型，将用于确定 PERK自磷酸化以及我们的分子干预的生理影响。如果成功，这些肽的开发将是治疗脑损伤的有吸引力的治疗工具。