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

• 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
• 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

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卒中（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在很大程度上是不成功的。我们发现，钙依赖性磷酸酶钙调神经磷酸酶（CN）可以结合PERK，内质网（ER）中的应激传感器，增加其自身磷酸化，并增强称为未折叠蛋白质反应（UPR）的细胞过程。UPR在应激期间减弱蛋白质翻译，并使细胞有更多的时间恢复。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增加了细胞培养物中缺血条件后的细胞活力。该R21提案的目标是开发可用于在体内特异性调节PERK自身磷酸化的分子干预。最终，从该提案中产生的数据将用于描述在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，Ca 2+作为辅助因子。这种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，从而减少了损伤后的脑损伤。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。2)描绘了体内CN与PERK结合的Ca 2+依赖性。将使用生化测定和生物物理技术来绘制CN和PERK的结合相互作用并产生肽片段。星形胶质细胞的原代培养物将用于体内测试这些分子干预的功效。将使用共聚焦显微镜对ER附近Ca 2+微区的变化进行成像。氧葡萄糖脱氢酶，一种缺血的体外模型，将用于确定PERK自磷酸化的生理影响以及我们的分子干预。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。