CNS Functions of Calpain 5

Calpain 5 的中枢神经系统功能

基本信息

批准号：
9343053
负责人：
James W. Geddes
金额：
$ 39.6万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2021-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9343053
关键词：
Actins Active Sites Acute Acute Promyelocytic Leukemia Afferent Neurons Affinity Affinity Chromatography Alzheimer&apos s Disease Basic Science Binding Binding Sites Biotin Brain C2 Domain CRISPR/Cas technology Caenorhabditis elegans Calcium Calpain Caspase Cell Line Cell Nucleus Cell membrane Cells Cessation of life Characteristics Chronic Cleaved cell Computer Simulation Cysteine Cytokinesis Data Dynein ATPase EF Hand Motifs EF-Hand Domain Family Genes Genetic Genetic Transcription Human Impairment In Vitro Knock-out Knockout Mice Lipids Long-Term Potentiation Mass Spectrum Analysis Membrane Membrane Lipids Messenger RNA Methods Microtubules Mitochondria Necrosis Nerve Degeneration Neurodegenerative Disorders Neurosciences Nomenclature Nuclear Pathologic Pentas Peptide Hydrolases Phenotype Phospholipids Physiological Property Proprotein Convertase 1 Proprotein Convertase 2 Protease Domain Protein Isoforms Proteins Research Role Signal Transduction Site-Directed Mutagenesis Spinal Cord Stroke Structure Techniques Toxic effect Traumatic Brain Injury Triad Acrylic Resin alpha Actinin alpha Tubulin base beta Tubulin in vivo inhibitor/antagonist insight interest knock-down member migration novel overexpression protein TDP-43 response sensor sex determination

项目摘要

Abstract: Calpains are Ca2+-activated, neutral (non-lysosomal) proteases implicated in neurodegeneration following acute insults such as stroke and traumatic brain injury, as well as in neurodegenerative disorders such as Alzheimer’s disease. Calpain research in the CNS has focused almost exclusively on the classical calpains, Calpains 1 and 2. Calpain 5 (CAPN5), which lacks the penta-EF hand domain of classical calpains, is the 2nd most highly expressed calpain in the CNS. Calpain 5 has homology to other calpains in the cysteine protease domain and also has a unique C2 domain at the C-terminus. C2 domains are involved in binding to membranes and lipids. Very little is known regarding the functions and substrates of Calpain 5. The C. elegans orthologue of Calpain 5, Tra-3, regulates transcriptional activity involved in sex determination. Tra-3 is also required for neurodegeneration induced by TDP-43 toxicity and for necrotic death of sensory neurons following calcium overload. At the subcellular level, CAPN5 is found in nuclear and crude mitochondrial fractions, and associated with promeylocytic nuclear bodies. The purpose of this proposal is gain insight into the CNS functions of Calpain 5. Aim 1 will identify Calpain 5 binding partners and substrates using three complimentary techniques: tandem affinity purification; proximity dependent localization; and in vitro affinity capture, with each technique paired with mass spectrometry for protein identification. The binding partners and substrates will be evaluated under both basal and elevated intracellular calcium conditions. The role of the C2 domain in Ca2+ binding along with CAPN5 activation and localization will be evaluated in Aim 2. Aim 3 will explore the physiological functions of CAPN5, utilizing knockout cell lines generated using CRISPR/Cas-9 and conditional knockout mice. Preliminary data support the feasibility of each Aim. Together, these results will provide essential information for deciphering the CNS function(s) of CAPN5.

抽象的：钙蛋白酶是 Ca2+ 激活的中性（非溶酶体）蛋白酶，与神经变性有关中风和创伤性脑损伤等急性损伤以及神经退行性疾病后阿尔茨海默病等疾病的钙蛋白酶研究几乎完全集中在中枢神经系统中。经典钙蛋白酶、钙蛋白酶 1 和 2。钙蛋白酶 5 (CAPN5)，缺少 penta-EF 手经典钙蛋白酶的结构域，是中枢神经系统中第二高表达的钙蛋白酶 5。半胱氨酸蛋白酶结构域与其他钙蛋白酶具有同源性，并且还具有独特的 C2 结构域 C 末端与膜和脂质的结合知之甚少。关于 Calpain 5 的功能和底物。C. elegans 的 Calpain 5、Tra-3、 Tra-3 也需要调节参与性别决定的转录活性。 TDP-43 毒性诱导的神经变性以及随后感觉神经元的坏死性死亡在亚细胞水平，CAPN5 存在于细胞核和粗线粒体中。分数，并与早幼细胞核体相关。该提案的目的是获得增益。深入了解 Calpain 5 的 CNS 功能。目标 1 将识别 Calpain 5 结合伙伴并使用三种互补技术的底物：串联亲和纯化；邻近依赖；定位；和体外亲和捕获，每种技术都与质谱分析相结合蛋白质鉴定将在基础和底物下进行评估。 C2 结构域在 Ca2+ 结合中的作用升高。 CAPN5 激活和定位将在目标 2 中进行评估。目标 3 将探索生理学 CAPN5 的功能，利用 CRISPR/Cas-9 和条件条件生成的敲除细胞系初步数据支持每个目标的可行性。提供破译 CAPN5 的 CNS 功能的重要信息。