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： CALPAIN是Ca2+活化的，中性的（非溶酶体）蛋白酶在神经变性中实现的急性损伤（例如中风和脑外伤）以及神经退行性阿尔茨海默氏病等疾病。 CNS中的Calpain研究几乎完全集中在在经典的Calpains上，Calpains 1和2。Calpain5（CAPN5），缺少Penta-ef Hand 经典钙的域是中枢神经系统中第二高表达的钙蛋白酶。 Calpain 5具有与半胱氨酸蛋白结构域中其他钙蛋白酶的同源性，并且在 C末端。 C2结构域与膜和脂质结合。鲜为人知关于钙蛋白酶5的功能和底物。调节与性别确定有关的转录活性。 Tra-3也需要 TDP-43毒性诱导的神经变性和在接下来的感觉神经元的坏死死亡钙超负荷。在亚细胞水平上，CAPN5在核和粗线粒体中发现分数，与丙梅细胞核体有关。该提议的目的是收获对Calpain 5的中枢神经系统功能的了解。AIM1将确定Calpain 5 Binding Partners和使用三种免费技术的底物：串联亲和力纯化；接近依赖性本土化;并在体外亲和力捕获，每种技术都与质谱法配对蛋白质鉴定。约束伙伴和底物将根据基本和细胞内钙条件升高。 C2域在Ca2+结合中的作用以及 CAPN5激活和定位将在AIM 2中评估。AIM3将探索生理 CAPN5的功能，使用使用CRISPR/CAS-9和条件生成的基因敲除细胞系淘汰老鼠。初步数据支持每个目标的可行性。这些结果将在一起提供基本信息，以解密CAPN5的CNS函数。