CNS Functions of Calpain 5

Calpain 5 的中枢神经系统功能

• 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
• 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' 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

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.

摘要： 钙蛋白酶是一种与神经退行性变有关的钙激活中性蛋白酶 在急性损伤如中风和创伤性脑损伤后，以及在神经退行性疾病中， 老年痴呆症等疾病。中枢神经系统中的钙蛋白酶研究几乎完全集中在 经典的钙蛋白酶，钙蛋白酶1和2钙蛋白酶5（CAPN 5），缺乏五EF手 经典钙蛋白酶的结构域，是CNS中第二高表达的钙蛋白酶。Calpain 5具有 在半胱氨酸蛋白酶结构域中与其他钙蛋白酶具有同源性，并且在半胱氨酸蛋白酶结构域中也具有独特的C2结构域。 C端C2结构域参与与膜和脂质的结合。知之甚少 关于钙蛋白酶5的功能和底物。梭钙蛋白酶5，Tra-3， 调节参与性别决定的转录活性。还需要Tra-3用于 TDP-43毒性诱导的神经变性和感觉神经元的坏死性死亡 钙超载在亚细胞水平，CAPN 5存在于细胞核和粗线粒体中， 部分，并与promeylocytic核机构。这个提议的目的是 深入了解Calpain 5的CNS功能。目的1将鉴定钙蛋白酶5结合伴侣， 使用三种互补技术：串联亲和纯化;邻近依赖性 定位;和体外亲和捕获，每种技术与质谱法配对， 蛋白质鉴定结合伴侣和底物将在基础和 细胞内钙升高的情况。C2结构域在Ca 2+结合中的作用沿着 CAPN 5激活和定位将在目标2中评价。目标3将探索生理 CAPN 5的功能，利用使用CRISPR/Cas-9和条件性 敲除小鼠初步数据支持每个目标的可行性。这些结果将 为破译CAPN 5的CNS功能提供必要信息。