"The Role of CAP and sNASP in Physiological Actin Depolymerization"

“CAP 和 sNASP 在生理肌动蛋白解聚中的作用”

• 批准号: 8538373
• 负责人: Kieran Patrick Normoyle
• 金额: $ 4.72万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8538373
• 关键词: Actin-Binding Protein; Actinin; Actins; Area; Binding; Biochemical; Biological Assay; Bundling; C-terminal; Cattle; Cell Nucleus; Cell division; Cell physiology; Cells; Chromatin Structure; Clinical; Complex; Cytoplasm; Cytoplasmic Protein; Cytoskeleton; Disease; Electron Microscope; Electron Microscopy; Electrons; Environment; F-Actin; Filament; Focal Segmental Glomerulosclerosis; Functional disorder; Histones; Human; Kidney Diseases; Link; Measures; Mediating; Microfilaments; Microscopy; Modeling; Nephrology; Nuclear; Nuclear Protein; Nuclear Structure; Physiological; Polymers; Process; Proteins; Reaction; Research; Role; Side; Specificity; System; Techniques; Tertiary Protein Structure; Testing; base; cell component structure; cell motility; chromatin remodeling; cofilin; coronin protein; depolymerization; filamin; gene function; genetic analysis; genetic manipulation; human disease; in vivo; knock-down; monomer; pre-clinical research; protein function; reconstitution; sperm protein

DESCRIPTION (provided by applicant): The actin cytoskeleton is a key component of cell structure necessary for many cellular processes such as cell division and cell motility. Actin exists at concentrations inside cells that favor assembly and pose serious challenges to disassembly; in fact, we currently do not know how the cell is able to disassembly actin filaments under physiological conditions. This proposal aims to understand physiological actin dynamics by identifying and characterizing the factors responsible for the least understood side of the actin cycle: disassembly. Such an understanding would have a major impact on any area of clinical or pre-clinical research where cellular problems are actin-dependent; one such area is nephrology. Genetic analysis has found actin-binding proteins prominent among those proteins responsible for familial kidney disorders: 1-actinin and formins in focal segmental glomerulosclerosis and filamin and other actin bundling protein in polycystic kindney disease. We have identified two previously unappreciated proteins using an activity-based biochemical reconstitution that augment the previously characterized cofilin-, coronin-, and AIP1- mediated actin depolymerization system. Cyclase associated protein (CAP) allows this triple-protein mix to depolymerize a fluorescent actin substrate in the presence of cellular concentrations of polymeric and monomeric actin when alone the triple-mix can only deal with physiological monomeric actin concentrations. We propose to use a range of techniques including fluorescent microscopy of bulk actin arrays and electron and fluorescent microscopy of single actin filaments to better characterize the mechanism by which this reaction is accomplished. Somatic nuclear autoantigenic sperm protein (sNASP) functions similarly to AIP1, though further analysis of this similarity is needed. While AIP1 is a cytoplasmic protein, sNASP is a nuclear protein that we have identified as an actin disassembly factor, and since it is known to bind histones and function in chromatin remodeling it represents a possible link between the cytoskeleton and chromatin structure. We plan to use many of the same techniques to characterize sNASP as we will use to characterize CAP, with additional genetic manipulations to test AIP1 and sNASP redundancy in vivo.

描述（由申请人提供）：肌动蛋白细胞骨架是许多细胞过程（例如细胞分裂和细胞运动性）所必需的细胞结构的关键组成部分。肌动蛋白存在于有利于组装的细胞内部的浓度下，并构成严重的挑战以拆卸。实际上，我们目前不知道该细胞在生理条件下如何拆卸肌动蛋白。该建议旨在通过识别和表征导致肌动蛋白周期中最不理解的一面的因素来理解生理肌动蛋白动力学：拆卸。这样的理解将对任何临床或临床前研究的任何领域产生重大影响。这样的领域是肾脏科。遗传分析发现，在负责家族性肾脏疾病的那些蛋白质中突出的肌动蛋白结合蛋白：局灶性节段性肾小球硬化症中的1-肌动蛋白和formins在多囊性金尼病中的其他肌动蛋白以及其他肌动蛋白捆绑蛋白。我们已经使用基于活性的生化重构鉴定了两种先前未批准的蛋白质，该蛋白质增强了先前表征的Cofilin-，Coronin-和AIP1-介导的肌动蛋白解聚系统。环化酶相关的蛋白（CAP）允许这种三蛋白混合在存在聚合物和单体肌动蛋白的细胞浓度的情况下将荧光肌动蛋白底物解散，而单独的三重混合只能处理生理单体肌动蛋白浓度。我们建议使用一系列技术，包括散装肌动蛋白阵列的荧光显微镜和单个肌动蛋白丝的荧光显微镜，以更好地表征完成该反应的机制。体细胞核自身抗原精子蛋白（SNASP）的作用类似于AIP1，尽管需要对这种相似性进行进一步的分析。虽然AIP1是一种细胞质蛋白，但SNASP是一种核蛋白，我们已确定为肌动蛋白拆卸因子，并且由于已知它在染色质重塑中结合组蛋白和功能，它代表了细胞骨架和染色质结构之间的可能联系。我们计划使用许多相同的技术来表征SNASP的表征CAP，并具有其他遗传操作来测试AIP1和SNASP冗余。