STRUCTURAL BASIS FOR HIP1 FUNCTION IN CLATHRIN MEDIATED ENDOCYTOSIS AND HUNTINGTO

网格蛋白介导的内吞作用和狩猎中 HIP1 功能的结构基础

基本信息

批准号：
7652098
负责人：
JOEL A YBE
金额：
$ 30.95万
依托单位：
TRUSTEES OF INDIANA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-09-30 至 2013-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7652098
关键词：
3-Dimensional 8 year old Acidic Amino Acids Address Affinity Alanine American Amino Acid Substitution Amino Acids Apoptosis Apoptotic Area Award Base Sequence Binding Binding Sites Biological Assay Boxing Brain C-terminal Caspase Cells Cerebral cortex Cessation of life Charge Circular Dichroism Classification Clathrin Clathrin Light Chains Clathrin-Coated Vesicles Cleaved cell Coiled-Coil Domain Communication Complex Congresses Country Crystallization Cysteine DEVDase Data Data Set Disease Electrostatics Emotional Emotional Disturbance Endocytosis Epitopes Equilibrium Event Faculty Fluorescence Funding Germany Glutamine Head Heating Hela Cells Hereditary Disease High Prevalence Homeostasis Hot Spot Huntington Disease Hybrids Immunoprecipitation Indiana Knockout Mice Learning Left Length Light Link Location Mammalian Cell Maps Mediating Methods Modeling Molecular Molecular Biology Movement Mutagenesis Mutate Mutation N-terminal National Institute of General Medical Sciences Neostriatum Nerve Degeneration Neurologic Neurons Nuclear Magnetic Resonance Pathway interactions Persons Play Positioning Attribute Property Protein Binding Protein Family Proteins Publishing Relative (related person)Research Resistance Resolution Risk Roentgen Rays Role Sampling Scanning Sequence Analysis Shapes Site-Directed Mutagenesis Solutions Stretching Structure Sum Surface Symptoms Tail Temperature Testing Thermodynamics Toxic effect Tryptophan X-Ray Crystallography base beamline caspase-3 caspase-8 cold temperature design dimer disulfide bond flexibility human Huntingtin protein in vivo insight melting mutant novel overexpression polyglutamine prevent pro-caspase-8 protein function research study response trafficking wasting web site

项目摘要

DESCRIPTION (provided by applicant): Huntington's disease (HD) is a late-onset neurodegenerative genetic disorder that produces uncontrolled physical movements, devastates intellectual faculties and triggers emotional disturbances. Those suffering from HD typically die within 10 years after symptoms surface and there is currently no cure. There is an emerging link between HD and clathrin-mediated endocytosis through Huntingtin-interacting protein 1 (HIP1). The purpose of this renewal application is to elucidate how the interaction between clathrin-coated vesicles and HIP1 (and HIP1R, R for related) is regulated in healthy cells to gain insights into the role of clathrin- mediated endocytosis in HD. A closely related purpose of this proposal is to find what structural determinants mediate the binding of HIP1 to Huntingtin-interacting protein 1-protein interactor (HIPPI), an accessory protein that modifies the activity of HIP1 in Huntington's disease. The first specific Aim is to determine what destabilizes the opened region of HIP1 to understand if molecular flexibility in the coiled-coil of this protein is important for its function. Site-directed mutagenesis will target specific positions in the coiled-coil to see if the opened region of HIP1 can be stabilized. Deletion mapping studies will be performed to pinpoint the location of "hard-wired" structure in the opened region that was found to be highly resistant to temperatures that would inactivate other proteins in the body. The powerful methods of X-ray crystallography and Nuclear Magnetic Resonance (NMR) will be used to study the static and dynamic properties of the stable sub-structure unit to assess if this feature is significant for HIP1 function. The second specific Aim is to determine if the binding dynamics of clathrin to HIP1 and HIP1R are regulated through molecular flexibility. First, the surfaces of HIP1 and HIP1R that make contact with the light chain subunit of clathrin will be fully characterized. Second, two binding models will be evaluated to determine if the binding determinants for clathrin light chain are spread between the two helices of dimeric HIP1 or if the determinants are contained entirely in one of the two helices. Third, a mutagenesis approach will determine if clathrin light chain and HIP proteins bind in a "head-to-head" or "head-to-tail" direction to deepen our understanding of how clathrin-coated vesicles build up in cells. The third specific Aim is to finish the 3.7 E crystal structure of the clathrin trimer domain to understand if this domain can interact productively with the N-terminus of HIP1. The fourth specific Aim is to investigate what structural determinants control the formation of the HIPPI/HIP1 complex that triggers the caspase-3/caspase-8 apoptotic pathway in Huntington's disease. Planned mutagenesis and in vivo studies will focus on two highly charged regions in the opened region of HIP1. The sum total of the information that will flow from the proposed research will deepen our understanding of how HIP proteins function in clathrin-mediated endocytosis and how HIP1, specifically, can potentiate the progress of Huntington's disease.

描述（由申请人提供）：亨廷顿氏病（HD）是一种晚期神经退行性遗传疾病，会产生不受控制的身体运动，破坏知识能力并触发情绪障碍。患有高清的人通常在症状表面后的10年内死亡，目前无法治愈。 HD与网格蛋白介导的内吞作用之间通过亨廷顿蛋白相互作用蛋白1（HIP1）存在着新的联系。这种更新应用的目的是阐明如何在健康细胞中调节网格蛋白包被囊泡与HIP1（以及HIP1R，R的HIP1R，R）之间的相互作用，以深入了解网li介导的HD中网状蛋白介导的内吞作用的作用。该提案的一个密切相关的目的是找到哪种结构决定因素介导了HIP1与亨廷汀相互作用蛋白1-蛋白质相互作用（HIPPI）的结合，这是一种辅助蛋白，可修饰HIP1在亨廷顿病中的活性。第一个具体目的是确定是什么破坏了HIP1的开放区域的稳定性，以了解该蛋白质盘绕螺旋中的分子柔韧性是否对其功能很重要。定点诱变将靶向盘绕圈中的特定位置，以查看是否可以稳定HIP1的打开区域。将进行删除映射研究，以查明开放区域中“硬连线”结构的位置，该区域被发现对会使体内其他蛋白质失活的温度具有高度抗性。 X射线晶体学和核磁共振（NMR）的强大方法将用于研究稳定子结构单元的静态和动态特性，以评估此功能对于HIP1功能是否重要。第二个具体目的是确定网格蛋白与HIP1和HIP1R的结合动力学是否通过分子柔韧性调节。首先，将充分表征与网格蛋白的轻链亚基接触的HIP1和HIP1R的表面。其次，将评估两个结合模型，以确定网格蛋白轻链的结合确定因素是否在二聚体HIP1的两个螺旋之间扩展，或者是否完全包含在两个螺旋中的一个螺旋中。第三，一种诱变方法将确定网格蛋白轻链和髋关节蛋白是否在“头对头”或“从头到尾”方向结合，以加深我们对细胞中细胞蛋白涂层囊泡如何堆积的理解。第三个具体目的是完成网格蛋白三聚体域的3.7 E晶体结构，以了解该域是否可以与HIP1的N端有效地相互作用。第四个具体目的是研究哪种结构决定因素控制了HIPPI/HIP1复合物的形成，从而触发了Huntington病中caspase-3/caspase-8凋亡途径。计划的诱变和体内研究将集中在HIP1开放区域中的两个高电荷区域。从提议的研究中流入的信息的总和将加深我们对髋蛋白在网格蛋白介导的内吞作用中的功能以及HIP1的理解，以及HIP1如何增强亨廷顿氏病的进步。