Elucidating the role of UBXD4 at the axon initial segment

阐明 UBXD4 在轴突起始段的作用

基本信息

批准号：
8329148
负责人：
Shelly A Buffington
金额：
$ 2.27万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-04-07 至 2012-10-05
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8329148
关键词：
Actins Action Potentials Adaptor Signaling Protein Affect Axon Binding Cells Chimeric Proteins Chronic Co-Immunoprecipitations Communication Cytoskeleton Defect Deubiquitinating Enzyme Disease Distal Electroporation Event Functional disorder Generations Half-Life Hippocampus (Brain)Hour Immunoblotting Immunofluorescence Immunologic In Vitro Injury Lead Length Location Maintenance Measures Mediating Membrane Molecular Monitor Natural regeneration Nervous System Physiology Nervous system structure Neurologic Neurons Peptide Hydrolases Phenotype Plastics Play Process Proteasome Inhibitor Proteins Recycling Role Scaffolding Protein Site Sodium Channel Spectrin Structure Synapses System Testing Therapeutic Ubiquitin Ubiquitination Work base density gain of function immunocytochemistry improved in utero in vivo knock-down loss of function multicatalytic endopeptidase complex neuronal cell body overexpression prevent protein complex protein degradation repaired research study response small hairpin RNA voltage

项目摘要

DESCRIPTION (provided by applicant): The axon initial segment (AIS) is critical for the generation of action potentials and the maintenance of neuronal polarity. Disruption of this domain due to disease or injury can lead to nervous system dysfunction. The voltage threshold for action potential initiation is lowest at the AIS due to the high- density clusters of voltage-gated sodium channels accumulated here through interaction with the cytoskeletal adaptor protein ankyrinG (ankG). The functional organization of the AIS depends on ankG. AIS proteins are remarkably stable with half-lives that can exceed two weeks. In contrast, the half-lives of many synaptic proteins are on the order of hours. Recent work strongly suggests that the AIS is also plastic and can adapt in response to changes in activity levels. The molecular mechanisms underlying AIS protein stability and how AIS protein levels are modulated in response to changes in neuronal activity, however, remain unknown. AIS stability may depend on events that interfere with protein degradation. One way to achieve such stability could be to prevent ubiquitination of AIS proteins. The newly identified ubiquitin-related protein UBXD4 is enriched at the AIS. UBXD4 is structurally similar to ubiquitin and is thought to either block the degradation domains of its interacting partners or to tether binding partners to a deubiquitinating enzyme. The objective of this project is three-fold: To (1) determine if protein recycling at the AIS is a ubiquitin proteasome system (UPS) mediated process, (2) determine if UBXD4 is involved in the assembly of the AIS, and (3) test the hypothesis that UBXD4 contributes to AIS stability by modulating AIS protein half-life. Action potential initiation threshold at the AIS can vary between neurons and may be modulated in an activity-dependent manner. Differences in ankG turnover rates and Nav expression could drive shifts in threshold potential. To determine if activity-dependent protein recycling at the AIS is a UPS-dependent event, the activity of cultured hippocampal neurons will be pharmacologically manipulated. Cell lysates will then be immunoblotted to detect changes in AIS protein levels and to quantify the extent to which AIS proteins are ubiquitinated. Protein levels will also be assessed by immunocytochemistry. Proteasome inhibitors will be administered to cells to determine if decreased protein degradation by the UPS increases AIS protein half-life. The role of UBXD4 in AIS assembly will be tested in cultured neurons and in vivo by introducing shRNA to silence UBXD4 expression. AIS assembly will be monitored by immunostaining. UBXD4 localization at the AIS depends on ankG. Co-immunoprecipitation and pull-down experiments with His-ankG and GST-UBXD4 fusion proteins will be used to determine if UBXD4 directly interacts with ankG. Finally, AIS protein stability will be measured after the silencing UBXD4 expression and overexpressing UBXD4. AIS protein turnover rates will be assessed by immunofluorescence. The results of this project will improve our understanding of the molecular mechanisms that stabilize the AIS and contribute to its structural plasticity in response to changes in activity.

描述（由申请人提供）：轴突初始段（AI）对于动作电位的产生和神经元极性的维持至关重要。由于疾病或受伤而导致的该领域的破坏会导致神经系统功能障碍。由于与细胞骨架适配器蛋白Ankyring（ANKG）相互作用，此处积累的电压门控钠通道的高密度簇（ANKG），AIS的电势启动的电压阈值在AIS处最低。 AIS的功能组织取决于ANKG。 AIS蛋白非常稳定，半衰期可能超过两周。相反，许多突触蛋白的半衰期在小时的顺序上。最近的工作强烈表明，AIS也是塑性的，可以响应活动水平的变化而适应。然而，AIS蛋白稳定性的分子机制以及如何根据神经元活性的变化调节AIS蛋白水平。 AIS稳定性可能取决于干扰蛋白质降解的事件。达到这种稳定性的一种方法可能是防止AIS蛋白的泛素化。新近鉴定的泛素相关蛋白UBXD4在AIS上富集。 UBXD4在结构上与泛素相似，被认为可以阻止其相互作用伴侣的降解域，或者将结合伴侣绑定到去泛素化酶。该项目的目的是三倍：（1）确定在AIS处的蛋白质回收是否是泛素蛋白酶体系统（UPS）介导的过程，（2）确定UBXD4是否参与了AIS的组装，（3）测试UBXD4对AIS稳定性的假设通过模块化AIS蛋白质的蛋白质蛋白蛋白寿命有助于AIS稳定性。 AIS处的动作电位启动阈值在神经元之间可能有所不同，并且可以以活动依赖性方式调节。 ANKG离职率和NAV表达的差异可能会驱动阈值潜力的转移。为了确定AIS在AIS处的活性依赖性蛋白质回收是否是UPS依赖性事件，将通过药理学操纵培养的海马神经元的活性。然后，将对细胞裂解物进行免疫印迹，以检测AIS蛋白水平的变化，并量化AIS蛋白泛素化的程度。蛋白质水平也将通过免疫细胞化学评估。蛋白酶体抑制剂将用于细胞，以确定UPS降低蛋白质降解是否会增加AIS蛋白的半衰期。 UBXD4在AIS组装中的作用将在培养的神经元和体内测试，通过引入SHRNA以使UBXD4表达沉默。 AIS组件将通过免疫染色来监测。 AIS处的UBXD4定位取决于ANKG。与His-ANKG和GST-UBXD4融合蛋白进行的共免疫沉淀和下拉实验将用于确定UBXD4是否直接与ANKG相互作用。最后，将在沉默的UBXD4表达和过表达UBXD4之后测量AIS蛋白稳定性。 AIS蛋白更新率将通过免疫荧光评估。该项目的结果将提高我们对稳定AI的分子机制的理解，并响应活动变化而导致其结构可塑性。