Elucidating the role of UBXD4 at the axon initial segment

阐明 UBXD4 在轴突起始段的作用

• 批准号: 8122860
• 负责人: Shelly A Buffington
• 金额: $ 3.54万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-07 至 2014-04-06
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8122860
• 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

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. PUBLIC HEALTH RELEVANCE: The axon initial segment is a specific neuronal domain that is essential for the initiation of neuronal communication. Thus, events that affect this structure or damage it can lead to altered nervous system function. The objectives of this project are to study the contribution of a newly identified axon initial segment protein, UBXD4, to maintaining the protein complex assembled here and to understand how neuronal activity levels affect axon initial segment structure and assembly.

描述（由申请人提供）：轴突起始段（AIS）对于动作电位的产生和神经元极性的维持至关重要。由于疾病或损伤而破坏该区域可能会导致神经系统功能障碍。动作电位启动的电压阈值在 AIS 处最低，因为通过与细胞骨架衔接蛋白锚蛋白 G (ankG) 相互作用，此处积累了高密度的电压门控钠通道簇。 AIS 的功能组织取决于 ankG。 AIS 蛋白非常稳定，半衰期可以超过两周。相比之下，许多突触蛋白的半衰期约为数小时。最近的研究强烈表明，AIS 也是可塑的，可以适应活动水平的变化。然而，AIS 蛋白稳定性的分子机制以及 AIS 蛋白水平如何响应神经元活动变化进行调节仍然未知。 AIS 稳定性可能取决于干扰蛋白质降解的事件。实现这种稳定性的一种方法是防止 AIS 蛋白泛素化。新鉴定的泛素相关蛋白 UBXD4 在 AIS 处富集。 UBXD4 在结构上与泛素相似，被认为可以阻断其相互作用伙伴的降解结构域或将结合伙伴与去泛素化酶连接。该项目的目标有三个：(1) 确定 AIS 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 AIS 的组装，以及 (3 ) 检验 UBXD4 通过调节 AIS 蛋白半衰期有助于 AIS 稳定性的假设。 AIS 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进行调节。 ankG 周转率和 Nav 表达的差异可能会导致阈值电位的变化。为了确定 AIS 的活性依赖性蛋白质回收是否是 UPS 依赖性事件，将对培养的海马神经元的活性进行药理学操作。然后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进行评估。将向细胞施用蛋白酶体抑制剂，以确定 UPS 减少的蛋白质降解是否会增加 AIS 蛋白质的半衰期。 UBXD4 在 AIS 组装中的作用将在培养的神经元中和体内通过引入 shRNA 来沉默 UBXD4 表达来测试。 AIS 组装将通过免疫染色进行监测。 UBXD4 在 AIS 上的定位取决于 ankG。 His-ankG 和 GST-UBXD4 融合蛋白的免疫共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，在沉默UBXD4表达和过表达UBXD4后测量AIS蛋白稳定性。 AIS 蛋白质周转率将通过免疫荧光进行评估。该项目的结果将提高我们对稳定 AIS 的分子机制的理解，并有助于其响应活动变化的结构可塑性。 公共健康相关性：轴突起始段是一个特定的神经元结构域，对于神经元通讯的启动至关重要。因此，影响该结构或损害该结构的事件可能导致神经系统功能改变。该项目的目标是研究新鉴定的轴突初始片段蛋白 UBXD4 对维持此处组装的蛋白质复合物的贡献，并了解神经元活动水平如何影响轴突初始片段结构和组装。