Defining Mechanisms of TDP-43 Ubiquitination for Quality Control and Aggregation

定义 TDP-43 泛素化的质量控制和聚集机制

基本信息

批准号：
8127409
负责人：
Eric Bernard Dammer
金额：
$ 5.13万
依托单位：
EMORY UNIVERSITY
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：
2011-09-30 至 2013-09-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8127409
关键词：
Accounting Adult Affect Age Age of Onset Aging Alzheimer&apos s Disease American Amyotrophic Lateral Sclerosis Autophagocytosis Autophagosome Autopsy Biochemical Biological Assay Biological Markers Cell Line Cells Characteristics Clinical Coupled DNA Damage DNA-Binding Proteins Data Degradation Pathway Dementia Deposition Disease Disease Progression Drug Delivery Systems Drug Metabolic Detoxication Engineering Environmental Risk Factor Etiology Failure Familial Amyotrophic Lateral Sclerosis Frontotemporal Dementia Frontotemporal Lobar Degenerations Future Half-Life Human Human Cell Line Immunohistochemistry In Vitro Kinetics Length Life Ligation Link Lysosomes Mass Spectrum Analysis Measurement Measures Mediating Mentors Methods Modeling Molecular Mutagenesis Mutation Nerve Degeneration Neurodegenerative Disorders Neuroglia Neurons Onset of illness Parkinson Disease Pathogenesis Pathology Persons Physiological Play Polyubiquitin Presenile Dementia Proteasome Inhibition Proteins Proteomics Publishing Quality Control RNA Research Role Signal Transduction Site Staining method Stains System Testing Tissue Model Tissues Ubiquitin Ubiquitination age related brain tissue disease characteristic disorder subtype enzyme activity frontal lobe frontotemporal lobar dementia-amyotrophic lateral sclerosis improved multicatalytic endopeptidase complex novel overexpression prophylactic protein TDP-43 protein aggregate protein aggregation protein complex protein degradation research study software development therapeutic target ubiquitin ligase ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): Neurodegeneration in frontotemporal lobar dementia with ubiquitinated inclusions (FTLD-U), amyotrophic lateral sclerosis (ALS), and other delayed-onset diseases associated with degeneration of aging neurons involves a deficit in the degradation of the TAR DNA binding protein (TDP-43). This deficit is apparent through the characteristic pathology of a cross-section of neurodegenerative diseases, i.e., accumulation and aggregation of protein inclusions positive for TDP-43 and ubiquitin (Ub) in ALS, FTLD-U, and a large fraction of Alzheimer's Disease (AD) and Parkinson's Disease (PD) cases. Ub is a small protein conjugated singly or in polymeric chains to substrate proteins like TDP-43 that has the capacity to alter substrate fate, e.g. degradation, (in) activation, or relocation out of specific cellular compartments or protein complexes. The proposed research identifies the determinants of TDP-43 degradation, and determines the role of K63-linked polyubiquitin in rerouting of TDP-43 from degradation pathways to disease-characteristic aggregates. Half-life studies in cortical neurons and glia and in human cell lines measure the contributions of cellular alternatives for protein degradation, the proteasome and lysosome, to the degradation kinetics of endogenously expressed TDP-43. In addition, K63-linked Ub chains on TDP-43 and their association with the cell's quality control and aggregation-control machinery are identified by experiments in a cell line engineered to express modified Ub. Components of cellular quality control machinery have been shown to engage TDP-43 in neurodegenerative tissue and in models of neurodegeneration, particularly those which test the effects of insufficient proteasome or lysosome activity on substrates such as TDP-43. This insufficiency may passively develop in neurons during the course of aging or may develop due to environmental factors that affect sporadic neurodegeneration. Significant quantitative differences in interactions between TDP-43 and the Ub-dependent proteasome, lysosome, and quality control systems of cells in disease tissue will be identified. Interactions relevant to disease progression (biomarkers) or etiology (therapeutic targets) are to be confirmed by immunohistochemical staining in postmortem tissue from FTLD-U and ALS neurodegenerative disease. Finally, biochemical assays will be employed to identify specific therapeutic target(s) that deposit K63-linked Ub chains onto TDP-43; K63 Ub linkages significantly increase in neurodegenerative disease and published models of TDP-43 proteinopathy. ALS-specific mutations of TDP-43 will be tested for changes in the propensity of the protein to interact with identified interaction partners, particularly the Ub ligase that deposits K63-linked Ub chains onto TDP-43. The proposed measurements of alternative Ub chains and identification of novel disease-related proteins will rely on mass spectrometry methods and software developed by the PI and his mentors at the Emory proteomics facility and Center for Neurodegenerative Disease, where the proposed research takes place. PUBLIC HEALTH RELEVANCE: Frontotemporal lobar dementia (FTLD) is the leading cause of adult-onset dementia in persons under the age of 60, affecting 250,000 Americans each year and accounting for about 20 percent of pre-senile dementia cases. In FTLD and amyotrophic lateral sclerosis (ALS) subtypes, the same proteins aggregate and accumulate by unknown mechanisms, instead of being degraded within certain neurons. The proposed research examines FTLD and ALS brain tissue and models in living cells relevant to disease via proteomic, and immunochemical methods to identify mechanisms of disease progression or initiation by identifying the mechanisms that affect insufficient degradation and accumulation of the disease-associated proteins thought to be an underlying cause of FTLD-U and ALS, and potentially a cross-section of other late-onset neurodegenerative diseases including Alzheimer and Parkinson diseases. Novel disease subtypes, markers, and specific protein interactions and enzyme activities relevant as drug targets will also be discovered.

描述（由申请人提供）：额颞Lobar痴呆中的神经退行性变性（FTLD-U）（FTLD-U），肌萎缩性侧面硬化症（ALS）和其他延迟性疾病与Aging Neuroons退化有关结合蛋白（TDP-43）。通过神经退行性疾病的横截面的特征病理学，即蛋白质夹杂物在ALS，FTLD-U中的TDP-43和泛素（UB）阳性的蛋白质夹杂物以及大量的阿尔茨海默氏病（ALZHEIMER氏病）中的蛋白质夹杂物（UB）（UB）阳性（UB）（UB）（UB）（UB）（UB）（UB）（UB）（UB）（UB）（UB）（UB）（UB）（Alzheimer病（Alzheimer氏病）（UB）（UB）（UB）（UB）（UB）（UB） AD）和帕金森氏病（PD）病例。 UB是一种单独或聚合物链中与TDP-43（例如TDP-43）的小蛋白质结合的小蛋白质，具有改变底物命运的能力，例如从特定的细胞室或蛋白质复合物中降解，（IN）激活或迁移。拟议的研究确定了TDP-43降解的决定因素，并确定了K63连接的多泛素在从降解途径中重新布置TDP-43在疾病特征聚合中的作用。在皮质神经元和神经胶质以及人类细胞系中的半衰期研究测量了细胞替代方案对蛋白质降解，蛋白酶体和溶酶体的贡献，对内源表达的TDP-43的降解动力学。此外，通过TDP-43上的K63连接的UB链及其与细胞的质量控制和聚集控制机械的关联，可以通过工程设计以表达经过修改的UB的细胞系中的实验来鉴定。细胞质量控制机制的成分已被证明可以在神经退行性组织和神经变性模型中参与TDP-43，尤其是那些测试蛋白酶体或溶酶体活性不足对TDP-43等底物的影响的组件。这种功能不全可能会在衰老过程中在神经元中被动发展，或者由于影响零星神经变性的环境因素而发展。将确定TDP-43与UB依赖性蛋白酶体，溶酶体和疾病组织中细胞的质量控制系统之间的相互作用之间的显着定量差异。与疾病进展有关（生物标志物）或病因（治疗靶标）相关的相互作用，可以通过FTLD-U和ALS神经退行性疾病的验尸组织中的免疫组织化学染色来证实。最后，将使用生化测定来确定将K63连接UB链沉积在TDP-43上的特定治疗靶标。 K63 UB链接显着增加了神经退行性疾病和TDP-43蛋白质病的模型。 TDP-43的ALS特异性突变将测试是否与已识别相互作用伙伴相互作用的蛋白质倾向的变化，尤其是将K63连接UB链沉积在TDP-43上的UB连接酶。提出的对替代UB链的测量以及与疾病相关蛋白质的新型UB链的鉴定将依赖于PI及其指导者在Emory蛋白质组学设施和神经退行性疾病中心开发的质谱法和软件，并进行了拟议的研究。公共卫生相关性：额叶小叶痴呆症（FTLD）是60岁以下的成人痴呆症的主要原因，每年影响25万名美国人，约占新年痴呆症前痴呆症病例的20％。在FTLD和肌萎缩性侧索硬化（ALS）亚型中，相同的蛋白质聚集并通过未知机制积累，而不是在某些神经元中降解。拟议的研究研究了通过蛋白质组学与疾病相关的生物细胞中的FTLD和ALS脑组织以及模型，以及通过鉴定影响疾病不足的降解和疾病相关蛋白质积累的机制来鉴定疾病进展或起始机制的免疫化学方法FTLD-U和ALS的根本原因，也可能是其他晚发神经退行性疾病的横截面，包括阿尔茨海默氏症和帕金森氏病。还将发现新型疾病亚型，标记和特定蛋白质相互作用以及与药物靶标相关的酶活性。