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Phosphorylation as a site-specific regulatory mechanism of the liquid-liquid phase separation of TDP-43 low complexity domain.

磷酸化作为 TDP-43 低复杂性结构域液-液相分离的位点特异性调节机制。

基本信息

批准号：
10620157
负责人：
Raza Haider
金额：
$ 5.27万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-05-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10620157
关键词：
Affect Aging Alzheimer like pathology Alzheimer&apos s Disease Alzheimer&apos s disease patient Amino Acids Amyloid Amyotrophic Lateral Sclerosis Antibodies Arginine Asparagine Aspartic Acid Behavior Brain C-terminal Cellular Stress Charge Complex Coupled Cytoplasmic Granules Data Deposition Detection Development Disease Electron Spin Resonance Spectroscopy Electrostatics Fluorescence Recovery After Photobleaching Fluorescence Resonance Energy Transfer Frontotemporal Lobar Degenerations Gel Immobilization In Vitro Knowledge Label Learning Liquid substance Mediating Membrane Messenger RNA Methods Microscopy Modeling Modification Molecular Monitor Mutate Mutation N-terminal NMR Spectroscopy Nerve Degeneration Neurodegenerative Disorders Nuclear Magnetic Resonance Organelles Pathologic Pathology Patients Phase Phosphorylation Phosphorylation Site Phosphotransferases Physiological Physiological Processes Play Post-Translational Protein Processing Process Property Proteins RNA RNA-Binding Proteins Resolution Role Sampling Scheme Sepharose Serine Signal Transduction Site Sodium Chloride Spectrum Analysis Spin Labels Stress Structure TDP-43 aggregation Techniques Testing Therapeutic Time Translations Variant Western Blotting age related age related neurodegeneration alpha helix expectation experimental study fluorophore frontotemporal lobar dementia amyotrophic lateral sclerosis gel electrophoresis in vivo insight intermolecular interaction light scattering mimetics monomer nucleic acid binding protein protein TDP-43 self assembly stress granule virtual

项目摘要

Project Summary The TAR DNA-binding protein of 43 kDa (TDP-43) is a nucleic-acid binding protein whose fragments have been detected in pathological amyloid inclusions in the brains of patients with age-related neurodegenerative disorders, including Alzheimer’s disease, amyotrophic lateral sclerosis, and frontotemporal lobar degeneration. These fragments contain a region of TDP-43 called the low complexity domain (LCD), which has low amino acid diversity and a propensity to form amyloid aggregates. The LCD also drives TDP-43 liquid-liquid phase separation (LLPS), a phenomenon in which the protein self-associates into a reversible, dynamic, droplet-like phase. LLPS is a physiologic process that mediates several functions of TDP-43, including its involvement in the cellular stress response, but prolonged LLPS has been shown to promote pathological aggregation, underscoring the need to understand how this process is regulated in vivo. Phosphorylation of TDP-43 LCD has been well-documented in Alzheimer’s disease and other age-related neurodegenerative disorders. Though the role of this modification remains largely unexplored for TDP-43, it does influence LLPS in similar RNA-binding proteins such as FUS. To determine if LCD phosphorylation can regulate TDP-43 LLPS, phosphomimetic variants of the LCD with Ser->Asp substitutions at known phosphorylation sites were prepared. Phase separation of variants was studied using light-scattering and microscopy, and it was found the phosphomimetic substitutions dramatically modulated LLPS in a site-specific manner. LCD variants with phosphomimetic substitutions in the α-helical region, a part of the protein involved in protein-protein contacts, displayed reduced LLPS-propensity; conversely, LCD variants with phosphomimetic substitutions in the C- terminal region (CTR), which is known to be phosphorylated in disease, displayed enhanced LLPS-propensity. To elucidate the mechanisms behind this site-specific modulation of LLPS, we propose a three-pronged approach using nuclear magnetic resonance (NMR) spectroscopy, electron paramagnetic resonance (EPR) spectroscopy and fluorescence resonance energy transfer (FRET). NMR will be employed to determine local secondary structure of the phosphomimetic α-helical variant. As the α-helical region is known to be altered by oligomerization, EPR will be used concurrently to see if the α-helical phosphomimetic substitution changes the oligomerization behavior of this variant. Preliminary data suggest the CTR variants have higher LLPS- propensities because of an intermolecular electrostatic interaction created by the phosphomimetic substitution at the CTR. Our second aim will thus use FRET to verify the presence of this hypothesized intermolecular interaction and probe which residues contribute to it. Furthermore, fluorescence recovery after photobleaching (FRAP) will be employed to determine how these phosphomimetic substitutions alter the material properties of TDP-43 LCD droplets. These approaches will provide mechanistic insights into how phosphorylation may site- specifically direct TDP-43 LLPS and contribute to neurodegenerative pathologies like Alzheimer’s disease.

项目摘要 43 kDa的TAR DNA结合蛋白(TDP-43)是一种核酸结合蛋白，其片段有在年龄相关性神经退行性变患者脑组织中检测到病理性淀粉样包涵体疾病，包括阿尔茨海默病、肌萎缩侧索硬化症和额颞叶变性。这些片段包含TDP-43的一个区域，称为低复杂结构域(LCD)，它具有低氨基酸多样性和形成淀粉样聚集体的倾向。LCD还驱动TDP-43液-液相分离(LLP)，一种蛋白质自我结合成可逆的、动态的、液滴状的现象相位。LLP是一种生理过程，它调节TDP-43的几个功能，包括它参与细胞应激反应，但延长LLP已被证明促进病理性聚集，强调有必要了解这一过程在体内是如何调控的。 TDP-43 LCD的磷酸化在阿尔茨海默病和其他与年龄相关的疾病中得到了很好的证明神经退行性疾病。尽管这种修饰对TDP-43的作用在很大程度上仍未被探索，但它确实如此影响类似的RNA结合蛋白中的LLP，如FUS。确定LCD磷酸化是否可以调节 TDP-43 LLP，LCD的仿磷变体，在已知的磷酸化位点上具有Ser-&Gt；Asp取代已经做好了准备。用光散射和显微镜研究了异构体的相分离，发现仿磷取代以一种位点特异性的方式显著地调节LLP。LCD变种，带 α-螺旋区中的仿磷取代，这是参与蛋白质-蛋白质接触的蛋白质的一部分，表现出降低的LLP倾向；相反，在C- 已知在疾病中被磷酸化的末端区域(CTR)显示出增强的LLPS倾向。为了阐明这种位点特异性调节LLP的机制，我们提出了一个三管齐下的机制使用核磁共振波谱、电子顺磁共振(EPR)的方法光谱和荧光共振能量转移(FRET)。将使用核磁共振来确定局部仿磷α-螺旋变异体的二级结构。因为已知α-螺旋区域由齐聚，EPR将同时用于观察α-螺旋仿磷取代是否改变该变体的寡聚行为。初步数据显示，CTR变种的LLP更高- 由于仿磷取代所产生的分子间静电相互作用而产生的倾向在CTR。因此，我们的第二个目标将使用FRET来验证这种假设的分子间的存在相互作用，并探索哪些残基对其有贡献。此外，光漂白后的荧光恢复 (FRAP)将被用来确定这些仿磷取代如何改变 TDP-43液晶屏液滴。这些方法将提供对磷酸化如何定位的机械性见解- 特别是指导TDP-43 LLP，并导致阿尔茨海默病等神经退行性病变。