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.

项目概要 43 kDa 的 TAR DNA 结合蛋白 (TDP-43) 是一种核酸结合蛋白，其片段具有在与年龄相关的神经退行性疾病患者的大脑中检测到病理性淀粉样蛋白内含物疾病，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，称为低复杂性结构域 (LCD)，其氨基酸含量较低 LCD 还驱动 TDP-43 液-液相。分离（LLPS），一种蛋白质自结合成可逆、动态、液滴状的现象 LLPS 是介导 TDP-43 多种功能的生理过程，包括参与 TDP-43 的功能。细胞应激反应，但延长的 LLPS 已被证明会促进病理聚集，强调需要了解这个过程在体内是如何调节的。 TDP-43 LCD 的磷酸化已在阿尔茨海默病和其他与年龄相关的疾病中得到充分记录尽管这种修饰对于 TDP-43 的作用在很大程度上尚未被探索，但它确实如此。影响 FUS 等类似 RNA 结合蛋白中的 LLPS 以确定 LCD 磷酸化是否可以调节。 TDP-43 LLPS，LCD 的磷酸模拟变体，在已知磷酸化位点具有 Ser->Asp 取代使用光散射和显微镜研究了变体的相分离，并发现了这一点。磷模拟取代以位点特异性方式显着调节 LLPS 的 LCD 变体。 α-螺旋区域中的磷模拟取代，α-螺旋区域是参与蛋白质-蛋白质接触的蛋白质的一部分，相反，在 C- 中具有磷模拟取代的 LCD 变体表现出降低的 LLPS 倾向；已知在疾病中被磷酸化的末端区域（CTR）表现出增强的 LLPS 倾向。为了阐明 LLPS 的位点特异性调节背后的机制，我们提出了三管齐下的方法使用核磁共振 (NMR) 光谱、电子顺磁共振 (EPR) 的方法光谱学和荧光共振能量转移（FRET）将用于确定局部。磷模拟α-螺旋变体的二级结构已知为α-螺旋区域。寡聚化，同时使用 EPR 来观察 α-螺旋拟磷取代是否改变了该变体的寡聚行为表明 CTR 变体具有更高的 LLPS-。由于拟磷取代产生的分子间静电相互作用而产生的倾向因此，我们的第二个目标是使用 FRET 来验证这种轻敲分子间的存在。相互作用和探针对其有贡献此外，光漂白后的荧光恢复。（FRAP）将用于确定这些磷模拟取代如何改变材料特性 TDP-43 LCD 液滴将提供有关磷酸化如何定位的机制见解。专门指导 TDP-43 LLPS 并导致阿尔茨海默病等神经退行性疾病。