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（TDP-43）的焦油DNA结合蛋白是一种核酸结合蛋白，其片段具有在患有与年龄相关的神经退行性患者的大脑的病理淀粉样蛋白包含物中检测到疾病，包括阿尔茨海默氏病，肌萎缩性侧索硬化和额颞叶变性。这些片段包含一个称为低复杂性域（LCD）的TDP-43区域，该区域的氨基酸低多样性和形成淀粉样蛋白聚集体的承诺。 LCD还驱动TDP-43液态相分离（LLP），一种现象，其中蛋白质自缔合成可逆，动态，液滴状的现象阶段。 LLP是一个生理过程，介导了TDP-43的多个功能，包括其参与细胞应激反应，但已显示长期LLP促进病理聚集，强调需要在体内调节此过程的需求。 TDP-43 LCD的磷酸化已在阿尔茨海默氏病和其他年龄有关神经退行性疾病。尽管对于TDP-43，这种修改的作用在很大程度上仍然是出乎意料的，但它确实如此在类似的RNA结合蛋白（例如FUS）中影响LLP。确定是否可以调节LCD光疗 TDP-43 LLP，LCD的磷酸化变体，在已知的磷酸化位点具有Ser-> asp取代准备好了。使用光散射和显微镜研究变体的相位分离，并发现磷酸化取代以特定于位置的方式显着调制了LLP。 LCD变体 α-螺旋区域的磷酸化取代，这是蛋白质蛋白接触的蛋白质的一部分，显示出降低的LLP-propity；相反，c-中具有磷酸化取代的LCD变体已知在疾病中被磷酸化的末端区域（CTR）显示出增强的LLPS-Protmisty。为了阐明该LLP的特定地点调制背后的机制，我们提出了一个三管齐下的使用核磁共振（NMR）光谱，电子顺磁共振（EPR）的方法光谱和荧光共振能量转移（FRET）。 NMR将进行确定本地磷酸化α-螺旋变体的二级结构。由于已知α-螺旋区域会改变寡聚化，EPR将同时使用，以查看α螺旋磷酸化取代是否改变了该变体的寡聚行为。初步数据表明CTR变体具有较高的LLP- 倾向是由于磷酸化取代产生的分子间静电相互作用在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的存在保留的相互作用和探测有助于它。此外，光漂白后的荧光恢复（FRAP）将用于确定这些磷酸化取代如何改变 TDP-43 LCD液滴。这些方法将提供有关磷酸化如何位点的机械见解。特别是直接的TDP-43 LLP，并有助于神经退行性病理，例如阿尔茨海默氏病。