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

项目摘要 43 kDa的TAR DNA结合蛋白（TDP-43）是一种核酸结合蛋白，其片段具有 在与年龄相关的神经退行性疾病患者大脑中的病理性淀粉样蛋白包涵体中检测到 这些疾病包括阿尔茨海默病、肌萎缩侧索硬化和额颞叶变性。 这些片段含有TDP-43的一个称为低复杂性结构域（LCD）的区域，其具有低氨基酸残基。 多样性和形成淀粉样蛋白聚集体的倾向。LCD还驱动TDP-43液-液相 分离（LLPS），一种蛋白质自缔合成可逆的、动态的、液滴状的蛋白质分子的现象。 相位LLPS是介导TDP-43的几种功能的生理过程，包括其参与 细胞应激反应，但已显示延长的LLPS促进病理性聚集， 强调需要了解这个过程是如何在体内调节。 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已有充分的记录。 神经退行性疾病虽然这种修饰的作用在TDP-43中仍然很大程度上未被探索，但它确实 影响类似RNA结合蛋白如FUS中的LLPS。为了确定LCD磷酸化是否可以调节 TDP-43 LLPS，在已知磷酸化位点具有Ser->Asp取代的LCD的磷酸化模拟变体 制备了使用光散射和显微镜研究了变体的相分离，发现 磷酸模拟物取代以位点特异性方式显著调节LLPS。LCD变体， 在α-螺旋区域中的磷酸模拟物取代，α-螺旋区域是参与蛋白质-蛋白质接触的蛋白质的一部分， 显示出降低的LLPS倾向;相反，在C- 已知在疾病中磷酸化的末端区（CTR）显示增强的LLPS倾向。 为了阐明LLPS位点特异性调节背后的机制，我们提出了三管齐下的方法。 方法使用核磁共振（NMR）光谱，电子顺磁共振（EPR） 光谱和荧光共振能量转移（FRET）。NMR将用于确定局部 拟磷酸化α-螺旋变体的二级结构。由于已知α-螺旋区会被 在寡聚化中，EPR将同时用于观察α-螺旋磷酸化模拟物取代是否改变了寡聚化， 该变体的寡聚化行为。初步数据表明CTR变体具有更高的LLPS- 由于磷酸化模拟物取代产生的分子间静电相互作用的倾向 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 并研究了光漂白后荧光的恢复 （FRAP）将被用来确定这些磷酸模拟物取代如何改变材料的性质。 TDP-43 LCD液滴。这些方法将提供关于磷酸化如何定位的机制见解- 特异性引导TDP-43 LLPS并导致神经退行性病变如阿尔茨海默病。

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.