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。

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.