ALS相关蛋白低复杂度结构域多肽自组装和相分离的分子模拟研究

低复杂度结构域（low-complexity domain：LCD）在TDP-43和FUS等肌萎缩侧索硬化症（ALS）等疾病相关蛋白中普遍存在，这些ALS相关蛋白及其低复杂度结构域均能通过液-液相分离（liquid-liquid phase separation：LLPS）形成动态可逆的具有液体扩散融合特性的凝聚体，该凝聚体随时间会失去其动态性，转变为不可逆的固态淀粉样纤维（对应液-固相变，liquid-solid phase separation：LSPS）。ALS相关蛋白的液-液相分离和液-固相变与细胞内重要的生理和病理过程密切相关，ALS相关氨基酸突变和磷酸化等翻译后对蛋白质的液-液和液-固相变具有调控作用。最新研究表明，低复杂度结构域多肽（称为LCD多肽）对整个蛋白和低复杂度结构域的液-液相分离至关重要。阐明ALS相关蛋白液-液和液-固相变的微观机制，对理解疾病发生机理和开发治疗策略具有极为重要的科学意义。由于ALS相关蛋白及其低复杂度结构域至少由上百个氨基酸组成，从原子水平直接模拟相分离过程中大量低复杂度结构域分子间相互作用仍具有很大的挑战性。本项目采用全原子与粗粒化模型相结合的分子动力学模拟方法，研究TDP-43等ALS相关蛋白中，具有可逆组装和不可逆组装特性、且氨基酸序列明显短于低复杂度结构域的LCD多肽的自组装以及相行为，探索ALS相关突变和磷酸化等翻译后修饰对组装动力学和热力学的影响，构建具有相分离能力的低复杂度结构域多肽及其ALS突变体的相图，揭示低复杂度结构域多肽发生可逆组装、不可逆组装以及相分离的本质和规律。本项目的研究将为阐明TDP-43等ALS相关蛋白的液-液和液-固相变的微观机制奠定基础、为理解其生理和病理机理提供线索，进而丰富生物大分子相变和相分离的理论体系。

Low complexity domains (LCD) are commonly found in ALS-associated proteins (such as TDP-43 and FUS). These ALS-associated proteins and their low complexity domains can form reversible liquid-like droplets through liquid-liquid phase separation (LLPS), and the maturation of phase-separated liquid droplets can lead to the formation of irreversible solid-like amyloid fibrils via liquid-to-solid phase transition (LSPS). The liquid-liquid phase separation of these ALS-associated proteins are related to important physiological and pathological processes in the cell. ALS-associated mutations and posttranslational modifications (such as phosphorylation) can modulate the liquid-liquid phase separation of LCDs and the ALS-associated proteins. Recent experimental studies demonstrated that peptides from low complexity domains (these peptides are denoted as LCD peptides) are crucial to the liquid-liquid phase separation of the full-length LCD and of the whole protein. Deciphering the microscopic mechanism of LLPS and LSPS of ALS-associated proteins is fundamentally important for understanding the pathogenesis of ALS-related diseases and the development of therapeutic strategies. As ALS-associated proteins and their LCDs consist of at least a hundred amino acid residues, it is quite challenging to simulate directly their phase separation and phase transition at atomic level of details. In this proposal, we utilize a combined all-atom and coarse-grained molecular dynamics simulation method to investigate the formation dynamics and thermodynamics of reversible/irreversible assemblies and phase behaviors of LCD peptides from different ALS-proteins. We also explore the effect of ALS-related mutations and phosphorylation on the self-assembly dynamics and phase behavior of LCD peptides. The coexistence phase diagram of LCD peptides and their ALS-related mutants will be constructed. Our aim is to reveal the physical determinants that drive LCD peptides to form reversible or irreversible assemblies and to undergo phase separation and phase transition. The results will pave the way for deep understanding the physical mechanisms of liquid-liquid phase separation and liquid-to-solid phase transition of the ALS-related proteins, and provide new clues for understanding their physiological and pathological processes, thus enrich the phase separation and phase transition theory of biomolecules.