CAREER: Deciphering molecular mechanisms underlying liquid-liquid phase separation of ubiquilin proteins

活细胞可以将化学反应或其他功能分离到单独的室室或细胞器中，其中大多数被脂质膜包围，脂质膜在细胞其余部分和细胞器之间形成屏障。相反，无膜细胞器是由大分子（如蛋白质）的液-液相分离（LLPS）形成的，大分子（如蛋白质）结合形成液体状液滴和不被脂质膜包围的细胞器。破译控制这些无膜细胞器组装和拆卸的生物物理规则对于理解它们的细胞功能是必要的。本项目将建立泛素蛋白作为研究LLPS组装和拆卸的模型系统。泛素是调节细胞内蛋白质质量控制机制的蛋白质。该项目的广泛影响将导致一种新的多尺度方法的发展，该方法使用核磁共振、小角度散射、显微镜和计算建模来研究LLPS，然后可以扩展到其他分子系统。此外，该教育计划旨在提高高中生的科学素养，扩大经济弱势和/或代表性不足人群在STEM领域的代表性，并培养STEM研究生的科学教学和沟通技巧。为了实现这些目标，高中生将参加雪城大学夏季科学研究所（SSI）的年度迷你研讨会。这些学生将对物理参数（如温度）如何调节蛋白质的LLPS进行实验，其结果将纳入项目的科学目标。本研究项目的重点是阐明UBQLN1、UBQLN2和UBQLN4等泛素蛋白液液相分离的分子决定因素。由于UBQLN2中的本序无序区和折叠结构域都可以调节其LLPS，因此UBQLN2是研究LLPS的一个相关模型系统。该项目的目标是：(1)利用缺失构建体、核磁共振波谱和显微镜研究蛋白质寡聚化在促进UBQLN蛋白质相分离中的作用；(2)利用小角度x射线和中子散射（SAXS， SANS）观察蛋白质液滴内部，并通过计算建模对相分离和非相分离状态进行结构表征。该项目的执行将直接有助于PI建立LLPS综合模型的长期目标。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Living cells can separate chemical reactions or other functions into separate compartments, or organelles, most of which are surrounded by lipid membranes that form a barrier between the rest of the cell and the organelle. Membrane-less organelles, instead, are formed by liquid-liquid phase separation (LLPS) of macromolecules (like proteins) that coalesce to form liquid-like droplets and organelles that are not surrounded by lipid membranes. Deciphering the biophysical rules governing the assembly and disassembly of these membrane-less organelles is necessary to understanding their cellular functions. This project will establish ubiquilin proteins as a model system by which the LLPS assembly and disassembly will be studied. Ubiquilins are proteins that regulate protein quality control mechanisms inside cells. The broader impact of this project will lead to the development of a new multiscale approach using nuclear magnetic resonance, small angle scattering, microscopy and computational modeling to study LLPS that can then be extended to other molecular systems. Additionally, the education plan aims to improve science literacy among high school students, broaden STEM representation from economically-disadvantaged and/or underrepresented populations, and develop science teaching and communication skills for STEM graduate students. To accomplish these goals, high school students will participate in a yearly mini-workshop as part of the Summer Science Institute (SSI) at Syracuse University. These students will experiment on how physical parameters (e.g. temperature) modulate LLPS of proteins, the results of which will be integrated into the project's scientific goals.The research project is focused on elucidating the molecular determinants of the liquid-liquid phase separation of ubiquilin proteins, specifically UBQLN1, UBQLN2 and UBQLN4. UBQLNs are a relevant model system for studying LLPS because both intrinsically-disordered regions and folded domains in UBQLN2 modulate its LLPS. The project's goals are to: (1) investigate the role of protein oligomerization in promoting phase separation of UBQLN proteins using deletion constructs, NMR spectroscopy, and microscopy, and (2) use small-angle X-ray and neutron scattering (SAXS, SANS) to look inside protein droplets, and structurally characterize the phase-separated and non-phase separated states with computational modeling. Execution of this project will directly contribute to the PI's long-term goal of building a comprehensive model of LLPS.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.