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

职业：破译泛素蛋白液-液相分离的分子机制

基本信息

批准号：
1750462
负责人：
Carlos Castaneda
金额：
$ 81.13万
依托单位：
Syracuse University
依托单位国家：
美国
项目类别：
Continuing Grant
财政年份：
2018
资助国家：
美国
起止时间：
2018-05-01 至 2024-04-30
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1750462&HistoricalAwards=false
关键词：
CAREER Deciphering molecular mechanisms underlying

项目摘要

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.

活细胞可以将化学反应或其他功能分离到单独的隔室或细胞器中，其中大部分被脂质膜包围，脂质膜在细胞的其余部分和细胞器之间形成屏障。相反，无膜细胞器是通过大分子（如蛋白质）的液-液相分离（LLPS）形成的，所述大分子聚结以形成不被脂质膜包围的液体状液滴和细胞器。破译这些无膜细胞器的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。本计画将建立泛素蛋白作为模型系统，研究LLPS的组装与分解。泛素是调节细胞内蛋白质质量控制机制的蛋白质。该项目的更广泛影响将导致开发一种新的多尺度方法，使用核磁共振，小角散射，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/或代表性不足人口的STEM代表性，并为STEM研究生发展科学教学和沟通技能。为了实现这些目标，高中生将参加一年一度的小型研讨会，作为锡拉丘兹大学夏季科学研究所（SSI）的一部分。这些学生将实验物理参数（如温度）如何调节蛋白质的LLPS，其结果将被整合到项目的科学目标中。该研究项目的重点是阐明泛素蛋白质，特别是UBQLN 1，UBQLN 2和UBQLN 4的液-液相分离的分子决定因素。UBQLN是研究LLPS的相关模型系统，因为UBQLN 2中的内在无序区域和折叠结构域都调节其LLPS。该项目的目标是：（1）研究蛋白质寡聚化在促进UBQLN蛋白质相分离中的作用，使用缺失构建体，NMR光谱学和显微镜，以及（2）使用小角X射线和中子散射（SAXS，SANS）来观察蛋白质液滴内部，并通过计算建模来表征相分离和非相分离状态。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NSF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。