The Role and Mechanisms of UBQLN2-mediated Phase Transitions in the Assembly and Disassembly of Biomolecular Condensates

项目总结/摘要： 生物分子凝聚物是动态的，无膜的隔间，时空调节生物分子的凝聚物。 从基因转录到细胞应激反应的无数细胞功能。液-液相 分离（LLPS）越来越多地被认为是这些冷凝物 集合。正确冷凝功能的关键是保持其动力学， 组装/拆卸过程，但对这些机制知之甚少。提示来自 一种疾病状态，其中冷凝物可能经历液体到固体的转变，形成细胞质内含物 其含有蛋白质质量控制组分，并且是蛋白质病的特征， 肌萎缩侧索硬化症我们已经鉴定了UBQLN 2，它是泛素介导的蛋白质的一个成员， 质量控制系统，作为冷凝功能的贡献者。我们最近发现UBQLN 2形成于 在体外凝聚，并被募集到应激颗粒，细胞质凝聚物， 压力UBQLN 2的多种功能是通过与蛋白酶体亚基的相互作用来驱动的， 多聚泛素链和客户蛋白。泛蛋白和聚泛蛋白， 通过降解和自噬维持蛋白质稳态，驱动UBQLN 2的分解 体外凝聚。这些观察结果对于相分离机制如何 调节蛋白质质量控制系统的功能。在这个项目中，我们的目标是确定分子 以及驱动UBQLN 2凝聚体组装和分解的细胞机制。要求1 确定结构域-结构域相互作用如何促进或抑制UBQLN 2的相分离， 构建来自UBQLN 2结构域缺失和UBQLN 3结构域缺失的组合的构建体的相图。 疾病相关突变这些结构域缺失将用于模拟UBQLN 2的不同“状态 当特定的结构域与结合伴侣结合并且不能促进LLPS时。我们将使用 UBQLN 2疾病相关突变作为自然提供的文库，以阐明分子内和分子间 UBQLN 2相互作用促进或抑制冷凝物组装并改变冷凝物形态， 在体外和哺乳动物细胞培养模型中的材料特性。目标2量化UBQLN 2 缩合物受到UBQLN 2与蛋白质质量控制组分的接合的影响，包括 蛋白酶体受体、客户蛋白和不同类型的聚泛素链。我们监测这些 体外和体内设计的突变体的效果。重要的是，我们开发了重组的UBQLN 2 冷凝物模型，以量化多聚泛素和多聚泛素化底物 与UBQLN 2接合以分解冷凝物。这些研究将为确定 相分离的生理作用，因为它涉及蛋白质稳态通过泛素- 介导的路径。

PROJECT SUMMARY/ABSTRACT: Biomolecular condensates are dynamic, membraneless compartments that spatiotemporally regulate a myriad of cellular functions from gene transcription to cellular stress response. Liquid-liquid phase separation (LLPS) is increasingly appreciated as the biophysical mechanism for how these condensates assemble. Key to proper condensate function is the maintenance of their dynamics and assembly/disassembly processes, but little is known about these mechanisms. Hints are provided from disease states whereby condensates may undergo liquid-to-solid transitions into cytoplasmic inclusions that contain protein quality control components and are characteristic of proteinopathies such as amyotrophic lateral sclerosis. We have identified UBQLN2, a member of ubiquitin-mediated protein quality control systems, as a contributor to condensate function. We recently showed that UBQLN2 forms condensates in vitro, and is recruited to stress granules, cytoplasmic condensates that form in response to stress. The multitude of UBQLN2 functions are driven through interactions with proteasomal subunits, polyubiquitin chains, and client proteins. Ubiquitin and polyubiquitin, biological signals for the maintenance of protein homeostasis through degradation and autophagy, drive disassembly of UBQLN2 condensates in vitro. These observations have broad implications for how phase separation mechanisms regulate the function of protein quality control systems. In this project, we aim to identify the molecular and cellular mechanisms that drive how UBQLN2 condensates assemble and disassemble. Aim 1 determines how domain-domain interactions promote or inhibit phase separation of UBQLN2 via construction of phase diagrams for constructs from a combination of UBQLN2 domain deletion and disease-linked mutations. These domain deletions will be used to mimic the different “states” of UBQLN2 when specific domains are engaged with binding partners and unable to contribute to LLPS. We will use UBQLN2 disease-linked mutations as a nature-provided library to elucidate how intra- and intermolecular UBQLN2 interactions promote or inhibit condensate assembly and alter condensate morphology and material properties both in vitro and in mammalian cell culture models. Aim 2 quantifies how UBQLN2 condensates are affected by UBQLN2 engagement with protein quality control components, including proteasomal receptors, client proteins, and different types of polyubiquitin chains. We monitor these effects in vitro and with designed mutants in vivo. Importantly, we develop a reconstituted UBQLN2 condensate model to quantify the parameters of how polyubiquitin and polyubiquitinated substrates engage with UBQLN2 to disassemble condensates. These studies will lay the foundation for determining the physiological roles of phase separation as it pertains to protein homeostasis through ubiquitin- mediated pathways.