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

项目摘要/摘要： 生物分子凝聚体是动态的、无膜的隔间，在时空上调节 从基因转录到细胞应激反应的无数细胞功能。液-液相 分离(LLP)作为这些凝析油的生物物理机制越来越受到重视 集合。正确的凝析油功能的关键是保持它们的动力学和 组装/拆卸过程，但对这些机制知之甚少。提示来自 凝析油可能经历液-固转变为细胞质包裹体的疾病状态 含有蛋白质质量控制成分，是蛋白质病的特征，如 肌萎缩侧索硬化症。我们已经鉴定了UBQLN2，它是泛素介导的蛋白质的成员 质量控制系统，作为凝析油功能的贡献者。我们最近证明了UBQLN2形成 在体外凝聚，并被招募到应激颗粒，作为反应形成的细胞质凝聚物 来施加压力。UBQLN2的众多功能是通过与蛋白酶体亚基的相互作用来驱动的， 多泛素链和客户蛋白。泛素和多泛素，生物信号 通过降解和自噬维持蛋白质的动态平衡，驱动UBQLN2的拆解 在体外凝聚。这些观察结果对相分离机制有广泛的影响 规范蛋白质质量控制系统的功能。在这个项目中，我们的目标是识别分子 以及驱动UBQLN2凝析物组装和分解的细胞机制。目标1 确定结构域-结构域相互作用如何通过以下方式促进或抑制UBQLN2的相分离 从UBQLN2域删除和UBQLN2域删除的组合构建构造物的相图 与疾病相关的突变。这些结构域的删除将被用来模拟UBQLN2的不同状态 当特定的域与绑定合作伙伴接触并且无法为LLP做出贡献时。我们将使用 UBQLN2疾病相关突变作为自然提供的文库来阐明分子内和分子间如何 UBQLN2相互作用促进或抑制凝析油聚集并改变凝析油形态和 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UBQLN2如何 凝析油受UBQLN2与蛋白质质量控制组件的接触影响，包括 蛋白酶体受体、客户蛋白和不同类型的多泛素链。我们监控这些 体外效应和体内设计的突变体。重要的是，我们开发了一个重组的UBQLN2 凝聚模型量化多泛素和多泛素化底物的参数 与UBQLN2接合以分解冷凝液。这些研究将为确定 相分离在蛋白质动态平衡中的生理作用是通过泛素- 中介途径。

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.