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

UBQLN2介导的相变在生物分子凝聚体组装和分解中的作用和机制

• 批准号: 10117270
• 负责人: Carlos Antonio Castaneda
• 金额: $ 30万
• 依托单位: SYRACUSE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10117270
• 关键词: Affect; Age; Amyotrophic Lateral Sclerosis; Autophagocytosis; Behavior; Binding; Biological; Biology; Biophysical Process; Cell Culture Techniques; Cell physiology; Cells; Cellular Stress Response; Characteristics; Client; Cytoplasmic Inclusion; Degradation Pathway; Disease; Foundations; Genetic Transcription; Goals; Image; In Vitro; Lead; Length; Libraries; Link; Liquid substance; Maintenance; Mammalian Cell; Mediating; Microscopy; Modeling; Molecular; Monitor; Morphology; Mutation; NMR Spectroscopy; Nature; Neurodegenerative Disorders; Organelles; Pathway interactions; Phase; Phase Transition; Physiological; Polyubiquitin; Process; Property; Proteins; Quality Control; RNA-Binding Proteins; Regulation; Research; Resolution; Role; S Phase; Signal Transduction; Signaling Protein; Solid; Stress; System; Tertiary Protein Structure; Therapeutic; Time; Ubiquitin; aqueous; base; deletion library; design; experimental study; in vivo; intermolecular interaction; member; multicatalytic endopeptidase complex; mutant; physical process; protein TDP-43; protein function; proteostasis; receptor; receptor binding; reconstitution; recruit; response; spatiotemporal; stress granule

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.

项目摘要/摘要： 生物分子冷凝物是动态的无膜隔室，可在空间上调节A 从基因转录到细胞应激反应的无数细胞功能。液态液相 分离（LLP）越来越多地被视为这些凝结物的生物物理机制 集合。适当的冷凝物功能的关键是维护其动力学和 组装/拆卸过程，但对这些机制知之甚少。提示来自 疾病状态，凝结物可能会经历液体到固醇转变为细胞质内包含物 包含蛋白质质量控​​制成分，并且是蛋白质的特征 肌萎缩性侧硬化症。我们已经确定了UBQLN2，泛素介导的蛋白的成员 质量控制系统，作为冷凝水功能的贡献。我们最近表明UBQLN2形成 体外冷凝物，并募集到应力颗粒，细胞质冷凝物中形成的响应 压力。众多的UBQLN2函数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，生物学信号 通过降解和自噬来维持蛋白质稳态，驱动ubqln2驱动器 体外凝结。这些观察结果对相分离机制具有广泛的影响 调节蛋白质质量控​​制系统的功能。在这个项目中，我们旨在确定分子 以及驱动UBQLN2凝结组装和拆卸的细胞机制。目标1 确定域域相互作用如何促进或抑制UBQLN2通过 从UBQLN2域删除和 疾病连接的突变。这些域删除将用于模仿UBQLN2的不同“状态” 当特定领域与约束伙伴互动并且无法为LLP贡献时。我们将使用 UBQLN2疾病连接的突变作为自然提供的文库，以阐明分子内和分子间 UBQLN2相互作用促进或抑制冷凝水组装并改变冷凝水的形态和 体外和哺乳动物细胞培养模型的材料特性。 AIM 2量化UBQLN2的方式 冷凝水受UBQLN2与蛋白质质量控​​制组件的互动的影响，包括 蛋白酶体受体，客户蛋白和不同类型的多泛素链。我们监视这些 在体外和设计突变体的体外作用。重要的是，我们开发了一个重构的UBQLN2 凝结物模型，以量化多泛素和多泛素化底物的参数 与UBQLN2互动以拆卸冷凝水。这些研究将为确定 相位分离的物理作用与蛋白质稳态通过泛素 - 介导的途径。