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: 10334494
• 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/10334494
• 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; 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： 生物分子凝聚物是动态的、无膜的隔室，可以时空调节 从基因转录到细胞应激反应的无数细胞功能。 分离（LLPS）作为这些冷凝物如何产生的生物物理机制越来越受到重视。 正确凝结物功能的关键是维持其动态和性能。 组装/拆卸过程，但对这些机制知之甚少。 疾病状态，其中凝结物可能经历液体到固体转变为细胞质内含物 含有蛋白质质量控​​制成分，是蛋白质病的特征，例如 我们已经鉴定出 UBQLN2，泛素介导蛋白的成员。 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 的形成。 体外凝结物，并被募集到应激颗粒，响应形成的细胞质凝结物 UBQLN2 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、泛素和多聚泛素、生物信号。 通过降解和自噬维持蛋白质稳态，驱动 UBQLN2 解体 这些观察结果对于相分离机制具有广泛的意义。 调节蛋白质质量控​​制系统的功能在这个项目中，我们的目标是识别分子。 以及驱动 UBQLN2 凝结组装和分解的细胞机制。 确定域-域相互作用如何促进或抑制 UBQLN2 的相分离 构建 UBQLN2 结构域删除和组合构建体的相图 这些结构域缺失将用于模拟 UBQLN2 的不同“状态”。 当特定域与绑定合作伙伴交互且无法为 LLPS 做出贡献时，我们将使用。 UBQLN2 疾病相关突变作为自然提供的库，用于阐明分子内和分子间如何 UBQLN2 相互作用促进或抑制冷凝物组装并改变冷凝物形态和 目标 2 量化 UBQLN2 的体外和哺乳动物细胞培养模型中的材料特性。 冷凝物受到 UBQLN2 与蛋白质质量控​​制成分结合的影响，包括 我们监测蛋白酶体受体、客户蛋白和不同类型的多聚泛素链。 重要的是，我们开发了重组的 UBQLN2。 凝聚模型来量化多泛素和多泛素化底物的参数 与 UBQLN2 合作分解凝结物这些研究将为确定奠定基础。 相分离的生理作用，因为它涉及通过泛素实现的蛋白质稳态 介导途径。