Rotation 1: Using engineered protein systems for studying LLPS in the cellular environment

第 1 轮：使用工程蛋白质系统研究细胞环境中的 LLPS

• 批准号: 2888268
• 金额: --
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2888268
• 关键词: Rotation; Using; engineered; protein; systems

BBSRC strategic theme: Bioscience for an integrated understanding of healthBackground: Biomolecular condensates (BCs) are discrete membraneless subcellular compartments that provide efficient and reversible spatiotemporal control of cellular processes in an energy-dependent manner. BCs facilitate a plethora of physiological processes due to their propensity to act as reaction crucibles by increasing the local concentrations of substrates and effectors such as enzymes, and their capacity to undergo dynamic exchange with the surrounding intracellular milieu. In contrast, in dysregulated phase separation (LLPS), BCs lose their dynamic nature such that there is progressive transformation from liquid-like states to gel-like and solid states. There is therefore increasing evidence that aberrant LLPS leads to pathological protein aggregates such as disease-related aggregates seen in neurodegenerative disorders. Recent evidence has therefore prompted the search for therapeutic interventions aimed at modulating LLPS and restoring normal physiological conditions.The overarching aim of the PhD project will be to apply designer LLPS-CTPRs to understand the molecular mechanisms by which neurodegenerative proteins behave in physiological LLPS environment and how protein mutations dysregulate the LLPS environment leading to irreversible aggregation. Aim 1: Use designer biomolecular condensates to understand how disease-related proteins behave in an LLPS environment.This will involve: a. in vitro characterisation of BCs formed from LLPS-CTPRs that can recruit a-synuclein and TDP-43 in condensate formation with the aim of determining the biophysical properties of droplets.b. in cellulo characterisation of protein droplet formation using SH-SY5Y cells overexpressing fluorescent-tagged a-synuclein and a TDP-43 tagged cell model of ALS developed by the Kumita lab.Aim 2: Determine how mutations in disease-related proteins accelerate transition from reversible LLPS to irreversible protein aggregation.In clonal cell lines that express a-synuclein and TDP-43 mutations I will repeat the same characterisation protocols to investigate changes in structural features within the LLPS/aggregate systems. This will allow for comparison of protein recruitment, and droplet formation between physiological and pathological states.Aim 3: Determine the therapeutic potential of using engineered proteins to enhance the degradation of disease-related proteins.Key information obtained from in vitro and in cellulo characterisation of droplet properties will direct reconciliation of protein recruitment with efficient targeted degradation. Once disease-related protein recruitment in LLPS formation is better understood, targeted degradation strategies being developed in the Kumita group can be utilised to determine if disease-related BCs can be targeted to the autophagy pathway. As aberrantly misfolded proteins are a pathological hallmark of neurodegenerative diseases, removal of these species through facilitated protein degradation may prove beneficial for therapeutic intervention. The results from this study will yield significant advances in understanding the underlying molecular mechanisms that link the beneficial LLPS formation with pathological amyloid formation. This will enable the development of therapeutic intervention strategies, such as targeted degradation of disease-related proteins.

背景：生物分子凝聚物（BCs）是离散的无膜亚细胞区室，以能量依赖的方式提供有效和可逆的细胞过程时空控制。由于BCs倾向于作为反应坩埚，通过增加底物和效应物（如酶）的局部浓度，以及它们与周围细胞内环境进行动态交换的能力，从而促进了大量的生理过程。相反，在失调相分离（LLPS）中，BCs失去了其动态性质，从而从类液体状态逐渐转变为凝胶状和固态。因此，越来越多的证据表明，异常LLPS导致病理性蛋白质聚集，如在神经退行性疾病中看到的疾病相关聚集。因此，最近的证据促使人们寻找旨在调节LLPS和恢复正常生理状况的治疗干预措施。博士项目的总体目标将是应用设计的LLPS- ctpr来了解神经退行性蛋白在生理LLPS环境中的行为的分子机制，以及蛋白质突变如何失调LLPS环境导致不可逆聚集。目的1：使用设计的生物分子凝聚物来了解疾病相关蛋白在LLPS环境中的行为。这将涉及：a.体外表征由llps - ctpr形成的bc，它可以在冷凝物形成中招募a-synuclein和TDP-43，目的是确定液滴的生物物理性质。使用过表达荧光标记的a-synuclein的SH-SY5Y细胞和Kumita实验室开发的TDP-43标记的ALS细胞模型来表征蛋白液滴形成的细胞特性。目的2：确定疾病相关蛋白的突变如何加速从可逆LLPS到不可逆蛋白聚集的转变。在表达a-synuclein和TDP-43突变的克隆细胞系中，我将重复相同的表征方案来研究LLPS/聚集体系统中结构特征的变化。这将允许比较蛋白质募集，以及生理和病理状态之间的液滴形成。目的3：确定利用工程蛋白增强疾病相关蛋白降解的治疗潜力。从体外和纤维素中获得的液滴特性的关键信息将指导蛋白质招募与有效靶向降解的协调。一旦对LLPS形成过程中与疾病相关的蛋白募集有了更好的了解，Kumita团队开发的靶向降解策略就可以用来确定与疾病相关的bc是否可以靶向自噬途径。由于异常错误折叠的蛋白质是神经退行性疾病的病理标志，通过促进蛋白质降解去除这些物种可能对治疗干预有益。这项研究的结果将在理解将有益的LLPS形成与病理性淀粉样蛋白形成联系起来的潜在分子机制方面取得重大进展。这将有助于制定治疗干预策略，例如靶向降解疾病相关蛋白质。