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

BBSRC战略主题：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时空控制细胞过程。由于它们倾向于通过增加底物和效应物（如酶）的局部浓度来充当反应坩埚，以及它们与周围细胞内环境进行动态交换的能力，因此BC促进过多的生理过程。相反，在失调相分离（LLPS）中，BC失去其动态性质，使得存在从液体样状态到凝胶样和固体状态的渐进转变。因此，越来越多的证据表明，异常LLPS导致病理性蛋白质聚集体，例如在神经退行性疾病中观察到的疾病相关聚集体。因此，最近的证据促使寻找治疗干预措施，旨在调节LLPS和恢复正常的生理conditions.The博士项目的总体目标将是应用设计师LLPS-CTPR来了解神经退行性蛋白在生理LLPS环境中表现的分子机制，以及蛋白质突变如何失调LLPS环境，导致不可逆的聚集。目的1：使用设计的生物分子缩合物来了解疾病相关蛋白质在LLPS环境中的行为。由LLPS-CTPR形成的BC的体外表征，所述LLPS-CTPR可以在冷凝物形成中募集α-突触核蛋白和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α-突触核蛋白的SH-SY 5 Y细胞和由Kumita实验室开发的ALS的TDP-43标记的细胞模型对蛋白液滴形成进行细胞内表征。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。我将重复相同的表征方案，以研究LLPS/聚集体系统内结构特征的变化。这将允许比较蛋白质的招聘，和液滴形成之间的生理和病理states.Aim 3：确定使用工程蛋白质的治疗潜力，以提高疾病相关的proteins.Key信息的降解液滴的性能在体外和在cellulo表征将直接和解的蛋白质招聘与有效的靶向降解。一旦更好地理解LLPS形成中的疾病相关蛋白质募集，Kumita小组正在开发的靶向降解策略可以用于确定疾病相关的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学标志，通过促进蛋白质降解去除这些物质可能有利于治疗干预。 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。这将有助于开发治疗干预策略，例如靶向降解疾病相关蛋白。

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.