Chemically Probing and Regulating Misfolding and Aggregation of Intrinsically Disordered Proteins in Membraneless Organelles

化学探测和调节无膜细胞器中内在无序蛋白质的错误折叠和聚集

• 批准号: 9797181
• 负责人: Xin Zhang
• 金额: $ 39.22万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9797181
• 关键词: Biochemical; Biological Assay; Cell physiology; Cells; Cellular Stress; Chemicals; Data; Detection; Diffusion; Discipline; Disease; Fluorescence; Goals; In Vitro; Knowledge; Liquid substance; Literature; Location; Methods; Monitor; Morphology; Names; Neurodegenerative Disorders; Neurons; Organelles; Phase; Physiological; Play; Prion Diseases; Process; Proteins; RNA; RNA Recognition Motif; RNA-Binding Proteins; Research; Resolution; Signal Transduction; Sugar Phosphates; Technology; Testing; Tube; base; cellular pathology; fluorescence imaging; human disease; imaging modality; mutant; novel; prevent; prion-like; protein aggregate; protein aggregation; small molecule; stress granule

PROJECT SUMMARY Membraneless organelles have important functions in cellular physiology and pathology. Recent studies show that these organelles are formed through liquid-liquid phase separation of intrinsically disordered proteins (IDPs) and RNA molecules. IDPs phase separate into liquid droplets in test tubes and form P bodies or stress granules in stressed cells. Both mutant and wild type forms of several IDPs are found aggregated in neurons and associated with neurodegenerative disorders. However, very little is known about how IDPs misfold and aggregate in these organelles and how this process can be regulated. Lack of this knowledge is attributed to the current method that is used to monitor membraneless organelles in live cells: this process is visualized through imaging fluorescent protein-tagged IDPs to analyze changes of their location and diffusion rate before and after organelle formation. Nonetheless, this method does not reveal whether IDPs misfold or aggregate within the organelle, because the morphology remains unchanged before and after IDPs aggregation. To overcome this challenge, the PI has developed a novel imaging method, hereinafter named AggTag (aggregation tag), to enable fluorogenic detection (turn-on fluorescence) of misfolded soluble oligomers both in test tubes and live cells. In this MIRA proposal, the PI plans to further develop the AggTag method with new probes that can distinguish soluble oligomers from insoluble aggregates using orthogonal fluorescent signals (Project 1). This unprecedented resolution will allow the PI to ask how IDPs misfold and aggregate in phase separated droplets. The PI have begun this direction with a focus on a group of intrinsically disordered RNA binding proteins (RBPs), which harbor RNA binding domains (RBD) and disordered prion-like domains (PLD). While PLD has been the primary focus in literatures, preliminary data have led to a novel hypothesis that whether RBD misfolds contributes to whether RBP misfolds during and after formation of droplets. This hypothesis will be tested both in vitro and in live cells, using a combination of the AggTag method and biochemical assays (Project 2). Finally, the PI will develop chemical strategies to control phase separation and membraneless organelles. Although LLPS can be prevented and dissolved by small molecules, disruption of the liquid droplets could obstruct their physiological functions. Till now, no small molecules have been discovered to promote formation of liquid droplets and prevent RBP misfolding. Preliminary data indicate that sugar phosphates are a novel class of molecules that promote droplet formation, stabilize liquid droplets, and prevent RBP misfolding in droplets. Based on these results, the PI will use efforts from multiple disciplines to understand the mechanisms underlying the observed effects of sugar phosphates and further develop them into a class of chemical regulators with proper selectivity and efficacy (Project 3). In summary, the proposed research will provide an enabling technology to visualize misfolding and aggregation of proteins in membraneless organelles and generate novel chemical compounds to regulate this disease-related process.

项目概要 无膜细胞器在细胞生理学和病理学中具有重要功能。最近的研究表明 这些细胞器是通过本质无序蛋白质（IDP）的液-液相分离形成的 和RNA分子。 IDPs在试管中相分离成液滴并形成P体或应力颗粒 在应激细胞中。发现几种 IDP 的突变型和野生型形式聚集在神经元中 与神经退行性疾病有关。然而，人们对国内流离失所者如何错误折叠和发生知之甚少。 聚集在这些细胞器中以及如何调节这个过程。缺乏这方面知识的原因是 当前用于监测活细胞中无膜细胞器的方法：该过程通过以下方式可视化 对荧光蛋白标记的 IDP 进行成像，分析其位置和扩散速率前后的变化 细胞器的形成。尽管如此，这种方法并没有揭示国内流离失所者是否在内部流离失所者中错误折叠或聚集。 细胞器，因为 IDP 聚集前后形态保持不变。为了克服这个 为了应对挑战，PI开发了一种新颖的成像方法，以下称为AggTag（聚合标签）， 能够对试管和活体中错误折叠的可溶性低聚物进行荧光检测（开启荧光） 细胞。在此 MIRA 提案中，PI 计划使用新探针进一步开发 AggTag 方法，该方法可以 使用正交荧光信号区分可溶性低聚物和不溶性聚集体（项目 1）。这 前所未有的分辨率将使 PI 能够了解 IDP 如何错误折叠并聚集在相分离的液滴中。 PI 已经开始这个方向，重点关注一组本质上无序的 RNA 结合蛋白 (RBP)， 其中包含 RNA 结合域 (RBD) 和无序朊病毒样域 (PLD)。虽然 PLD 已 作为文献的主要关注点，初步数据得出了一个新的假设，即 RBD 是否会错误折叠 影响 RBP 在液滴形成期间和之后是否错误折叠。这个假设将被检验 结合使用 AggTag 方法和生化检测（项目 2），在体外和活细胞中进行实验。最后， 首席研究员将开发化学策略来控制相分离和无膜细胞器。虽然 LLPS 可以通过小分子来预防和溶解，液滴的破坏可能会阻碍其 生理功能。迄今为止，尚未发现小分子能够促进液体形成 液滴并防止 RBP 错误折叠。初步数据表明糖磷酸盐是一类新型的 促进液滴形成、稳定液滴并防止 RBP 在液滴中错误折叠的分子。基于 根据这些结果，PI 将利用多个学科的努力来了解潜在的机制 观察了糖磷酸盐的作用，并将其进一步开发成一类具有适当作用的化学调节剂 选择性和功效（项目 3）。总之，拟议的研究将提供一种使能技术 可视化无膜细胞器中蛋白质的错误折叠和聚集并产生新的化学物质 调节这种疾病相关过程的化合物。