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

项目摘要 无膜细胞器在细胞生理和病理学中具有重要的功能。最近的研究表明 这些细胞器是通过内在无序蛋白质（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，形成P体或应力颗粒 在应激细胞中。发现几种IDP的突变体和野生型形式都聚集在神经元中， 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地折叠和 以及这个过程是如何被调节的。缺乏这方面的知识是由于 目前用于监测活细胞中无膜细胞器的方法：该过程通过 成像荧光蛋白标记的IDP，以分析它们的位置和扩散率的变化前后 细胞器形成尽管如此，这种方法并没有揭示国内流离失所者是否错误地折叠或聚集在 细胞器，因为形态保持不变之前和之后的国内流离失所者聚集。为了克服这个 为了应对挑战，PI开发了一种新的成像方法，下文称为AggTag（聚合标签）， 能够在试管和活体中对错误折叠的可溶性低聚物进行荧光检测（开启荧光） 细胞在该MIRA提案中，PI计划使用新探针进一步开发AggTag方法， 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允许PI询问IDP如何在相分离的液滴中错误折叠和聚集。 PI已经开始关注一组内在无序的RNA结合蛋白（RBP）， 其具有RNA结合结构域（RBD）和无序朊病毒样结构域（PLD）。虽然PLD一直是 在文献的主要焦点，初步的数据导致了一个新的假设，是否RBD错误折叠 有助于RBP在液滴形成期间和之后是否错误折叠。这一假设将在以下两个方面得到检验： 在体外和活细胞中，使用AggTag方法和生物化学测定的组合（项目2）。最后， PI将开发控制相分离和无膜细胞器的化学策略。虽然 LLPS可以被小分子阻止和溶解，液滴的破坏可以阻碍它们的溶解。 生理功能。到目前为止，还没有发现小分子促进液体的形成 液滴和防止RBP错误折叠。初步数据表明，糖磷酸盐是一类新的 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 在这些结果的基础上，PI将利用多学科的努力来了解 观察到糖磷酸盐的影响，并进一步将其发展为一类具有适当 选择性和有效性（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 化合物来调节这种疾病相关的过程。

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.