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

项目总结 无膜细胞器在细胞生理和病理中具有重要的功能。最近的研究表明 这些细胞器是通过固有无序蛋白质(Idp)的液-液相分离而形成的。 和RNA分子。IDPs相在试管中分离成液滴并形成P体或应力颗粒 在压力较大的细胞中。几种IDPs的突变型和野生型都被发现聚集在神经元和 与神经退行性疾病有关。然而，人们对境内流离失所者是如何错折和 这些细胞器中的聚集以及如何调节这一过程。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：这一过程通过 荧光蛋白标记的IDPs成像前后位置和扩散速度的变化分析 细胞器形成。尽管如此，这种方法并不能揭示国内流离失所者是否在 细胞器，因为在IDPs聚集前后形态保持不变。要克服这一点 挑战，PI开发了一种新的成像方法，以下称为AggTag(聚合标签)，以 启用试管和活体中错误折叠的可溶性低聚物的荧光检测(开启荧光) 细胞。在这个Mira提案中，PI计划进一步开发AggTag方法，使用新的探测器，可以 使用正交荧光信号区分可溶低聚物和不溶聚集体(项目1)。这 史无前例的分辨率将允许国际和平研究所询问国内流离失所者如何错误折叠和聚集相分离的液滴。 PI已经开始了这个方向，专注于一组内在无序的RNA结合蛋白(RBPs)， 它含有RNA结合域(RBD)和无序的普恩样结构域(PLD)。而可编程逻辑器件一直是 在文献中主要关注的是，初步数据导致了一个新的假设，即RBD是否会出错 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方法和生化分析相结合(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 LLP可以被小分子阻止和溶解，液滴的破坏可能会阻碍LLP 生理功能。到目前为止，还没有发现促进液体形成的小分子。 并防止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。基座 根据这些结果，PI将使用来自多个学科的努力来了解 观察糖磷酸盐的作用效果，并进一步将其发展成为一类具有适当 选择性和有效性(项目3)。总而言之，拟议的研究将提供一种使技术能够 可视化蛋白质在无膜细胞器中的错误折叠和聚集并产生新的化学物质 调节这一疾病相关过程的化合物。

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.