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

基本信息

批准号：
10207682
负责人：
Xin Zhang
金额：
$ 39.16万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2022-09-22
项目状态：
已结题

项目摘要

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.

项目摘要无膜细胞器在细胞生理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固有无序蛋白（IDP）的液态液相分离而形成的和RNA分子。 IDPS相分为测试管中的液滴并形成p身体或应力颗粒在压力细胞中。在神经元和与神经退行性疾病有关。但是，关于IDP如何错误折叠和这些细胞器的汇总以及如何调节此过程。缺乏这种知识归因于当前用于监视活细胞中无膜细胞器的方法：通过成像荧光蛋白标签的IDP，以分析其位置的变化和扩散率之前和之后细胞器形成。但是，此方法并未揭示IDP在 Organelle，因为在IDP聚集之前和之后的形态保持不变。克服这一点挑战，PI开发了一种新颖的成像方法，以下是称为Aggtag（聚合标签），在测试管中启用错误折叠的可溶性低聚物的荧光检测（转开荧光）细胞。在此Mira提案中，PI计划通过可以使用新的探针进一步开发Aggtag方法使用正交荧光信号（项目1）区分可溶性低聚物与不溶性聚集体。这前所未有的分辨率将使PI询问IDP如何在相分离的液滴中错误折叠和聚集。 PI已经开始了这个方向，重点是一组本质上无序的RNA结合蛋白（RBP），携带RNA结合结构域（RBD）和无序的prion样域（PLD）。而PLD一直是文献中的主要重点，初步数据导致了一个新的假设，即RBD是否折叠有助于RBP在液滴形成期间和之后是否错误折叠。该假设将两者都检验在体外和活细胞中，结合了Aggtag方法和生化测定法（项目2）。最后， PI将制定化学策略来控制相分离和膜细胞器。虽然 LLP可以通过小分子来预防和溶解，液滴的破坏可能会阻碍生理功能。到现在为止，已经发现没有小分子促进液体的形成液滴并防止RBP错误折叠。初步数据表明糖磷酸盐是一类新的促进液滴形成，稳定液滴并防止液滴中RBP折叠的分子。基于在这些结果上，PI将利用多个学科的努力来了解该机制观察到磷酸盐的作用，并将它们进一步发展为具有正确的化学调节剂选择性和功效（项目3）。总而言之，拟议的研究将为技术提供一种促进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型化学物质化合物调节这种与疾病相关的过程。