Building a Toolbox of Sensors and Approaches to Monitor the Proteostasis Network Core B

核心B-Frydman/Finkbeiner-蛋白质稳定性分析的传感器和工具 摘要： 该核心将开发工具来定义衰老过程中的蛋白质平衡网络(PN) 并将为项目1-4提供技术支持。 资源还将用于测试和验证Core D的化合物。Core B将有两个接近的站点 地理上的接近。斯坦福大学的弗莱德曼团队将建造分子传感器和分析来测量 蛋白质在老化过程中的变化，首先是为酵母伴侣机制开发的记者和 泛素-蛋白酶体系统(UPS)。加州大学格拉德斯通分校的芬克贝纳团队将建立在哺乳动物的基础上 表达和分析工具，以评估蛋白质聚集和自噬，并帮助解决特定的 他们的多路纵向单细胞成像平台对哪些问题至关重要。通过携手合作- 手动并测试传感器在所有人使用的实验中报告不同蛋白质平衡事件的能力 项目，我们将产生一套化验，以提供任何细胞的PN的全面看法。工具将是 可供研究界使用。核心B的主要任务将是开发分子传感器， 可靠地、定量地报告业务流程的每一个分支以及在老龄化和 与疾病相关的错误折叠蛋白质。成像传感器还将使我们能够检查蛋白质平衡 机制从一个细胞传递到另一个细胞。对这些问题的回答将提供关键的见解 研究衰老过程中蛋白质平衡功能障碍和蛋白质聚集的机制，将开辟新的途径 用于维持健康和治疗疾病。我们建议：(1)开发和验证一个小组 蛋白质平衡传感器和记者以及(2)协助项目负责人(PL)分析PN 传感器：对任何细胞的PN状态进行全面检查。 我们的最终目标是建立一套全面的传感器，与384孔板兼容，可以使用 作为一个集成的面板，审问PN的所有方面。通过综合知识的集体主体 将蛋白质平衡网络转化为一组基于荧光的传感器和记者，核心B将产生 整个蛋白质平衡领域的无价工具箱，将促进进展，而不仅仅是在相关的实验室 有了这种PPG，也有其他实验室对蛋白质平衡功能障碍与衰老和疾病之间的联系感兴趣。

Core B – Frydman/Finkbeiner - Sensors and Tools for Proteostasis Analyses Summary: This Core will develop tools to define the proteostasis network (PN) during aging and in the context of disease-associated aggregation-prone proteins and will provide technical support to Projects 1–4. The resources will also be used to test and validate compounds from Core D. Core B will have two sites in close geographical proximity. The Frydman group at Stanford will build molecular sensors and assays to measure proteostasis changes during aging, starting with reporters developed for yeast chaperone machinery and the ubiquitin-proteasome system (UPS). The Finkbeiner group at Gladstone/UCSF will build on mammalian expression and analysis tools to assess protein aggregation and autophagy and help PLs address specific questions for which their multiplexed longitudinal single-cell imaging platform is critical. By working hand-in- hand and testing the ability of the sensors to report on different proteostasis events in experiments used by all Projects, we will produce a set of assays that provide a comprehensive view of the PN of any cell. Tools will be available to the research community. A primary task of Core B will be to develop molecular sensors that robustly and quantitatively report on each branch of the PN and that change in aging and in the context of disease-associated misfolded proteins. Imaging sensors will also allow us to examine how proteostasis mechanisms are communicated from one cell to another. Answers to these questions will provide key insights into mechanisms of proteostasis dysfunction and protein aggregation during aging and will open new avenues for maintaining health and treating disease. We Propose to: (1) Develop and validate a panel of proteostasis sensors and reporters and (2) Assist project leaders (PLs) in the analysis of the PN sensors: towards a comprehensive exam of the PN status of any cell. Our ultimate goal is to build a comprehensive set of sensors compatible with 384-well plates that could be used as an integrated panel to interrogate all aspects of the PN. By synthesizing the collective body of knowledge of the proteostasis network into a set of fluorescence-based sensors and reporters, Core B will produce an invaluable toolbox for the whole proteostasis field that will facilitate progress, not only in the labs associated with this PPG but also in other labs interested in linking proteostasis dysfunction with aging and disease.