Miles Jay, van Oosten-Hawle Patricija
School of Molecular and Cell Biology & Astbury Centre for Structural Molecular Biology, University of Leeds.
School of Molecular and Cell Biology & Astbury Centre for Structural Molecular Biology, University of Leeds;
J Vis Exp. 2020 May 16(159). doi: 10.3791/61357.
Over the past decade there has been a transformative increase in knowledge surrounding the regulation of protein quality control processes, unveiling the importance of intercellular signaling processes in the regulation of cell-nonautonomous proteostasis. Recent studies are now beginning to uncover signaling components and pathways that coordinate protein quality control from one tissue to another. It is therefore important to identify mechanisms and components of the cell-nonautonomous proteostasis network (PN) and its relevance for aging, stress responses and protein misfolding diseases. In the laboratory, we use genetic knockdown by tissue-specific RNAi in combination with stress reporters and tissue-specific proteostasis sensors to study this. We describe methodologies to examine and to identify components of the cell-nonautonomous PN that can act in tissues perceiving a stress condition and in responding cells to activate a protective response. We first describe how to generate hairpin RNAi constructs for constitutive genetic knockdown in specific tissues and how to perform tissue-specific genetic knockdown by feeding RNAi at different life stages. Stress reporters and behavioral assays function as valuable readouts that enable the fast screening of genes and conditions modifying systemic stress signaling processes. Finally, proteostasis sensors expressed in different tissues are utilized to determine changes in the tissue-specific capacity of the PN at different stages of development and aging. Thus, these tools should help clarify and allow monitoring the capacity of PN in specific tissues, while helping to identify components that function in different tissues to mediate cell-nonautonomous PN in an organism.
在过去十年中，围绕蛋白质质量控制过程调控的知识有了变革性的增长，揭示了细胞间信号传导过程在细胞非自主性蛋白质稳态调控中的重要性。最近的研究开始揭示协调从一个组织到另一个组织蛋白质质量控制的信号成分和途径。因此，识别细胞非自主性蛋白质稳态网络（PN）的机制和成分及其与衰老、应激反应和蛋白质错误折叠疾病的相关性很重要。在实验室中，我们使用组织特异性RNAi进行基因敲低，并结合应激报告基因和组织特异性蛋白质稳态传感器来研究这一问题。我们描述了用于检查和识别细胞非自主性PN成分的方法，这些成分可在感知应激条件的组织中起作用，并在反应细胞中激活保护性反应。我们首先描述如何生成用于特定组织中组成型基因敲低的发夹RNAi构建体，以及如何通过喂食RNAi进行组织特异性基因敲低。应激报告基因和行为分析作为有价值的读数，能够快速筛选改变全身应激信号传导过程 的基因和条件。最后，利用在不同组织中表达的蛋白质稳态传感器来确定PN在发育和衰老不同阶段的组织特异性能力变化。因此，这些工具应有助于阐明并监测特定组织中PN的能力，同时有助于识别在不同组织中起作用以介导生物体中细胞非自主性PN的成分。
