CLPS1-CLPF ADAPTORS FOR PROTEASE SUBSTRATE SELECTION IN CHLOROPLASTS

蛋白质分解，即蛋白质或多肽分解成氨基酸，对于去除不需要的或受损的蛋白质和调节细胞过程(如新陈代谢)至关重要。叶绿体是重要的植物细胞器，在生物量生产力和农业生产中起着重要作用。然而，叶绿体蛋白质寿命和降解的决定因素却知之甚少。细胞和细胞器包含数百种蛋白质分解系统。这项研究将深入了解中央叶绿体CLP蛋白酶系统的作用，该系统由蛋白质伴侣、蛋白酶核心复合体和几种蛋白质组成，这些蛋白质参与特定CLP酶底物的选择和递送。这项研究成果还可以应用于分子农业和合成生物学(例如，通过可调的蛋白质降解)，因为叶绿体等细胞隔间有利于过度表达具有营养或药用价值的产品。这项研究的结果将允许更合理的蛋白质设计，以稳定积累叶绿体蛋白质，从而直接影响这些应用。该项目将在本科生、研究生和博士后水平上提供蛋白质组学、质谱学、分子遗传学、生物化学、结构生物学方面的培训。暑期实习将通过我们的NSF赞助的REU计划提供。该项目建立在对生物材料的广泛投资基础上，如拟南芥蛋白酶突变体、先前确定的候选CLP底物或CLP适配器，以及在质谱学、蛋白质组学和结构生物学方面的专业知识。这项建议的中心目标是揭示一个新的ClpS1-ClpF适配器系统的功能、结构和机制细节，包括底物选择、底物半衰期以及与CLP伴侣的相互作用。它还可能导致发现其他CLP适配器。我们将使用我们的体内工具来测试ClpS1和ClpF在底物选择中的生理作用。体内降解测试将测试CLP成分对候选底物半衰期的影响。将通过X射线结晶学或低温电子显微镜获得ClpS1和ClpF及其功能伙伴的高分辨率结构信息。体外和植物中的相互作用分析和功能互补将决定蛋白质结构域的作用。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Proteolysis, the breakdown of proteins or peptides to amino acids, is critical for removal of unwanted or damaged proteins and regulation of cellular processes such as metabolism. Chloroplasts are essential plant organelles that are important in biomass productivity and agriculture. However, the determinants of chloroplast protein life-time and degradation are poorly understood. Cells and organelles contain hundreds of proteolytic systems. This research will provide insight into the role of the central chloroplast Clp protease system, consisting of protein chaperones, a protease core complex and several proteins involved in selection and delivery of specific Clp protease substrates. The research findings can also be implemented in molecular farming and synthetic biology (e.g. through tunable protein degradation), since cellular compartments such as chloroplasts are favored for overexpression of products with nutritional or pharmaceutical value. The outcome of this research will allow more rational protein design for stable accumulation of chloroplast proteins, thus directly impacting these applications. This project will provide training in proteomics, mass spectrometry, molecular genetics, biochemistry, structural biology at the undergraduate, graduate and post-doctoral levels. Summer internships will be offered through our NSF-sponsored REU programs. This project is built on extensive investments in biological materials such as Arabidopsis protease mutants, previously identified candidate Clp substrate or Clp adaptors, as well as expertise in mass spectrometry, proteomics and structural biology. The central objective of this proposal is to unravel the functional, structural, and mechanistic details of a novel ClpS1-ClpF adaptor system, including substrate selection, substrate half-life, and interactions with Clp chaperones. It may also lead to the discovery of additional Clp adaptors. We will use our in vivo tools to test the physiological role of ClpS1 and ClpF in substrate selection. In vivo degradation assays will test the impact of Clp components on the half-life of candidate substrates. High-resolution structural information through X-ray crystallography or CryoEM will be obtained for ClpS1 and ClpF, with their functional partners. In vitro and in planta interaction assays and functional complementation will determine the role of protein domains.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.