CLPS1-CLPF ADAPTORS FOR PROTEASE SUBSTRATE SELECTION IN CHLOROPLASTS

蛋白质水解，蛋白质或肽分解为氨基酸，对于去除不需要的或受损的蛋白质和调节细胞过程如代谢至关重要。叶绿体是重要的植物细胞器，在生物质生产和农业中非常重要。 然而，叶绿体蛋白质寿命和降解的决定因素知之甚少。细胞和细胞器包含数百个蛋白水解系统。这项研究将提供深入了解中央叶绿体Clp蛋白酶系统的作用，包括蛋白伴侣，蛋白酶核心复合物和几种蛋白质参与选择和交付特定的Clp蛋白酶底物。研究结果也可以在分子农业和合成生物学中实施（例如通过可调蛋白质降解），因为细胞区室如叶绿体有利于具有营养或药用价值的产品的过表达。本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。该项目将提供本科、研究生和博士后水平的蛋白质组学、质谱法、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之上，如拟南芥蛋白酶突变体、先前确定的候选Clp底物或Clp衔接子，以及质谱、蛋白质组学和结构生物学方面的专业知识。该提案的中心目标是解开一种新的ClpS 1-ClpF适配器系统的功能，结构和机制的细节，包括底物选择，底物半衰期，和与Clp分子伴侣的相互作用。它还可能导致发现额外的Clp衔接子。我们将使用我们的体内工具来测试ClpS 1和ClpF在底物选择中的生理作用。体内降解试验将测试Clp组分对候选底物半衰期的影响。通过X射线晶体学或CryoEM将获得ClpS 1和ClpF及其功能伙伴的高分辨率结构信息。在体外和在植物相互作用试验和功能互补将确定蛋白质结构域的作用。这个奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.