DETERMINATION OF THE N-DEGRON PATHWAY AND ITS SUBSTRATES IN PLANT CHLOROPLASTS

植物叶绿体中N-DEGRON途径及其底物的测定

• 批准号: 2322813
• 负责人: Klaas van Wijk
• 金额: $ 95万
• 依托单位: Cornell University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-15 至 2026-06-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2322813&HistoricalAwards=false
• 关键词: DETERMINATION; DEGRON; PATHWAY; ITS; SUBSTRATES

Protein amino (N) termini (i.e., the start of the linear sequence of amino acids in a protein) are major determinants of protein stability. This is conceptualized in the N-degron pathway which states that certain amino acids, when exposed abnormally at the N-terminus of a protein, act as triggers (degrons) for protein degradation. Plant chloroplasts have an ancient relationship with prokaryotes and their N-degron pathway involves the bacterial-like Clp chaperone-protease system, with the N-recognin ClpS1 playing a central role in recognition, binding, and delivery of N-degron substrates to Clp chaperones. This research will determine the mechanistic features of this important pathway. The Broader impacts of this research include its intrinsic merit as plant chloroplasts play a central role in energy transduction on the planet and the work may facilitate the rational design of stable transgenic proteins targeted to chloroplasts with applications in pharmaceutical and biofortification products. Additional activities include interdisciplinary training, bridging cell biology, molecular genetics, quantitative imaging, bioinformatics, and biochemistry. Summer internships will be offered through our NSF-sponsored REU programs.A main impediment for determining the molecular details of a chloroplast N-degron pathway is an in planta quantitative reporter system that can test specific N-termini of interest. The research will employ our newly developed in vivo reporter system to determine N-degrons in chloroplasts and the involvement of chloroplast ClpS1 and the Clp chaperone-protease system. Independently, our in vivo ClpC1 chaperone trapping approach will determine the impact of ClpS1 and ClpF on substrate selection and delivery to the Clp chaperone-protease system. Identified ClpS1/ClpF dependent substrates will be investigated for their degrons. We will also determine how the N-domain of chloroplast glutamate t-RNA reductase is recognized as a conditional substrate by ClpS1 and ClpF in vivo using a unique transgenic reporter.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.

蛋白质氨基 (N) 末端（即蛋白质中氨基酸线性序列的起点）是蛋白质稳定性的主要决定因素。这是在 N-降解决定子途径中概念化的，该途径指出某些氨基酸在蛋白质的 N 末端异常暴露时，会充当蛋白质降解的触发器（降解决定子）。植物叶绿体与原核生物有着古老的关系，它们的 N-降解决定子途径涉及类似细菌的 Clp 伴侣蛋白酶系统，其中 N-识别蛋白 ClpS1 在 N-降解决定子底物识别、结合和递送到 Clp 伴侣中发挥着核心作用。这项研究将确定这一重要途径的机制特征。这项研究的更广泛影响包括其内在优点，因为植物叶绿体在地球上的能量转换中发挥着核心作用，这项工作可能有助于合理设计针对叶绿体的稳定转基因蛋白，并应用于制药和生物强化产品。其他活动包括跨学科培训、桥接细胞生物学、分子遗传学、定量成像、生物信息学和生物化学。暑期实习将通过我们的 NSF 赞助的 REU 项目提供。确定叶绿体 N-降解决定子途径的分子细节的一个主要障碍是植物内定量报告系统，该系统可以测试感兴趣的特定 N 末端。该研究将采用我们新开发的体内报告系统来确定叶绿体中的 N-降解决定子以及叶绿体 ClpS1 和 Clp 分子伴侣蛋白酶系统的参与。独立地，我们的体内 ClpC1 伴侣捕获方法将确定 ClpS1 和 ClpF 对底物选择和向 Clp 伴侣蛋白酶系统的递送的影响。将研究已鉴定的 ClpS1/ClpF 依赖性底物的降解决定子。我们还将使用独特的转基因报告基因确定叶绿体谷氨酸 t-RNA 还原酶的 N 结构域如何在体内被 ClpS1 和 ClpF 识别为条件底物。该奖项反映了 NSF 的法定使命，并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，被认为值得支持。