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。这在N-降解子途径中被概念化，该途径指出，当某些氨基酸在蛋白质的N-末端异常暴露时，就会作为蛋白质降解的触发物(退化)。植物叶绿体与原核生物有着古老的亲缘关系，它们的N-降解子途径涉及类细菌的CLP伴侣-蛋白酶系统，其中N-识别蛋白ClpS1在识别、结合N-降解子底物和将N-降解子底物输送到CLP伴侣的过程中发挥核心作用。这项研究将确定这一重要途径的机制特征。这项研究的更广泛的影响包括它的内在价值，因为植物叶绿体在地球上的能量传递中发挥着核心作用，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，并将其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计划提供。确定叶绿体N-降解子途径的分子细节的主要障碍是可以测试感兴趣的特定N-末端的植物内定量报告系统。这项研究将利用我们新开发的在体报告系统来确定叶绿体中的N-降解物以及叶绿体ClpS1和CLP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对底物选择和递送到CLP伴侣-蛋白酶系统的影响。确定的依赖ClpS1/ClpF的底物将被研究其降解情况。我们还将使用独特的转基因记者在体内确定叶绿体谷氨酸t-RNA还原酶的N-结构域如何被ClpS1和ClpF识别为条件底物。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。