EAGER: Quantitative reporter systems for in vivo testing of an N-end rule for protein stability in plant chloroplasts

EAGER：用于体内测试植物叶绿体中蛋白质稳定性 N 端规则的定量报告系统

• 批准号: 1834636
• 负责人: Klaas van Wijk
• 金额: $ 30万
• 依托单位: Cornell University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1834636&HistoricalAwards=false
• 关键词: EAGER; Quantitative; reporter; systems; vivo

Quantitative reporter systems for in vivo testing of an N-end rule for protein stability in plant chloroplasts ? Klaas van Wijk, Cornell University Short abstract EAGER application 1834636Part 1: A nontechnical description of the project. Plants and algae are different from other organisms, such as humans and bacteria, in that they have an extra type of organelle in their cells, namely chloroplasts. These chloroplasts contain several thousand different proteins that fulfill many critical functions, including photosynthesis. The chloroplast also contains several enzymatic systems, named proteases, which selectively degrade chloroplast proteins once they are no longer needed. This project aims to build an experimental system that will allow to determine the signals within proteins that lead to their removal by proteases. A better understanding of the stability rules of proteins is important for improvement of crops and also will help in a better design of chloroplast proteins that generate products of pharmaceutical value and biofortification of crops. The project will also serve to educate and train students and postdocs in STEM, including members of underrepresented groups.Part 2: A technical description of the project. Protein amino (N) termini are prone to modifications and are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts and non-photosynthetic plastids. The role of the N-terminus in protein stability is conceptualized in the N-end rule, which states that certain amino acids, when exposed at the N-terminus of a protein, act as triggers (degrons) for degradation. Based on several types of indirect experimental evidence, as well as evolutionary arguments, we postulate that an N-end rule in plastids must exist, however that has not been proven. The main impediment for testing for an N-end rule in chloroplasts is a suitable in vivo quantitative reporter system. This EAGER proposal aims to generate such a system based on a combination of novel quantitative tools and techniques developed for other biological systems. The proposed research will then use this system to directly test possible N-degrons in chloroplasts and the involvement of chloroplast ClpS1 and the Clp protease and chaperone system in Arabidopsis. Therefore, this work could make a transformative contribution to chloroplast proteostasis and the evolutionary adaptation of the N-end rule for protein stability in organelles. Identification of such an N-end rule would be highly predictive for chloroplast protein stability.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.

植物叶绿体中蛋白质稳定性n端规则的体内定量报告系统Klaas van Wijk， Cornell University简短摘要EAGER应用程序1834636第1部分：项目的非技术描述。植物和藻类不同于其他生物，如人类和细菌，因为它们的细胞中有一种额外的细胞器，即叶绿体。这些叶绿体含有数千种不同的蛋白质，这些蛋白质完成许多关键功能，包括光合作用。叶绿体还含有几种酶系统，称为蛋白酶，一旦不再需要叶绿体蛋白质，它们就会选择性地降解。该项目旨在建立一个实验系统，以确定蛋白质内部导致蛋白酶去除它们的信号。更好地了解蛋白质的稳定性规律对作物的改良具有重要意义，也将有助于更好地设计叶绿体蛋白质，从而产生具有药用价值的产品和作物的生物强化。该项目还将教育和培训STEM领域的学生和博士后，包括代表性不足的群体的成员。第2部分：项目的技术描述。蛋白质氨基末端容易发生修饰，是细菌、真核生物、叶绿体和非光合质体中蛋白质稳定性的主要决定因素。n端在蛋白质稳定性中的作用在n端规则中被概念化，该规则指出，当暴露在蛋白质的n端时，某些氨基酸充当降解的触发器（degrons）。基于几种类型的间接实验证据，以及进化论点，我们假设在质体中一定存在n端规则，但这尚未得到证实。在叶绿体中检测n端规则的主要障碍是合适的体内定量报告系统。这项EAGER提案旨在基于为其他生物系统开发的新型定量工具和技术的组合来生成这样一个系统。接下来的研究将使用该系统直接测试拟南芥叶绿体中可能存在的n -degron以及叶绿体ClpS1和Clp蛋白酶和伴侣系统的参与。因此，这项工作可能对叶绿体蛋白质静止和n端规则在细胞器中蛋白质稳定性的进化适应做出革命性的贡献。这种n端规则的鉴定将高度预测叶绿体蛋白的稳定性。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。