CHLOROPLAST SOLUBLE PROTEASES AND THEIR PHYSIOLOGICAL SUBSTRATES: An integrated genetic and targeted systems analysis of chloroplast proteolysis

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。叶绿体是植物生产力和农业生产中必不可少的细胞器，但对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质分解系统；这些系统必须通过顺序、并行和/或共享蛋白质底物来相互补充。这项研究将为深入了解叶绿体蛋白水解酶的网络提供依据，并为发现其他亚细胞中的蛋白水解酶网络提供一个范例。这些研究成果也可以应用于分子农业和合成生物学，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、生物化学和生物信息学方面的培训。暑期实习将通过NSF赞助的REU计划提供，并通过康奈尔大学4H延期计划提供给高中生。为了帮助培训下一代科学家，将在第二年组织一次植物蛋白分解研讨会，邀请蛋白酶和降解技术方面的专家参加。这项研究建立在对拟南芥蛋白酶单一和更高顺序突变体、先前确定的蛋白酶底物、特定生化工具和用于检测蛋白质降解事件(例如尾巴)的基于质谱学的工作流程的投资的基础上。在目标1中，研究人员将研究主要叶绿体代谢途径的关键酶(如四吡咯和莽草酸)是如何在体内被CLP蛋白酶系统识别和降解的。这个必需的CLP系统是最复杂和最丰富的可溶性叶绿体寡聚体蛋白酶，由16个不同的基因产物组成，包括底物选择器和伴侣。在AIM 2中，将使用亲和和捕获方法鉴定额外的CLP候选底物和可能的接头，随后将进行体内生理和体外相互作用研究。目的3.在体外分析的基础上，提出了另外两种叶绿体多肽酶，即PREP和OOP，用于降解较小的蛋白质和蛋白质片段，包括裂解的叶绿体转运肽。初步的分子遗传学数据表明CLP、PREP和OOP之间存在功能相互作用。这些多肽酶在体内的生理作用将通过非靶向和靶向的方法来确定，并调查它们是否以及如何形成一个功能性的蛋白酶网络。

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 organelles in plant productivity and agriculture, but determinants of chloroplast protein life-time and degradation are poorly understood. Cells and organelles contain hundreds of proteolytic systems; these must complement each other, by acting in sequence, in parallel and/or by sharing protein substrates. This research will provide insight into the network of chloroplast protease and serve as an example for protease network discovery in other subcellular compartments. The research findings can also be implemented in molecular farming and synthetic biology, 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 and bioinformatics at the undergraduate, graduate and post-doctoral levels. Summer internships will be offered through our NSF-sponsored REU program and to high school students through the Cornell 4H extension program. To help to train the next generation scientists, a plant proteolysis workshop with invited experts on proteases and degradomics technology will be organized in year 2.This research is built upon investments in Arabidopsis protease single and higher order mutants, previously identified protease substrates, specific biochemical tools and mass spectrometry-based workflows for detection of protein degradation events (e.g. TAILS). In AIM 1, investigators will study how key enzymes of major chloroplast metabolic pathways (e.g. tetrapyrroles and shikimate) are recognized and degraded by the Clp protease system in vivo. This essential Clp system is the most complex and abundant soluble chloroplast oligomeric protease consisting of 16 different gene products, including substrate selectors and chaperones. In AIM 2, additional Clp candidate substrates and perhaps adaptors will be identified, using affinity and trapping approaches, followed by in vivo physiological and in vitro interaction studies. AIM 3. Based on in vitro analysis, two additional chloroplast peptidases, PREP and OOP were proposed to degrade smaller proteins and protein fragments, including cleaved chloroplast transit peptides. Preliminary molecular genetics data indicate functional interactions between Clp, PREP and OOP. The in vivo physiological role of these peptidases will be determined by untargeted and targeted approaches and investigate if and how they form a functional protease network.