Structure-Function Analysis of Type II Metacaspases to Reveal Distinct Activation Mechanisms

II 型元半胱天冬酶的结构功能分析揭示独特的激活机制

• 批准号: 2052997
• 负责人: Eric Lam
• 金额: $ 135.81万
• 依托单位: Rutgers University New Brunswick
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2052997&HistoricalAwards=false
• 关键词: Structure; Function; Analysis; Type; II

Plant stresses caused by diseases, insect pests, heat, cold or drought are major causes of crop loss globally. Understanding how plants respond and recover from stress can greatly facilitate the effort to improve crop productivity through improved breeding and bioengineering approaches. Highly conserved proteases, enzymes that can cleave other proteins, are important in mediating stress resistance as well as developmental pathways in plants. This research will address how plant metacaspases, one type of these conserved proteases, contribute and control the developmental processes associated with stress response. Using advances in visualization techniques of crystallography and cryo-Electron Microscopy techniques, the structures for key members of the plant metacaspase family will be clarified to atomic resolution. In parallel, studies of variants for these metacaspases in transgenic plants will inform on their effects on stress response pathways. Together, these advances will likely have important impacts on agriculture through engineering crops in the future to enhance pathogen resistance and stress tolerance by modifying metacaspase functions. This project will provide diversified, interdisciplinary training to postdoctoral researchers and graduate students to enrich the scientific workforce. In addition, involvement of undergraduate students in experiential training in our multi-disciplinary project should contribute to science education at large. Genetic studies have shown that the metacaspase (MC) proteases are involved in abiotic and biotic stresses-induced cell death in higher plants. Two classes of Type II MCs are exemplified by MC4 and MC9 from Arabidopsis with their activity dependent on calcium ion and mildly acidic pH, respectively. The crystal structure of AtMC4 has been reported at 2.8 Å resolution and uncovered evidence for a multi-step autolytic process in the activation and maturation of the AtMC4 zymogen upon calcium induction. In this project, the structure for AtMC9 will be solved by using crystallography and cryo-Electron Microscopy (cryo-EM), which will enable discovery of the molecular determinants that underlie its activation at acidic pH and independence from calcium. In addition, the structure will be solved for a chimeric protein created from swapping the linker domain between AtMC4 and AtMC9 that resulted in an active proteases which no longer show autolytic cleavage upon its activation by calcium. These new protein structures should help rationalize their observed biochemical differences. To complement these structural studies, in vivo function of mutated and chimeric Type II MCs that have novel combinations of regulatory characteristics will be examined using atmc4 and atmc9 knockout mutants as genetic backgrounds. Lastly, proposed active translocation of AtMC9 zymogen to the acidic apoplastic space upon stresses will be tested by using fusions with acid-stable GFP variants. Together, these in vitro and in vivo trait analysis will reveal the detailed molecular basis for activation of these two metacaspases to induce plant stress response.This award is co-funded by the Cellular Dynamics and Function cluster in the Division of Molecular and Cellular Biosciences and the Plant, Fungal and Microbial Developmental Mechanisms Program in the Division of Integrative Organismal Systems.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.

由疾病，绝缘害虫，热，冷或干旱引起的植物压力是全球作物损失的主要原因。了解植物如何反应和从压力中恢复，可以通过改善繁殖和生物工程方法来大大支持提高农作物生产力的努力。高度保守的蛋白质，可以清除其他蛋白质的酶，对于介导抗压力和植物的发育途径很重要。这项研究将解决植物元叠酶（这些保守蛋白酶的一种类型）如何贡献和控制与压力反应相关的发育过程。利用晶体学和冷冻电子显微镜技术的可视化技术的进步，将阐明植物肠胃痛酶家族的主要成员的结构，以阐明原子分辨率。同时，对转基因植物中这些肠胃菜酶变体的研究将告知其对应激反应途径的影响。这些进步总之可能会在未来通过工程作物对农业产生重要影响，以通过修饰肠胃菜酶功能来增强病原体的耐药性和胁迫耐受性。该项目将为博士后研究人员和研究生提供多元化的跨学科培训，以丰富科学劳动力。此外，本科生参与我们多学科项目的经验丰富的培训应为整个科学教育做出贡献。遗传研究表明，肠烟酶（MC）蛋白酶与非生物胁迫诱导的较高植物中的细胞死亡有关。 MC4和MC9从拟南芥的活性分别取决于钙离子和轻度酸性pH，用MC4和MC9示例两类。已经报道了ATMC4的晶体结构以2.8Å分辨率，并发现了在钙诱导后ATMC4 Zymogen激活和成熟的多步体自溶过程的证据。在该项目中，ATMC9的结构将通过使用晶体学和冷冻电子显微镜（Cryo-EM）来解决，这将使能够发现其在酸性pH和钙独立性下激活的分子确定剂。此外，将解决通过将接头域在ATMC4和ATMC9之间交换的嵌合蛋白的结构，该蛋白会导致活性蛋白，该蛋白不再在钙激活后显示自动溶解裂解。这些新的蛋白质结构应有助于合理化其观察到的生化差异。为了补充这些结构研究，将使用ATMC4和ATMC9基因敲除突变体作为遗传背景的突变和嵌合II型MC的体内功能，具有新颖的调节特征组合。最后，通过使用与酸稳定的GFP变体的融合来测试应力时提出的ATMC9 Zymogen在应力下向酸性塑料空间的主动易位。这些体外和体内性状分析共同揭示了激活这两个元座空间的详细分子基础，以诱导植物压力反应。该奖项是由细胞动力学和功能集群共同授予的分子和细胞生物科学的划分，以及植物和微生物发展机制的植物和微生物的统计机制，以反映植物和微生物的制定机制。使用基金会的智力优点和更广泛的影响标准，认为通过评估被认为是宝贵的支持。