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、虫害、高温、寒冷或干旱引起的植物胁迫是全球作物损失的主要原因，了解植物如何应对胁迫并从胁迫中恢复可以极大地促进通过改进育种和生物工程方法来提高作物生产力。可以裂解其他蛋白质的酶对于介导植物的抗逆性以及发育途径非常重要。这项研究将解决植物元半胱天冬酶（这些保守蛋白酶的一种）如何促进和控制与胁迫反应相关的发育过程。利用晶体学可视化技术和冷冻电子显微镜技术的进步，植物元半胱天冬酶家族关键成员的结构将被阐明到原子分辨率，同时，对转基因植物中这些元半胱天冬酶变体的研究将揭示它们对胁迫的影响。总之，这些进展可能会对未来的农业产生重要影响，通过改变元半胱天冬酶的耐受功能来增强病原体的抗性和应激能力。此外，本科生参与我们的多学科项目的体验式培训应该有助于整个科学教育。遗传学研究表明，元半胱天冬酶 (MC) 蛋白酶涉及非生物和生物。高等植物中应激诱导的细胞死亡有两类，例如来自拟南芥的 MC4 和 MC9，它们的活性分别依赖于钙离子和弱酸性 pH 值。 AtMC4 的分辨率为 2.8 Å，并发现了钙诱导后 AtMC4 酶原激活和成熟过程中多步骤自溶过程的证据。在该项目中，将通过使用晶体学和冷冻电子显微镜来解析 AtMC9 的结构。 （冷冻电镜），这将能够发现其在酸性 pH 下激活且不依赖于钙的分子决定因素。此外，还将解析由其产生的嵌合蛋白的结构。交换 AtMC4 和 AtMC9 之间的连接结构域，导致活性蛋白酶在被钙激活后不再表现出自溶裂解，这些新的蛋白质结构应该有助于合理化它们观察到的生化差异，以补充突变和突变的体内功能。将使用 atmc4 和 atmc9 敲除突变体作为遗传背景来检查具有新颖的调控特征组合的嵌合 II 型 MC，最后，提出了 AtMC9 酶原的主动易位。将通过使用与酸稳定的 GFP 变体的融合来测试胁迫下的酸性质外体空间，这些体外和体内特征分析将揭示激活这两种元半胱天冬酶以诱导植物胁迫反应的详细分子基础。由分子和细胞生物科学部的细胞动力学和功能集群以及综合有机系统部的植物、真菌和微生物发育机制项目共同资助。授予 NSF 的法定使命，并通过评估反映使用基金会的智力优点和更广泛的影响审查标准，被认为值得支持。