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

由病虫害、高温、寒冷或干旱引起的植物逆境是全球农作物损失的主要原因。了解植物如何应对和恢复胁迫，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的努力。高度保守的蛋白酶是一种可以裂解其他蛋白质的酶，在植物的抗逆性和发育途径中起着重要的作用。这项研究将探讨这些保守的蛋白水解酶中的一种--植物中的偏半胱氨酸酶如何参与和控制与逆境反应相关的发育过程。利用结晶学可视化技术和低温电子显微镜技术的进展，将阐明植物Metacaspase家族关键成员的结构，使其达到原子分辨率。同时，对转基因植物中这些元天冬氨酸酶的变异的研究将有助于了解它们对胁迫反应途径的影响。综上所述，这些进展可能会对未来的农业产生重要影响，通过改变Metacaspase功能来提高作物对病原菌的抗性和抗逆性。该项目将为博士后研究人员和研究生提供多样化的跨学科培训，以丰富科学队伍。此外，在我们的多学科项目中，本科生参与体验式培训应该有助于整个科学教育。遗传研究表明，在高等植物中，Metacaspase(MC)酶参与了非生物和生物胁迫诱导的细胞死亡。拟南芥的MC4和MC9是两类II型MC的典型代表，它们的活性分别依赖于钙离子和中等酸性的pH。AtMC4的晶体结构已被报道为2.8？分辨率，并发现了在钙诱导下AtMC4酶原的激活和成熟过程中存在多步骤自溶过程的证据。在这个项目中，AtMC9的结构将通过使用结晶学和冷冻电子显微镜(Cryo-EM)来解决，这将使我们能够发现支撑其在酸性pH下激活并独立于钙的分子决定因素。此外，通过交换AtMC4和AtMC9之间的连接域而创建的嵌合蛋白的结构将得到解决，该嵌合蛋白导致了一种活性的蛋白酶，当其被钙激活时不再显示自溶切割。这些新的蛋白质结构应该有助于使它们观察到的生化差异合理化。为了补充这些结构研究，具有新的调节特性组合的突变和嵌合型II型MC的体内功能将以atmc4和atmc9敲除突变体作为遗传背景进行检测。最后，所提出的AtMC9酶原在胁迫下主动移位到酸性质外体空间的建议将通过与酸性稳定的GFP变异体的融合来测试。这些体外和体内的特性分析将揭示激活这两种偏半胱氨酸酶以诱导植物应激反应的详细分子基础。该奖项由分子和细胞生物科学部的细胞动力学和功能簇以及综合组织系统部的植物、真菌和微生物发育机制计划共同资助。该奖项反映了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。

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.