EAGER: Optimization of proximity labeling approach to understand composition and regulation of the plant ubiquitin ligases interactome

该项目的目标是使用一种新技术来鉴定与细胞蛋白质降解机制的一个组成部分相互作用的蛋白质。蛋白质降解机器最著名的是泛素蛋白酶体系统（UPS）已经成为几乎所有生物过程的中心。UPS介导的蛋白质降解是一个高度调节的过程，不能及时降解特定蛋白质将对生物体产生有害影响。在植物中，UPS控制许多过程，包括生长和发育，激素信号，以及对生物和非生物胁迫的反应。已知植物适应UPS以促进响应和耐受不利环境条件所需的细胞变化。由于基本的生物学过程（如对环境变化的反应）的机制通常在植物中是保守的，因此我们在该项目中的发现将广泛适用于不同的植物物种。此外，该项目将通过以下方式对科学和教育产生超越当前目标的影响：（1）为本科生和高级研究人员提供指导，研究经验和新颖的平台，（2）在更大的社区中宣传植物科学的重要性，以及（3）通过推进新兴植物功能蛋白质组学领域的前沿方法的知识。尽管在植物功能蛋白质组学领域取得了进展，尽管植物中的蛋白水解调节是已知的，但关于植物泛素（Ub）连接酶的组成和调节，特别是在不同环境条件下的组成和调节知之甚少。虽然越来越多的基因组研究暗示了Ub连接酶在调节生物和非生物胁迫信号传导中的关键作用，但在蛋白质水平上的全面研究仍然没有定位。要全面了解工厂中的UPS流程，需要确定Ub系统目标和其他调节器（例如，去泛素化酶、泛素样蛋白和泛素相关蛋白）。到目前为止，由于捕获动态Ub连接酶复合物的技术挑战，在蛋白质水平上仅进行了非常有限的尝试来表征来自植物细胞的Ub连接酶。因此，在EAGER项目中，我们将具体：（1）开发和优化邻近标记工具，以分离Cullin-RINGs（CRL）Ub连接酶及其相互作用物，（2）使用优化的方法和材料，在正常生长条件下分离特定的Ub连接酶相互作用物。此外，我们将使用MAX2 F-box蛋白作为测试案例，以证明接近标记方法在捕获MAX2的相互作用物以响应独脚金内酯激素治疗中的实用性。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

The goal of this project is to use a novel technology to identify proteins that interact with a component of the cellular protein degradation machinery. The protein degradation machine best known as the Ubiquitin Proteasome System (UPS) has emerged to be at the center of virtually every biological process. UPS-mediated protein degradation is a highly regulated process and failure to degrade specific proteins in a timely fashion will have a deleterious effect on the organism. In plants, UPS control numerous processes including growth and development, hormone signaling, and responses to biotic and abiotic stress. Plants are known to adapt the UPS to facilitate cellular changes required to respond to and tolerate adverse environmental conditions. Since the mechanisms underlying fundamental biological processes such as responses to environmental changes are often conserved in plants, our findings from this project will be broadly applicable across different plant species. Furthermore, the project will impact science and education beyond the immediate goals by: (1) providing mentorship, research experiences, and novel platform for undergraduate students and senior researchers, (2) propagating the importance of plant sciences in the larger community, and (3) by advancing the knowledge in cutting edge approaches of the emerging plant functional proteomic field.Despite the advances made in the field of proteolytic regulation in plants, little is known about the composition and regulation of plant ubiquitin (Ub) ligase enzymes, in particular under diverse environmental conditions. While increasing number of genomic studies implicate the critical role for Ub ligase in regulating biotic and abiotic stress signaling, comprehensive studies at the protein levels remain unmapped. A full understanding of UPS processes in plants demands the identification of Ub system targets and other regulators (e.g., deubiquitinating enzymes, ubiquitin-like and ubiquitin associated proteins). Thus far, only very limited attempts at the protein levels have been made to characterize Ub ligases from plant cells because of technical challenges in capturing dynamic Ub ligase complexes. Therefore, in this EAGER project, we will specifically: (1) Develop and optimize proximity labeling tools to isolate Cullin-RINGs (CRLs) Ub ligases and their interactors in planta and, (2) Use optimized methods and materials to isolate specific Ub ligase interactomes under normal growth conditions. In addition, we will use the MAX2 F-box protein as a test case to demonstrate the utility of the proximity labeling approach in capturing interactors of MAX2 in response to strigolactone hormone treatment.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.