Defining a new realm of proteolysis activated protein function

这项申请描述了将改变我们对植物蛋白酶的生物学作用、它们对植物细胞内蛋白质的作用以及它们作用的生物学后果的理解的工作。它旨在解决两个相关的问题：蛋白质被细胞中的蛋白酶切割后会发生什么？而且，重要的生物功能是否通过新产生的蛋白质片段的稳定性来调节？内切酶是一种在特定的内部位置裂解蛋白质，从而产生两个或两个以上新的多肽的蛋白酶。虽然植物基因组中含有许多内切酶，但已知的靶标很少，切割靶标蛋白的生物学后果也知之甚少。这一应用将检验中心假设，即细胞中特定蛋白质的功能是由内肽酶通过称为N-degron通路的特定蛋白质降解机制来调节的。在被内肽酶切割后，新的氨基端残基被显示在被切割的蛋白质中，这些残基作为促进新产生的多肽降解的信号。我们将使用从项目合作伙伴那里获得的已发表和未发表的数据来研究一种内肽酶家族，即Metacaspase，它允许识别N-degron途径的可能底物。在拟议的研究计划中，将研究N-降解子途径的三个原理。首先，我们将证明底物被Metacaspase切割后被引入到N-degron途径中。在初步工作中，我们已经鉴定了超过25个具有不同氨基末端残基的候选蛋白质片段，代表了N-degron通路的许多分支。其次，我们将研究被偏半胱氨酸酶切割的蛋白质的命运。在这里，我们将定义多肽是在内切酶裂解后立即降解，还是在裂解后降解是有条件的，并可能取决于细胞内的变化，例如N-degron途径酶活性的变化。初步数据显示，对于我们已经确定的一些Metacaspase靶蛋白，降解受到特定环境变化的抑制。最后，我们将调查Metacaspase对蛋白质的切割是否具有重要的生物学后果。作为一个例子，我们以前证明了植物氧感应系统，它是由Metap蛋白酶切割ERFVII转录因子启动的，并通过N-degron途径控制，表现出这三个原理(Gibbs等人自然2011，Abbas等自然2022)，因此我们希望在这个项目中找到许多重要的例子。在拟议的工作中，我们将通过N-降解子途径发现植物蛋白酶在调节蛋白质活性方面的新成分和新机制。通过提供完全重新定义N-降解子途径作用的新信息，这项工作还将促进创造新的资源和方法，以解决与多种环境胁迫耐受性相关的农艺问题。该项目的中心将是跨越质谱学、酶学、遗传学和植物生理学的跨学科实验方法的组合，这只有由于生物学家和化学家之间拟议的合作才有可能。因此，该项目为新的跨学科培训提供了巨大的潜力。拟议的工作是及时的，建立在我们初步数据的基础上，并提供了通过定义N-降解子途径底物的广度、调节其稳定性的条件性以及条件稳定性的表型意义来直接解决主要知识差距的机会。

This application describes work that will transform our understanding of the biological roles of plant proteases, their action on proteins within plant cells and the biological consequences of their action. It aims to address two related questions; what happens to proteins after they are cleaved by proteases in the cell? And, is important biological function regulated through the stability of the newly produced protein fragments? Endoproteases are a type of protease that cleave proteins at specific internal sites resulting in the production of two or more new peptides. Although plant genomes contain many endoproteases, very few targets are known and the biological consequences of cutting target proteins are poorly understood. This application will test the central hypothesis that the function of specific proteins in cells is regulated by endopeptideases through a specific mechanism of protein degradation called the N-degron pathways. Following cutting by endopeptidases new amino-terminal residues are revealed in cleaved proteins, and these residues act as a signal promoting degradation of the newly produced peptides. We will use published and unpublished data derived from project partners for one type of endopeptidase family, metacaspases, that allows the identification of possible substrates of the N-degron pathways. Three principles of N-degron pathway function will be investigated in the proposed research programme. Firstly, we will prove that substrates are introduced into the N-degron pathways following their cleavage by metacaspases. In preliminary work we have already identified over 25 candidate protein fragments with different amino-terminal residues, representing many of the branches of the N-degron pathways. Secondly, we will investigate the fate of proteins cleaved by metacaspases. Here we will define whether peptides are degraded immediately after endopeptidase cleavage, or whether degradation after cleavage is conditional, and might depend on changes within the cell, for example changes in the activities of enzymes of the N-degron pathways. Preliminary data shows that for some metacaspase target proteins that we have identified, degradation is inhibited by specific environmental changes. Finally, we will investigate whether cleavage of proteins by metacaspases has biologically important consequences. As an example, we previously showed that the plant oxygen sensing system, that is initiated by cleavage of ERFVII transcription factors by MetAP proteases and is controlled through an N-degron pathway, exhibits these three principles (Gibbs et al Nature 2011, Abbas et al Nature 2022), and we therefore expect to find many important examples in this project. In the proposed work we will discover new components and mechanisms of plant protease function in regulating protein activity though the N-degron pathways. By providing new information that will completely redefine the role of the N-degron pathways, the work will also facilitate the creation of novel resources and approaches to address agronomic problems associated with multiple environmental stress tolerances. Central to the project will be the combination of inter-disciplinary experimental approaches spanning Mass Spectrometry, enzymology, genetics and plant physiology, only possible because of the proposed collaboration between biologists and chemists. The project therefore provides great potential for novel interdisciplinary training. The proposed work is timely and builds on our preliminary data, and offers the opportunity to directly address a major knowledge gap by defining the breadth of N-degron pathway substrates, the conditionality of the regulation of their stability, and the phenotypic significance of conditional stability.