Defining a new realm of proteolysis activated protein function

定义蛋白水解激活蛋白功能的新领域

• 批准号: BB/X014258/1
• 负责人: Michael Holdsworth
• 金额: $ 78.75万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX014258%2F1
• 关键词: Defining; new; realm; proteolysis; activated

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 We will use published and unpublished data derived from project partners for one type of endopeptidase family, metacaspases, that allows the identification of possible substrates of the N-degron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.

本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的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？重要的生物学功能是通过新产生的蛋白质片段的稳定性来调节的吗？内切蛋白酶是一种蛋白酶，其在特定的内部位点切割蛋白质，导致产生两种或更多种新肽。虽然植物基因组含有许多内切蛋白酶，但已知的靶点很少，切割靶蛋白的生物学后果也知之甚少。该应用将测试中心假设，即细胞中特定蛋白质的功能由内肽酶通过称为N-降解决定子途径的蛋白质降解的特定机制来调节。在被内肽酶切割后，在切割的蛋白质中显示新的氨基末端残基，并且这些残基充当促进新产生的肽降解的信号。我们将使用来自项目合作伙伴的已发表和未发表的数据，用于一种类型的内肽酶家族，metacaspases，可以识别N-降解决定子途径的可能底物。在拟议的研究计划中，将研究N-降解决定子途径功能的三个原则。首先，我们将证明，底物被引入到N-降解决定子途径后，他们的切割metacaspases。在初步工作中，我们已经确定了超过25个具有不同氨基末端残基的候选蛋白片段，代表了N-降解决定子途径的许多分支。其次，我们将研究被后半胱氨酸天冬氨酸蛋白酶切割的蛋白质的命运。在这里，我们将定义肽是否在内肽酶切割后立即降解，或者切割后的降解是否是有条件的，并且可能取决于细胞内的变化，例如N-降解决定子途径的酶活性的变化。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，我们将调查是否由metacaspases蛋白质切割具有生物学上的重要后果。作为一个例子，我们以前表明，植物氧传感系统，这是由ERFVII转录因子的切割由MetAP蛋白酶启动，并通过N-降解决定子途径控制，表现出这三个原则（吉布斯等自然2011，阿巴斯等自然2022），因此，我们希望在这个项目中找到许多重要的例子。本论文的工作将揭示植物蛋白酶通过N-降解决定子途径调控蛋白质活性的新组分和机制。通过提供新的信息，将完全重新定义N-降解决定子途径的作用，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学问题。该项目的核心将是跨学科实验方法的结合，包括质谱，酶学，遗传学和植物生理学，只有生物学家和化学家之间的合作才有可能。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，并建立在我们的初步数据，并提供了机会，直接解决一个主要的知识差距，通过定义的广度N-降解决定子途径底物，调节其稳定性的条件性，和条件稳定性的表型意义。