Defining a new realm of proteolysis activated protein function

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 We will use published and unpublished data derived from project partners for one type of endopeptidase family, metacaspases, that allows the identification of possible substrates of the N-degron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

Defining the role of proteostasis in controlling protein activity is a key goal towards understanding proteome function, and targeted protease action on specific proteins is a fundamental mechanism regulating proteostasis. Plant genomes contain many proteases, but their targets and biological function remain largely unknown. This proposal aims to address this major knowledge gap by defining important targets of the metacaspase endopeptidases and understanding biological consequences of protein cleavage by metacaspases. Targeted endopeptidase proteolysis provides a dynamic mechanism to alter proteome composition and function by generating novel proteoforms. Following cleavage of proteins by endopeptidases, amino (N) and carboxy (C) terminal proteoform fragments are produced. For C-terminal fragments this results in a novel N-terminal residue. N-degron pathways regulate C-terminal proteoform half-life dependent on the amino-terminal residue revealed after cleavage. Artificial substrates have been used to show that N-degron pathways exist in plants, but only 4 physiological substrates, restricted to 2 branches, have been identified. In three work packages we will investigate three principles underlying endopeptidase action and subsequent N-degron pathway function. We will demonstrate that novel substrates of N-degron pathways are revealed after metacaspase cleavage, we will show that conditional stability of substrates occurs in cells, and that conditional stability has biological importance. The proposed work is timely, building on preliminary data developed by the project partners, and provides the promise of defining a new realm of plant protein biochemistry with important biological effects in relation to environmental interactions. The project involves a combination of inter-disciplinary approaches spanning Mass Spectrometry, enzymology, genetics and plant physiology, possible through collaboration of biologists and chemists, providing excellent training potential