Quantitative proteomics in plant protease substrate identification

Abstract

Contents Summary936I.Introduction936II.The quest for plant protease substrates – proteomics to the rescue?937III.Quantitative proteome comparison reveals candidate substrates938IV.Dynamic metabolic stable isotope labeling to measure protein turnover in vivo938V.Terminomics – large‐scale identification of protease cleavage sites939VI.Substrate or not substrate, that is the question940VII.Concluding remarks941Acknowledgements941References941 Summary Proteolysis is a central regulatory mechanism of protein homeostasis and protein function that affects all aspects of plant life. Higher plants encode for hundreds of proteases, but their physiological substrates and hence their molecular functions remain mostly unknown. Current quantitative mass spectrometry‐based proteomics enables unbiased large‐scale interrogation of the proteome and its modifications. Here we provide an overview of proteomics techniques that allow profiling of changes in protein abundance, measurement of proteome turnover rates, identification of protease cleavage sites in vivo and in vitro and determination of protease sequence specificity. We discuss how these techniques can help to reveal protease substrates and determine plant protease function, illustrated by recent studies on selected plant proteases.

The iTRAQ-based chloroplast proteomic analysis of Triticum aestivum L. leaves subjected to drought stress and 5-aminolevulinic acid alleviation reveals several proteins involved in the protection of photosynthesis

“…For example, the histones H2A, H2B, H3, and H4 were all down-regulated by the exogenous application of ALA under drought stress, which were coincident with results reported in rice under cold stress [62]. The non-chloroplast protein contaminations should be attributed to the Percoll-based chloroplast purification protocol that cannot fully exclude cross-contaminations originating from other plastids and cell compartments, even it has been well established [7,8]. Histones are prone to reversible posttranslational modifications such as phosphorylation, ubiquitination, acetylation, methylation, and glycosylation, which allow the proteins to respond flexibly to stimuli [63].…”

“…Therefore, whole leaf-based studies have a limitation in attempting to reveal the function of chloroplast proteins. A proteomic analysis following chloroplast isolation provides a promising approach for elucidating chloroplast-located protein regulation and its relationship with stress resistance [7,8]. Until now, the strategy of chloroplast proteomics has been employed to investigate the possible molecular regulatory pathways in response to biotic or abiotic stress in a variety of plants, such as the chlorosis mechanisms of Nicotiana tabacum leaves induced by viral infection [9], the response mechanism of tomato (Solanum lycopersicum L.) to drought stress and recovery [10], and the pathways associated with salt tolerance in Kandelia candel [11].…”

Plant proteases during developmental programmed cell death

“…So far, only a few publications describe the dynamic subcellular localization of proteases, for instance by fusion proteins that take activation cleavage sites into account (Hierl et al, 2014). Additionally, strategies to identify the protease targets in quantitative degradomics studies (Demir et al, 2018;Huesgen and Overall, 2012) will be an important path to a functional understanding of the role proteases play in the control and execution of dPCD in plants. Coordinated with cellular differentiation, dPCD competency is achieved at least partly on the transcriptional level.…”

Ten Prominent Host Proteases in Plant-Pathogen Interactions

“…Our current knowledge of the exact roles of proteases places them as key players in many facets of pathogen responses. Future efforts in this field will need to address the lack of known substrates e.g., by applying novel mass-spectrometry based methods [ 84 , 85 ], the assignment of subcellular localisation, and the role of proteases in interactions with different pathogens. Despite these limitations, proteases are now well established as important contributors to host defence.…”