The Increasing Impact of Activity-Based Protein Profiling in Plant Science

Morimoto, Kanehisa; Hoorn, Renier A. L. van der

doi:10.1093/pcp/pcw003

Cited by 61 publications

(54 citation statements)

References 81 publications

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“…Suitable experimental approaches are highly dependent on the target, but include confirmation of coexpression and co‐localization in vivo (Tsiatsiani et al ., ; Bhuiyan et al ., ), tests for direct interaction in vivo and in vitro (Nishimura et al ., ; Bhuiyan et al ., ), evaluation whether complementation lines shows the expected opposite effect (Tsiatsiani et al ., ; Zimmermann et al ., ) and evaluation whether the protease is active under physiological conditions. Activity based protein profiling (ABPP) with class‐specific chemical probes can monitor protease activity in vivo and in vitro and, as a chemical proteomics method (Table S1), reveal the proteases that participate in the active protease web of a given tissue or proteome (Morimoto & van der Hoorn, ).…”

Section: Substrate or Not Substrate That Is The Questionmentioning

confidence: 99%

Quantitative proteomics in plant protease substrate identification

et al. 2017

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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.

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Section: Substrate or Not Substrate That Is The Questionmentioning

confidence: 99%

Quantitative proteomics in plant protease substrate identification

et al. 2017

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“…To study protease activities, rather than transcript or protein accumulation, we applied activity-based protease profiling (ABPP). ABPP is based on the use of fluorescent or biotinylated chemical probes that react irreversibly with the active site of enzymes in a mechanism-dependent manner [39–41]. Here, we applied ABPP to study protease activities during leaf senescence induced by individually darkening leaves of Arabidopsis and we used PLCP and VPE mutants and over expressing lines to confirm the origin of these signals and determine the relative contribution of these proteases to leaf senescence.…”

Section: Introductionmentioning

confidence: 99%

Major Cys protease activities are not essential for senescence in individually darkened Arabidopsis leaves

et al. 2017

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BackgroundPapain-like Cys Proteases (PLCPs) and Vacuolar Processing Enzymes (VPEs) are amongst the most highly expressed proteases during leaf senescence in Arabidopsis. Using activity-based protein profiling (ABPP), a method that enables detection of active enzymes within a complex sample using chemical probes, the activities of PLCPs and VPEs were investigated in individually darkened leaves of Arabidopsis, and their role in senescence was tested in null mutants.ResultsABPP and mass spectrometry revealed an increased activity of several PLCPs, particularly RD21A and AALP. By contrast, despite increased VPE transcript levels, active VPE decreased in individually darkened leaves. Eight protease knock-out lines and two protease over expressing lines were subjected to senescence phenotype analysis to determine the importance of individual protease activities to senescence. Unexpectedly, despite the absence of dominating PLCP activities in these plants, the rubisco and chlorophyll decline in individually darkened leaves and the onset of whole plant senescence were unaltered. However, a significant delay in progression of whole plant senescence was observed in aalp-1 and rd21A-1/aalp-1 mutants, visible in the reduced number of senescent leaves.ConclusionsMajor Cys protease activities are not essential for dark-induced and developmental senescence and only a knock out line lacking AALP shows a slight but significant delay in plant senescence.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0955-5) contains supplementary material, which is available to authorized users.

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“…We performed activity‐based protein profiling (ABPP) to determine the inhibitory spectrum of Sl CYS8 and proteomics to identify target proteases. ABPP is increasingly used in plant science to monitor the activity of hundreds of proteins using tagged chemical probes that react covalently and irreversibly with the active site of proteins (Morimoto and van der Hoorn, ). Here, we used ABPP to show that Sl CYS8 selectively inhibits nine papain‐like cysteine proteases in N. benthamiana .…”

Section: Introductionmentioning

confidence: 99%

Activity‐based proteomics reveals nine target proteases for the recombinant protein‐stabilizing inhibitor SlCYS8 in Nicotiana benthamiana

Jutras

Grosse‐Holz

Kaschani

et al. 2019

Plant Biotechnology Journal

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Summary Co‐expression of protease inhibitors like the tomato cystatin Sl CYS 8 is useful to increase recombinant protein production in plants, but key proteases involved in protein proteolysis are still unknown. Here, we performed activity‐based protein profiling to identify proteases that are inhibited by Sl CYS 8 in agroinfiltrated Nicotiana benthamiana . We discovered that Sl CYS 8 selectively suppresses papain‐like cysteine protease ( PLCP ) activity in both apoplastic fluids and total leaf extracts, while not affecting vacuolar‐processing enzyme and serine hydrolase activity. A robust concentration‐dependent inhibition of PLCP s occurred in vitro when purified Sl CYS 8 was added to leaf extracts, indicating direct cystatin– PLCP interactions. Activity‐based proteomics revealed that nine different Cathepsin‐L/‐F‐like PLCP s are strongly inhibited by Sl CYS 8 in leaves. By contrast, the activity of five other Cathepsin‐B/‐H‐like PLCP s, as well as 87 Ser hydrolases, was unaffected by Sl CYS 8. Sl CYS 8 expression prevented protein degradation by inhibiting intermediate and mature isoforms of granulin‐containing proteases from the Resistant‐to‐Desiccation‐21 ( RD 21) P LCP subfamily. Our data underline the key role of endogenous PLCP s on recombinant protein degradation and reveal candidate proteases for depletion strategies.

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The Increasing Impact of Activity-Based Protein Profiling in Plant Science

Cited by 61 publications

References 81 publications

Quantitative proteomics in plant protease substrate identification

Quantitative proteomics in plant protease substrate identification

Major Cys protease activities are not essential for senescence in individually darkened Arabidopsis leaves

Activity‐based proteomics reveals nine target proteases for the recombinant protein‐stabilizing inhibitor SlCYS8 in Nicotiana benthamiana

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