Activity profiling of papain-like cysteine proteases in plants

R.A.L. van der Hoorn, M.A. Leeuwenburgh, M. Bogyo, M.H.A.J. Joosten, S.C. Peck

Research output: Contribution to journalArticleAcademicpeer-review

103 Citations (Scopus)

Abstract

Transcriptomic and proteomic technologies are generating a wealth of data that are frequently used by scientists to predict the function of proteins based on their expression or presence. However, activity of many proteins, such as transcription factors, kinases, and proteases, depends on posttranslational modifications that frequently are not detected by these technologies. Therefore, to monitor activity of proteases rather than their abundance, we introduce protease activity profiling in plants. This technology is based on the use of biotinylated, irreversible protease inhibitors that react with active proteases in a mechanism-based manner. Using a biotinylated derivative of the Cys protease inhibitor E-64, we display simultaneous activities of many papain-like Cys proteases in extracts from various tissues and from different plant species. Labeling is pH dependent, stimulated with reducing agents, and inhibited specifically by Cys protease inhibitors but not by inhibitors of other protease classes. Using one-step affinity capture of bintinylated proteases followed by sequencing mass spectrometry, we identified proteases that include xylem-specific XCP2, desiccation-induced RD21, and cathepsin B- and aleurain-like proteases. Together, these results demonstrate that this technology can identify differentially activated proteases and/or characterize the activity of a particular protease within complex mixtures.
Original languageEnglish
Pages (from-to)1170-1178
JournalPlant Physiology
Volume135
Issue number3
DOIs
Publication statusPublished - 2004

Fingerprint

Papain
Cysteine Proteases
papain
cysteine proteinases
Peptide Hydrolases
proteinases
proteinase inhibitors
Protease Inhibitors
Technology
cathepsin B
Xylem
Desiccation
Cathepsin B
Tissue Extracts
reducing agents
Reducing Agents
post-translational modification
Post Translational Protein Processing
Complex Mixtures
transcriptomics

Keywords

  • large-scale identification
  • programmed cell-death
  • arabidopsis-thaliana
  • proteolytic-enzymes
  • expression
  • proteins
  • proteome
  • serine
  • genes
  • senescence

Cite this

van der Hoorn, R. A. L., Leeuwenburgh, M. A., Bogyo, M., Joosten, M. H. A. J., & Peck, S. C. (2004). Activity profiling of papain-like cysteine proteases in plants. Plant Physiology, 135(3), 1170-1178. https://doi.org/10.1104/pp.104.041467
van der Hoorn, R.A.L. ; Leeuwenburgh, M.A. ; Bogyo, M. ; Joosten, M.H.A.J. ; Peck, S.C. / Activity profiling of papain-like cysteine proteases in plants. In: Plant Physiology. 2004 ; Vol. 135, No. 3. pp. 1170-1178.
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van der Hoorn, RAL, Leeuwenburgh, MA, Bogyo, M, Joosten, MHAJ & Peck, SC 2004, 'Activity profiling of papain-like cysteine proteases in plants', Plant Physiology, vol. 135, no. 3, pp. 1170-1178. https://doi.org/10.1104/pp.104.041467

Activity profiling of papain-like cysteine proteases in plants. / van der Hoorn, R.A.L.; Leeuwenburgh, M.A.; Bogyo, M.; Joosten, M.H.A.J.; Peck, S.C.

In: Plant Physiology, Vol. 135, No. 3, 2004, p. 1170-1178.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Activity profiling of papain-like cysteine proteases in plants

AU - van der Hoorn, R.A.L.

AU - Leeuwenburgh, M.A.

AU - Bogyo, M.

AU - Joosten, M.H.A.J.

AU - Peck, S.C.

PY - 2004

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AB - Transcriptomic and proteomic technologies are generating a wealth of data that are frequently used by scientists to predict the function of proteins based on their expression or presence. However, activity of many proteins, such as transcription factors, kinases, and proteases, depends on posttranslational modifications that frequently are not detected by these technologies. Therefore, to monitor activity of proteases rather than their abundance, we introduce protease activity profiling in plants. This technology is based on the use of biotinylated, irreversible protease inhibitors that react with active proteases in a mechanism-based manner. Using a biotinylated derivative of the Cys protease inhibitor E-64, we display simultaneous activities of many papain-like Cys proteases in extracts from various tissues and from different plant species. Labeling is pH dependent, stimulated with reducing agents, and inhibited specifically by Cys protease inhibitors but not by inhibitors of other protease classes. Using one-step affinity capture of bintinylated proteases followed by sequencing mass spectrometry, we identified proteases that include xylem-specific XCP2, desiccation-induced RD21, and cathepsin B- and aleurain-like proteases. Together, these results demonstrate that this technology can identify differentially activated proteases and/or characterize the activity of a particular protease within complex mixtures.

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KW - arabidopsis-thaliana

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KW - serine

KW - genes

KW - senescence

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