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Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b
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Inhibitor Discovery by Convolution ABPP.

Balakumaran Chandrasekar1,2, Tram Ngoc Hong1,2, Renier A L van der Hoorn3

  • 1The Plant Chemetics Laboratory, Max Planck Institute for Plant Breeding Research, Carl-von-Linne Weg 10, 50829, Cologne, Germany.

Methods in Molecular Biology (Clifton, N.J.)
|October 26, 2016
PubMed
Summary

Convolution activity-based protein profiling (ABPP) detects protein inhibitors by comparing labeling before and after mixing proteomes. This method identified a beta-galactosidase inhibitor in plant-pathogen interactions.

Keywords:
Activity-based protein profiling (ABPP)Beta-galactosidaseConvolution ABPPCyclophellitol-aziridineGalactostatinNicotiana benthamianaPseudomonas syringaeSecreted proteomes

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Area of Science:

  • Proteomics
  • Biochemistry
  • Chemical Biology

Background:

  • Activity-based protein profiling (ABPP) is a proteomic technique utilizing chemical probes to label active proteins in their native biological context.
  • Traditional ABPP methods include comparative and competitive approaches, each with limitations in inhibitor detection.
  • There is a need for methods that can specifically identify the presence of inhibitors affecting protein activity.

Purpose of the Study:

  • To introduce and validate a novel ABPP method, termed convolution ABPP, for detecting inhibitors in proteomes.
  • To demonstrate the utility of convolution ABPP in identifying inhibitors by comparing labeling intensities in mixed and unmixed proteomes.
  • To showcase the application of convolution ABPP in a biological system, specifically in plant-pathogen interactions.

Main Methods:

  • Convolution ABPP involves comparing protein labeling intensities between two conditions: labeling proteomes before mixing (label-and-mix) and labeling after mixing (mix-and-label).
  • A decrease in labeling signal in the mix-and-label sample compared to the label-and-mix sample indicates the presence of an inhibitor in one of the original proteomes.
  • The method was applied to analyze secreted proteomes from Pseudomonas syringae-infected Nicotiana benthamiana leaves.

Main Results:

  • Convolution ABPP successfully identified a reduction in labeling signal in the mix-and-label sample, indicating the presence of inhibitors.
  • The study demonstrated the presence of a beta-galactosidase inhibitor in the secreted proteomes of Pseudomonas syringae-infected Nicotiana benthamiana leaves.
  • This proof-of-concept application highlights the method's effectiveness in complex biological samples.

Conclusions:

  • Convolution ABPP is a robust and broadly applicable method for detecting inhibitors of protein activity within proteomes.
  • The technique offers a valuable tool for understanding the mechanisms of inhibition in various biological systems, including plant immunity.
  • This approach enhances the capabilities of activity-based protein profiling for inhibitor discovery and characterization.