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Related Concept Videos

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DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Sigma's Non-specific Protease Activity Assay - Casein as a Substrate
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Profiling DUBs and Ubl-specific proteases with activity-based probes.

Paul P Geurink1, Gerbrand J van der Heden van Noort1, Monique P C Mulder1

  • 1Department of Cell and Chemical Biology, Chemical Immunology, Oncode Institute, Leiden University Medical Centre, Leiden, The Netherlands.

Methods in Enzymology
|March 10, 2019
PubMed
Summary
This summary is machine-generated.

Activity-based probes (ABPs) are valuable tools for studying deubiquitinating enzymes (DUBs) and ubiquitin-like protein (Ubl) proteases. ABPs help characterize enzyme activity, substrate preferences, and structural features, offering insights into cellular processes and pathogen interactions.

Keywords:
(di)ubiquitin-based probesActivity-based probesDeubiquitinating enzymesMERS-CoV PLproSARS-CoV PLproUb linkage specificityUbl-specific proteasesUb–Ubl crosstalkViral DUBs

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Protein (poly-)ubiquitination is a crucial posttranslational modification regulating cellular processes.
  • Deubiquitinating enzymes (DUBs) reverse ubiquitination, and their dysfunction is linked to human diseases.
  • The ubiquitin system interacts with ubiquitin-like proteins (Ubls), forming heterogeneous chains and exhibiting cross-reactivity with DUBs.

Purpose of the Study:

  • To explore the utility of activity-based probes (ABPs) for studying deubiquitinating enzymes (DUBs) and ubiquitin-like (Ubl) proteases.
  • To demonstrate the application of ABPs in characterizing enzyme activity, substrate preferences, and structural features.
  • To highlight the role of ABPs in understanding viral protease activity and DUB substrate specificity.

Main Methods:

  • Utilizing activity-based probes (ABPs) to covalently label and report on active DUBs and Ubl proteases.
  • Assessing substrate preferences and structural characteristics of deconjugating enzymes using ABPs.
  • Characterizing the activity of viral proteases against ubiquitin and Ubls with ABPs.

Main Results:

  • ABPs provide a direct measure of enzyme activity, independent of protein abundance.
  • ABPs enable detailed analysis of substrate specificity and structural determinants in DUBs.
  • ABPs are effective in studying both human and pathogen-derived deconjugating enzymes.

Conclusions:

  • Activity-based probes are powerful tools for investigating the function and regulation of DUBs and Ubl proteases.
  • ABPs offer fundamental insights into the physiological roles of deconjugating enzymes and their involvement in disease and host-pathogen interactions.
  • The application of ABPs advances our understanding of enzyme mechanisms and substrate recognition in the ubiquitin system.