title: Detection of ubiquitin-proteasome enzymatic activities in cells: application of activity-based probes to inhibitor development.
abstract: BACKGROUND
  Synthetic probes that mimic natural substrates can enable the detection of enzymatic activities in a cellular environment. One area where such activity-based probes have been applied is the ubiquitin-proteasome pathway, which is emerging as an important therapeutic target. A family of reagents has been developed that specifically label deubiquitylating enzymes (DUBs) and facilitate characterization of their inhibitors.
  
  
  METHODS
  Here we focus on the application of probes for intracellular DUBs, a group of specific proteases involved in the ubiquitin proteasome system. In particular, the functional characterization of the active subunits of this family of proteases that specifically recognize ubiquitin and ubiquitin-like proteins will be discussed. In addition we present the potential and design of activity-based probes targeting kinases and phosphatases to study phosphorylation.
  
  
  CONCLUSIONS
  Synthetic molecular probes have increased our understanding of the functional role of DUBs in living cells. In addition to the detection of enzymatic activities of known members, activity-based probes have contributed to a number of functional assignments of previously uncharacterized enzymes. This method enables cellular validation of the specificity of small molecule DUB inhibitors.
  
  
  CONCLUSIONS
  Molecular probes combined with mass spectrometry-based proteomics and cellular assays represent a powerful approach for discovery and functional validation, a concept that can be expanded to other enzyme classes. This addresses a need for more informative cell-based assays that are required to accelerate the drug development process. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.
authors.affiliation: Department of Physiology, Anatomy & Genetics, University of Oxford, Oxford OX1 3QX, UK.
authors.forename: Holger B
authors.initials: HB
authors.lastname: Kramer
authors.forename: Benjamin
authors.initials: B
authors.lastname: Nicholson
authors.forename: Benedikt M
authors.initials: BM
authors.lastname: Kessler
authors.forename: Mikael
authors.initials: M
authors.lastname: Altun
mesh_terms: D000818:Animals; D015195:Drug Design; D006801:Humans; D015335:Molecular Probes; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D015335:Molecular Probes; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex