A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate

Biochim Biophys Acta. 2012 Nov;1823(11):2069-78. doi: 10.1016/j.bbamcr.2012.05.006. Epub 2012 May 22.

Abstract

Substrate ubiquitylation is a reversible process critical to cellular homeostasis that is often dysregulated in many human pathologies including cancer and neurodegeneration. Elucidating the mechanistic details of this pathway could unlock a large store of information useful to the design of diagnostic and therapeutic interventions. Proteomic approaches to the questions at hand have generally utilized mass spectrometry (MS), which has been successful in identifying both ubiquitylation substrates and profiling pan-cellular chain linkages, but is generally unable to connect the two. Interacting partners of the deubiquitylating enzymes (DUBs) have also been reported by MS, although substrates of catalytically competent DUBs generally cannot be. Where they have been used towards the study of ubiquitylation, protein microarrays have usually functioned as platforms for the identification of substrates for specific E3 ubiquitin ligases. Here, we report on the first use of protein microarrays to identify substrates of DUBs, and in so doing demonstrate the first example of microarray proteomics involving multiple (i.e., distinct, sequential and opposing) enzymatic activities. This technique demonstrates the selectivity of DUBs for both substrate and type (mono- versus poly-) of ubiquitylation. This work shows that the vast majority of DUBs are monoubiquitylated in vitro, and are incapable of removing this modification from themselves. This work also underscores the critical role of utilizing both ubiquitin chains and substrates when attempting to characterize DUBs. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Endopeptidases / metabolism*
  • Humans
  • Proteasome Endopeptidase Complex / metabolism*
  • Protein Array Analysis*
  • Proteomics*
  • Substrate Specificity
  • Ubiquitin / metabolism*
  • Ubiquitin-Protein Ligases / metabolism*
  • Ubiquitinated Proteins / metabolism*
  • Ubiquitination

Substances

  • Ubiquitin
  • Ubiquitinated Proteins
  • Ubiquitin-Protein Ligases
  • Endopeptidases
  • Proteasome Endopeptidase Complex