Understanding cullin-RING E3 biology through proteomics-based substrate identification

Abstract

Protein turnover through the ubiquitin-proteasome pathway controls numerous developmental decisions and biochemical processes in eukaryotes. Central to protein ubiquitylation are ubiquitin ligases, which provide specificity in targeted ubiquitylation. With more than 600 ubiquitin ligases encoded by the human genome, many of which remain to be studied, considerable effort is being placed on the development of methods for identifying substrates of specific ubiquitin ligases. In this review, we describe proteomic technologies for the identification of ubiquitin ligase targets, with a particular focus on members of the cullin-RING E3 class of ubiquitin ligases, which use F-box proteins as substrate specific adaptor proteins. Various proteomic methods are described and are compared with genetic approaches that are available. The continued development of such methods is likely to have a substantial impact on the ubiquitin-proteasome field.

Fig. 1.

Application of CompPASS-based proteomics for identification of substrates and binding partners of SCFβ-TRCP2. Dot plot depicting known β-TrCP2 interactors, including substrates, identified after IP-LC/MS and CompPASS analysis. Red dots indicate proteins with increased spectral counts in proteasome inhibitor (Bortezomib) treated sample relative to the untreated sample. Proteins that were not detected in the absence of Bortezomib are labeled in bold type.

Fig. 2.

Methods for enrichment of substrates in association with F-box proteins for identification by IP-LC/MS. A, diagram showing the ubiquitylation and subsequent degradation of SCF substrates. B, diagram showing that inhibiting the proteasome by treating cells with proteasomal inhibitors such as MG132 or Bortezomib results in the accumulation of SCF substrates and increases the fraction of substrates bound to the cognate FBP. C, diagram showing how the introduction of an FBP lacking the F-box domain can increase the cellular pool of its substrate, allowing more substrates to bind this mutant FBP.

Fig. 3.

Quantitative MS approach using SILAC. A, diagram showing how different treatments can alter the abundance and number of diGly remnants found on tryptic peptides for a ubiquitylated CRL substrate. B, outline of the quantitative MS approach using SILAC together with the anti-diGly antibody.