Comprehensive Identification of Substrates for F-box Proteins by Differential Proteomics Analysis

Although elucidation of enzyme–substrate relations
is fundamental
to the advancement of biology, universal approaches to the identification
of substrates for a given enzyme have not been established. It is
especially difficult to identify substrates for ubiquitin ligases,
given that most such substrates are immediately ubiquitylated and
degraded as a result of their association with the enzyme. We here
describe the development of a new approach, DiPIUS (differential proteomics-based
identification of ubiquitylation substrates), to the discovery of
substrates for ubiquitin ligases. We applied DiPIUS to Fbxw7α,
Skp2, and Fbxl5, three of the most well-characterized F-box proteins,
and identified candidate substrates including previously known targets.
DiPIUS is thus a powerful tool for unbiased and comprehensive screening
for substrates of ubiquitin ligases.