Ubiquitylation is an important area in cancer research since many ubiquitylation enzymes and substrates are either tumor suppressors or oncogenes: examples are p53, E2F-1, the cyclins, VHL, BRCA-1, E6, and Mdm2. Current genomic approaches for studying ubiquitylation rely on motif capture and mass spectrometry, and are biased towards abundant proteins and do not reveal which proteins whose turnover is generally regulated by ubiquitylation. Here we describe a systematic approach for identifying new ubiquitylation targets, substrates for known E3 ligases, and E3 ligases for known substrates. Our approach relies on the construction of a fusion library between the human ORFeome (developed by Marc Vidal) and luciferase. The fusion library will be transfected into cells on a well-by-well basis (1 cDNA per well), and two different experimental conditions will be compared to see which ORFs are upregulated by the test condition. Our first example utilizes ts20 cells, a cell line that bears a temperature sensitive mutation in the E1 ubiquitin-activating enzyme, upon which all ubiquitylation depends. Second, we will use siRNA for VHL to see which proteins are upregulated when this tumor suppressor E3 is knocked down. Third, we will use siRNA for the EglNs, a family of prolyl hydroxylases that are known to target hypoxia inducible factor (Hif). In our future directions we discuss approaches to characterize proteins identified in our screens, as well as techniques to identify E3 ligases that regulate known substrates. ? ? ? ?
