The conjugation of proteins with ubiquitin involves the sequential action of proteins known as E1s (ubiquitin activating enzymes), E2s or UBCs (ubiquitin conjugating enzymes), and E3s (ubiquitin protein ligases). We have characterized a family of closely related E2s and gone on to establish that Nedd4, which is related to E6-AP (the E3 that ubiquitinates p53 in the presence of human papilloma virus E6) in its carboxyl-terminus, is an enzymatically active E3. Other ongoing studies, being carried out together with Dr. Michael Kuehn (EIB), have involved the use of Nedd4 as bait in a yeast two-hybrid screen. This has resulted in the isolation of several proteins that both bind to, and are substrates for, ubiquitination by Nedd4. More recently we have utilized an E2 enzyme (UbcH5B) in the yeast two-hybrid system in the hope of isolating novel E3s. This has resulted in the isolation of cDNAs encoding a protein that binds UbcH5B and related E2s both in vivo and in vitro. This protein, AO7, is notable for a RING finger that is required for its E2 binding and to mediate its own ubiquitination. This led to the determination that a number of otherwise unrelated RING finger-containing proteins including BRCA1, Siah-1, and c-Cbl have the capacity to mediate ubiquitination. We also have evidence that the RING finger of Mdm2 provides the molecular basis for the E3 activity of Mdm2 both towards itself and toward p53. Structure-function analysis of RING finger proteins suggests conserved residues other than the zinc-coordinating Cys and His are required for function, and additional analysis suggests that other divalent cations can substitute for zinc in active RINGs. One area of ongoing study is oriented towards the structural characterization of the Mdm2 RING finger and in the identification of both naturally occurring and small molecule inhibitors of Mdm2 E3 activity.
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