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. 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. Our work on the structure of the Mdm2 has been a technical challenge and has now evolved into attempts to co-purify the Mdm2 RING finger along with that of MdmX, with which it dimerizes. Our studies looking for inhibitors of Mdm2 have resulted in the identification of a family of compounds that inhibit Mdm2 E3 activity both in vitro and in cells. Additionally, our screens have also resulted in the identification of a cell-permeant E1 inhibitor, which should be of great value for experimental purposes and may also serve as the basis for new therapeutics with functions similar to proteasome inhibitors, which are now in clinical trials. In addition to the work on Mdm2 other structure-functions studies are oriented towards understand the function or RNF6 and RLIM and of BRAP2.
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