In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF(Skp2) substrate p27(Kip1). CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.

Cullin-based E3 ubiquitin ligases are activated through modification of the cullin subunit with the ubiquitin-like protein Nedd8. Dcn1 regulates cullin neddylation and thus ubiquitin ligase activity. Here we describe the 1.9 A X-ray crystal structure of yeast Dcn1 encompassing an N-terminal ubiquitin-binding (UBA) domain and a C-terminal domain of unique architecture, which we termed PONY domain. A conserved surface on Dcn1 is required for direct binding to cullins and for neddylation. The reciprocal binding site for Dcn1 on Cdc53 is located approximately 18 A from the site of neddylation. Dcn1 does not require cysteine residues for catalytic function, and directly interacts with the Nedd8 E2 Ubc12 on a surface that overlaps with the E1-binding site. We show that Dcn1 is necessary and sufficient for cullin neddylation in a purified recombinant system. Taken together, these data demonstrate that Dcn1 is a scaffold-like E3 ligase for cullin neddylation.

Cullin ͉ DCUN1D ͉ Nedd8 ͉ ubiquitin ͉ squamous cell carcinoma-related oncogene

Background:The IpaH ubiquitin ligases are bacterial type III effectors that ubiquitinate and inactivate eukaryotic host proteins. Results: Two distinct modes of autoinhibition confer substrate-dependent activation of IpaH enzymes. Conclusion: Conserved intramolecular interactions regulate IpaH activity. Significance: IpaH autoregulatory mechanisms may be potential targets for therapeutic intervention.The IpaH family of novel E3 ligase (NEL) enzymes occur in a variety of pathogenic and commensal bacteria that interact with eukaryotic hosts. We demonstrate that the leucine-rich repeat (LRR) substrate recognition domains of different IpaH enzymes autoinhibit the enzymatic activity of the adjacent catalytic novel E3 ligase domain by two distinct but conserved structural mechanisms. Autoinhibition is required for the in vivo biological activity of two IpaH enzymes in a eukaryotic model system. Autoinhibition was retro-engineered into a constitutively active IpaH enzyme from Yersinia pestis by introduction of single site substitutions, thereby demonstrating the conservation of autoregulatory infrastructure across the IpaH enzyme family.The ubiquitin system regulates diverse cellular processes in all eukaryotes through the covalent attachment of ubiquitin to acceptor lysine residues on target substrates, thereby dictating protein stability, activity, and localization. Ubiquitin is activated and conjugated to its substrates by a sequential enzymatic cascade, E1 3 E2 3 E3 (1). E3 enzymes, also known as ubiquitin ligases, specifically recognize their substrates via protein interaction domains. The eukaryotic E3 enzymes fall into two major classes. The HECT 4 domain class facilitates transfer to substrates via a catalytic cysteine residue, whereas the RING/ U-box domain class serves as a platform to bridge the E2 enzyme to the substrate.The crucial role of the ubiquitin system in the regulation of host immune surveillance poises ubiquitin-dependent processes as a pervasive target for a wide range of pathogens (2). Several type III secretion virulence effectors in Gram-negative pathogenic bacteria possess ubiquitin ligase activity, including AvrPtoB from Pseudomonas syringae (3), SopA from Salmonella enterica typhimurium (4), NIeG from enterohemorrhagic Escherichia coli (5), and the IpaH family members from Shigella flexneri and Salmonella enterica (6). AvrPtoB and NIeG are structural mimics of RING/U-box domain ubiquitin ligases, whereas SopA is a structural mimic of HECT domain ubiquitin ligases (5,7,8). In contrast, IpaH enzymes possess a novel catalytic fold that does not resemble known eukaryotic E3 enzymes (9 -11). The substrates for most pathogen-encoded E3 enzymes remain to be identified but are likely to function in host defense systems. For example, AvrPtoB and IpaH9.8, respectively, target the Fen and NF-B essential modulator/IB kinase ␥ (NEMO/IKK␥) protein kinases, both of which are key mediators of the innate immune response (12,13).The domain architecture of the IpaH family consists of a N-terminal leucine-ric...

Noncancer patients have a shorter LOS than cancer patients and a similar likelihood of being discharged alive from a PCU than cancer patients, and the diagnosis of cancer did not correlate with survival in our study population. Our findings demonstrate that noncancer patients are not "oversurviving," and that referring physicians and PCUs should not reject or restrict noncancer referrals out of concern that these patients are having a detrimental impact on PCU bed availability.