The E6AP ubiquitin-protein ligase (E3) mediates the human papillomavirus-induced degradation of the p53 tumor suppressor in cervical cancer and is mutated in Angelman syndrome, a neurological disorder. The crystal structure of the catalytic hect domain of E6AP reveals a bilobal structure with a broad catalytic cleft at the junction of the two lobes. The cleft consists of conserved residues whose mutation interferes with ubiquitin-thioester bond formation and is the site of Angelman syndrome mutations. The crystal structure of the E6AP hect domain bound to the UbcH7 ubiquitin-conjugating enzyme (E2) reveals the determinants of E2-E3 specificity and provides insights into the transfer of ubiquitin from the E2 to the E3.

VP40, the putative matrix protein of both Ebola and Marburg viruses, possesses a conserved proline-rich motif (PY motif) at its N terminus. We demonstrate that the VP40 protein can mediate its own release from mammalian cells, and that the PY motif is important for this self-exocytosis (budding) function. In addition, we used Western-ligand blotting to demonstrate that the PY motif of VP40 can mediate interactions with specific cellular proteins that have type I WW-domains, including the mammalian ubiquitin ligase, Nedd4. Single point mutations that disrupted the PY motif of VP40 abolished the PY͞WW-domain interactions. Significantly, the full-length VP40 protein was shown to interact both physically and functionally with full-length Rsp5, a ubiquitin ligase of yeast and homolog of Nedd4. The VP40 protein was multiubiquitinated by Rsp5 in a PY-dependent manner in an in vitro ubiquitination assay. These data demonstrate that the VP40 protein of Ebola virus possesses a PY motif that is functionally similar to those described previously for Gag and M proteins of specific retroviruses and rhabdoviruses, respectively. Last, these studies imply that VP40 likely plays an important role in filovirus budding, and that budding of retroviruses, rhabdoviruses, and filoviruses may proceed via analogous mechanisms.

The matrix (M) proteins of vesicular stomatitis virus (VSV) and rabies virus (RV) play a key role in both assembly and budding of progeny virions. A PPPY motif (PY motif or late-budding domain) is conserved in the M proteins of VSV and RV. These PY motifs are important for virus budding and for mediating interactions with specific cellular proteins containing WW domains. The PY motif and flanking sequences of the M protein of VSV were used as bait to screen a mouse embryo cDNA library for cellular interactors. The mouse Nedd4 protein, a membrane-localized ubiquitin ligase containing multiple WW domains, was identified from this screen. Ubiquitin ligase Rsp5, the yeast homolog of Nedd4, was able to interact both physically and functionally with full-length VSV M protein in a PY-dependent manner. Indeed, the VSV M protein was multiubiquitinated by Rsp5 in an in vitro ubiquitination assay. To demonstrate further that ubiquitin may be involved in the budding process of rhabdoviruses, proteasome inhibitors (e.g., MG132) were used to decrease the level of free ubiquitin in VSV-and RV-infected cells. Viral titers measured from MG132-treated cells were reproducibly 10-to 20-fold lower than those measured from untreated control cells, suggesting that free ubiquitin is important for efficient virus budding. Last, release of a VSV PY mutant was not inhibited in the presence of MG132, signifying that the functional L domain of VSV is required for the inhibitory effect exhibited by MG132. These data suggest that the cellular ubiquitin-proteasome machinery is involved in the budding process of VSV and RV.

We demonstrate herein a newly developed lable-free photoelectrochemical immunosensor using a CdS quantum dots (QDs) multilayer film coupled with a biospecific interaction. The CdS QDs multilayer film was prepared by layer-by-layer assembling positively charged poly(dimethyldiallylammonium chloride) (PDDA) and thioglycolic acid (TGA)-capped water-soluble CdS QDs with negative charges on the surface of an indium−tin oxide (ITO) electrode. Ascorbic acid (AA) was exploited as an efficient and nontoxic electron donor for scavenging photogenerated holes under mild solution medium. The photoexcitation of CdS QDs modified electrode potentiostated at 0 V (vs. Ag/AgCl) in the presence of 0.1 M AA led to a stable anodic photocurrent. To perform the immunoassay, goat antimouse IgG was conjugated onto CdS QDs modified electrode by using the classic EDC coupling reactions between COOH groups on the surfaces of the TGA capped CdS QDs and NH2 groups of the antibody. The concentrations of mouse IgG were measured through the decrease in photocurrent intensity resulting from the increase in steric hindrances due to the formation of the immunocomplex. The synthetic conditions (different Cd/S ratio and different pH) of CdS QDs and the number of PDDA/CdS bilayers could influence the photoelectrochemical properties of CdS QDs modified electrodes used for immunosensor construction. Under the optimal conditions, a linear relationship between photocurrent decrease and mouse IgG concentration was obtained in the range of 10 pg/mL to 100 ng/mL with a detection limit of 8.0 pg/mL. This strategy opens a new perspective for the application of QDs, which might be of great significance for QDs in photoelectrochemical bioanalysis in the future.

Rsp5 is an E3 ubiquitin-protein ligase of Saccharomyces cerevisiae that belongs to the hect domain family of E3 proteins. We have previously shown that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II, Rpb1, in vitro. We show here that Rpb1 ubiquitination and degradation are induced in vivo by UV irradiation and by the UV-mimetic compound 4-nitroquinoline-1-oxide (4-NQO) and that a functional RSP5 gene product is required for this effect. The 26S proteasome is also required; a mutation of SEN3/RPN2 (sen3-1), which encodes an essential regulatory subunit of the 26S proteasome, partially blocks 4-NQO-induced degradation of Rpb1. These results suggest that Rsp5-mediated ubiquitination and degradation of Rpb1 are components of the response to DNA damage. A human WW domain-containing hect (WW-hect) E3 protein closely related to Rsp5, Rpf1/hNedd4, also binds and ubiquitinates both yeast and human Rpb1 in vitro, suggesting that Rpf1 and/or another WW-hect E3 protein mediates UV-induced degradation of the large subunit of polymerase II in human cells.Ubiquitin-dependent proteolysis involves the covalent ligation of ubiquitin to substrate proteins, which are then recognized and degraded by the 26S proteasome. While many of the components involved in catalyzing protein ubiquitination have been identified and characterized biochemically, we are only beginning to understand how the system specifically recognizes appropriate substrates. At least three classes of activities, known as E1 (ubiquitin-activating), E2 (ubiquitin-conjugating), and E3 (ubiquitin-protein ligase) enzymes, cooperate in catalyzing protein ubiquitination (34). The enzymatic mechanisms and functions of the E1 and E2 proteins have been well characterized. In contrast, the E3 enzymes are a diverse and less-well-characterized group of activities, and many lines of evidence indicate that E3 activities play a major role in determining the substrate specificity of the ubiquitination pathway (14,28,34).The hect (homologous to E6-AP carboxyl terminus) domain defines a family of E3 proteins that were discovered through the characterization of human E6-AP (17). The interaction of E6-AP with the E6 protein of the cervical cancer-associated human papillomavirus types causes E6-AP to associate with and ubiquitinate p53, suggesting that E6 functions in promoting cellular immortalization by, at least in part, stimulating the destruction of this important tumor suppressor protein (16). The hect E3 molecular masses range from 92 to over 500 kDa, with the hect domain comprising the approximately 350 carboxyl-terminal amino acids (17, 34). Exactly five hect E3s are encoded by the Saccharomyces cerevisiae genome, and over 30 have been identified so far in mammalian species. An obligatory intermediate in the ubiquitination reactions catalyzed by hect E3s is a ubiquitin-thioester formed between the thiol group of an absolutely conserved cysteine within the hect domain and the terminal carboxyl group of ubiquitin (33). E3 becomes "charged" with ubiquitin vi...