Jon M. Huibregtse

The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53

The E6 protein encoded by the oncogenic human papillomavirus types 16 and 18 is one of two viral products expressed in HPV-associated cancers. E6 is an oncoprotein which cooperates with E7 to immortalize primary human keratinocytes. Insight into the mechanism by which E6 functions in oncogenesis is provided by the observation that the E6 protein encoded by HPV-16 and HPV-18 can complex the wild-type p53 protein in vitro. Wild-type p53 gene has tumor suppressor properties, and is a target for several of the oncoproteins encoded by DNA tumor viruses. In this study we demonstrate that the E6 proteins of the oncogenic HPVs that bind p53 stimulate the degradation of p53. The E6-promoted degradation of p53 is ATP dependent and involves the ubiquitin-dependent protease system. Selective degradation of cellular proteins such as p53 with negative regulatory functions provides a novel mechanism of action for dominant-acting oncoproteins.

The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53

The ubiquitin-dependent proteolytic pathway plays a major role in selective protein degradation. Ubiquitination of proteins requires the sequential action of the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2), and in some cases ubiquitin-protein ligases (E3s). The oncogenic human papillomavirus (HPV) types 16 and 18 utilize this cellular proteolytic system to target the tumor suppressor protein p53. The HPV E6 oncoprotein binds to a cellular protein of 100 kd, termed E6-associated protein (E6-AP). The E6-E6-AP complex specifically interacts with p53, resulting in the rapid ubiquitin-dependent degradation of p53. Here we report the purification and identification of the factors necessary for the E6-E6-AP-mediated ubiquitination of p53. The ubiquitination of p53 requires the E1 enzyme and a novel E2 in mammalian cells, while E3 activity is conferred by the E6-E6-AP complex. Furthermore, E6-AP appears to have ubiquitin-protein ligase activity in the absence of E6.

A cellular protein mediates association of p53 with the E6 oncoprotein of human papillomavirus types 16 or 18.

The E6 protein of human papillomavirus types 16 and 18 (HPV‐16 and HPV‐18) can stably associate with the p53 protein in vitro. In the presence of rabbit reticulocyte lysate, this association leads to the specific degradation of p53 through the ubiquitin‐dependent proteolysis system. We have examined the E6‐p53 complex in more detail and have found that association of E6 with p53 is mediated by an additional cellular factor. This factor is present in rabbit reticulocyte lysate, primary human keratinocytes and in each of five human cell lines examined. The factor is designated E6‐AP, for E6‐associated protein, based on the observation that the E6 proteins of HPV‐16 and 18 can form a stable complex with the factor in the absence of p53, whereas p53 association with the factor can be detected only in the presence of E6. Gel filtration and coprecipitation experiments indicate that E6‐AP is a monomeric protein of approximately 100 kDa.

Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53

The E6 oncoproteins of the cancer-associated or high-risk human papillomaviruses (HPVs) target the cellular p53 protein. The association of E6 with p53 leads to the specific ubiquitination and degradation of p53 in vitro, suggesting a model by which E6 deregulates cell growth control by the elimination of the p53 tumor suppressor protein. Complex formation between E6 and p53 requires an additional cellular factor, designated E6-AP (E6-associated protein), which has a native and subunit molecular mass of approximately 100 kDa. Here we report the purification of E6-AP and the cloning of its corresponding cDNA, which contains a novel open reading frame encoding 865 amino acids. E6-AP, translated in vitro, has the following properties: (i) it associates with wild-type p53 in the presence of the HPV16 E6 protein and simultaneously stimulates the association of E6 with p53, (ii) it associates with the high-risk HPV16 and HPV18 E6 proteins in the absence of p53, and (iii) it induces the E6- and ubiquitin-dependent degradation of p53 in vitro.

Human Scribble (Vartul) Is Targeted for Ubiquitin-Mediated Degradation by the High-Risk Papillomavirus E6 Proteins and the E6AP Ubiquitin-Protein Ligase

ABSTRACT The high-risk human papillomavirus (HPV) E6 proteins stimulate the ubiquitination and degradation of p53, dependent on the E6AP ubiquitin-protein ligase. Other proteins have also been shown to be targeted for degradation by E6, including hDlg, the human homolog of the Drosophila melanogaster Discs large (Dlg) tumor suppressor. We show here that the human homolog of theDrosophila Scribble (Vartul) (hScrib) tumor suppressor protein is also targeted for ubiquitination by the E6-E6AP complex in vitro and that expression of E6 induces degradation of hScrib in vivo. Characterization of the E6AP-E6-hScrib complex indicated that hScrib binds directly to E6 and that the binding is mediated by the PDZ domains of hScrib and a carboxyl-terminal epitope conserved among the high-risk HPV E6 proteins. Green fluorescent protein-hScrib was localized to the periphery of MDCK cells, where it colocalized with ZO-1, a component of tight junctions. E6 expression resulted in loss of integrity of tight junctions, as measured by ZO-1 localization, and this effect was dependent on the PDZ binding epitope of E6. Thus, the high-risk HPV E6 proteins induce the degradation of the human homologs of two Drosophila PDZ domain-containing tumor suppressor proteins, hDlg and hScrib, both of which are associated with cell junction complexes. The fact that Scrib/Vart and Dlg appear to cooperate in a pathway that controls Drosophila epithelial cell growth suggests that the combined targeting of hScrib and hDlg is an important component of the biologic activity of high-risk HPV E6 proteins.

Localization of the E6-AP regions that direct human papillomavirus E6 binding, association with p53, and ubiquitination of associated proteins.

E6-AP is a 100-kDa cellular protein that mediates the interaction of the human papillomavirus type 16 and 18 E6 proteins with p53. The association of p53 with E6 and E6-AP promotes the specific ubiquitination and subsequent proteolytic degradation of p53 in vitro. We recently isolated a cDNA encoding E6-AP and have now mapped functional domains of E6-AP involved in binding E6, association with p53, and ubiquitination of p53. The E6 binding domain consists of an 18-amino-acid region within the central portion of the molecule. Deletion of these 18 amino acids from E6-AP results in loss of both E6 and p53 binding activities. The region that directs p53 binding spans the E6 binding domain and consists of approximately 500 amino acids. E6-AP sequences in addition to those required for formation of a stable ternary complex with E6 and p53 are necessary to stimulate the ubiquitination of p53. These sequences lie within the C-terminal 84 amino acids of E6-AP. The entire region required for E6-dependent ubiquitination of p53 is also required for the ubiquitination of an artificial E6 fusion protein.

Mycobacterial disease and impaired IFN-γ immunity in humans with inherited ISG15 deficiency.

Tuberculosis Vaccine Conundrum Some children experience severe clinical disease when they are vaccinated against tuberculosis, an attenuated live vaccine that is normally innocuous in humans. Several germline mutations have been identified that account for this susceptibility, and now Bogunovic et al. (p. 1684, published online 2 August) add another to the list—ISG15. Uncovering this mutation, which is inherited in an autosomal recessive manner, was a surprise because studies with mice deficient in ISG15 showed enhanced susceptibility to some viral, but not bacterial, infections. Nevertheless, patients lacking ISG15 were not able to produce adequate amounts of interferon-γ, a cytokine critical for clearance of the bacteria. A mutation that accounts for adverse reactions to the Bacille Calmette-Guérin vaccine against tuberculosis is identified. ISG15 is an interferon (IFN)-α/β–inducible, ubiquitin-like intracellular protein. Its conjugation to various proteins (ISGylation) contributes to antiviral immunity in mice. Here, we describe human patients with inherited ISG15 deficiency and mycobacterial, but not viral, diseases. The lack of intracellular ISG15 production and protein ISGylation was not associated with cellular susceptibility to any viruses that we tested, consistent with the lack of viral diseases in these patients. By contrast, the lack of mycobacterium-induced ISG15 secretion by leukocytes—granulocyte, in particular—reduced the production of IFN-γ by lymphocytes, including natural killer cells, probably accounting for the enhanced susceptibility to mycobacterial disease. This experiment of nature shows that human ISGylation is largely redundant for antiviral immunity, but that ISG15 plays an essential role as an IFN-γ–inducing secreted molecule for optimal antimycobacterial immunity.

Herc5, an Interferon-induced HECT E3 Enzyme, Is Required for Conjugation of ISG15 in Human Cells

ISG15 is an interferon (IFN)-α/β-induced ubiquitin-like protein that is conjugated to cellular proteins during innate immune responses to viral and bacterial infections. A recent proteomics study identified 158 human proteins targeted for ISG15 conjugation, including the ISG15 E1 and E2 enzymes (Ube1L and UbcH8, respectively) and a HECT E3 enzyme, Herc5. Like the genes encoding Ube1L and UbcH8, expression of Herc5 was also induced by IFN-β, suggesting that Herc5 might be a component of the ISG15 conjugation system. Consistent with this, small interfering RNAs targeting Herc5 had a dramatic effect on overall ISG15 conjugation in human cells, abrogating conjugation to the vast majority of ISG15 target proteins in vivo. In addition, co-transfection of plasmids expressing ISG15, Ube1L, UbcH8, and Herc5 resulted in robust ISG15 conjugation in non-IFN-treated cells, while the active-site cysteine mutant of Herc5 or a mutant lacking the RCC1 repeat region did not support ISG15 conjugation. These results demonstrate that Herc5 is required for conjugation of ISG15 to a broad spectrum of target proteins in human cells.

Polyubiquitination by HECT E3s and the Determinants of Chain Type Specificity

ABSTRACT Polyubiquitination can mediate several different biochemical functions, determined in part by which lysine of ubiquitin is used to link the polyubiquitin chain. Among the HECT domain ubiquitin ligases, some, such as human E6AP, preferentially catalyze the formation of K48-linked polyubiquitin chains, while others, including Saccharomyces cerevisiae Rsp5 and human Itch, preferentially catalyze the formation of K63-linked chains. The features of HECT E3s that determine their chain type specificities have not been identified. We show here that chain type specificity is a function solely of the Rsp5 HECT domain, that the identity of the cooperating E2 protein does not influence the chain type specificity, that single chains produced by Rsp5 contain between 12 and 30 ubiquitin moieties, and that the determinants of chain type specificity are located within the last 60 amino acids of the C lobe of the HECT domain. Our results are also consistent with a simple sequential-addition mechanism for polyubiquitination by Rsp5, rather than a mechanism involving the formation of either E2- or E3-linked polyubiquitin chain transfers.

The Rsp5 ubiquitin ligase is coupled to and antagonized by the Ubp2 deubiquitinating enzyme

Saccharomyces cerevisiae Rsp5 is an essential HECT ubiquitin ligase involved in several biological processes. To gain further insight into regulation of this enzyme, we identified proteins that copurified with epitope‐tagged Rsp5. Ubp2, a deubiquitinating enzyme, was a prominent copurifying protein. Rup1, a previously uncharacterized UBA domain protein, was required for binding of Rsp5 to Ubp2 both in vitro and in vivo. Overexpression of Ubp2 or Rup1 in the rsp5‐1 mutant elicited a strong growth defect, while overexpression of a catalytically inactive Ubp2 mutant or Rup1 deleted of the UBA domain did not, suggesting an antagonistic relationship between Rsp5 and the Ubp2/Rup1 complex. Consistent with this model, rsp5‐1 temperature sensitivity was suppressed by either ubp2Δ or rup1Δ mutations. Ubp2 reversed Rsp5‐catalyzed substrate ubiquitination in vitro, and Rsp5 and Ubp2 preferentially assembled and disassembled, respectively, K63‐linked polyubiquitin chains. Together, these results indicate that Rsp5 activity is modulated by being physically coupled to the Rup1/Ubp2 deubiquitinating enzyme complex, representing a novel mode of regulation for an HECT ubiquitin ligase.