The role of the ubiquitin ligase E6-AP in human papillomavirus E6-mediated degradation of PDZ domain-containing proteins

The E6 oncoprotein of human papillomaviruses associated with cervical cancer targets the tumor suppressor p53 and several other cellular proteins including the human homologs of Dlg and Scribble for degradation via the ubiquitin-proteasome system. Similar to p53 degradation, E6-induced degradation of Scribble is mediated by the ubiquitin ligase E6-AP. In contrast, degradation of Dlg in vitro and within cells has been reported to be independent of E6-AP, suggesting that the E6 oncoprotein has the ability to interact with ubiquitin ligases other than E6-AP. Furthermore, the ability of the E6 oncoprotein to interact with these yet unidentified ubiquitin ligases may be shared by the E6 protein of so-called low risk human papillomaviruses that are not associated with cervical cancer. In this study, we used the RNA interference technology and mouse embryo fibroblasts derived from E6-AP-deficient mice to obtain information about the identity of the ubiquitin ligase(s) involved in E6-mediated degradation of Dlg. We report that, within cells, E6-mediated degradation of Dlg depends on the presence of functional E6-AP and provide evidence that the E6 protein of low risk human papillomaviruses functionally interacts with E6-AP. Based on these data, we propose that, in general, the proteolytic properties of human papillomavirus E6 proteins are mediated by interaction with E6-AP.     

Growth suppression induced by downregulation of E6-AP expression in human papillomavirus-positive cancer cell lines depends on p53

The ubiquitin-protein ligase E6-AP is utilized by the E6 oncoprotein of human papillomaviruses (HPVs) associated with cervical cancer to target the tumor suppressor p53 for degradation. Here, we report that downregulation of E6-AP expression by RNA interference results in both the accumulation of p53 and growth suppression of the HPV-positive cervical cancer cell lines HeLa and SiHa. In addition, HeLa cells, in which p53 expression was suppressed by RNA interference, are significantly less sensitive to the downregulation of E6-AP expression with respect to growth suppression than parental HeLa cells. These data indicate that the anti-growth-suppressive properties of E6-AP in HPV-positive cells depend on its ability to induce p53 degradation.     

Inhibition of E6-induced degradation of its cellular substrates by novel blocking peptides

The E6 oncoprotein derived from the tumour-associated human papillomavirus (HPV) types induces the ubiquitin-mediated degradation of several cellular proteins by conjugating them with the cellular ubiquitin ligase E6-AP. This is a HECT domain-containing ligase that was originally identified through its involvement in the E6-mediated degradation of the cellular tumour suppressor protein p53. Here we have investigated, in more detail, the nature of the E6/E6-AP interaction using binding peptides isolated from an E6-specific library. The selected peptides were either predicted or shown to have an alpha-helical core resembling the E6-binding motif on E6-AP, as well as amino acid alterations that increased their affinity for E6. These peptides were potent inhibitors of the E6/E6-AP interaction. Further analysis of the effects of these peptides on the ability of E6 to direct the proteolytic degradation of its various substrates, including p53, Dlg and the MAGI family of proteins, as well as using E6-AP immunodepletion, revealed striking differences in the mechanism by which E6 targets its cellular substrates for degradation. These results suggest that the site on E6 bound by E6-AP is also most likely occupied by other, as yet unidentified, ubiquitin ligases.     

Human papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligase

The E6 protein of the high-risk human papillomaviruses (HPVs) and the cellular ubiquitin-protein ligase E6AP form a complex which causes the ubiquitination and degradation of p53. We show here that HPV16 E6 promotes the ubiquitination and degradation of E6AP itself. The half-life of E6AP is shorter in HPV-positive cervical cancer cells than in HPV-negative cervical cancer cells, and E6AP is stabilized in HPV-positive cancer cells when expression of the viral oncoproteins is repressed. Expression of HPV16 E6 in cells results in a threefold decrease in the half-life of transfected E6AP. E6-mediated degradation of E6AP requires (i) the binding of E6 to E6AP, (ii) the catalytic activity of E6AP, and (iii) activity of the 26S proteasome, suggesting that E6-E6AP interaction results in E6AP self-ubiquitination and degradation. In addition, both in vitro and in vivo experiments indicate that E6AP self-ubiquitination results primarily from an intramolecular transfer of ubiquitin from the active-site cysteine to one or more lysine residues; however, intermolecular transfer can also occur in the context of an E6-mediated E6AP multimer. Finally, we demonstrate that an E6 mutant that is able to immortalize human mammary epithelial cells but is unable to degrade p53 retains its ability to bind and degrade E6AP, raising the possibility that E6-mediated degradation of E6AP contributes to its ability to transform mammalian cells.     

UBE4B,a ubiquitin chain assembly factor,is required for MDM2-mediated p53 polyubiquitination and degradation

Although MDM2 is known to be a critical negative regulator of p53, MDM2 only catalyzes p53 mono- or multiple monoubiquitination in vitro and in vivo, which is insufficient for the initiation of proteasomal degradation. MDM2 does not polyubiquitinate p53 in vitro, however, which indicates that the activity of other ubiquitin ligase(s) or cofactor(s) is required for MDM2-mediated p53 polyubiquitination and degradation. In our recent study, we demonstrated that UBE4B, an E3 and E4 ubiquitin ligase with a U-box domain, interacts physically with both p53 and MDM2. Our findings revealed that UBE4B negatively regulates the level of p53 and inhibits p53-dependent transactivation and apoptosis. We propose that inhibition of MDM2 binding to UBE4B may provide another approach to inhibit MDM2 E3 ligase activity for tumor suppressor p53. It could lead to novel anticancer therapies, with the possibility of reducing the public health burden from cancer.Key words: ubiquitination, MDM2, UBE4B, p53, degradation     

MDM2 is a negative regulator of p21WAF1/CIP1, independent of p53

The MDM2 oncogene has both p53-dependent and p53-independent activities. We have previously reported that antisense MDM2 inhibitors have significant anti-tumor activity in multiple human cancer models with various p53 statuses (Zhang, Z., Li, M., Wang, H., Agrawal, S., and Zhang, R. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 11636-11641). We have also provided evidence that MDM2 has a direct role in the regulation of p21, a cyclin-dependent kinase inhibitor. Here we provide evidence supporting functional interaction between MDM2 and p21 in vitro and in vivo. The inhibition of MDM2 with anti-MDM2 antisense oligonucleotide or Short Interference RNA targeting MDM2 significantly elevated p21 protein levels in PC3 cells (p53 null). In contrast, overexpression of MDM2 diminished the p21 level in the same cells by shortening the p21 half-life, an effect reversed by MDM2 antisense inhibition. MDM2 facilitates p21 degradation independent of ubiquitination and the E3 ligase function of MDM2. Instead, MDM2 promotes p21 degradation by facilitating binding of p21 with the proteasomal C8 subunit. The physical interaction between p21 and MDM2 was demonstrated both in vitro and in vivo with the binding region in amino acids 180-298 of the MDM2 protein. In summary, we provide evidence supporting a physical interaction between MDM2 and p21. We also demonstrate that, by reducing p21 protein stability via proteasome-mediated degradation, MDM2 functions as a negative regulator of p21, an effect independent of both p53 and ubiquitination.     

Proteasomal degradation of p53 by human papillomavirus E6 oncoprotein relies on the structural integrity of p53 core domain

The E6 oncoprotein produced by high-risk mucosal HPV stimulates ubiquitinylation and proteasome-dependent degradation of the tumour suppressor p53 via formation of a trimeric complex comprising E6, p53, and E6-AP. p53 is also degraded by its main cellular regulator MDM2. The main binding site of p53 to MDM2 is situated in the natively unfolded N-terminal region of p53. By contrast, the regions of p53 implicated in the degradation by viral E6 are not fully identified to date. Here we generated a series of mutations (Y103G, Y107G, T155A, T155V, T155D, L264A, L265A) targeting the central folded core domain of p53 within a region opposite to its DNA-binding site. We analysed by in vitro and in vivo assays the impact of these mutations on p53 degradation mediated by viral E6 oncoprotein. Whereas all mutants remained susceptible to MDM2-mediated degradation, several of them (Y103G, Y107G, T155D, L265A) became resistant to E6-mediated degradation, confirming previous works that pointed to the core domain as an essential region for the degradation of p53. In parallel, we systematically checked the impact of the mutations on the transactivation activity of p53 as well as on the conformation of p53, analysed by Nuclear Magnetic Resonance (NMR), circular dichroism (CD), and antibody probing. These measurements suggested that the conformational integrity of the core domain is an essential parameter for the degradation of p53 by E6, while it is not essential for the degradation of p53 by MDM2. Thus, the intracellular stability of a protein may or may not rely on its biophysical stability depending on the degradation pathway taken into consideration.     

Solution structure analysis of the HPV16 E6 oncoprotein reveals a self-association mechanism required for E6-mediated degradation of p53

The viral oncoprotein E6 is an essential factor for cervical cancers induced by "high-risk" mucosal HPV. Among other oncogenic activities, E6 recruits the ubiquitin ligase E6AP to promote the ubiquitination and subsequent proteasomal degradation of p53. E6 is prone to self-association, which long precluded its structural analysis. Here we found that E6 specifically dimerizes through its N-terminal domain and that disruption of the dimer interface strongly increases E6 solubility. This allowed us to raise structural data covering the entire HPV16 E6 protein, including the high-resolution NMR structures of the two zinc-binding domains of E6 and a robust data-driven model structure of the N-terminal domain homodimer. Interestingly, homodimer interface mutations that disrupt E6 self-association also inactivate E6-mediated p53 degradation. These data suggest that E6 needs to self-associate via its N-terminal domain to promote the polyubiquitination of p53 by E6AP.     

Human papillomavirus E6 regulates the cytoskeleton dynamics of keratinocytes through targeted degradation of p53

The attachment and spreading of keratinocyte cells result from interactions between integrins and immobilized extracellular matrix molecules. Human papillomavirus type 16 (HPV-16) E6 augmented the kinetics of cell spreading, while E6 genes from HPV-11 or bovine papillomavirus type 1 did not. The ability of E6 to interact with the E6AP ubiquitin ligase and target p53 degradation was required to augment cell-spreading kinetics; dominant negative p53 alleles also enhanced the kinetics of cell spreading and the level of attachment of cells to hydrophobic surfaces. The targeted degradation of p53 by E6 may contribute to the invasive phenotype exhibited by cervical cells that contain high-risk HPV types.     

Targeting Retinoblastoma Protein for Degradation by Proteasomes

Inactivation of retinoblastoma protein (Rb) plays a key role in human tumorigenesis.Although the regulation of Rb by phosphorylation has been extensively studied, the regulationfor proteasome-mediated Rb protein degradation is largely unknown. Viral oncoprotein E7,Epstein-Barr virus nuclear antigen 3C (EBNA3C), human cytomegalovirus pp71 and cellularoncoprotein gankyrin all contain the L-x-C-x-E Rb-binding motif and target Rb protein fordegradation in either ubiquitin-dependent or ubiquitin-independent proteasome pathways. Themolecular mechanisms, however, remain elusive. The MDM2 oncoprotein is overexpressed in avariety of human cancers. MDM2 functions as an ubiquitin E3 ligase and induces p53 proteindegradation through ubiquitination-proteasome pathway. Both MDM2 central acidic domain andthe C-terminal RING domain are critical for p53 degradation. MDM2 also interacts with Rbthrough its central acidic domain and inhibits Rb function in part by blocking Rb-E2F-DNAcomplex formation. Recently, we show that MDM2 binds to C8 subunit of 20S proteasome andpromotes Rb-C8 interaction, leading to a proteasome-dependent ubiquitin-independentdegradation of Rb. Knockdown of MDM2 results in accumulation of hypophosphorylated Rband inhibition of DNA synthesis. Taken together, we suggest that targeting Rb protein fordegradation by proteasomes may represent a common neoplastic strategy during human cancerdevelopment.     

Human papillomavirus type 16 E7 oncoprotein associates with the cullin 2 ubiquitin ligase complex, which contributes to degradation of the retinoblastoma tumor suppressor

Human papillomavirus type 16 (HPV16) and other high-risk HPVs are etiologically linked to the development of cervical carcinomas and contribute to a number of other tumors of the anogenital tract, as well as oral cancers. The high-risk HPV E6 and E7 oncoproteins are consistently expressed in cervical cancer cells and are necessary for the induction and maintenance of the transformed phenotype. An important aspect of HPV16 E7's oncogenic activities is destabilization of the retinoblastoma tumor suppressor (pRB) through a ubiquitin/proteasome-dependent mechanism, although the exact molecular mechanism is unknown. Here, we report that HPV16 E7 is associated with an enzymatically active cullin 2 ubiquitin ligase complex and that the HPV16 E7/pRB complex contains cullin 2. Depletion of cullin 2 by RNA interference causes increased steady-state levels and stability of pRB in HPV16 E7-expressing cells, and ectopic expression of HPV16 E7 and the cullin 2 complex leads to pRB ubiquitination in vivo. Hence, we propose that the HPV16 E7-associated cullin 2 ubiquitin ligase complex contributes to aberrant degradation of the pRB tumor suppressor in HPV16 E7-expressing cells.     

Critical contribution of the MDM2 acidic domain to p53 ubiquitination

MDM2 is an E3 ubiquitin ligase that targets p53 for proteasomal degradation. Recent studies have shown, however, that the ring-finger domain (RFD) of MDM2, where the ubiquitin E3 ligase activity resides, is necessary but not sufficient for p53 ubiquitination, suggesting that an additional activity of MDM2 might be required. To test this possibility, we generated a series of MDM2/MDMX chimeric proteins to assess the contribution of each domain of MDM2 to the ubiquitination process. MDMX is a close structural homolog of MDM2 that nevertheless lacks the E3 ligase activity in vivo. We demonstrate here that MDMX gains self-ubiquitination activity and becomes extremely unstable upon introduction of the MDM2 RFD, indicating that the RFD is essential for self-ubiquitination. This MDMX chimeric protein, however, is unable to ubiquitinate p53 in vivo despite its E3 ligase activity and binding to p53, separating the self-ubiquitination activity of MDM2 from its ability to ubiquitinate p53. Significantly, fusion of the central acidic domain (AD) of MDM2 to the MDMX chimeric protein renders the protein fully capable of ubiquitinating p53, and p53 ubiquitination is associated with p53 degradation and nuclear export. Moreover, the AD mini protein expressed in trans can functionally rescue the AD-lacking MDM2 mutant, further supporting a critical role for the AD in MDM2-mediated p53 ubiquitination.     

Wogonin induces apoptosis by suppressing E6 and E7 expressions and activating intrinsic signaling pathways in HPV-16 cervical cancer cells

Wogonin is a flavonoid compound extracted from Scutellaria baicalensis and is well known as a benzodiazepine receptor ligand with anxiolytic effects. Many recent studies have demonstrated that wogonin modulates angiogenesis, proliferation, invasion, and tumor progress in various cancer tissues. We further explored the mechanism of action of wogonin on cervical cancer cells that contain or lack human papillomavirus (HPV) DNA. Wogonin was cytotoxic to HPV 16 (+) cervical cancer cells, SiHa and CaSki, but not to HPV-negative cells. We demonstrated that wogonin induced apoptosis by suppressing the expressions of the E6 and E7 viral oncogenes in HPV-infected cervical cancer CaSki and SiHa cells. The modulation of p53 and protein retinoblastoma (pRb) were also triggered by the suppression of E6 and E7 expressions. However, p53 was not altered in HPV-negative cervical cancer C33A cells. Moreover, wogonin modulated the mitochondrial membrane potential and the expression of pro- and anti-apoptotic factors such as Bax and Bcl-2. Wogonin also provoked the cleavage of caspase-3, caspase-9, and poly ADP ribose polymerase. After transfection of siRNAs to target E6 and E7, additional restoration of p53 and pRb was not induced, but processing of caspases and PARP was increased compared with wogonin treatment alone. Together, our findings demonstrated that wogonin effectively promotes apoptosis by downregulating E6 and E7 expressions and promoting intrinsic apoptosis in human cervical cancer cells.