The E6 oncoprotein derived from tumour-associated human papillomaviruses (HPVs) binds to and induces the degradation of the cellular tumour-suppressor protein p53. A common polymorphism that occurs in the p53 amino-acid sequence results in the presence of either a proline or an arginine at position 72. The effect of this polymorphism on the susceptibility of p53 to E6-mediated degradation has been investigated and the arginine form of p53 was found to be significantly more susceptible than the proline form. Moreover, allelic analysis of patients with HPV-associated tumours revealed a striking overrepresentation of homozygous arginine-72 p53 compared with the normal population, which indicated that individuals homozygous for arginine 72 are about seven times more susceptible to HPV-associated tumorigenesis than heterozygotes. The arginine-encoding allele therefore represents a significant risk factor in the development of HPV-associated cancers.

Previous studies have shown that the oncogenic HPV E6 proteins form a complex with the human homologue of the Drosophila tumour suppressor protein, discs large (Dlg). This is mediated by the carboxy terminus of the E6 proteins and involves recognition of at least one PDZ domain of Dlg. This region of E6 is not conserved amongst E6 proteins from the low risk papillomavirus types and, hence, binding of HPV E6 proteins to Dlg correlates with the oncogenic potential of these viruses. We have performed studies to investigate the consequences of the interaction between E6 and Dlg. Mutational analysis of both the HPV18 E6 and Dlg proteins has further de®ned the regions of E6 and Dlg necessary for complex formation. Strikingly, co-expression of wild type HPV18 E6 with Dlg in vitro or in vivo results in a dramatic decrease in the amount of Dlg protein, whereas mutants of E6 which fail to complex with Dlg have minimal e ect on Dlg protein levels. The oncogenic HPV16 E6 also decreased the Dlg levels, but this was not observed with the low risk HPV11 E6 protein. Moreover, a region within the ®rst 544 amino acids of Dlg containing the three PDZ domains confers susceptibility to E6 mediated degradation. Finally, treatment of cells with a proteasome inhibitor overrides the capacity of E6 to degrade Dlg. These results demonstrate that Dlg is targeted by high risk HPV E6 proteins for proteasome mediated degradation.

Loss of cell polarity is one of the hallmarks of malignant carcinomas. Most of the understanding about the link between cell polarity and proliferation control comes from studies on the Drosophila tumor suppressors discs large (Dlg), scribble (Scrib) and lethal giant larvae (lgl). Mammalian homologues of these proteins have been described and are conserved in sequence and function. Human Dlg (hDlg) and Scrib were independently shown to be down-regulated during malignant progression. This, and other lines of evidence, points toward the participation of both hDlg and hScrib in a common pathway involved in polarity control and tumor suppression. We investigated the correlation between the expression of both proteins in tissues and their relative contributions to the maintenance of tissue architecture during colon cancer development. We analyzed the levels and distribution of hDlg and hScrib by immunohistochemistry, using serial sections of the same sample. We used normal and neoplastic colon mucosa, since it offers a good model for analyzing these features in progressive dysplastic stages. The results demonstrate that both proteins localize at the same regions in polarized colon epithelia, and that in normal samples the proteins' distribution varies as cells differentiate at the surface mucosa. In neoplasia, alterations in the expression pattern of hDlg and of hScrib increase during tumor progression; down-regulation of both proteins being associated with lack of epithelial cell polarity and disorganized tissue architecture. The results, therefore, demonstrate that there is an inverse relationship between the levels of hDlg and hScrib expression and the loss of cell polarity and tissue architecture in the colon. ' 2006 Wiley-Liss, Inc.Key words: hDlg and Scribble tumor suppressors; colon cancer; tissue architecture; immunohistochemistry Epithelial cells display an apico-basal polarity that is required for their correct function. Polarity is mediated by the presence of different cell junctions that depend on the formation of multiprotein networks at the cell membrane. Moreover, loss of epithelial polarity and tissue architecture is one of the hallmarks of malignant carcinomas. Many observations have suggested that cell polarity and proliferation control are coupled cellular processes. 1 Compelling evidence and most of the understanding of this linkage comes from studies on the proteins encoded by the Drosophila neoplastic tumor suppressor genes discs large (Dlg), scribble (Scrib) and lethal giant larvae (lgl). 2 These proteins are functionally linked and mutations in any of them cause very similar phenotypes, involving hyperproliferation and loss of proper tissue architecture, ultimately leading to neoplasia. 3 The Dlg, Scrib and Lgl tumor suppressors are conserved in both sequence and function among species and the mammalian homologues of these proteins have also been shown to complement the equivalent Drosophila mutants, suggesting an evolutionarily conserved role in regulating cell polarity and tumor suppression...

The Discs Large (DLG) tumour suppressor protein is targeted for ubiquitin mediated degradation by the high risk human papillomavirus (HPV) E6 proteins. In this study we have used a mutational analysis of E6 in order to investigate the mechanism by which this occurs. We ®rst show that the dierences in the anities of HPV-16 and of HPV-18 E6 proteins for binding DLG is re¯ected in their respective abilities to target DLG for degradation. A mutational analysis of HPV-18 E6 has enabled us to de®ne regions within the carboxy terminal half of the protein which are essential for the ability of E6 to direct the degradation of DLG. Mutants within the amino terminal portion of E6 which have lost the ability to bind the E6-AP ubiquitin ligase, as measured by their ability to degrade p53, nonetheless retain the ability to degrade DLG. Signi®cant levels of DLG degradation are also obtained using wheat germ extracts which lack E6-AP. Finally, we show that the transfer of the DLG binding domain onto the low risk HPV-6 E6 confers DLG binding activity to that protein and, most signi®cantly, allows HPV-6 E6 to target DLG for degradation. These results indicate that E6 mediated degradation of DLG does not involve the E6-AP ubiquitin ligase and, in addition, shows that the high and low risk HPV E6 proteins most likely share a common cellular intermediary in the ubiquitin pathway. Oncogene (2000) 19, 719 ± 725.

High-risk HPVs play a causal role in the development of cervical cancer, and their E6 oncoproteins target h-Dlg for ubiquitin-mediated proteolysis. The h-Dlg oncosuppressor is associated with cell-cell interactions, and deregulation of these structures leads to defective cell adhesion, loss of cell polarity and unregulated proliferation. We evaluated the contribution of this E6 activity in the progression to malignancy in HPV infections by analyzing h-Dlg expression in HPV-associated lesions. We analyzed h-Dlg in cervical, laryngeal, vulvar, colon and kidney histologic samples by Dlg immunohistochemistry. HPV association was ascertained by a PCR-colorimetric method. Although Dlg was certainly expressed in intraepithelial cervical, vulvar and laryngeal HPVassociated lesions, its cellular and tissue distribution patterns were altered compared to normal tissue. However, marked reduction in Dlg levels was observed in HPV-positive invasive cervical carcinomas. To elucidate whether the loss of Dlg was significant for carcinogenesis in general, we investigated Dlg expression in tumors not associated with HPV. In colon and kidney carcinomas, Dlg was expressed, albeit with a different pattern of distribution with respect to the normal tissue. The loss of Dlg may be considered a late-stage marker in cervical carcinogenesis, but alterations in its expression and localization take place during the different dysplastic stages. Dlg downregulation and/or alterations in its localization may contribute to transformation and may explain some of the characteristics of the malignant cells, such as loss of polarity and high migration ability. © 2004 Wiley-Liss, Inc. Key words: human papillomavirus; E6 protein; hDlg oncosuppressor; proteolysis; immunohistochemistryA large number of epidemiologic studies have demonstrated high-risk HPV association in almost 100% of invasive carcinomas of the uterine cervix and in the development of precursor intraepithelial neoplastic lesions. 1 The principal oncogenic effects of HPVs are mediated by the E6 and E7 proteins, which target and interfere with the function of key regulatory cellular proteins. 2 E6 oncoproteins have a great number of cellular targets involved in the regulation of cell proliferation, differentiation, apoptosis and adhesion. E6 stimulates the degradation of many of its partners, such as p53, bak, Mcm7, h-Dlg1 and h-scrib, by ubiquitin-mediated proteolysis. [3][4][5][6][7] Dlg, human homologue of Drosophila Dlg-A, was the first reported target of E6 related to cell-cell interactions and polarity. 6 Dlg 8,9 is a member of the MAGuK family of proteins, characterized by specific protein recognition domains including SH3, PDZ and homologous GuK regions. 8 -10 In Drosophila, Dlg is involved in cell growth control, maintenance of cell adhesion and polarity and functions that block cell invasion during development. 11 Dlg and another 2 related tumor suppressors, scribble and lgl, act cooperatively in regulating cell polarity and proliferation, suggesting an important connection betw...

The protein Kinase A (PKA) pathway was found to selectively regulate the function of oncogenic but not non-oncogenic E6 proteins. High risk E6 proteins are phosphorylated at their Dlg/PDZ binding motif at the C-terminus by a PKA like activity. This PKA and PDZ binding module is found only for human PV, is strictly conserved in all the transforming HPVs and is absent in all the low risk HPV types. We present evidence of a conditional regulation of E6 induced degradation of Dlg. HPV18E6 positive but not HPV negative keratinocytes exhibit increased Dlg steady state levels under conditions of high PKA activity, with a concomitant increase in the presence of Dlg at tight junctions. In vitro binding experiments show that E6 phosphorylation by PKA reduces its binding to Dlg and molecular modelling can explain this observation in a structural context. E6 dependent degradation of Dlg in cells with high PKA levels is inhibited and this is dependent on phosphorylation of the PDZ binding site in E6. In contrast, the degradation of p53 induced by E6 is not a ected by PKA. We propose a di erential regulation of E6 for the ubiquitin mediated degradation of speci®c E6 target proteins. Oncogene (2000) 19, 5884 ± 5891.

The genes encoding two subunits of acetyl coenzyme A carboxylase, biotin carboxyl carrier protein, and biotin carboxylase have been cloned from Bacillus subtilis. DNA sequencing and RNA blot hybridization studies indicated that the B. subtilis accB homolog which encodes biotin carboxyl carrier protein, is part of an operon that includes accC, the gene encoding the biotin carboxylase subunit of acetyl coenzyme A carboxylase.Our knowledge of lipid metabolism in Bacillus subtilis is scant. Although B. subtilis is often considered the paradigmatic gram-positive organism, the mechanisms involved in lipid biosynthesis have been little studied, and much is argued by analogy with Escherichia coli (5). Recent studies indicate that fatty acids might act as signalling molecules that are important for cellular differentiation in B. subtilis (19), which prompted us to identify genes involved in fatty acid synthesis. Since the first enzymatic step in a metabolic pathway is often rate limiting, we sought to isolate and characterize the genes encoding subunits of the acetyl coenzyme A (acetyl-CoA) carboxylase (ACC), which is the enzyme catalyzing the first committed step of fatty acid synthesis, i.e., the ATP-dependent carboxylation of acetylCoA to malonyl-CoA (12, 13). In E. coli, carboxylation of acetyl-CoA proceeds through two distinct reactions and involves an enzyme composed of four subunits: biotin carboxyl carrier protein (BCCP), biotin carboxylase (BC), and carboxyl transferase (CT), a tetramer composed of two nonidentical subunits (12, 13). Cloning of prokaryotic ACCs subunits in the gram-negative bacteria E. coli (12, 13), Anabaena species (8), and Pseudomonas aeruginosa species (2) and in mycobacteria (16) has been reported; however, no acc genes from Bacillus species or other gram-positive species have been reported. The genes studied encode protein products similar to those of the E. coli BCCP and BC subunits. However, these gene arrangements have been reported to differ. In E. coli (12) and P. aeruginosa (2), the BCCP and BC genes (accB and accC, respectively) form a two-gene operon, whereas in Anabena species, the genes encoding BCCP and BC are unlinked (8). In contrast, genes from Mycobacterium leprae and Mycobacterium tuberculosis encode biotinylated proteins that in these organisms have both BCCP and BC functions (16), which is an arrangement that is also seen in the ␣ subunit of the mammalian mitochondrial propionyl-CoA carboxylase (3, 11). The genes coding for the CT ␣ and ␤ subunits (accA and accD, respectively) have been cloned only for E. coli (13).Here, we report that a DNA fragment containing the gene encoding the BCCP subunit of B. subtilis complements an E. coli strain with a mutant BCCP subunit. Moreover, we present the nucleotide sequence of an approximately 2.5-kb fragment that includes the B. subtilis accB and accC genes, and we demonstrate that the accB and accC genes form part of an operon located 220Њ downstream of spoIIIA (in the sin-ahrC interval) of the B. subtilis chromosome.Cloning of B. s...

A unique feature of the cancer-causing mucosotropic human papillomaviruses (HPVs) is the ability of their E6 proteins to interact with a number of PDZ domain-containing cellular substrates, including the cell polarity regulators hDlg and hScrib. These interactions are essential for the ability of these viruses to induce malignant progression. Rhesus papillomaviruses (RhPV) are similar to their human counterparts in that they also cause anogenital malignancy in their host, the Rhesus Macaque. However, unlike HPV E6, the RhPV E6 has no PDZ-binding motif. We now show that such a motif is present on the RhPV E7 oncoprotein. This motif specifically confers PDZ-binding activity and directs the interaction of RhPV E7 with the cell polarity regulator Par3, which it targets for proteasome-mediated degradation. These results demonstrate an amazing evolutionary conservation of function between the RhPV and the HPV oncoproteins, where both target proteins of the same cell polarity control network, although through different components and pathways.