Identification of the regions of the HPV 5 E6 protein involved in Bak degradation and inhibition of apoptosis

Abstract

UVB induced DNA damage is the major aetiological agent in NMSC development, but mounting evidence suggests a role for human papillomaviruses (HPV) from genus beta, including HPV 5 and HPV 8, in the development of NMSC on sun exposed body sites. We have previously shown that UVB activates Bak, an apoptogenic mitochondrial factor that, following an apoptotic stimulus, undergoes a conformational change that leads to pore formation in the mitochondrial membrane that releases apoptotic factors. The HPV E6 protein effectively inhibits UVB-induced apoptosis and targets Bak for proteolytic degradation. We have now identified the regions of the HPV5 E6 that are required to mediate Bak proteolysis and contribute toward the antiapoptotic activity of the protein. Interestingly, while wild-type HPV5 E6 does not bind or target p53 for proteolysis, we have isolated specific HPV5 E6 mutants that switch target specificity from Bak to p53 in a p53 codon 72 isoform-dependent manner. Furthermore, we demonstrate that the ability of wild-type HPV5 E6 to target Bak or specific E6 mutants to target p53 for proteolysis is not dependent on the E6-AP ubiquitin ligase. ' 2008 Wiley-Liss, Inc.Key words: skin cancer; HPV; Bak; E6; UV; light Chronic exposure to solar UV irradiation has been identified as the primary causative agent in the development of nonmelanoma skin cancer (NMSC).1 The formation of ''sunburn cells'' frequently observed in epidermis treated with UVB display apoptotic characteristic such as condensed nuclei, 2,3 and the response to UVB radiation is in part dependent upon the expression of p53. 4This p53-driven response, often termed ''cellular proofreading'', eliminates rather than repairs severely damaged cells, however p53-independent pathways have also been described. [5][6][7] However, studies indicate that Li Fraumeni patients, who have only one copy of p53, and patients with regions of mutated p53 on sunexposed sites, are not predisposed to tumour development 8,9 indicating that p53-independent mechanisms also play an important role in eliminating damaged cells.Studies on Epidermodysplasia Verruciformis (EV) patients have identified a link between extensive wart infection and the development of skin tumours on sun-exposed sites.10 Epidemiological studies also invoke a role for HPV in NMSC development in both immunocompromised organ transplant recipients and immunocompetent individuals. 11,12 There is increasing evidence that b-HPV types facilitate the persistence of DNA-damaged cells within the epithelium, following UVB-exposure, chiefly through interfering with DNA damage responses and inhibiting apoptotic pathways, reviewed by Akg€ ul et al. 13The Bak protein is a key apoptogenic factor located in the outer mitochondrial membrane.14 Following UV exposure, Bak is activated and stabilized in a p53-independent manner 7 by BH3 domain-containing proteins. 15,16 Bak activation involves a change in conformation at the N-terminus of the prote in 17 leading to Bak multimerization, [18][19][20] that is believed to form pores...

p53 Degradation Activity, Expression, and Subcellular Localization of E6 Proteins from 29 Human Papillomavirus Genotypes

“…HPV16 E6 was shown to interact in vitro with the karyopherins ␣2, ␤1, and ␤2 (37), suggesting that E6 is actively imported in the nucleus. Our subcellular localization of the 29 HPV E6 proteins used in this study, as fusions with YFP, showed that the E6 proteins from HPV types 11,16,18,26,30,31,33,34,35,39,42,45,51,52,53,56,58,59,66,68,69,70,73,82,83, and 97 accumulate predominantly in the nucleus after transfection (Fig. 5).…”

“…Accordingly, transgenic mice expressing E6 in the skin develop malignant skin tumors, whereas E7 expression induces primarily benign hyperplasia (25,54,55). In addition to its ability to degrade p53, E6 was also shown to target several other proteins, among which is the multidomain proapoptotic protein Bak (14,30,51,64,65,69). This activity is thought to be important to prevent apoptosis of infected cells and to favor the transformation process.…”

A conserved C-terminal sequence of high-risk cutaneous beta-human papillomavirus E6 proteins alters localization and signalling of β1-integrin to promote cell migration

“…E6 proteins encoded by b-HPVs do not in general share this function; however, a recent report suggested that the HPV49 E6 protein is able to target p53 for degradation to some degree (Cornet et al, 2012), but whether this is dependent on the p53 isoform (Storey et al, 1998), as noted previously for specific mutants of HPV5 E6 (Simmonds & Storey, 2008), remains to be determined. Nevertheless, transgenic mice expressing the HPV8 E6 protein mimic expression of the complete early region of the HPV8 genome, in that E6 expression leads to papilloma formation, which can progress spontaneously to SCC (Marcuzzi et al, 2009).…”

“…2b). Whilst other mutations generated across the HPV5 E6 protein do not affect its stability (Simmonds & Storey, 2008), we cannot rule out at this time whether or not these mutations at the C terminus alter E6 structure or stability. Empty lanes were left between each sample on the gel to limit sample cross-contamination (indicated by the background staining observed).…”

Human Papillomaviruses Oncoproteins

“…the intrinsic pathway, which triggers sensing apoptotic signals that arise within the cell (DNA damage, oxidative stress) [54]. The lr/hrE6 oncoproteins block intrinsic apoptotic signaling interacting with Bak, inducing its proteasomal-dependent degradation [55], or using a mechanism depending or not on E6AP and E3 ubiquitin ligases [56].…”