HPV E6 Protein

HPV E6 proteins consist of approximately 150 amino acids and contain two zinc finger domains each of them composed of two Cys-X-X-Cys motifs. In contrast to low risk E6 proteins, high risk E6 proteins possess a PDZ binding motif (X-(S/T)-X-(V/I/L)) at their very C terminus, which facilitates binding to PDZ domain-containing proteins (figure 4) (reviewed in Ghittoni et al., 2010).

Figure 4: Schematic map of E6 proteins

Low and high risk E6 proteins contain two zinc finger motifs. In addition, high risk E6 proteins have a C-terminal PDZ binding motive (modified from Ghittoni et al., 2010).

The best studied function of high risk HPV E6 proteins is the degradation of the tumor suppressor p53 (Scheffner et al., 1990). p53 is a transcription factor with critical regulatory functions in a cell including the induction of apoptosis. Simplified, p53 is activated in response to DNA damage, nucleotide depletion, hypoxia or other triggers, resulting in induction of genes involved in cell cycle regulation and apoptosis (reviewed in Efeyan and Serrano, 2007). p53 levels increase due to the action of E7 by binding to pRB and other cell

cycle regulators (see 1.1.5.1). As a consequence, these cells have an increased susceptibility to apoptosis (Jones et al., 1997b), which is counteracted by E6 via degradation of p53. E6 binds to p53 only in the presence of the cellular E3 ubiquitin ligase E6AP (E6 associated protein) forming a ternary complex (Huibregtse et al., 1991, 1993a). The E6/E6AP complex ubiquitylates p53, thereby leading to its proteasomal degradation (see 1.2), which contributes to the oncogenic potential of high risk HPVs. (Huibregtse et al., 1993b; Scheffner et al., 1993). In the absence of E6, E6AP is not involved in p53 degradation. As E6 mutants unable to induce degradation of p53 are competent to transform human mammary epithelial cells (HMEC) as efficiently as wild type E6, additional E6 functions must be necessary for their oncogenic potential (Liu et al., 1999).

Numerous cellular proteins have been reported to interact with high risk E6 proteins (reviewed in Pim and Banks, 2010) including PDZ domain-containing proteins (for details see 1.3) like hDlg, MAGI-1, -2, -3, and Scribble. The name PDZ derives from the first three proteins identified containing PDZ domains: PSD-95 (postsynaptic density-95 protein), DLG (Drosophila disc large protein) and ZO-1 (zonula occludens 1 protein) (reviewed in Harris and Lim, 2001). MAGUK proteins, including for example MAGI-1, -2, -3 and hDlg, are a group of PDZ domain-containing proteins which affect processes like cell polarity and maintenance of cell-to-cell interactions. Interaction of high risk E6 proteins with PDZ domain-containing proteins is mediated by the C-terminal PDZ binding motif of E6 (Kiyono et al., 1997).

Importantly, this PDZ binding motif is not required for binding to E6AP or p53. However, high risk E6 proteins target PDZ domain-containing proteins like hDlg for proteasomal degradation in an E6AP dependent manner (Kuballa et al., 2007). The fact that the PDZ binding motif of E6 proteins is required for transformation in transgenic mice underlines its functional importance (Simonson et al., 2005).

Immortalization experiments revealed additional important functions of high risk E6 proteins, in particular the induction of telomerase activity (Gewin et al., 2004; Oh et al., 2001;

Veldman et al., 2001; Veldman et al., 2003). Telomerase is only expressed in a small subset of normal cells (stem cells), however it is also expressed in tumor tissues and tumor derived cell lines (Kim et al., 1994b). E6 binds to hTERT (telomerase reverse transcriptase) promoter and induces its expression, which results in the activation of the telomerase complex. This activation is independent of the capability of E6 to degrade p53 and PDZ domain-containing proteins, but it is dependent on the binding of E6 to E6AP (Gewin et al., 2004; Kiyono et al., 1998; Liu et al., 2005). Two critical targets of E6/E6AP are known, namely Myc and NFX1, which act as transactivator and transrepressor of the hTERT promoter, respectively (Gewin et al., 2004; Katzenellenbogen et al., 2009; Liu et al., 2008). Furthermore, E6 can directly interact with the hTERT protein, which indicates that E6 may activate telomerase by two dinstinct mechanisms (Liu et al., 2009). However, the exact mechanisms how high risk E6

proteins activate telomerase are not completely understood so far.

Moreover, E6 proteins as well as E7 proteins induce genomic instability, precisely expression of high risk E6 proteins is associated with unaligned or lagging chromosomes (Duensing and Munger, 2002).

In contrast to high risk HPV E6 proteins, the functions of low risk HPV E6 proteins are only poorly characterized. Indeed they can interact with the cellular E3 ligase E6AP just like high risk E6 proteins (Brimer et al., 2007; Kuballa et al., 2007), but no targets for ubiquitylation are known so far. Low risk E6 proteins do not lead to the degradation of p53 (Scheffner et al., 1990) and as they do not contain a PDZ binding motif (figure 3), they presumably cannot interact and degrade PDZ domain-containing proteins, either (Brimer et al., 2007). Chimeric E6 proteins consisting of the N terminus of low risk E6 proteins and the very C terminus of high risk E6 proteins including the PDZ binding motif are able to bind and degrade PDZ domain-containing proteins, showing that the PDZ binding motif is sufficient to bind to these PDZ domain-containing proteins (Kuballa et al., 2007; Pim et al., 2002).

Some of the identified interaction partners and targets of high risk E6 proteins, e.g. Bak (Bcl-2 homologous antagonist/killer) have also been reported to be regulated by low risk E6 proteins (Kuhne and Banks, 1998; Thomas and Banks, 1999). However, in our experiments we neither could show binding of E6 to Bak nor E6-dependent ubiquitylation and degradation of Bak (Scheffner group, unpublished data).

As mentioned above, low risk and high risk E6 proteins bind to E6AP (Brimer et al., 2007;

Kuballa et al., 2007). Both are stabilized by this complex formation, and it is believed that binding of E6AP to E6 protects it from ubiquitylation and proteasomal degradation (Tomaic et al., 2009; Weber, 2009). However, the responsible E3 ligase for E6 ubiquitylation remains unknown.

Another HECT E3 ligase, EDD (E3 ubiquitin ligase identified by differential display) was identified as an interaction partner of 18E6 (Tomaic et al., 2011). In the same study it was shown that EDD also interacts with low risk 11E6 and high risk 16E6 but with a lower affinity than 18E6. In addition, EDD interacts with E6AP in an E6 independent manner and thereby regulates the expression levels of E6AP, resulting in the modulation of the E3 ligase activity of the E6/E6AP complex (Tomaic et al., 2011).

Another published interaction partner for high risk and low risk E6 proteins is the tumor suppressor TIP60 (Tat-interaction protein 60 kDa) (Jha et al., 2010), a histone acetyltransferase which is involved in transcriptional regulation, check-point activation and p53-directed proapototic pathways (reviewed in Sapountzi et al., 2006). Both low risk and high risk E6 proteins destabilize TIP60 by proteasomal degradation in an E6AP independent manner. As TIP60 represses the early HPV promoter, the destabilization of TIP60 by E6 proteins leads to derepression of the promotor (Jha et al., 2010).

Finally, further investigations are necessary to elucidate different and common properties of high risk and low risk E6 proteins.