There are more than 90 human DUBs (2
). The largest family of these enzymes includes the ubiquitin-specific proteases, characterized by prototypic Cys and His box motifs. DUBs play important roles in a wide variety of cellular processes. These include protecting substrates from K48-linked ubiquitin-mediated degradation by the proteasome and altering signaling and localization of proteins through the removal of K63-linked ubiquitin moieties. USP15 was first characterized a decade ago (3
); however, only recently have some of its functions and targets been elucidated.
In this study, we report that the HPV16 E6 oncoprotein and USP15 interact and demonstrate that USP15 is involved in regulating E6 protein stability. Overexpression of USP15 stabilizes E6, and siRNA-mediated knockdown of USP15 results in decreased levels of E6 protein. The catalytic activity of USP15 was required for increased E6 protein levels, suggesting that ubiquitylated E6 may itself be a target for USP15 deubiquitylation.
There are previous reports in the literature of viral proteins interacting with cellular DUBs. For instance, USP7 (or HAUSP) interacts with the herpes simplex virus type I immediate early protein ICP0 (4
) as well as the Epstein-Barr virus protein EBNA1 (13
). USP7 is known to regulate the cellular turnover of p53 (26
), suggesting that these herpesviruses may be able to manipulate the apoptotic status of infected cells through interaction with USP7. Binding of USP11 has been reported to stabilize HPV16 E7 through deubiquitylation, extending its half-life and enhancing the downstream degradation of Rb (27
). In addition, HPV16 and HPV18 E6 proteins have been implicated in the ubiquitylation and degradation of the CYLD DUB, resulting in the sustained hypoxia-induced activation of NF-κB (1
). That study demonstrated CYLD in complex with E6 but did not provide evidence of a direct interaction between E6 and the CYLD DUB (1
No link between USP15 and cancer has yet been established. USP15 was found to be active in various tumor cell lines, including those derived from human cervix, colon, lung, brain, and kidney cancers, as well as in some human lymphomas (31
). A subsequent study compared the activities of a variety of USPs from primary cervical carcinoma tissue and matched normal cervical tissue; no consistent up- or downregulation of USP15 activity was detected in samples from 27 patients (34
In our study, we examined whether the decrease in E6 following USP15 knockdown resulted in increased p53 levels. We did not observe any effect up to 96 h after introduction of the siRNAs. The lack of effect could be due to the previously observed phenomenon that when E6 is removed as the major regulator of p53 in HPV-positive cells, the “natural” regulators MDM2, COP1, and Pirh2 resume control of p53 levels (21
). Alternatively, the reduction in E6 levels might not have been sufficient to affect the overall cellular E6/E6AP ubiquitylation activity. It is also possible that the E6-USP15 interaction has functional consequences in a pathway other than that defined for p53.
Identification of the substrate targets for USP15 is ongoing. USP15 associates with the COP9 signalosome (CSN) (12
), a multiprotein complex involved in regulating the ubiquitin-proteasome pathway predominantly though interaction with cullin-based E3 ligases. In this context, USP15 has been shown to deubiquitylate the E3 ligase Rbx1, presumably to protect Rbx1 from autoubiquitylation (12
). More recently, IκBα was shown to be deubiquitylated by USP15 in association with the CSN after tumor necrosis factor alpha-mediated stimulation of the NF-κB pathway (37
). Our preliminary data suggest that E6 may affect the ability of USP15 to break down ubiquitin chains, but no link to any of the aforementioned pathways has been established (our unpublished data).
Future efforts will focus on understanding the normal cellular role of USP15 and how its function may be affected by the presence of E6, as well as evaluating a larger subset of known E6-interacting proteins to determine if USP15 may be involved in their regulation. We were unable to establish cell lines with either stable overexpression or stable knockdown by short hairpin RNA of USP15, indicating that USP15 likely plays a vital role within the cell and that its expression levels are tightly regulated. Insights into the relevance for carcinogenesis will also be gained by investigating whether both high-risk and low-risk HPV E6 proteins can interact with and are stabilized by USP15.