Viral Oncology, Cancer Cell Biology
i) Regulation of ubiquitin-proteasome and DNA damage signalling pathways by adenovirus
In recent years it has become apparent that in order to replicate efficiently viruses have evolved to selectively activate/inactivate DNA damage response signalling pathways in order to promote viral replication. In this regard we have determined that different Ad serotypes differentially regulate DNA damage signalling pathways during infection (Blackford et al., 2008; Forrester et al., 2011). These studies have also determined that the adenovirus E1B55K-binding protein, E1B-AP5 (also known as hnRNPUL1) is an integral component of ATR signalling pathways activated during infection (Blackford et al., 2008).
It has previously been determined that Ad5 utilizes a cullin 5-RING ubiquitin ligase to target p53 and the Mre11-Rad50-NBS1 component, Mre11 for proteasomal-mediated degradation during infection in order to inactivate p53 and ATM signalling pathways. Recent work from our lab has determined that Ad12 preferentially utilizes a cullin 2-RING ubiquitin ligase to target p53 for degradation during infection, and that Ad12 also uses this ubiquitin ligase to target the ATR activator, TopBP1 for proteasomal degradation during infection (Blackford et al., 2010). Ad12-mediated degradation of TopBP1 ensures that the checkpoint kinase, Chk1 is not activated during infection.
We are currently employing proteomic techniques to identify novel adenovirus early region-binding proteins in adenovirus-infected and adenovirus-transformed cells. We anticipate that this approach will help identify proteins that function in DNA damage response pathways or anti-viral/stress response pathways and that are targeted by adenovirus in order to facilitate viral replication and/or promote adenovirus-mediated cellular transformation. In order to determine to what extent adenovirus targets the ubiquitin-proteasome pathway during infection we also employ proteomic techniques in order to identify those proteins whose expression is affected significantly by adenovirus infection or during the process of adenovirus-mediated cellular transformation.
ii) Regulation of the Anaphase-Promoting Complex/Cyclosome (APC/C)
The anaphase-promoting complex/cyclosome (APC/C) is a multicomponent E3 ubiquitin ligase that, by targeting protein substrates for 26S proteasome-mediated degradation through ubiquitylation, coordinates the temporal progression of eukaryotic cells through mitosis and the subsequent G1 phase of the cell cycle. Work in our lab established that the APC/C subunits APC5 and APC7 possess within their primary sequence CBP and p300 protein-protein interaction domains that are evolutionarily conserved in adenovirus E1A (Turnell et al., 2005) In this regard we determined that the APC/C and CBP/p300 cooperate to regulate transcription and cell cycle progression and that E1A targets APC/C-CBP/p300 complexes to promote cellular transformation.
In collaboration with Dr Grant Stewart we recently investigated the role of the DNA damage response protein, Mediator of DNA damage Checkpoint 1 (MDC1) in mitosis. We determined that MDC1 regulates metaphase-to-anaphase transition through its ability to bind directly to the APC/C-Cdc20 and modulate its E3 ubiquitin ligase activity (Townsend et al., 2009). The ability of MDC1 to regulate metaphase-to-anaphase transition was found to be independent of both the Spindle Assembly Checkpoint and ATM/ATR activation, but depended upon its ability to regulate the Cdc20-dependent activation of the APC/C (Townsend et al., 2009).
In light of these studies, current work in the lab is focused towards establishing how CBP/p300 and DNA damage signalling pathways regulate APC/C function, and determining the role of the APC/C in CBP/p300 transcription programmes and DNA damage signalling pathways. We have also identified novel APC/C-interacting proteins by mass spectrometry and are consequently interested in establishing the role of these proteins in cell cycle control as well as determining their functional relationship to the APC/C.
Sedgwick G. G., Townsend K., Martin A., Shimwell N. J., Grand R. J. A., Stewart G. S., Nilsson J., and Turnell A. S. (2012) Transcriptional Intermediary Factor 1γ binds to the Anaphase-Promoting Complex/Cyclosome and promotes mitosis. Oncogene. doi: 10.1038/onc.2012.501. [Epub ahead of print].
Turnell A. S. and Grand R. J. (2012). DNA viruses and the cellular DNA-damage response. J. Gen. Virol. 93:2076-2097.
Forrester N. A., Patel R. N., Speiseder T., Groitl P., Sedgwick G. G., Shimwell N. J., Seed R. I., Ó Catnaigh P, McCabe C. J., Stewart G. S., Dobner T., Grand R. J., Martin, A. and Turnell A. S. (2012). Adenovirus E4orf3 targets Transcriptional Intermediary Factor 1γ for proteasome-dependent degradation during infection. J. Virol. 86:3167-3179.
Forrester N. A., Sedgwick G.G., Thomas A,. Blackford A.N., Speiseder T., Dobner T., Byrd P.J., Stewart G.S., Turnell A.S. and Grand R.J. (2011). Serotype-specific inactivation of the cellular DNA damage response during adenovirus infection. J Virol. 85:2201-2211.
Blackford A. N., Patel R. N., Forrester, N. A., Theil K., Groitl P., Stewart G. S., Taylor A. M. R., Morgan I. M., Dobner T., Grand R. J. A. and Turnell A. S. (2010) Adenovirus 12 E4orf6 inhibits ATR activation by promoting TOPBP1 degradation Proc. Natl. Acad. Sci. USA. 107:12251-12256.
Townsend K., Mason H., Blackford A. N., Miller E. S., Chapman J.R., Sedgwick G. G., Barone G., Turnell A. S. and Stewart G. S. (2009) MDC1 regulates mitotic progression. J. Biol. Chem. 284:33939-48.
Blackford A. N., Stewart G. S., Taylor A. M. R., Dobner T., Grand R. J. and Turnell A. S. (2008) A role for E1B-AP5 in ATR kinase signalling pathways during adenovirus infection J. Virol. 82:7640-7652.
Bruton R. K., Rasti M., Mapp K. L., Young N., Carter R. Z., Abramowicz I. A., Onion D. F., Shuen D. F., Mymryk J. S., Turnell A. S. and Grand R. J. A. (2007) CtIP binds directly to AdE1A through its N terminal region and CR3. Oncogene 26:7467-7479.
Rasti M., Grand R. J. A., Yousef Y. F., Shuen M., Mymryk J. S., Gallimore P. H. and Turnell A. S. (2006) Roles for APIS and the 20S proteasome in adenovirus E1A-dependent transcription. EMBO J. 25:2710-2722.
Turnell A. S., Stewart G. S., Grand R. J. A., Rookes S. M., Martin A., Yamano H., Elledge S. J. and Gallimore P. H. (2005) The APC/C and CBP/p300 cooperate to regulate transcription and cell-cycle progression. Nature 438:690-695.
Rasti M., Grand R. J. A., Mymryk J. S., Gallimore P. H. and Turnell A. S. (2005) Recruitment of CBP/p300, TBP and S8 to distinct regions at the N-terminus of adenovirus E1A. J. Virol. 79:5594-5605.
Turnell A. S. , Grand R. J. A., Gorbea C., Xhang X., Wang W., Mymryk J. S. and Gallimore P. H. (2000) Regulation of the 26S proteasome by adenovirus E1A. EMBO J. 19:4759-73.
Hutton F. G., Turnell A. S., Gallimore P. H. and Grand R. J. A. (2000) Consequences of disruption of the interaction between p53 and the larger adenovirus early region 1B protein in adenovirus E1 transformed cells. Oncogene 19:452-62.
Turnell A. S., Grand R. J. A. and Gallimore P. H. (1999) The replicative capacities of large E1B-null group A and group C adenoviruses are independent of host cell p53 status. J. Virol. 73:2074-83.