High risk forms of the Human papillomavirus (HPV) are responsible for virtually all instances of cervical cancer. While there are preventative vaccines available, there is a significant unmet medical need for the treatment of current infections. By the age of 50, approximately 80% of women in the United States will have contracted at least one strain of genital HPV, resulting in an estimated 5 million deaths from cervical cancer over the next 20 years. HPV, and similar viruses, cause tumor growth by activating host cell genes required for viral replication, thus deregulating the host cell cycle. CBP (CREB-binding protein) and its nearest homolog p300, are members of a family of transcriptional co-activators involved in cell growth and differentiation, acting as both activators and repressors for multiple cellular pathways. They are therefore targeted by many cancer-causing viruses, including HPV, the Human T-cell Leukemia virus type-1 (HTLV-1), and Adenovirus E1A. In the case of high risk forms of HPV, which lead to cervical cancer, the oncoprotein E7 targets the TAZ2 domain of CBP/p300 in order to promote cell proliferation, ultimately resulting in tumor formation. This project will determine the structural basis by which E7 hijacks CBP/p300, leading to cervical cancer by elucidation of the E7:TAZ2 complex structure. The first step will be to identify the minimal domain of E7 necessary to bind TAZ2, followed by expression in bacteria and subsequent purification. The structures of the E7:TAZ2 complex, for both high risk HPV16 and low risk HPV6, will then be determined by NMR spectroscopy, and compared in order to ascertain structural and functional differences. Binding constants will be measured for high and low risk E7, with both the TAZ2 and TAZ1 domains, using NMR analysis and isothermal titration calorimetry (ITC), in order to quantitate differences in affinity. A detailed structural comparison will also be made to the previously solved E1A:TAZ2 complex to better understand viral oncoprotein mechanisms. Utilizing NMR chemical shift perturbation, it will be determined if E7 competes with the tumor suppressor p53 for interaction with TAZ2, as is the case for E1A. NMR chemical shift perturbation also will be employed to determine if high and low risk E7 bind simultaneously to TAZ2 and the tumor suppressor retinoblastoma protein (pRb). If so, NMR experiments will be used to measure whether the interaction is synergistic, as it is for E1A, and a model will be generated for the trimeric pRb:E7:TAZ2 complex, using methods previously employed to evaluate pRb:E1A:TAZ2. The results from this study will provide crucial information regarding the mechanism viruses like HPV use to hijack host cells, ultimately resulting in cervical cancer. Furthermore, this study will provide fundamental information regarding the ability of viral cancer-causing proteins to bind multiple partners within host cells. Understanding this mechanism has the potential to lead to treatments for viral-induced cancer.

Public Health Relevance

This project will determine the structural basis by which the HPV oncoprotein E7 hijacks CBP (CREB- binding Protein)/p300, resulting in cervical cancer, by elucidation of the E7:TAZ2 complex structure by NMR spectroscopy. The results from this study will provide fundamental information regarding the ability of oncogenic proteins, such as E7, to bind multiple partners within host cells. This information may ultimately lead to treatments for viral-induced cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32GM095118-01
Application #
8002568
Study Section
Special Emphasis Panel (ZRG1-F04B-B (20))
Program Officer
Flicker, Paula F
Project Start
2010-09-01
Project End
2013-09-29
Budget Start
2010-09-01
Budget End
2011-09-29
Support Year
1
Fiscal Year
2010
Total Cost
$45,590
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037