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Production of functional, stable, unmutated recombinant human papillomavirus E6 oncoprotein: implications for HPV-tumor diagnosis and therapy
BACKGROUND: High-risk human papillomaviruses (HR-HPVs) types 16 and 18 are the main etiological agents of cervical cancer, with more than 550,000 new cases each year worldwide. HPVs are also associated with other ano-genital and head-and-neck tumors. The HR-HPV E6 and E7 oncoproteins are responsible...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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BioMed Central
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4963926/ https://www.ncbi.nlm.nih.gov/pubmed/27465494 http://dx.doi.org/10.1186/s12967-016-0978-6 |
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author | Illiano, Elena Demurtas, Olivia Costantina Massa, Silvia Di Bonito, Paola Consalvi, Valerio Chiaraluce, Roberta Zanotto, Carlo De Giuli Morghen, Carlo Radaelli, Antonia Venuti, Aldo Franconi, Rosella |
author_facet | Illiano, Elena Demurtas, Olivia Costantina Massa, Silvia Di Bonito, Paola Consalvi, Valerio Chiaraluce, Roberta Zanotto, Carlo De Giuli Morghen, Carlo Radaelli, Antonia Venuti, Aldo Franconi, Rosella |
author_sort | Illiano, Elena |
collection | PubMed |
description | BACKGROUND: High-risk human papillomaviruses (HR-HPVs) types 16 and 18 are the main etiological agents of cervical cancer, with more than 550,000 new cases each year worldwide. HPVs are also associated with other ano-genital and head-and-neck tumors. The HR-HPV E6 and E7 oncoproteins are responsible for onset and maintenance of the cell transformation state, and they represent appropriate targets for development of diagnostic and therapeutic tools. METHODS: The unmutated E6 gene from HPV16 and HPV18 and from low-risk HPV11 was cloned in a prokaryotic expression vector for expression of the Histidine-tagged E6 protein (His(6)-E6), according to a novel procedure. The structural properties were determined using circular dichroism and fluorescence spectroscopy. His(6)-E6 oncoprotein immunogenicity was assessed in a mouse model, and its functionality was determined using in vitro GST pull-down and protein degradation assays. RESULTS: The His(6)-tagged E6 proteins from HPV16, HPV18, and HPV11 E6 genes, without any further modification in the amino-acid sequence, were produced in bacteria as soluble and stable molecules. Structural analyses of HPV16 His(6)-E6 suggests that it maintains correct folding and conformational properties. C57BL/6 mice immunized with HPV16 His(6)-E6 developed significant humoral immune responses. The E6 proteins from HPV16, HPV18, and HPV11 were purified according to a new procedure, and investigated for protein–protein interactions. HR-HPV His(6)-E6 bound p53, the PDZ1 motif from MAGI-1 proteins, the human discs large tumor suppressor, and the human ubiquitin ligase E6-associated protein, thus suggesting that it is biologically active. The purified HR-HPV E6 proteins also targeted the MAGI-3 and p53 proteins for degradation. CONCLUSIONS: This new procedure generates a stable, unmutated HPV16 E6 protein, which maintains the E6 properties in in vitro binding assays. This will be useful for basic studies, and for development of diagnostic kits and immunotherapies in preclinical mouse models of HPV-related tumorigenesis. |
format | Online Article Text |
id | pubmed-4963926 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-49639262016-07-29 Production of functional, stable, unmutated recombinant human papillomavirus E6 oncoprotein: implications for HPV-tumor diagnosis and therapy Illiano, Elena Demurtas, Olivia Costantina Massa, Silvia Di Bonito, Paola Consalvi, Valerio Chiaraluce, Roberta Zanotto, Carlo De Giuli Morghen, Carlo Radaelli, Antonia Venuti, Aldo Franconi, Rosella J Transl Med Research BACKGROUND: High-risk human papillomaviruses (HR-HPVs) types 16 and 18 are the main etiological agents of cervical cancer, with more than 550,000 new cases each year worldwide. HPVs are also associated with other ano-genital and head-and-neck tumors. The HR-HPV E6 and E7 oncoproteins are responsible for onset and maintenance of the cell transformation state, and they represent appropriate targets for development of diagnostic and therapeutic tools. METHODS: The unmutated E6 gene from HPV16 and HPV18 and from low-risk HPV11 was cloned in a prokaryotic expression vector for expression of the Histidine-tagged E6 protein (His(6)-E6), according to a novel procedure. The structural properties were determined using circular dichroism and fluorescence spectroscopy. His(6)-E6 oncoprotein immunogenicity was assessed in a mouse model, and its functionality was determined using in vitro GST pull-down and protein degradation assays. RESULTS: The His(6)-tagged E6 proteins from HPV16, HPV18, and HPV11 E6 genes, without any further modification in the amino-acid sequence, were produced in bacteria as soluble and stable molecules. Structural analyses of HPV16 His(6)-E6 suggests that it maintains correct folding and conformational properties. C57BL/6 mice immunized with HPV16 His(6)-E6 developed significant humoral immune responses. The E6 proteins from HPV16, HPV18, and HPV11 were purified according to a new procedure, and investigated for protein–protein interactions. HR-HPV His(6)-E6 bound p53, the PDZ1 motif from MAGI-1 proteins, the human discs large tumor suppressor, and the human ubiquitin ligase E6-associated protein, thus suggesting that it is biologically active. The purified HR-HPV E6 proteins also targeted the MAGI-3 and p53 proteins for degradation. CONCLUSIONS: This new procedure generates a stable, unmutated HPV16 E6 protein, which maintains the E6 properties in in vitro binding assays. This will be useful for basic studies, and for development of diagnostic kits and immunotherapies in preclinical mouse models of HPV-related tumorigenesis. BioMed Central 2016-07-28 /pmc/articles/PMC4963926/ /pubmed/27465494 http://dx.doi.org/10.1186/s12967-016-0978-6 Text en © The Author(s) 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Illiano, Elena Demurtas, Olivia Costantina Massa, Silvia Di Bonito, Paola Consalvi, Valerio Chiaraluce, Roberta Zanotto, Carlo De Giuli Morghen, Carlo Radaelli, Antonia Venuti, Aldo Franconi, Rosella Production of functional, stable, unmutated recombinant human papillomavirus E6 oncoprotein: implications for HPV-tumor diagnosis and therapy |
title | Production of functional, stable, unmutated recombinant human papillomavirus E6 oncoprotein: implications for HPV-tumor diagnosis and therapy |
title_full | Production of functional, stable, unmutated recombinant human papillomavirus E6 oncoprotein: implications for HPV-tumor diagnosis and therapy |
title_fullStr | Production of functional, stable, unmutated recombinant human papillomavirus E6 oncoprotein: implications for HPV-tumor diagnosis and therapy |
title_full_unstemmed | Production of functional, stable, unmutated recombinant human papillomavirus E6 oncoprotein: implications for HPV-tumor diagnosis and therapy |
title_short | Production of functional, stable, unmutated recombinant human papillomavirus E6 oncoprotein: implications for HPV-tumor diagnosis and therapy |
title_sort | production of functional, stable, unmutated recombinant human papillomavirus e6 oncoprotein: implications for hpv-tumor diagnosis and therapy |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4963926/ https://www.ncbi.nlm.nih.gov/pubmed/27465494 http://dx.doi.org/10.1186/s12967-016-0978-6 |
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