Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2016
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
_version_ 1782445009326309376
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
work_keys_str_mv AT illianoelena productionoffunctionalstableunmutatedrecombinanthumanpapillomaviruse6oncoproteinimplicationsforhpvtumordiagnosisandtherapy
AT demurtasoliviacostantina productionoffunctionalstableunmutatedrecombinanthumanpapillomaviruse6oncoproteinimplicationsforhpvtumordiagnosisandtherapy
AT massasilvia productionoffunctionalstableunmutatedrecombinanthumanpapillomaviruse6oncoproteinimplicationsforhpvtumordiagnosisandtherapy
AT dibonitopaola productionoffunctionalstableunmutatedrecombinanthumanpapillomaviruse6oncoproteinimplicationsforhpvtumordiagnosisandtherapy
AT consalvivalerio productionoffunctionalstableunmutatedrecombinanthumanpapillomaviruse6oncoproteinimplicationsforhpvtumordiagnosisandtherapy
AT chiaraluceroberta productionoffunctionalstableunmutatedrecombinanthumanpapillomaviruse6oncoproteinimplicationsforhpvtumordiagnosisandtherapy
AT zanottocarlo productionoffunctionalstableunmutatedrecombinanthumanpapillomaviruse6oncoproteinimplicationsforhpvtumordiagnosisandtherapy
AT degiulimorghencarlo productionoffunctionalstableunmutatedrecombinanthumanpapillomaviruse6oncoproteinimplicationsforhpvtumordiagnosisandtherapy
AT radaelliantonia productionoffunctionalstableunmutatedrecombinanthumanpapillomaviruse6oncoproteinimplicationsforhpvtumordiagnosisandtherapy
AT venutialdo productionoffunctionalstableunmutatedrecombinanthumanpapillomaviruse6oncoproteinimplicationsforhpvtumordiagnosisandtherapy
AT franconirosella productionoffunctionalstableunmutatedrecombinanthumanpapillomaviruse6oncoproteinimplicationsforhpvtumordiagnosisandtherapy