The human papillomavirus E7-E2 interaction mechanism in vitro reveals a finely tuned system for modulating available E7 and E2 proteins

Transcription of the human papillomavirus E7 oncoprotein is negatively controlled by the viral E2 protein, and loss of this repression leads to irreversible transformation and carcinogenesis. Here we show that interaction of the HPV16 E7 protein with the DNA binding domain of the E2 protein (E2C) le...

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Autores principales:	Smal, C., Wetzler, D.E., Dantur, K.I., Chemes, L.B., Garcia-Alai, M.M., Dellarole, M., Alonso, L.G., Gaston, K., De Prat-Gay, G.
Formato:	JOUR
Materias:	Different sizes DNA binding DNA binding domain DNA duplexes E7 oncoprotein Gel electrophoresis Hetero-oligomerization Human papillomavirus In-vitro Interaction mechanisms Intracellular levels N-terminal domains Oligomeric complexes Phosphorylation sites Protein-protein interface Site directed mutagenesis Soluble complexes Viral proteins Binding sites Complexation DNA Electrophoresis Enzyme activity Genes Ionic strength Nucleic acids Oligomerization Oligomers Phosphorylation Titration Transcription Proteins protein E7 protein VP2 amino terminal sequence article gel electrophoresis human in vitro study ionic strength light scattering nonhuman oligomerization phosphorylation priority journal protein interaction titrimetry Wart virus Amino Acid Sequence Cell Line, Tumor Cell Proliferation DNA-Binding Proteins Human papillomavirus 16 Humans Molecular Sequence Data Oncogene Proteins, Viral Protein Structure, Tertiary ras Proteins Virus Integration
Acceso en línea:	http://hdl.handle.net/20.500.12110/paper_00062960_v48_n50_p11939_Smal
Aporte de:	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires

id	todo:paper_00062960_v48_n50_p11939_Smal
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spelling	todo:paper_00062960_v48_n50_p11939_Smal2023-10-03T14:04:30Z The human papillomavirus E7-E2 interaction mechanism in vitro reveals a finely tuned system for modulating available E7 and E2 proteins Smal, C. Wetzler, D.E. Dantur, K.I. Chemes, L.B. Garcia-Alai, M.M. Dellarole, M. Alonso, L.G. Gaston, K. De Prat-Gay, G. Different sizes DNA binding DNA binding domain DNA duplexes E7 oncoprotein Gel electrophoresis Hetero-oligomerization Human papillomavirus In-vitro Interaction mechanisms Intracellular levels N-terminal domains Oligomeric complexes Phosphorylation sites Protein-protein interface Site directed mutagenesis Soluble complexes Viral proteins Binding sites Complexation DNA Electrophoresis Enzyme activity Genes Ionic strength Nucleic acids Oligomerization Oligomers Phosphorylation Titration Transcription Proteins protein E7 protein VP2 amino terminal sequence article DNA binding gel electrophoresis human in vitro study ionic strength light scattering nonhuman oligomerization phosphorylation priority journal protein interaction titrimetry Wart virus Amino Acid Sequence Cell Line, Tumor Cell Proliferation DNA-Binding Proteins Human papillomavirus 16 Humans Molecular Sequence Data Oncogene Proteins, Viral Protein Structure, Tertiary ras Proteins Virus Integration Human papillomavirus Transcription of the human papillomavirus E7 oncoprotein is negatively controlled by the viral E2 protein, and loss of this repression leads to irreversible transformation and carcinogenesis. Here we show that interaction of the HPV16 E7 protein with the DNA binding domain of the E2 protein (E2C) leads to ionic strength-dependent hetero-oligomerization even at the lowest concentrations measurable. Titration experiments followed by light scattering and native gel electrophoresis show insoluble oligomeric complexes with a ≥2000 nm diameter and intermediate soluble complexes 40 and 115 nm in diameter, respectively, formed in excess of E2C. Adiscrete oligomeric soluble complex formed in excess of E7 displays a diameter of 12 nm. The N-terminal domain of E7 interacts with E2C with a KD of 0.1 μM, where the stretch of residues 25-40 of E7, encompassing both a PEST motif and phosphorylation sites, is sufficient for the interaction. Displacement of the soluble E7-E2C complex by an E2 site DNA duplex and site-directed mutagenesis indicate that the protein-protein interface involves the DNA binding helix of E2. The formation of complexes of different sizes and properties in excess of either of the viral proteins reveals a finely tuned mechanism that could regulate the intracellular levels of both proteins as infection and transformation progress. Sequestering E2 into E7-E2 oligomers provides a possible additional route to uncontrolled E7 expression, in addition and prior to the disruption of the E2 gene during viral integration into the host genome. © 2009 American Chemical Society. Fil:Smal, C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Wetzler, D.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Chemes, L.B. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Garcia-Alai, M.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Dellarole, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Alonso, L.G. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:De Prat-Gay, G. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_00062960_v48_n50_p11939_Smal
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-134
collection	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
topic	Different sizes DNA binding DNA binding domain DNA duplexes E7 oncoprotein Gel electrophoresis Hetero-oligomerization Human papillomavirus In-vitro Interaction mechanisms Intracellular levels N-terminal domains Oligomeric complexes Phosphorylation sites Protein-protein interface Site directed mutagenesis Soluble complexes Viral proteins Binding sites Complexation DNA Electrophoresis Enzyme activity Genes Ionic strength Nucleic acids Oligomerization Oligomers Phosphorylation Titration Transcription Proteins protein E7 protein VP2 amino terminal sequence article DNA binding gel electrophoresis human in vitro study ionic strength light scattering nonhuman oligomerization phosphorylation priority journal protein interaction titrimetry Wart virus Amino Acid Sequence Cell Line, Tumor Cell Proliferation DNA-Binding Proteins Human papillomavirus 16 Humans Molecular Sequence Data Oncogene Proteins, Viral Protein Structure, Tertiary ras Proteins Virus Integration Human papillomavirus
spellingShingle	Different sizes DNA binding DNA binding domain DNA duplexes E7 oncoprotein Gel electrophoresis Hetero-oligomerization Human papillomavirus In-vitro Interaction mechanisms Intracellular levels N-terminal domains Oligomeric complexes Phosphorylation sites Protein-protein interface Site directed mutagenesis Soluble complexes Viral proteins Binding sites Complexation DNA Electrophoresis Enzyme activity Genes Ionic strength Nucleic acids Oligomerization Oligomers Phosphorylation Titration Transcription Proteins protein E7 protein VP2 amino terminal sequence article DNA binding gel electrophoresis human in vitro study ionic strength light scattering nonhuman oligomerization phosphorylation priority journal protein interaction titrimetry Wart virus Amino Acid Sequence Cell Line, Tumor Cell Proliferation DNA-Binding Proteins Human papillomavirus 16 Humans Molecular Sequence Data Oncogene Proteins, Viral Protein Structure, Tertiary ras Proteins Virus Integration Human papillomavirus Smal, C. Wetzler, D.E. Dantur, K.I. Chemes, L.B. Garcia-Alai, M.M. Dellarole, M. Alonso, L.G. Gaston, K. De Prat-Gay, G. The human papillomavirus E7-E2 interaction mechanism in vitro reveals a finely tuned system for modulating available E7 and E2 proteins
topic_facet	Different sizes DNA binding DNA binding domain DNA duplexes E7 oncoprotein Gel electrophoresis Hetero-oligomerization Human papillomavirus In-vitro Interaction mechanisms Intracellular levels N-terminal domains Oligomeric complexes Phosphorylation sites Protein-protein interface Site directed mutagenesis Soluble complexes Viral proteins Binding sites Complexation DNA Electrophoresis Enzyme activity Genes Ionic strength Nucleic acids Oligomerization Oligomers Phosphorylation Titration Transcription Proteins protein E7 protein VP2 amino terminal sequence article DNA binding gel electrophoresis human in vitro study ionic strength light scattering nonhuman oligomerization phosphorylation priority journal protein interaction titrimetry Wart virus Amino Acid Sequence Cell Line, Tumor Cell Proliferation DNA-Binding Proteins Human papillomavirus 16 Humans Molecular Sequence Data Oncogene Proteins, Viral Protein Structure, Tertiary ras Proteins Virus Integration Human papillomavirus
description	Transcription of the human papillomavirus E7 oncoprotein is negatively controlled by the viral E2 protein, and loss of this repression leads to irreversible transformation and carcinogenesis. Here we show that interaction of the HPV16 E7 protein with the DNA binding domain of the E2 protein (E2C) leads to ionic strength-dependent hetero-oligomerization even at the lowest concentrations measurable. Titration experiments followed by light scattering and native gel electrophoresis show insoluble oligomeric complexes with a ≥2000 nm diameter and intermediate soluble complexes 40 and 115 nm in diameter, respectively, formed in excess of E2C. Adiscrete oligomeric soluble complex formed in excess of E7 displays a diameter of 12 nm. The N-terminal domain of E7 interacts with E2C with a KD of 0.1 μM, where the stretch of residues 25-40 of E7, encompassing both a PEST motif and phosphorylation sites, is sufficient for the interaction. Displacement of the soluble E7-E2C complex by an E2 site DNA duplex and site-directed mutagenesis indicate that the protein-protein interface involves the DNA binding helix of E2. The formation of complexes of different sizes and properties in excess of either of the viral proteins reveals a finely tuned mechanism that could regulate the intracellular levels of both proteins as infection and transformation progress. Sequestering E2 into E7-E2 oligomers provides a possible additional route to uncontrolled E7 expression, in addition and prior to the disruption of the E2 gene during viral integration into the host genome. © 2009 American Chemical Society.
format	JOUR
author	Smal, C. Wetzler, D.E. Dantur, K.I. Chemes, L.B. Garcia-Alai, M.M. Dellarole, M. Alonso, L.G. Gaston, K. De Prat-Gay, G.
author_facet	Smal, C. Wetzler, D.E. Dantur, K.I. Chemes, L.B. Garcia-Alai, M.M. Dellarole, M. Alonso, L.G. Gaston, K. De Prat-Gay, G.
author_sort	Smal, C.
title	The human papillomavirus E7-E2 interaction mechanism in vitro reveals a finely tuned system for modulating available E7 and E2 proteins
title_short	The human papillomavirus E7-E2 interaction mechanism in vitro reveals a finely tuned system for modulating available E7 and E2 proteins
title_full	The human papillomavirus E7-E2 interaction mechanism in vitro reveals a finely tuned system for modulating available E7 and E2 proteins
title_fullStr	The human papillomavirus E7-E2 interaction mechanism in vitro reveals a finely tuned system for modulating available E7 and E2 proteins
title_full_unstemmed	The human papillomavirus E7-E2 interaction mechanism in vitro reveals a finely tuned system for modulating available E7 and E2 proteins
title_sort	human papillomavirus e7-e2 interaction mechanism in vitro reveals a finely tuned system for modulating available e7 and e2 proteins
url	http://hdl.handle.net/20.500.12110/paper_00062960_v48_n50_p11939_Smal
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The human papillomavirus E7-E2 interaction mechanism in vitro reveals a finely tuned system for modulating available E7 and E2 proteins

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