Design and characterization of an enhanced repressor of human papillomavirus E2 protein
ABSTRACT Papillomaviruses are causative agents of cervical and anogenital cancers. The viral E2 protein mediates viral DNA replication and transactivation of viral oncogenes and thus represents a specific target for therapeutic intervention. Short forms of E2, E2R, contain only the C‐terminal dimeri...
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| Veröffentlicht in: | The FASEB journal 2011-07, Vol.25 (7), p.2354-2361 |
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| description | ABSTRACT
Papillomaviruses are causative agents of cervical and anogenital cancers. The viral E2 protein mediates viral DNA replication and transactivation of viral oncogenes and thus represents a specific target for therapeutic intervention. Short forms of E2, E2R, contain only the C‐terminal dimerization domain, and repress the normal function of E2 due to formation of an inactive heterodimer. Using structure‐guided design, we replaced conserved residues at the dimer interface to design a heterodimer with increased stability. One E2R mutant in which histidine was replaced by a glutamate residue showed preferential heterodimer formation in vitro, as well as an increase in plasticity at the interface, as a result of histidine‐glutamate pair formation, as observed spectroscopically and in the crystal structure, determined to 2.2‐Å resolution. In addition, the enhanced E2R showed greater repression of transcription from E2‐responsive reporter plasmids in mammalian cell culture. Recent advances in protein delivery into the cell raise the possibility of using exogenously added proteins as therapeutic agents. More generally, this approach may be used to target the subunit interfaces of any multisubunit protein having a similar mechanism of action.—Bose, K., Meinke, G., Bohm, A., Baleja, J. D. Design and characterization of an enhanced repressor of human papillomavirus E2 protein. FASEB J. 25, 2354–2361 (2011). www.fasebj.org |
| doi_str_mv | 10.1096/fj.10-176461 |
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Papillomaviruses are causative agents of cervical and anogenital cancers. The viral E2 protein mediates viral DNA replication and transactivation of viral oncogenes and thus represents a specific target for therapeutic intervention. Short forms of E2, E2R, contain only the C‐terminal dimerization domain, and repress the normal function of E2 due to formation of an inactive heterodimer. Using structure‐guided design, we replaced conserved residues at the dimer interface to design a heterodimer with increased stability. One E2R mutant in which histidine was replaced by a glutamate residue showed preferential heterodimer formation in vitro, as well as an increase in plasticity at the interface, as a result of histidine‐glutamate pair formation, as observed spectroscopically and in the crystal structure, determined to 2.2‐Å resolution. In addition, the enhanced E2R showed greater repression of transcription from E2‐responsive reporter plasmids in mammalian cell culture. Recent advances in protein delivery into the cell raise the possibility of using exogenously added proteins as therapeutic agents. More generally, this approach may be used to target the subunit interfaces of any multisubunit protein having a similar mechanism of action.—Bose, K., Meinke, G., Bohm, A., Baleja, J. D. Design and characterization of an enhanced repressor of human papillomavirus E2 protein. FASEB J. 25, 2354–2361 (2011). www.fasebj.org</description><identifier>ISSN: 0892-6638</identifier><identifier>ISSN: 1530-6860</identifier><identifier>EISSN: 1530-6860</identifier><identifier>DOI: 10.1096/fj.10-176461</identifier><identifier>PMID: 21482558</identifier><language>eng</language><publisher>United States: Federation of American Societies for Experimental Biology</publisher><subject>Amino Acid Substitution ; Binding Sites - genetics ; Circular Dichroism ; crystallography ; Crystallography, X-Ray ; DNA-Binding Proteins - chemistry ; DNA-Binding Proteins - genetics ; DNA-Binding Proteins - metabolism ; HEK293 Cells ; Human papillomavirus ; Humans ; Kinetics ; Models, Molecular ; Mutation ; Oncogene Proteins, Viral - chemistry ; Oncogene Proteins, Viral - genetics ; Oncogene Proteins, Viral - metabolism ; Protein Binding ; Protein Conformation ; Protein Multimerization ; Protein Structure, Secondary ; Protein Structure, Tertiary ; Protein Unfolding ; protein‐DNA interaction ; Repressor Proteins - chemistry ; Repressor Proteins - genetics ; Repressor Proteins - metabolism ; Research Communications ; Spectrometry, Fluorescence ; subunit interface ; transactivation ; Transcriptional Activation</subject><ispartof>The FASEB journal, 2011-07, Vol.25 (7), p.2354-2361</ispartof><rights>2011 FASEB</rights><rights>FASEB 2011</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4637-c8bf20b22941ec48d13efa9e7bd707d18b69ddc8e1aa7b96f076d1911a174f3a3</citedby><cites>FETCH-LOGICAL-c4637-c8bf20b22941ec48d13efa9e7bd707d18b69ddc8e1aa7b96f076d1911a174f3a3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1096%2Ffj.10-176461$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1096%2Ffj.10-176461$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>230,314,780,784,885,1417,27924,27925,45574,45575</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21482558$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Bose, Kakoli</creatorcontrib><creatorcontrib>Meinke, Gretchen</creatorcontrib><creatorcontrib>Bohm, Andrew</creatorcontrib><creatorcontrib>Baleja, James D.</creatorcontrib><title>Design and characterization of an enhanced repressor of human papillomavirus E2 protein</title><title>The FASEB journal</title><addtitle>FASEB J</addtitle><description>ABSTRACT
Papillomaviruses are causative agents of cervical and anogenital cancers. The viral E2 protein mediates viral DNA replication and transactivation of viral oncogenes and thus represents a specific target for therapeutic intervention. Short forms of E2, E2R, contain only the C‐terminal dimerization domain, and repress the normal function of E2 due to formation of an inactive heterodimer. Using structure‐guided design, we replaced conserved residues at the dimer interface to design a heterodimer with increased stability. One E2R mutant in which histidine was replaced by a glutamate residue showed preferential heterodimer formation in vitro, as well as an increase in plasticity at the interface, as a result of histidine‐glutamate pair formation, as observed spectroscopically and in the crystal structure, determined to 2.2‐Å resolution. In addition, the enhanced E2R showed greater repression of transcription from E2‐responsive reporter plasmids in mammalian cell culture. Recent advances in protein delivery into the cell raise the possibility of using exogenously added proteins as therapeutic agents. More generally, this approach may be used to target the subunit interfaces of any multisubunit protein having a similar mechanism of action.—Bose, K., Meinke, G., Bohm, A., Baleja, J. D. Design and characterization of an enhanced repressor of human papillomavirus E2 protein. FASEB J. 25, 2354–2361 (2011). www.fasebj.org</description><subject>Amino Acid Substitution</subject><subject>Binding Sites - genetics</subject><subject>Circular Dichroism</subject><subject>crystallography</subject><subject>Crystallography, X-Ray</subject><subject>DNA-Binding Proteins - chemistry</subject><subject>DNA-Binding Proteins - genetics</subject><subject>DNA-Binding Proteins - metabolism</subject><subject>HEK293 Cells</subject><subject>Human papillomavirus</subject><subject>Humans</subject><subject>Kinetics</subject><subject>Models, Molecular</subject><subject>Mutation</subject><subject>Oncogene Proteins, Viral - chemistry</subject><subject>Oncogene Proteins, Viral - genetics</subject><subject>Oncogene Proteins, Viral - metabolism</subject><subject>Protein Binding</subject><subject>Protein Conformation</subject><subject>Protein Multimerization</subject><subject>Protein Structure, Secondary</subject><subject>Protein Structure, Tertiary</subject><subject>Protein Unfolding</subject><subject>protein‐DNA interaction</subject><subject>Repressor Proteins - chemistry</subject><subject>Repressor Proteins - genetics</subject><subject>Repressor Proteins - metabolism</subject><subject>Research Communications</subject><subject>Spectrometry, Fluorescence</subject><subject>subunit interface</subject><subject>transactivation</subject><subject>Transcriptional Activation</subject><issn>0892-6638</issn><issn>1530-6860</issn><issn>1530-6860</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkUFv1DAQhS1ERZfCjTPKjQuhHsexnQsSlBaQKnEAxNFy7HHXq8QOdlJUfj1pt1RwaU8zmvfp6Y0eIS-AvgHaiWO_W2cNUnABj8gG2obWQgn6mGyo6lgtRKMOydNSdpRSoCCekEMGXLG2VRvy4wOWcBErE11ltyYbO2MOv80cUqySX-8Vxq2JFl2VccpYSsrXwnYZV20yUxiGNJrLkJdSnbJqymnGEJ-RA2-Ggs9v5xH5fnb67eRTff7l4-eTd-e15aKRtVW9Z7RnrOOAlisHDXrToeydpNKB6kXnnFUIxsi-E55K4aADMCC5b0xzRN7ufaelH9FZjHM2g55yGE2-0skE_b8Sw1ZfpEvdAPC2YavBq1uDnH4uWGY9hmJxGEzEtBTdUc7Vmk4-SCq5kkIyvpKv96TNqZSM_i4PUH1dmva7m_WmtBV_-e8Pd_DfllZA7oFfYcCre8302df3jLKWUkmZbP4APdOkZA</recordid><startdate>201107</startdate><enddate>201107</enddate><creator>Bose, Kakoli</creator><creator>Meinke, Gretchen</creator><creator>Bohm, Andrew</creator><creator>Baleja, James D.</creator><general>Federation of American Societies for Experimental Biology</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>7U9</scope><scope>H94</scope><scope>5PM</scope></search><sort><creationdate>201107</creationdate><title>Design and characterization of an enhanced repressor of human papillomavirus E2 protein</title><author>Bose, Kakoli ; Meinke, Gretchen ; Bohm, Andrew ; Baleja, James D.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4637-c8bf20b22941ec48d13efa9e7bd707d18b69ddc8e1aa7b96f076d1911a174f3a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Amino Acid Substitution</topic><topic>Binding Sites - genetics</topic><topic>Circular Dichroism</topic><topic>crystallography</topic><topic>Crystallography, X-Ray</topic><topic>DNA-Binding Proteins - chemistry</topic><topic>DNA-Binding Proteins - genetics</topic><topic>DNA-Binding Proteins - metabolism</topic><topic>HEK293 Cells</topic><topic>Human papillomavirus</topic><topic>Humans</topic><topic>Kinetics</topic><topic>Models, Molecular</topic><topic>Mutation</topic><topic>Oncogene Proteins, Viral - chemistry</topic><topic>Oncogene Proteins, Viral - genetics</topic><topic>Oncogene Proteins, Viral - metabolism</topic><topic>Protein Binding</topic><topic>Protein Conformation</topic><topic>Protein Multimerization</topic><topic>Protein Structure, Secondary</topic><topic>Protein Structure, Tertiary</topic><topic>Protein Unfolding</topic><topic>protein‐DNA interaction</topic><topic>Repressor Proteins - chemistry</topic><topic>Repressor Proteins - genetics</topic><topic>Repressor Proteins - metabolism</topic><topic>Research Communications</topic><topic>Spectrometry, Fluorescence</topic><topic>subunit interface</topic><topic>transactivation</topic><topic>Transcriptional Activation</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Bose, Kakoli</creatorcontrib><creatorcontrib>Meinke, Gretchen</creatorcontrib><creatorcontrib>Bohm, Andrew</creatorcontrib><creatorcontrib>Baleja, James D.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Virology and AIDS Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>The FASEB journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Bose, Kakoli</au><au>Meinke, Gretchen</au><au>Bohm, Andrew</au><au>Baleja, James D.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Design and characterization of an enhanced repressor of human papillomavirus E2 protein</atitle><jtitle>The FASEB journal</jtitle><addtitle>FASEB J</addtitle><date>2011-07</date><risdate>2011</risdate><volume>25</volume><issue>7</issue><spage>2354</spage><epage>2361</epage><pages>2354-2361</pages><issn>0892-6638</issn><issn>1530-6860</issn><eissn>1530-6860</eissn><abstract>ABSTRACT
Papillomaviruses are causative agents of cervical and anogenital cancers. The viral E2 protein mediates viral DNA replication and transactivation of viral oncogenes and thus represents a specific target for therapeutic intervention. Short forms of E2, E2R, contain only the C‐terminal dimerization domain, and repress the normal function of E2 due to formation of an inactive heterodimer. Using structure‐guided design, we replaced conserved residues at the dimer interface to design a heterodimer with increased stability. One E2R mutant in which histidine was replaced by a glutamate residue showed preferential heterodimer formation in vitro, as well as an increase in plasticity at the interface, as a result of histidine‐glutamate pair formation, as observed spectroscopically and in the crystal structure, determined to 2.2‐Å resolution. In addition, the enhanced E2R showed greater repression of transcription from E2‐responsive reporter plasmids in mammalian cell culture. Recent advances in protein delivery into the cell raise the possibility of using exogenously added proteins as therapeutic agents. More generally, this approach may be used to target the subunit interfaces of any multisubunit protein having a similar mechanism of action.—Bose, K., Meinke, G., Bohm, A., Baleja, J. D. Design and characterization of an enhanced repressor of human papillomavirus E2 protein. FASEB J. 25, 2354–2361 (2011). www.fasebj.org</abstract><cop>United States</cop><pub>Federation of American Societies for Experimental Biology</pub><pmid>21482558</pmid><doi>10.1096/fj.10-176461</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Amino Acid Substitution Binding Sites - genetics Circular Dichroism crystallography Crystallography, X-Ray DNA-Binding Proteins - chemistry DNA-Binding Proteins - genetics DNA-Binding Proteins - metabolism HEK293 Cells Human papillomavirus Humans Kinetics Models, Molecular Mutation Oncogene Proteins, Viral - chemistry Oncogene Proteins, Viral - genetics Oncogene Proteins, Viral - metabolism Protein Binding Protein Conformation Protein Multimerization Protein Structure, Secondary Protein Structure, Tertiary Protein Unfolding protein‐DNA interaction Repressor Proteins - chemistry Repressor Proteins - genetics Repressor Proteins - metabolism Research Communications Spectrometry, Fluorescence subunit interface transactivation Transcriptional Activation |
| title | Design and characterization of an enhanced repressor of human papillomavirus E2 protein |
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