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Intracellular Analysis of the Interaction between the Human Papillomavirus Type 16 E6 Oncoprotein and Inhibitory Peptides.

Stutz C, Reinz E, Honegger A, Bulkescher J, Schweizer J, Zanier K, Travé G, Lohrey C, Hoppe-Seyler K, Hoppe-Seyler F - PLoS ONE (2015)

Bottom Line: Although they do not encompass the LxxLL binding motif found in cellular HPV16 E6 interaction partners, such as E6AP, the pep11 variants strongly bind to HPV16 E6 by contacting the recently identified E6AP binding pocket.These findings indicate that peptides, which do not contain the LxxLL motif, can reshape E6 to enable its interaction with p53.The formation of the trimeric HPV16 E6 / peptide / p53 complex was associated with an increase of endogenous HPV16 E6 protein amounts.

View Article: PubMed Central - PubMed

Affiliation: Molecular Therapy of Virus-Associated Cancers (F065), Program Infection and Cancer, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.

ABSTRACT
Oncogenic types of human papillomaviruses (HPVs) cause cervical cancer and other malignancies in humans. The HPV E6 oncoprotein is considered to be an attractive therapeutic target since its inhibition can lead to the apoptotic cell death of HPV-positive cancer cells. The HPV type 16 (HPV16) E6-binding peptide pep11, and variants thereof, induce cell death specifically in HPV16-positive cancer cells. Although they do not encompass the LxxLL binding motif found in cellular HPV16 E6 interaction partners, such as E6AP, the pep11 variants strongly bind to HPV16 E6 by contacting the recently identified E6AP binding pocket. Thus, these peptides can serve as prototype E6-inhibitory molecules which target the E6AP pocket. We here analyzed their intracellular interaction with HPV16 E6. By comprehensive intracellular binding studies and GST pull-down assays, we show that E6-binding competent pep11 variants induce the formation of a trimeric complex, consisting of pep11, HPV16 E6 and p53. These findings indicate that peptides, which do not contain the LxxLL motif, can reshape E6 to enable its interaction with p53. The formation of the trimeric HPV16 E6 / peptide / p53 complex was associated with an increase of endogenous HPV16 E6 protein amounts. Yet, total cellular p53 amounts were also increased, indicating that the E6 / E6AP-mediated degradation of p53 is blocked. These findings suggest that inhibition of oncogenic activities by targeting the E6AP pocket on HPV16 E6 could be a strategy for therapeutic intervention.

No MeSH data available.


Related in: MedlinePlus

HPV16 E6 mediating intracellular formation of a trimeric complex with E6-binding peptides and p53.(A) Schematic illustration of the employed modified mammalian two hybrid assay. Upper panel: Individual peptides are linked to GAL4-BD, p53 is linked to VP16-AD, E6 proteins are expressed from a co-transfected expression vector. Lower panel: Trimeric complex formation is detected by activation of the luciferase reporter. GAL4 BS, GAL4 binding sites. (B) Co-expression of individual peptides linked to the GAL4-BD, as indicated, together with p53-VP16-AD and HPV16 E6 or HPV 11 E6, respectively. Empty expression vector pNCMV served as negative control. Indicated are relative luciferase activities (RLA) above those of control-transfected cells, expressing the corresponding peptide-GAL4-BD fusions together with the co-transfected empty vectors pACT and pNCMV; values are arbitrarily set at 1.0. Results were obtained from three individual experiments, each performed in duplicates. Standard deviations are indicated. Asterisks above the columns indicate significant differences above those of cells expressing the corresponding peptides linked to GAL4-BD together with p53-VP16-AD, in the absence of E6 (co-transfected empty vector pNCMV), with p-values of ≤0.001 (***) and ≤0.05 (*). (C) Immunoblot analyses of expression levels of Flag-tagged HPV16 E6 and HPV11 E6 and of (D) individual peptides linked to GAL4-DB. Loading of protein extracts was normalized for equal transfection efficiencies, as determined by a co-transfected β-galactosidase expression vector. β-Gal, β-galactosidase; α-Tub, α-tubulin.
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pone.0132339.g004: HPV16 E6 mediating intracellular formation of a trimeric complex with E6-binding peptides and p53.(A) Schematic illustration of the employed modified mammalian two hybrid assay. Upper panel: Individual peptides are linked to GAL4-BD, p53 is linked to VP16-AD, E6 proteins are expressed from a co-transfected expression vector. Lower panel: Trimeric complex formation is detected by activation of the luciferase reporter. GAL4 BS, GAL4 binding sites. (B) Co-expression of individual peptides linked to the GAL4-BD, as indicated, together with p53-VP16-AD and HPV16 E6 or HPV 11 E6, respectively. Empty expression vector pNCMV served as negative control. Indicated are relative luciferase activities (RLA) above those of control-transfected cells, expressing the corresponding peptide-GAL4-BD fusions together with the co-transfected empty vectors pACT and pNCMV; values are arbitrarily set at 1.0. Results were obtained from three individual experiments, each performed in duplicates. Standard deviations are indicated. Asterisks above the columns indicate significant differences above those of cells expressing the corresponding peptides linked to GAL4-BD together with p53-VP16-AD, in the absence of E6 (co-transfected empty vector pNCMV), with p-values of ≤0.001 (***) and ≤0.05 (*). (C) Immunoblot analyses of expression levels of Flag-tagged HPV16 E6 and HPV11 E6 and of (D) individual peptides linked to GAL4-DB. Loading of protein extracts was normalized for equal transfection efficiencies, as determined by a co-transfected β-galactosidase expression vector. β-Gal, β-galactosidase; α-Tub, α-tubulin.

Mentions: Therefore, we tested the ability of E6 to undergo trimeric complex formation with p53, in the presence of E6-binding competent and E6-binding defective pep11 variants. To study this under intracellular conditions, we performed modified mammalian two-hybrid analyses in HeLa cells, by co-expressing: (i) individual peptides (E6APpep, pep11**, pep11**m, pep11’, pep11’m) linked to the GAL4 DNA binding domain (GAL4BD), (ii) p53 fused to the VP16 transactivation domain (VP16AD), and (iii) flag-tagged HPV16 or HPV11 E6 protein. If a trimeric complex is formed by these components, E6 could bridge the peptides linked to the GAL4BD with p53 fused to VP16AD, resulting in the activation of a co-transfected luciferase reporter plasmid under transcriptional control of GAL4 binding sites (Fig 4A).


Intracellular Analysis of the Interaction between the Human Papillomavirus Type 16 E6 Oncoprotein and Inhibitory Peptides.

Stutz C, Reinz E, Honegger A, Bulkescher J, Schweizer J, Zanier K, Travé G, Lohrey C, Hoppe-Seyler K, Hoppe-Seyler F - PLoS ONE (2015)

HPV16 E6 mediating intracellular formation of a trimeric complex with E6-binding peptides and p53.(A) Schematic illustration of the employed modified mammalian two hybrid assay. Upper panel: Individual peptides are linked to GAL4-BD, p53 is linked to VP16-AD, E6 proteins are expressed from a co-transfected expression vector. Lower panel: Trimeric complex formation is detected by activation of the luciferase reporter. GAL4 BS, GAL4 binding sites. (B) Co-expression of individual peptides linked to the GAL4-BD, as indicated, together with p53-VP16-AD and HPV16 E6 or HPV 11 E6, respectively. Empty expression vector pNCMV served as negative control. Indicated are relative luciferase activities (RLA) above those of control-transfected cells, expressing the corresponding peptide-GAL4-BD fusions together with the co-transfected empty vectors pACT and pNCMV; values are arbitrarily set at 1.0. Results were obtained from three individual experiments, each performed in duplicates. Standard deviations are indicated. Asterisks above the columns indicate significant differences above those of cells expressing the corresponding peptides linked to GAL4-BD together with p53-VP16-AD, in the absence of E6 (co-transfected empty vector pNCMV), with p-values of ≤0.001 (***) and ≤0.05 (*). (C) Immunoblot analyses of expression levels of Flag-tagged HPV16 E6 and HPV11 E6 and of (D) individual peptides linked to GAL4-DB. Loading of protein extracts was normalized for equal transfection efficiencies, as determined by a co-transfected β-galactosidase expression vector. β-Gal, β-galactosidase; α-Tub, α-tubulin.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4495056&req=5

pone.0132339.g004: HPV16 E6 mediating intracellular formation of a trimeric complex with E6-binding peptides and p53.(A) Schematic illustration of the employed modified mammalian two hybrid assay. Upper panel: Individual peptides are linked to GAL4-BD, p53 is linked to VP16-AD, E6 proteins are expressed from a co-transfected expression vector. Lower panel: Trimeric complex formation is detected by activation of the luciferase reporter. GAL4 BS, GAL4 binding sites. (B) Co-expression of individual peptides linked to the GAL4-BD, as indicated, together with p53-VP16-AD and HPV16 E6 or HPV 11 E6, respectively. Empty expression vector pNCMV served as negative control. Indicated are relative luciferase activities (RLA) above those of control-transfected cells, expressing the corresponding peptide-GAL4-BD fusions together with the co-transfected empty vectors pACT and pNCMV; values are arbitrarily set at 1.0. Results were obtained from three individual experiments, each performed in duplicates. Standard deviations are indicated. Asterisks above the columns indicate significant differences above those of cells expressing the corresponding peptides linked to GAL4-BD together with p53-VP16-AD, in the absence of E6 (co-transfected empty vector pNCMV), with p-values of ≤0.001 (***) and ≤0.05 (*). (C) Immunoblot analyses of expression levels of Flag-tagged HPV16 E6 and HPV11 E6 and of (D) individual peptides linked to GAL4-DB. Loading of protein extracts was normalized for equal transfection efficiencies, as determined by a co-transfected β-galactosidase expression vector. β-Gal, β-galactosidase; α-Tub, α-tubulin.
Mentions: Therefore, we tested the ability of E6 to undergo trimeric complex formation with p53, in the presence of E6-binding competent and E6-binding defective pep11 variants. To study this under intracellular conditions, we performed modified mammalian two-hybrid analyses in HeLa cells, by co-expressing: (i) individual peptides (E6APpep, pep11**, pep11**m, pep11’, pep11’m) linked to the GAL4 DNA binding domain (GAL4BD), (ii) p53 fused to the VP16 transactivation domain (VP16AD), and (iii) flag-tagged HPV16 or HPV11 E6 protein. If a trimeric complex is formed by these components, E6 could bridge the peptides linked to the GAL4BD with p53 fused to VP16AD, resulting in the activation of a co-transfected luciferase reporter plasmid under transcriptional control of GAL4 binding sites (Fig 4A).

Bottom Line: Although they do not encompass the LxxLL binding motif found in cellular HPV16 E6 interaction partners, such as E6AP, the pep11 variants strongly bind to HPV16 E6 by contacting the recently identified E6AP binding pocket.These findings indicate that peptides, which do not contain the LxxLL motif, can reshape E6 to enable its interaction with p53.The formation of the trimeric HPV16 E6 / peptide / p53 complex was associated with an increase of endogenous HPV16 E6 protein amounts.

View Article: PubMed Central - PubMed

Affiliation: Molecular Therapy of Virus-Associated Cancers (F065), Program Infection and Cancer, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.

ABSTRACT
Oncogenic types of human papillomaviruses (HPVs) cause cervical cancer and other malignancies in humans. The HPV E6 oncoprotein is considered to be an attractive therapeutic target since its inhibition can lead to the apoptotic cell death of HPV-positive cancer cells. The HPV type 16 (HPV16) E6-binding peptide pep11, and variants thereof, induce cell death specifically in HPV16-positive cancer cells. Although they do not encompass the LxxLL binding motif found in cellular HPV16 E6 interaction partners, such as E6AP, the pep11 variants strongly bind to HPV16 E6 by contacting the recently identified E6AP binding pocket. Thus, these peptides can serve as prototype E6-inhibitory molecules which target the E6AP pocket. We here analyzed their intracellular interaction with HPV16 E6. By comprehensive intracellular binding studies and GST pull-down assays, we show that E6-binding competent pep11 variants induce the formation of a trimeric complex, consisting of pep11, HPV16 E6 and p53. These findings indicate that peptides, which do not contain the LxxLL motif, can reshape E6 to enable its interaction with p53. The formation of the trimeric HPV16 E6 / peptide / p53 complex was associated with an increase of endogenous HPV16 E6 protein amounts. Yet, total cellular p53 amounts were also increased, indicating that the E6 / E6AP-mediated degradation of p53 is blocked. These findings suggest that inhibition of oncogenic activities by targeting the E6AP pocket on HPV16 E6 could be a strategy for therapeutic intervention.

No MeSH data available.


Related in: MedlinePlus