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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

Trimeric complex formation between HPV16 E6, p53 and pep11** or E6AP, in GST pull-down analyses.(A) p53 was expressed in H1299/K3 cells and pull-down assays were performed with GST-tagged HPV16 E6 (GST-16E6) in the presence of no peptide, synthetic pep11** or synthetic pep11**m. E6AP and p53 bound to GST-16 E6 are indicated. (B) The H1299/K3 lysate was pre-cleared from endogenous E6AP protein by pre-incubation with GST-16 E6. E6AP and p53 bound to GST-16 E6 are indicated.
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pone.0132339.g006: Trimeric complex formation between HPV16 E6, p53 and pep11** or E6AP, in GST pull-down analyses.(A) p53 was expressed in H1299/K3 cells and pull-down assays were performed with GST-tagged HPV16 E6 (GST-16E6) in the presence of no peptide, synthetic pep11** or synthetic pep11**m. E6AP and p53 bound to GST-16 E6 are indicated. (B) The H1299/K3 lysate was pre-cleared from endogenous E6AP protein by pre-incubation with GST-16 E6. E6AP and p53 bound to GST-16 E6 are indicated.

Mentions: To further confirm the complex formation between HPV16 E6, pep11** and p53 by an independent experimental approach, we performed pull-down experiments. First, we used lysate of p53- H1299/K3 cells in which endogenous E6AP expression is repressed, but not completely abolished by stable RNA interference [21]. We expressed p53 in these cells and added GST-HIS or GST-HPV16 E6 to the cell lysates, in the absence or presence of either chemically synthesized pep11** or pep11**m. We found that both E6AP and p53 were bound to GST-HPV16 E6 in the absence of the peptides. This indicates that the residual endogenous concentrations of E6AP in H1299/K3 cells were sufficient to support formation of the HPV16 E6 / E6AP / p53 complex. In the presence of pep11**, the amount of HPV16 E6-bound E6AP decreased whereas the level of bound p53 strongly increased (Fig 6A). This was not observed upon adding the HPV16 E6-binding defective pep11**m control. These findings indicate that pep11** can compete for E6AP binding to HPV16 E6, which is linked to strongly increased p53 amounts in the trimeric complex.


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)

Trimeric complex formation between HPV16 E6, p53 and pep11** or E6AP, in GST pull-down analyses.(A) p53 was expressed in H1299/K3 cells and pull-down assays were performed with GST-tagged HPV16 E6 (GST-16E6) in the presence of no peptide, synthetic pep11** or synthetic pep11**m. E6AP and p53 bound to GST-16 E6 are indicated. (B) The H1299/K3 lysate was pre-cleared from endogenous E6AP protein by pre-incubation with GST-16 E6. E6AP and p53 bound to GST-16 E6 are indicated.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132339.g006: Trimeric complex formation between HPV16 E6, p53 and pep11** or E6AP, in GST pull-down analyses.(A) p53 was expressed in H1299/K3 cells and pull-down assays were performed with GST-tagged HPV16 E6 (GST-16E6) in the presence of no peptide, synthetic pep11** or synthetic pep11**m. E6AP and p53 bound to GST-16 E6 are indicated. (B) The H1299/K3 lysate was pre-cleared from endogenous E6AP protein by pre-incubation with GST-16 E6. E6AP and p53 bound to GST-16 E6 are indicated.
Mentions: To further confirm the complex formation between HPV16 E6, pep11** and p53 by an independent experimental approach, we performed pull-down experiments. First, we used lysate of p53- H1299/K3 cells in which endogenous E6AP expression is repressed, but not completely abolished by stable RNA interference [21]. We expressed p53 in these cells and added GST-HIS or GST-HPV16 E6 to the cell lysates, in the absence or presence of either chemically synthesized pep11** or pep11**m. We found that both E6AP and p53 were bound to GST-HPV16 E6 in the absence of the peptides. This indicates that the residual endogenous concentrations of E6AP in H1299/K3 cells were sufficient to support formation of the HPV16 E6 / E6AP / p53 complex. In the presence of pep11**, the amount of HPV16 E6-bound E6AP decreased whereas the level of bound p53 strongly increased (Fig 6A). This was not observed upon adding the HPV16 E6-binding defective pep11**m control. These findings indicate that pep11** can compete for E6AP binding to HPV16 E6, which is linked to strongly increased p53 amounts in the trimeric complex.

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