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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

Immunoblot analyses of endogenous HPV16 E6, upon intracellular expression of E6-targeting peptides.Expression of either hrGFP-linked peptides pep11**m or pep11’m (both E6-binding defective), or pep11**, pep11’ or E6APpep (all E6-binding competent) in HPV-16 positive MRI-H186 cells. Soluble and insoluble protein fractions are indicated. Loading of protein extracts was normalized for equal transfection efficiencies, as determined by the activities of a co-transfected β-galactosidase expression vector. Expression levels of endogenous E6AP, of p53, and of individual peptide-hrGFP fusion proteins are indicated. α-Tub, α-tubulin; s. exp., short exposure; l. exp., long exposure.
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pone.0132339.g003: Immunoblot analyses of endogenous HPV16 E6, upon intracellular expression of E6-targeting peptides.Expression of either hrGFP-linked peptides pep11**m or pep11’m (both E6-binding defective), or pep11**, pep11’ or E6APpep (all E6-binding competent) in HPV-16 positive MRI-H186 cells. Soluble and insoluble protein fractions are indicated. Loading of protein extracts was normalized for equal transfection efficiencies, as determined by the activities of a co-transfected β-galactosidase expression vector. Expression levels of endogenous E6AP, of p53, and of individual peptide-hrGFP fusion proteins are indicated. α-Tub, α-tubulin; s. exp., short exposure; l. exp., long exposure.

Mentions: Next, we extended these investigations and analyzed endogenous E6 protein levels in HPV16-positive cancer cells. For this, hrGFP-linked E6APpep, pep11** or pep11’, and the E6-binding defective mutants pep11**m or pep11’m, were expressed in HPV16-positive MRI-H186 cells. Under these experimental conditions, only little E6 was detectable in the insoluble protein fraction, even after long exposure times of the immunoblots. Importantly, and as seen in the ectopic co-expression experiments (Fig 2), endogenous HPV16 E6 levels increased upon intracellular pep11** or pep11’ expression, as also observed upon expression of E6APpep (Fig 3). This increase of endogenous E6 amounts upon expression of E6-binding competent pep11 variants is also detectable in other HPV16-positive cervical cancer cells, as shown for SiHa cells (S2 Fig). Taken together, these findings indicate that, alike E6APpep-containing peptides [31], binding of pep11 variants to HPV16 E6 may stabilize the E6 protein. These results furthermore raise the question whether the E6-binding competent pep11 variants may induce a conformational change of HPV16 E6 that enables complex formation with p53, even though they show distinct binding differences compared to the HPV16 E6 / E6APpep interaction and do not contain the LxxLL motif [16,19].


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)

Immunoblot analyses of endogenous HPV16 E6, upon intracellular expression of E6-targeting peptides.Expression of either hrGFP-linked peptides pep11**m or pep11’m (both E6-binding defective), or pep11**, pep11’ or E6APpep (all E6-binding competent) in HPV-16 positive MRI-H186 cells. Soluble and insoluble protein fractions are indicated. Loading of protein extracts was normalized for equal transfection efficiencies, as determined by the activities of a co-transfected β-galactosidase expression vector. Expression levels of endogenous E6AP, of p53, and of individual peptide-hrGFP fusion proteins are indicated. α-Tub, α-tubulin; s. exp., short exposure; l. exp., long exposure.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132339.g003: Immunoblot analyses of endogenous HPV16 E6, upon intracellular expression of E6-targeting peptides.Expression of either hrGFP-linked peptides pep11**m or pep11’m (both E6-binding defective), or pep11**, pep11’ or E6APpep (all E6-binding competent) in HPV-16 positive MRI-H186 cells. Soluble and insoluble protein fractions are indicated. Loading of protein extracts was normalized for equal transfection efficiencies, as determined by the activities of a co-transfected β-galactosidase expression vector. Expression levels of endogenous E6AP, of p53, and of individual peptide-hrGFP fusion proteins are indicated. α-Tub, α-tubulin; s. exp., short exposure; l. exp., long exposure.
Mentions: Next, we extended these investigations and analyzed endogenous E6 protein levels in HPV16-positive cancer cells. For this, hrGFP-linked E6APpep, pep11** or pep11’, and the E6-binding defective mutants pep11**m or pep11’m, were expressed in HPV16-positive MRI-H186 cells. Under these experimental conditions, only little E6 was detectable in the insoluble protein fraction, even after long exposure times of the immunoblots. Importantly, and as seen in the ectopic co-expression experiments (Fig 2), endogenous HPV16 E6 levels increased upon intracellular pep11** or pep11’ expression, as also observed upon expression of E6APpep (Fig 3). This increase of endogenous E6 amounts upon expression of E6-binding competent pep11 variants is also detectable in other HPV16-positive cervical cancer cells, as shown for SiHa cells (S2 Fig). Taken together, these findings indicate that, alike E6APpep-containing peptides [31], binding of pep11 variants to HPV16 E6 may stabilize the E6 protein. These results furthermore raise the question whether the E6-binding competent pep11 variants may induce a conformational change of HPV16 E6 that enables complex formation with p53, even though they show distinct binding differences compared to the HPV16 E6 / E6APpep interaction and do not contain the LxxLL motif [16,19].

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