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

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.

The E6AP binding pocket of the HPV16 E6 oncoprotein provides a docking site for a small inhibitory peptide unrelated to E6AP, indicating druggability of E6.

The HPV E6 oncoprotein maintains the malignant phenotype of HPV-positive cancer cells and represents an attractive therapeutic target. E6 forms a complex with the cellular E6AP ubiquitin ligase, ultimately leading to p53 degradation. The recently elucidated x-ray structure of a HPV16 E6/E6AP complex showed that HPV16 E6 forms a distinct binding pocket for E6AP. This discovery raises the question whether the E6AP binding pocket is druggable, i. e. whether it provides a docking site for functional E6 inhibitors. To address these issues, we performed a detailed analysis of the HPV16 E6 interactions with two small peptides: (i) E6APpep, corresponding to the E6 binding domain of E6AP, and (ii) pep11**, a peptide that binds to HPV16 E6 and, in contrast to E6APpep, induces apoptosis, specifically in HPV16-positive cancer cells. Surface plasmon resonance, NMR chemical shift perturbation, and mammalian two-hybrid analyses coupled to mutagenesis indicate that E6APpep contacts HPV16 E6 amino acid residues within the E6AP pocket, both in vitro and intracellularly. Many of these amino acids were also important for binding to pep11**, suggesting that the binding sites for the two peptides on HPV16 E6 overlap. Yet, few E6 amino acids were differentially involved which may contribute to the higher binding affinity of pep11**. Data from the HPV16 E6/pep11** interaction allowed the rational design of single amino acid exchanges in HPV18 and HPV31 E6 that enabled their binding to pep11**. Taken together, these results suggest that E6 molecular surfaces mediating E6APpep binding can also accommodate pro-apoptotic peptides that belong to different sequence families. As proof of concept, this study provides the first experimental evidence that the E6AP binding pocket is druggable, opening new possibilities for rational, structure-based drug design.

Myeloid-derived suppressor cells predict survival of patients with advanced melanoma: comparison with regulatory T cells and NY-ESO-1- or melan-A-specific T cells.

PURPOSE: To analyze the prognostic relevance and relative impact of circulating myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) compared with functional tumor antigen-specific T cells in patients with melanoma with distant metastasis. EXPERIMENTAL DESIGN: The percentage of CD14(+)CD11b(+)HLA-DR(-/low) MDSCs, CD4(+)CD25(+)FoxP3(+) Tregs, and the presence of NY-ESO-1- or Melan-A-specific T cells was analyzed in 94 patients and validated in an additional cohort of 39 patients by flow cytometry. Univariate survival differences were calculated according to Kaplan-Meier and log-rank tests. Multivariate analyses were performed using Cox regression models. RESULTS: NY-ESO-1-specific T cells, the M-category, and the frequency of MDSCs were associated with survival. The absence of NY-ESO-1-specific T cells and the M-category M1c independently increased the risk of death. In a second Cox model not considering results on antigen-specific T cells, a frequency of >11% MDSCs showed independent impact. Its association with survival was confirmed in the additional patient cohort. Median survival of patients with a lower frequency of MDSCs was 13 months versus 8 months for others (P < 0.001, combined cohorts). We observed a strong correlation between high levels of MDSCs and the absence of melanoma antigen-specific T cells implying a causal and clinically relevant interaction. No prognostic impact was observed for Tregs. CONCLUSIONS: Circulating CD14(+)CD11b(+)HLA-DR(-/low) MDSCs have a negative impact on survival and inversely correlate with the presence of functional antigen-specific T cells in patients with advanced melanoma. Our findings provide a rationale to investigate MDSC-depleting strategies in the therapeutic setting especially in combination with vaccination or T-cell transfer approaches.

Measuring rotational diffusion of macromolecules by fluorescence correlation spectroscopy.

We describe a novel method to measure rotational diffusion of large biomolecules in solution based on fluorescence correlation on the nanosecond time scale. In contrast to conventional fluorescence anisotropy measurements, a correlation-based method will also work if the rotational diffusion time is much longer than the fluorescence decay time. Thus, the method is suited to study the rotational diffusion of macromolecules having rotational diffusion times of dozens to hundreds of nanoseconds, which is considerably larger than the fluorescence lifetime of most commercially available dyes or auto-fluorescent proteins. A pulsed interleaved excitation scheme with crossed excitation polarization maximizes the time-dependent amplitude of the measured correlation curve as caused by rotational diffusion. Using the determined rotational diffusion coefficient, precise values of the hydrodynamic radius can be obtained. The method is exemplified on sizing a set of common globular proteins.