The degradation of p53 is a hallmark of “high-risk” HPV types of the alpha genus and HPV-related carcinogenicity. The oncoprotein E6 forms a ternary complex with the E3 ubiquitin ligase E6-associated protein (E6AP) and tumor suppressor protein p53 targeting p53 for ubiquitination. The extent of p53 degradation by different E6 proteins varies greatly, even for the closely-related HPV 16 and 31. 16E6 and 31E6 display high sequence identity (∼67 %). We report here for the first time the structure of HPV31 E6 bound to the LxxLL motif of E6AP. 16E6 and 31E6 are structurally very similar in agreement with the high sequence conservation. Both E6 proteins bind E6AP and degrade p53. However, the binding affinities of 31E6 to E6AP and p53, respectively, are reduced 2-fold and 5.4-fold as compared to 16E6. The affinity of E6-E6AP-p53 ternary complex formation parallels the efficacy of the subsequent reaction, namely degradation of p53. Therefore, closely-related E6 proteins addressing the same cellular targets may still diverge in their binding efficiencies, possibly explaining their different phenotypic or pathologic impact. IMPORTANCE Variations of carcinogenicity of Human papillomaviruses are related to variations of the E6 and E7 interactome. While different HPV species and genera are known to target distinct host proteins, the fine differences between E6 and E7 of closely-related HPV types, supposed to target the same cellular protein pools, remain to be addressed. We compare the oncogenic E6 proteins of the closely related “high-risk” HPV types 31 and 16 with regard to their structure and their efficiency of ternary complex formation with their cellular targets p53 and E6AP, which results in p53 degradation. We solved the crystal structure of 31E6 bound to the E6AP LxxLL-motif. 16E6 and 31E6 structures are highly similar but a few sequence variations lead to different protein contacts within the ternary complex and, as quantified here, an overall lower binding affinity of 31E6 compared with 16E6. These results align with the observed lower p53-degradation potential of 31E6.
Förster resonance energy transfer (FRET) is a widespread technology used to analyze and quantify protein interactions in multiple settings. While FRET is traditionally measured by microscopy, flow cytometry based-FRET is becoming popular within the last decade and more commonly used. Flow cytometry based-FRET offers the possibility to assess FRET in a short time-frame in a high number of cells thereby allowing stringent and statistically robust quantification of FRET in multiple samples. Furthermore, established, simple and easy to implement gating strategies facilitate the adaptation of flow cytometry based-FRET measurements to most common flow cytometers. We here summarize the basics of flow cytometry based-FRET, highlight recent novel developments in this field and emphasize on exciting future perspectives.
Co‐immunoprecipitation (Co‐IP) is a straightforward method that is widely used in studying direct protein‐protein interactions in physiological environments. This technique is based on the antigen‐antibody interaction: the protein of interest (bait) is captured by a specific antibody, followed by antibody‐bait precipitation. The proteins interacting with the bait protein (prey) co‐precipitate with the antibody‐bait complex from a cell lysate as an antibody‐bait/prey complex. Nowadays, a variety of surface‐functionalized materials with antibodies immobilized on agarose or magnetic beads are available, replacing the precipitation of antibodies and simplifying the application. However, unspecific binding of cellular proteins to matrix surfaces and/or antibodies has become a common issue. Unspecific binding that leads to false‐positive signals and a high background can hamper further analysis. Our protocol describes a strategy to tremendously reduce unspecific background when isolating native proteins and protein complexes. Instead of eluting our samples under denaturing conditions, we elute triple hemagglutinin (3×HA)‐tagged bait/prey complexes in their native form with a competitive peptide simulating the 3×HA tag of the bait protein. Matrix‐unspecific interacting proteins and Co‐IP antibodies remain on the matrix instead of being eluted under conventionally applied denaturing conditions. We optimized the elution by altering incubation time, eluent concentration, and temperature. These improvements result in more pure proteins. This strategy not only reduces background in SDS‐PAGE and western blot but also allows complex characterization in vitro. © 2021 Wiley Periodicals LLC.
Human papillomaviruses are DNA tumor viruses. A persistent infection with high-risk HPV types is the necessary risk factor for the development of anogenital carcinoma. The E6 protein is a viral oncoprotein that directly interacts with different cellular regulatory proteins mainly affecting the cell cycle, cellular differentiation and polarization of epithelial cells. In dependency of the phylogenetic classification of HPV different interaction partners of E6 have been described. The Notch pathway seems to be one common target of HPV, which can be up or down regulated by different E6 proteins. Our novel triple fluorescence flow-cytometry-based assay allows a semi-quantitative comparison of the E6 proteins´ effect on the Notch pathway using a Notch-responsive reporter plasmid. As a result, all E6 proteins of beta-HPV repressed the Notch reporter expression, of which HPV38 E6 showed the greatest repression potential. In contrast, alpha-HPV E6 of HPV16, activates the reporter expression most significantly, whereas E6 of HPV31 and low-risk HPV6b showed significant activation only in a p53-null cell line. Interestingly, HPV18 E6, with the second highest carcinogenic risk, shows no effect. This high divergence within different genus of HPV is important for targeting the Notch pathway regarding a potential HPV therapy.
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