Spatio-temporäre Analyse des epidermalen Wachstumsfaktor-Rezeptors : Modulation der Signaltransduktion durch das humane Papillomavirus Typ 16 E5-Protein

Ursächlich für die Entstehung eines Zervixkarzinoms ist eine persistierende Infektion mit humanpathogenen Papillomaviren (HPV) des sogenannten Hochrisiko-Typs, deren DNA-Sequenzen in mehr als 65% aller Zervixkarzinome nachgewiesen werden konnten. Die HPV-Familie besteht aus mehr als 100 Typen, von denen vorwiegend die Typen 16 und 18 mit der tumoralen Entwicklung assoziiert sind. Verantwortlich für die Malignisierung des Epithels sind drei Onkogene des HPV-Typs 16, dazu zählt das kleine, hydrophobe Protein E5, welches hauptsächlich in der Membran von Golgi-Apparat und endosomaler Kompartimente lokalisiert ist. Das Protein besitzt selbst nur schwache transformierende Eigenschaften, ist aber in der Lage die Onkogenizität der beiden anderen Onkogene E6 und E7 zu potenzieren. Der Haupteffekt des E5-Onkogens ist eine Überaktivierung des EGF-Rezeptors (EGFR), deren Folge eine gesteigerte Transkription mit anschließender Mitose ist. Ziel dieser Arbeit war die Identifizierung der zellbiologischen Mechanismen, die durch E5 moduliert werden und eine verstärkte Aktivierung der EGF-Rezeptoren bewirken. Die quantitative Analyse mittels Immunblot und On-Cell Western Blot zeigte in Anwesenheit von HPV16 E5 eine Zunahme an Oberflächen-assoziierten EGF-Rezeptoren. Folglich wurde im Vergleich zu den Kontroll-Zellen eine Verstärkung der Liganden-vermittelten EGFR-Aktivierung hervorgerufen, die über einen langen Zeitraum nach Zugabe von EGF anhielt und unabhängig von der Internalisierungsrate war. Zusätzlich zu den klassischen molekular- und zellbiologischen Methoden wurde eine neue Technologie eingesetzt, die eine spatio-temporäre Analyse der E5-bedingten Modulation der EGFR-Signaltransduktion ermöglichte. Mit Hilfe der automatisierten, quantitativen Analyse von drei-dimensionalen Multikanalbildern, die am konfokalen Mikroskop generiert wurden, konnte eine genaue Charakterisierung des endozytotischen Transports von aktivierten und Gesamt-EGFR in Leervektor- und E5-transduzierten Zellen erfolgen. Der Einsatz von Vesikel-spezifischen Markern ermöglichte zudem die genaue Lokalisation der Rezeptoren innerhalb eines spezifischen zellulären Kompartiments. In Anwesenheit von E5 konnte eine stärkere und schnellere Fusion von EGFR-positiven Vesikeln mit dem frühen Endosomen nachgewiesen werden. Durch das zügige Fortschreiten des endozytotischen Transports erreichte eine deutliche höhere Anzahl von phospho-EGFR-positiven Vesikeln frühzeitig die perinuklear lokalisierten multivesikulären Körperchen (MVB), ohne zuvor die zytoplasmatischen MVBs zu passieren. Zu den späten Zeitpunkten der EGF-Stimulation konnte eine Akkumulation von aktivierten Rezeptoren in perinuklearen Endosomen-ähnlichen Kompartimenten heterogener Struktur dokumentiert werden, die mit einer verminderten Dephosphorylierung von aktivierten Rezeptoren verbunden war und schließlich zu einer langanhaltenden Überaktivierung von EGF-Rezeptoren führte. Durch die Behandlung mit dem Recycling-Inhibitor Monensin konnte im Vergleich zu den Kontrollen keine Beeinträchtigung der Phosphorylierung von EGFR in E5-exprimierenden Zellen dokumentiert werden. Damit kann eine potentielle Zunahme des Recyclings von EGF-Rezeptoren als Ursache der E5-vermittelten Überaktivierung ausgeschlossen werden. Die hier dargestellten Ergebnisse beweisen, dass die verstärkte und langanhaltende Aktivierung von EGFR durch eine modifizierte Endozytose und Dephosphorylierung während des Transports der Rezeptoren von frühen Endosomen zu den multivesikulären Körperchen verursacht wird. Dadurch umgehen die Rezeptoren den Abbau in den Lysosomen und bewirken eine gesteigerte Transkription früher Gene im Nukleus, die in einer EGFR-vermittelten Deregulation der Zellproliferation, -differenzierung und Apoptose resultiert

Focal delivery of AAV2/1-transgenes into the rat brain by localized ultrasound-induced BBB opening

Delivery of drugs and macromolecules to the central nervous system (CNS) is hindered by the blood-brain barrier (BBB). Several approaches have been used to overcome this hindrance to facilitate the treatment of various CNS diseases. We now present results showing that chimeric adeno-associated virus 2/1 (AAV2/1) particles containing the coding region for the LacZ gene are efficiently delivered into the rat brain upon intravenous (IV) administration after BBB opening by focused ultrasound in the presence of vascular acoustic resonators. We show that the transgene is correctly and efficiently expressed in cells located in the neighborhood of the insonated focus, especially in the vicinity of small vessels and capillaries. Histochemical LacZ staining allows the identification of large amounts of cells expressing the enzymatically active protein. Using double immunofluorescence (IF) with antibodies against tubulinIII and bacterial LacZ, we identified these cells to be mostly neurons. A small proportion of the transduced cells was recognized as glial cells, reacting positive in the IF with antibodies against astrocytic markers. These results demonstrate that our approach allows a very specific, localized, and efficient expression of intravenously administered transgenes in the brain of rats upon ultrasound-induced BBB opening

Disentangling ERBB Signaling in Breast Cancer Subtypes—A Model-Based Analysis

Targeted therapies have shown striking success in the treatment of cancer over the last years. However, their specific effects on an individual tumor appear to be varying and difficult to predict. Using an integrative modeling approach that combines mechanistic and regression modeling, we gained insights into the response mechanisms of breast cancer cells due to different ligand–drug combinations. The multi-pathway model, capturing ERBB receptor signaling as well as downstream MAPK and PI3K pathways was calibrated on time-resolved data of the luminal breast cancer cell lines MCF7 and T47D across an array of four ligands and five drugs. The same model was then successfully applied to triple negative and HER2-positive breast cancer cell lines, requiring adjustments mostly for the respective receptor compositions within these cell lines. The additional relevance of cell-line-specific mutations in the MAPK and PI3K pathway components was identified via L1 regularization, where the impact of these mutations on pathway activation was uncovered. Finally, we predicted and experimentally validated the proliferation response of cells to drug co-treatments. We developed a unified mathematical model that can describe the ERBB receptor and downstream signaling in response to therapeutic drugs targeting this clinically relevant signaling network in cell line that represent three major subtypes of breast cancer. Our data and model suggest that alterations in this network could render anti-HER therapies relevant beyond the HER2-positive subtype

Reorganization of gap junctions after focused ultrasound blood–brain barrier opening in the rat brain

Ultrasound-induced opening of the blood–brain barrier (BBB) is an emerging technique for targeted drug delivery to the central nervous system. Gap junctions allow transfer of information between adjacent cells and are responsible for tissue homeostasis. We examined the effect of ultrasound-induced BBB opening on the structure of gap junctions in cortical neurons, expressing Connexin 36, and astrocytes, expressing Connexin 43, after focused 1-MHz ultrasound exposure at 1.25 MPa of one hemisphere together with intravenous microbubble (Optison, Oslo, Norway) application. Quantification of immunofluorescence signals revealed that, compared with noninsonicated hemispheres, small-sized Connexin 43 and 36 gap-junctional plaques were markedly reduced in areas with BBB breakdown after 3 to 6 hours (34.02±6.04% versus 66.49±2.16%, P=0.02 for Connexin 43; 33.80±1.24% versus 36.77±3.43%, P=0.07 for Connexin 36). Complementing this finding, we found significant increases in large-sized gap-junctional plaques (5.76±0.96% versus 1.02±0.84%, P=0.05 for Connexin 43; 5.62±0.22% versus 4.65±0.80%, P=0.02 for Connexin 36). This effect was reversible at 24 hours after ultrasound exposure. Western blot analyses did not show any change in the total connexin amount. These results indicate that ultrasound-induced BBB opening leads to a reorganization of gap-junctional plaques in both neurons and astrocytes. The plaque-size increase may be a cellular response to imbalances in extracellular homeostasis after BBB leakage

SPR based kinetic analyses of the HPV16 E6/E6APpep and HPV16 E6/pep11** interactions.

<p>(<i>A</i>) Sensorgrams obtained by injecting purified F47R 4C/4S E6 at the indicated concentrations on a surface capturing E6APpep. RU stands for SPR response units. Color lines represent analyte protein injections at the indicated concentrations. (<i>B</i>) Interaction of E6 F47R 4C/4S with surface-captured pep11**. Color and black lines indicate analyte injections and global fits to a 1∶1 binding model, respectively.</p

Intracellular analyses of the HPV16 E6/E6APpep and HPV16 E6/pep11** interactions.

<p>(<i>A</i>) Mammalian two-hybrid analyses in HeLa cells expressing individual peptides, as indicated, linked to GAL4-BD, and wt HPV16 E6 linked to the VP16-AD. pep11**m does not bind to HPV16 E6 and served as a negative control. Shown are relative activities of the co-transfected luciferase reporter plasmid under transcriptional control of GAL4-binding sites above those of control-transfected cells (expressing corresponding peptide-GAL4-BD fusions together with empty control vector pACT; values arbitrarily set at 1.0). Results were obtained from three individual experiments, each performed in duplicates. Standard deviations are indicated. Asterisks above horizontal lines indicate statistically significant differences from pACT-transfected cells, with p-values of ≤0.001 (***). (<i>B</i>) and (<i>C</i>) Mutational analyses of HPV16 E6 to identify amino acid residues that contribute to E6APpep (<i>B</i>) or pep11** (<i>C</i>) binding. The luciferase signal for the interaction of wt HPV16 E6 with E6APpep or pep11** was set at 100% (left columns in (<i>B</i>) and (<i>C</i>), respectively). The horizontal line indicates 1.5-fold inhibition. Individual amino acid exchanges are indicated below each column. Error bars indicate standard deviation values. (<i>D</i>) Statistical analysis of the differential binding behaviour of HPV16 E6 mutants 16E6R55A and 16E6Y70A. The values for the interaction between wt HPV16 E6 protein with E6APpep or pep11**, respectively, were set at 100%. Asterisks above the horizontal lines indicate statistically significant reductions of luciferase activities, with p-values of ≤0.01 (**) or ≤0.001 (***). Error bars indicate standard deviation values.</p

NMR analyses of the HPV16 E6/E6APpep and HPV16 E6/pep11** interactions.

<p>(<i>A</i>) ¹H,¹⁵N SOFAST-HMQC spectra of 100 µM ¹⁵N E6 F47R 4C/4S samples in the absence (black spectra) and presence of 1∶1 stoichiometric ratios (cyan and red spectra) of unlabeled E6APpep (upper panel) or pep11** (lower panel). Cyan spectra were recorded immediately after peptide addition (t = 0 h), whereas red spectra were recorded after 3 h of incubation in the presence of the peptide. Note that in the case of spectra of E6/pep11** samples some signals have lower intensity in the red spectrum than in the cyan spectrum (see enlarged view of lower panel). This decrease in signal intensity is concomitant with the appearance of a white precipitate of E6 in the NMR tube. (<i>B</i>) pep11** induced E6 aggregation monitored by the decrease of the intensity of the W132 indole cross-peak. Intensity changes are expressed as a I_bound/I_ref ratio, where I_bound is the cross-peak intensity in the peptide-bound spectra and I_ref the cross-peak intensity in the reference (unbound) spectrum. W132 indole intensities were derived from ¹H,¹⁵N SOFAST-HMQC spectra recorded at regular time intervals on three different samples containing ¹⁵N labeled E6 F47R 4C/4S and unlabeled pep11** mixtures with concentration ratios adjusted to 1∶0.5 (gray diamonds), 1∶1 (cyan squares) and 1∶2 (green triangles). E6 concentrations in all three samples were adjusted to 100 µM. X-axis error bars correspond to the duration of the NMR experiment (i.e. error bar: 15 min), whereas y-axis error bars report on the signal-to-noise (S/N) ratio in the reference spectra, which, in this case, is expressed as the inverse of the S/N for sake of clarity (i.e. error bar: ± I_noise/I_ref). Estimates of the noise were obtained using the program NMRpipe <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112514#pone.0112514-Delaglio1" target="_blank">[32]</a>.</p

E6-binding peptides mediating intracellular formation of a trimeric complex with HPV16 E6 and p53.

<p>(A) Schematic illustration of the employed modified mammalian two hybrid assay. Upper panel: HPV16 E6 is linked to GAL4-BD, p53 is linked to VP16-AD, individual peptides are expressed from a co-transfected expression vector, in fusion with hrGFP. Lower panel: Trimeric complex formation is detected by activation of the luciferase reporter. GAL4 BS, GAL4 binding sites. (B) Expression of HPV16 E6-GAL4-BD with p53-VP16-AD, together with hrGFP-linked peptides, as indicated, or, as negative control, hrGFP alone. Shown are relative luciferase activities (RLA) above those of control-transfected cells, expressing HPV16 E6-GAL4-BD fusions together with the co-transfected basic vectors pACT and pCEP4hrGFP; 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 HPV16 E6-GAL4-BD together with p53-VP16-AD, in the absence of the peptides (co-transfected basic vector pCEP4hrGFP), with p-values of ≤0.001 (***) and ≤0.05 (*). (C) Immunoblot analyses of expression levels of individual peptides linked to hrGFP. 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.</p

NMR interface analyses of the HPV16 E6/E6APpep and HPV16/pep11** interactions.

<p>Indicated are the changes in the intensities of backbone amide cross-peaks of E6 F47R 4C/4S residues upon addition of a 1-fold excess of unlabeled E6APpep (<i>A</i>) or pep11** (<i>B</i>). I_bound/I_ref thresholds of 0.7 and 0.6 are indicated by colored horizontal lines (light green/dark green and pink/purple for the E6APpep and pep11** interactions, respectively). Error bars (I_noise/I_ref ) report on the signal-to-noise (S/N) ratio in the reference spectra (see legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112514#pone-0112514-g002" target="_blank">Figure 2</a>). Amide cross-peaks with a I_noise/I_ref >0.25 are considered noisy peaks. Cyan shaded areas indicate unassigned residues or prolines (which are additionally highlighted by yellow dots). HPV16 E6 secondary structure elements deduced from the HPV16 E6/E6APpep x-ray structure are indicated above the histograms.</p

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

<div><p>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 <i>in vitro</i> 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.</p></div