Nachweis und Analyse der Interaktion von Sprouty-Proteinen mit adenoviralen E1A-Onkoproteinen

Die adenoviralen E1A-Proteine greifen über spezifische Protein-Protein-Interaktionen in die Regulation zellulärer Prozesse ein. Die Identifikation und funktionelle Charakterisierung dieser zellulären Interaktionspartner von E1A tragen dazu bei, die Vorgänge zellulärer Mechanismen zur Regulation der Zellphysiologie aufzuklären. In der hier vorliegenden Arbeit konnte eine Interaktion der E1A-Proteine des Adenovirus Serotyps 12 mit dem zytoplasmatisch lokalisierten Sprouty 1-Protein gezeigt und charakterisiert werden. Zusätzlich ließen sich auch Interaktionen zwischen den hoch-onkogenen E1A-Proteinen und Sprouty 2 sowie Sprouty 3 feststellen. Weitere Interaktionsanalysen belegen außerdem, dass Sprouty 1 sowohl mit dem nicht-onkogenen E1A13S-Protein des Adenovirus-Serotyps 2 als auch mit dem E7-Onkoprotein des humanen Papillomavirustyp 16 interagiert. Die Analysen mit Deletionsmutanten zeigen, dass die Interaktion zu Sprouty 1 über den Aminoterminus und die CR3-Domäne der E1A-Proteine vermittelt wird. Die carboxyterminale Hälfte von Sprouty 1, in der sich die konservierte Sprouty-Domäne befindet, ist nachweislich für die Interaktion mit den hoch-onkogenen E1A-Proteinen essentiell. In Immunfluoreszenzuntersuchungen konnte eine Kolokalisation der E1A13S-Proteine des Adenovirus-Serotyps 12 mit Sprouty 1 im Zytoplasma nachgewiesen werden. Die funktionellen Analysen mit den Responseelementen TRE und SRE zeigen, dass die Koexpression des hoch-onkogenen E1A13S-Proteins mit Sprouty 1 eine Reduktion der E1A13S-induzierten Genexpression zur Folge hat. In den Analysen mit der aminoterminalen Deletionsmutante von E1A13S, die nur mit einer geringen Affinität an Sprouty 1 bindet, konnte keine signifikante Inhibition der ΔNE1A13S-induzierten Genexpression und der ERK1/2-Kinasen-Phosphorylierung durch Sprouty 1 detektiert werden. Diese Daten stützen die Vermutung einer funktionellen Interaktion von E1A13S mit Sprouty 1 im Zytoplasma, zur Modifikation des Ras/ERK MAP Kinase Signalweges

Sprouty is a cytoplasmic target of adenoviral E1A oncoproteins to regulate the receptor tyrosine kinase signalling pathway

<p>Abstract</p> <p>Background</p> <p>Oncoproteins encoded by the early region of adenoviruses have been shown to be powerful tools to study gene regulatory mechanisms, which affect major cellular events such as proliferation, differentiation, apoptosis and oncogenic transformation. They are possesing a key role to favor viral replication via their interaction with multiple cellular proteins. In a yeast two-hybrid screen we have identified Sprouty1 (Spry1) as a target of adenoviral E1A Oncoproteins. Spry proteins are central and complex regulators of the receptor tyrosine kinase (RTK) signalling pathway. The deregulation of Spry family members is often associated with alterations of the RTK signalling and its downstream effectors, leading to the ERK pathway.</p> <p>Results</p> <p>Here, we confirm our yeast two-hybrid data, showing the interaction between Spry1 and E1A in GST pull-down and immunoprecipitation assays. We also demonstrated the interaction of E1A with two further Spry isoforms. Using deletion mutants we identified the N-terminus and the CR conserved region (CR) 3 of E1A- and the C-terminal half of Spry1, which contains the highly conserved Spry domain, as the essential sites for direct interaction between Spry and E1A. Immunofluorescent microscopy data revealed a co-localization of E1A_13Swith Spry1 in the cytoplasm. SRE and TRE reporter assays demonstrated that co-expression of Spry1 with E1A_13Sabolishes the inhibitory function of Spry1 in RTK signalling, which is consequently accompanied with a decrease of E1A_13S-induced gene expression.</p> <p>Conclusions</p> <p>These results establish Spry1 as a cytoplasmic localized cellular target for E1A oncoproteins to regulate the RTK signalling pathway, and consequently cellular events downstream of RTK that are essential for viral replication and transformation.</p

Protein tyrosine phosphatases expression during development of mouse superior colliculus

Protein tyrosine phosphatases (PTPs) are key regulators of different processes during development of the central nervous system. However, expression patterns and potential roles of PTPs in the developing superior colliculus remain poorly investigated. In this study, a degenerate primer-based reverse transcription-polymerase chain reaction (RT-PCR) approach was used to isolate seven different intracellular PTPs and nine different receptor-type PTPs (RPTPs) from embryonic E15 mouse superior colliculus. Subsequently, the expression patterns of 11 PTPs (TC-PTP, PTP1C, PTP1D, PTP-MEG2, PTP-PEST, RPTPJ, RPTPε, RPTPRR, RPTPσ, RPTPκ and RPTPγ) were further analyzed in detail in superior colliculus from embryonic E13 to postnatal P20 stages by quantitative real-time RT-PCR, Western blotting and immunohistochemistry. Each of the 11 PTPs exhibits distinct spatiotemporal regulation of mRNAs and proteins in the developing superior colliculus suggesting their versatile roles in genesis of neuronal and glial cells and retinocollicular topographic mapping. At E13, additional double-immunohistochemical analysis revealed the expression of PTPs in collicular nestin-positive neural progenitor cells and RC-2-immunoreactive radial glia cells, indicating the potential functional importance of PTPs in neurogenesis and gliogenesis

Development of Chicken Embryos in Double-Yolk Eggs: Fertility, Hatchability, Embryo Malposition and Time of Embryonic Mortality

Fertility rate and hatchability rate are low for all types of double-yolk (DY) eggs in comparison to single-yolk eggs (SY), but these parameters also depend on the number of developing embryos in the egg. The hatchability rate of double-yolk eggs containing two developing embryos (DY2F) is vastly lower than in the case of double-yolk eggs containing only one embryo (DY1F). The aim of the study was to determine the differences between egg fertility rate, hatchability rate, time of embryonic mortality, and embryo malposition during incubation in three types of eggs from Hy-Line Brown hens: SY, DY1F and DY2F. In addition, the quality of the hatched chicks was assessed using the Pasgar©score. Following a 21-day incubation, chicks were obtained from DY1F and SY eggs. No chicks were obtained from DY2F eggs, although the embryos in these eggs developed up to the late stage of incubation. Early (≤7 d of incubation), middle (8–14 d), and late (≥15 d) embryonic mortality was significantly higher in DY eggs than in SY eggs. The embryonic mortality rate during early incubation was the same for DY1F and DY2F eggs, but middle and late embryonic mortality were significantly higher for DY2F eggs. Based on evaluation of embryo position according to Landauer, only three types of malposition that could potentially lead to embryonic death were noted. There were fewer malpositioned embryos in double-yolk eggs containing one embryo. Quality assessment of chicks (Pasgar©score) showed no differences between chicks hatched from eggs containing one yolk and those hatched from double-yolk eggs with one developing embryo, but chicks from double-yolk eggs were significantly heavier. The results of the research will contribute to a better understanding of the development and mortality of embryos in double-yolk eggs

Doxycycline Inducible Melanogenic Vaccinia Virus as Theranostic Anti-Cancer Agent

We reported earlier the diagnostic potential of a melanogenic vaccinia virus based system in magnetic resonance (MRI) and optoacoustic deep tissue imaging (MSOT). Since melanin overproduction lead to attenuated virus replication, we constructed a novel recombinant vaccinia virus strain (rVACV), GLV-1h462, which expressed the key enzyme of melanogenesis (tyrosinase) under the control of an inducible promoter-system. In this study melanin production was detected after exogenous addition of doxycycline in two different tumor xenograft mouse models. Furthermore, it was confirmed that this novel vaccinia virus strain still facilitated signal enhancement as detected by MRI and optoacoustic tomography. At the same time we demonstrated an enhanced oncolytic potential compared to the constitutively melanin synthesizing rVACV system