Untersuchungen an neuartigen Serin-Threonin-Kinase-Inhibitoren für die In-vivo-Visualisierung von Signaltransduktionswegen des quergestreiften Muskels
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Date
2003-07-29
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Universität Dortmund
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Abstract
Das Protein Titin ist in quergestreiften Muskeln neben Aktin und Myosin das dritthäufigste Muskelprotein. Titin besitzt neben einer Vielzahl von Bindungsstellen für andere Muskelproteine zusätzlich eine Serin/Threonin-Kinase-Domäne, die als Titin-Kinase bezeichnet wird. Es wird vermutet, dass Titin und insbesondere die Titin-Kinase maßgeblich an der Signaltransduktion des quergestreiften Muskels beteiligt sind. Protein-Kinase-Inhibitoren stellen geeignete biochemische Werkzeuge dar, um solche Signaltransduktionswege zu untersuchen. Stellvertretend für die Titin-Kinase konnten für ausgewählte Enzyme aus der Gruppe der Serin/Threonin-Kinasen erstmalig fluoreszenzmarkierte bifunktionelle Inhibitoren synthetisiert werden, welche aus einer Nukleotid- und einer fluoreszenzmarkierten Peptid-Komponente bestehen, die durch einen kovalenten Linker in einem geeigneten Abstand zueinander fixiert werden. Es konnte gezeigt werden, dass die synthetisierten Verbindungen zu einer Inhibition im unteren mikromolaren Bereich führen und unter zellulären Bedingungen spezifisch mit der jeweiligen Ziel-Kinase kolokalisieren. Die Grundvoraussetzungen für eine Visualisierung von Signaltransduktionswegen mittels solcher bifunktioneller Inhibitoren sind somit erfüllt. Darauf aufbauend konnte ein entsprechender potentieller bifunktioneller Inhibitor für die Titin-Kinase synthetisiert werden, der nun für Untersuchungen der Signalwege der Titin-Kinase im quergestreiften Muskel sowie für die Kristallisation verwendet werden kann. Weiterhin konnte eine neuartige bifunktionelle Inhibitor-Matrix für die Affinitätschromatographie generiert werden, mit der es möglich war, eine rekombinante Kinase aus einem komplexen Zelllysatgemisch zu isolieren.
Besides actin and myosin, titin is the third-most prevalent muscle protein in the striated muscle. Titin contains a number of binding sites for other muscle proteins and an additional serine/threonine-kinase domain, called the titin kinase. It is supposed that titin and especially titin kinase play an important role in the signal transduction of striated muscle. Protein kinase inhibitors are suitable biochemical tools for the investigation of such signalling pathways. In this work, in a representative manner to titin kinase, fluorescently labeled bi-substrate inhibitors for selected enzymes from the family of serine/threonine-kinases were synthesized for the first time. The inhibitors were composed of a nucleotide and afluorescently labeled peptide fragment and were kept in the right distance by an appropriate covalent linker. It could be shown that these inhibitors lead to inhibition in the low micromolar range and co-localize specifically with their respective kinases under cellular conditions. So, we could fulfill the prerequisites for the use of such bi-substrate inhibitors for the visualization of signalling pathways. Based on this approach an appropriate bi-substrate inhibitor for titin kinase was synthesized which can now be used for the investigation of signalling pathways of titin kinase in striated muscle and also for the co-crystallization with titin kinase. Furthermore, an innovative bi-substrate inhibitor matrix for affinity chromatography was synthesized which allows the isolation of a recombinant protein kinase from a complex cell lysate.
Besides actin and myosin, titin is the third-most prevalent muscle protein in the striated muscle. Titin contains a number of binding sites for other muscle proteins and an additional serine/threonine-kinase domain, called the titin kinase. It is supposed that titin and especially titin kinase play an important role in the signal transduction of striated muscle. Protein kinase inhibitors are suitable biochemical tools for the investigation of such signalling pathways. In this work, in a representative manner to titin kinase, fluorescently labeled bi-substrate inhibitors for selected enzymes from the family of serine/threonine-kinases were synthesized for the first time. The inhibitors were composed of a nucleotide and afluorescently labeled peptide fragment and were kept in the right distance by an appropriate covalent linker. It could be shown that these inhibitors lead to inhibition in the low micromolar range and co-localize specifically with their respective kinases under cellular conditions. So, we could fulfill the prerequisites for the use of such bi-substrate inhibitors for the visualization of signalling pathways. Based on this approach an appropriate bi-substrate inhibitor for titin kinase was synthesized which can now be used for the investigation of signalling pathways of titin kinase in striated muscle and also for the co-crystallization with titin kinase. Furthermore, an innovative bi-substrate inhibitor matrix for affinity chromatography was synthesized which allows the isolation of a recombinant protein kinase from a complex cell lysate.
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Quergestreifter Muskel, Sarkomer, Titin, Titin-Kinase, cAMP-abhängige Proteinkinase, Myosin-Leichtketten-Kinase, Signaltransduktion, Protein-Kinase-Inhibitor, bifunktioneller Inhibitor, Fluorophor, Fluoreszenzmikroskopie, HeLa-Zellen, Herzmuskelzellen, Permeabilisierung, Streptolysin-O, Mikroinjektion, striated muscle, sarcomere, titin, titin kinase, cAMP-dependent protein kinase, myosin light-chain kinase, signal transduction, Protein kinase inhibitor, Bi-substrate inhibitor, Fluorophor, HeLa cells, Cardiomyocytes, Permeabilisation, Streptolysin-O, microinjection, fluorescence microscopy