Publikationsserver der Universitätsbibliothek Marburg
Titel:Targeting Bid for mitoprotection - Bid crystallization, new mechanisms and inhibitory compounds
Autor:Oppermann, Sina
Weitere Beteiligte: Culmsee, Carsten (Prof. Dr.)
Veröffentlicht:2014
URI:https://archiv.ub.uni-marburg.de/diss/z2014/0353
DOI: https://doi.org/10.17192/z2014.0353
URN: urn:nbn:de:hebis:04-z2014-03535
DDC:500 Naturwissenschaften
Titel (trans.):Bid als Zielstruktur zur Erhaltung der mitochondrialen Funktion - Bid Kristallisation, neue Mechanismen und Bid Inhibitoren
Publikationsdatum:2014-07-02
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
crystallization, Protein-Protein Interaktionen, neuroprotection, mitochondrium, mitochondria, Inhibitoren, Bid, Neuroprotektion, protein interactions, Kristallisation, Mitochondrien, Bid, inhibitory compounds, drugs

Summary:
Mitochondriale Prozesse des Zelltods spielen eine entscheidende Rolle für den progressiven Verlust von Neuronen bei neurodegenerativen Erkrankungen (M. Alzheimer, M. Parkinson) und nach akuter Hirnschädigung durch Schädel-Hirn-Trauma oder zerebraler Ischämie. Eine Schlüsselfunktion nimmt hierbei das pro-apoptotische Bcl-2 Protein Bid ein. Durch die Aktivierung und mitochondriale Translokation von Bid kommt es zur Schädigung und Fragmentierung von Mitochondrien und letztlich zur Freisetzung von weiteren pro-apoptotischen Faktoren (AIF, Cytochrom C, Smac/DIABLO), die den Untergang der Neurone steuern. Um in Zukunft Bid als potentielles Target für die Therapie von akuten und chronischen neurologischen sowie nicht neurologischen Erkrankungen nutzen zu können, müssen die bisher ungeklärten Mechanismen der Bid-induzierten mitochondrialen Schädigung, sowie die Interaktionen von aktiviertem Bid mit weiteren am Zelltod beteiligten Proteine aufgeklärt werden. Ziel dieser Arbeit war daher die Untersuchung Bid-abhängiger, mitochondrialer Zelltod Mechanismen und beteiligter Protein-Wechselwirkungen. Darüber hinaus stand die Entwicklung neuer Leitstrukturen für protektive Bid-Inhibitoren sowie die Etablierung erster Kristallisationsansätze verschiedener Bid Konstrukte im Fokus dieser Arbeit. Als Modellsyteme mitochondrialer Prozesse des Zelltods dienten vor allem immortalisierte hippokampale Neurone (HT-22 Zellen), in denen eine Behandlung mit Glutamat den durch oxidativen Stress gekennzeichneten Zelltod induziert. Als weiteres Schädigungsmodell wurde die Überexpression von aktiviertem Bid (tBid) eingesetzt. Zum Nachweis von Proteininteraktionen wurden weiterhin eine primäre neuronale Zellkultur und ein in vivo-Modell der zerebralen Ischämie verwendet, sowie verschiedene Untersuchung mit rekombinanten Proteinen durchgeführt. Neue niedermolekulare Bid-Inhibitoren aus drei strukturell verschiedenen Substanzklassen wurden mittels Zellviabilitätsmessungen auf neuroprotektive Effekte geprüft. Sieben Substanzen zeigten nicht nur deutliche Protektion gegenüber dem Glutamat- und tBid-induzierten Zelltod, sondern konnten ebenso die Bid-abhängige mitochondriale Schädigung verhindern. Weiterhin bietet die Arbeit Einblicke in die Kristallisation von rekombinantem Bid Protein, welche als Basis für ein grundlegendes Verständnis der molekularen Proteinfunktion sowie der Struktur-basierten Wirkstoffentwicklung dient. Durch Verfolgung wichtiger Strategien im Konstruktdesign konnte eines der verwendeten Bid Konstrukte erfolgreich kristallisiert werden und lieferte eine Strukturauflösung von 3.75 bis 3.95 Ǻ unter Synchrotronstrahlung. Weiterhin wurde der Effekt der rekombinanten Proteine Bid, cBid und Bax auf Fluoreszenz-Liposomen getestet, um die Mechanismen der Bid-abhängigen mitochondrialen Membranpermeabilisierung zu untersuchen. Es konnte hier eine Schlüsselrolle für das mitochondriale Lipid Cardiolipin gezeigt werden, in dessen Abhängigkeit Caspase-8 aktiviertes Bid (cBid) eine Membrandestabilisierung vermittelte, welche durch die Koexistenz von Bax gesteigert werden konnte. Letztlich konnte erstmals eine direkte Interaktion zwischen Bid und dem mitochondrialen Porin VDAC1 in kultivierten Neuronen sowie in einem in vivo Modell der zerebralen Ischämie nachweisen. Die Inhibition von VDAC1 mittels Einsatz des Anionen-Kanal-Blockers DIDS sowie der Verwendung von VDAC1 siRNA konnte sowohl die Funktion als auch die Integrität der Mitochondrien nach Glutamat- und tBid-induzierter Schädigung schützen und bestätigte somit die essentielle Rolle von VDAC1 im Bid-abhängigen Zelltod. Weitere Untersuchungen zeigten erstmalig, dass beide Proteine, Bid und VDAC1, gleichermaßen im neuronalen Zelltod involviert sind und offensichtlich zusammen eine Schädigung der mitochondrialen Membran induzieren. Im Gegensatz dazu konnte gezeigt werden, dass die Isoform VDAC2 nur eine untergeordnete Rolle im Bid-induzierten Zelltod in Neuronen spielt. Die nachgewiesene Bid-VDAC1 Wechselwirkung besitzt hohe Relevanz für die mitochondrialen Prozesse des Zelltods. Damit verbindet die vorliegende Arbeit die bisher kontrovers betrachten Mechanismen der mitochodrialen Membranschädigung, die zuvor entweder auf die alleinige Aktivität der Bcl-2 Proteine oder auf VDACs zurückgeführt wurde. Mit der Identifizierung neuer potentieller Bid-Inhibitoren sowie durch Etablierung wichtiger Grundlagen für die Aufklärung der Kristallstruktur von Bid, stellt die Arbeit einen wesentlichen Beitrat für die strukturbasierte Wirkstoffentwicklung und Therapie verschiedener neurologischer und nicht neurologischer Erkrankungen dar, in welchen Bid-abhängige mitochondriale Prozesse des Zelltods eine wesentliche Rolle spielen.

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