Publikationsserver der Universitätsbibliothek Marburg
Titel:Charakterisierungsstudien der biologischen und neurotrophen Eigenschaften des cerebral dopamine neurotrophic factor (CDNF)
Autor:Barkholz, Michael
Weitere Beteiligte: Petersen, Maike (Prof.)
Veröffentlicht:2011
URI:https://archiv.ub.uni-marburg.de/diss/z2012/0792
DOI: https://doi.org/10.17192/z2012.0792
URN: urn:nbn:de:hebis:04-z2012-07920
DDC: Medizin
Titel (trans.):Characterization studies on the biological and neurotrophic effects of cerebral dopamine neurotrophic factor (CDNF)
Publikationsdatum:2012-08-29
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Brain-derived neurotrophic factor, Mesc-Zellen, Production of recombinant proteins, Ciliary neurotrophic factor, Mesc cells, ER-Retention, LUHMES-Zellen, HT-22 Zellen, Cerebral dopamine neurotrphic factor, LUHMES cells, HT-22 cells, 6-Hydroxydopamin, 6-hydroxydopamine, Herstellung rekombinanter Proteine

Zusammenfassung:
Neurotrophe Faktoren ermöglichen das Überleben von Neuronen auch unter pathologischen Stressbedingungen sowie eine erneute Proliferation neuronaler Zellstrukturen nach Beschädigung. Neurotrophe Faktoren sind definiert als sekretierte Proteine. Sie werden nach Strukturhomologien, Rezeptoren und Signaltransduktionswegen in verschiedene Familien unterteilt. Die Bezeichnung „neurotropher Faktor“ umfasst Proteine mit unterschiedlichsten Funktionen. Die neurotrophen Eigenschaften von CDNF, das zur CDNF/MANF Proteinfamilie gehört, wurden durch intrastriatale Injektion und darauf folgende Protektion und Proliferation von Neuronen in einem in vivo Schädigungsmodell nachgewiesen. Diese Proteinfamilie könnte somit einen neuen Ansatz zu einer Therapiemöglichkeit von neurodegenerativen Krankheiten bieten. CDNF und MANF haben ein Molekulargewicht von ungefähr 21 kDa. Der Aminoterminus enthält eine globuläre Saposin-ähnliche (saposin like protein SAPLIP)-Domäne, und der Carboxyterminus ist verwandt mit der Sequenz von scaffold attachment factors (SAFs) und enthält analog zu SAFs eine redoxaktive Cysteinbrücke eingebettet in ein CXXC-Motiv. Die biologische Funktion, Rezeptoren und Wirkmechanismus der Neuroprotektion der CDNF/MANF Proteinfamilie sind bisher unbekannt. In dieser Arbeit wurden unterschiedliche Versuche zur Charakterisierung der Eigenschaften von CDNF und dessen SAPLIP-Domäne unternommen. Für alle Versuche waren umfangreiche biotechnologische Vorarbeiten notwendig, die in der Forschungsabteilung von CSL Behring, Marburg vorgenommen wurden. Der erste Teil der Arbeit befasst sich mit der Überprüfung der These, dass CDNF, ausgehend von seiner SAPLIP-Domäne, transduktorische Eigenschaften besitzen könnte: Makromoleküle in Zellen zu schleusen, ist durch die selektiv durchlässige Zellmembran nur sehr beschränkt möglich. Diese Restriktionen erlauben nur einem sehr kleinen Teil möglicher therapeutischer Substanzen den Zugang ins Zellinnere. Die natürliche Struktur von beispielsweise Proteinen verhindert das passive Eindringen dieser Stoffe in die Zelle. In den 1980er Jahren wurden jedoch Peptidsequenzen entdeckt, die als Molekültransporter Makromoleküle in das Zellinnere schleusen können. Die N-terminalen Domäne von CDNF besitzt nicht die klassische Struktur proteinogener Molekültransporter, sie ist jedoch verwandt mit Saposinen. Saposine und Saposin-ähnliche Domänen weiterer Proteine können Lipide binden oder Zellmembranen permeabilisieren. Diese Eigenschaften führten zu der These, dass auch die SAPLIP-Domäne von CDNF potentielle transduktorische Funktionen ausüben könnte. Zur Überprüfung der These wurden CDNF-Fusionsproteine biotechnologisch produziert und auf ihre transduktorischen Eigenschaften untersucht. Es konnte aber keine Transduktion von CDNF oder Assoziation mit der Zellmembran detektiert werden. Der zweite Teil der Arbeit zeigt die zelluläre Lokalisation von nativem CDNF. CDNF wird als sekretierter neurotropher Faktor beschrieben. Die C-terminale KTEL-Sequenz von CDNF weist jedoch eine große Ähnlichkeit zum allgemeinen ER-Retentionssignal KDEL auf. Wir zeigen in dieser Arbeit mit unterschiedlichen Methoden zum ersten Mal, dass CDNF tatsächlich im ER retardiert wird. Es wurden weiterhin CDNF-Mutanten mit verändertem CTerminus produziert. Die CDNF-Mutante mit C-terminaler KDEL-Sequenz und natives CDNF konnten nicht im Überstand von transfizierten Säugetier-Zellkulturen detektiert werden. Im Gegensatz zu CDNF-Mutanten mit deletiertem, maskiertem oder anderweitig verändertem C-Terminus, die im Kulturüberstand nachweisbar waren. Dieser Vergleich mit unterschiedlichen CDNF-Mutanten zeigt die wichtige Funktion einer freien C-terminalen KTEL-Sequenz bei der zellulären Lokalisation von CDNF. Der dritte Teil der Arbeit beschäftigt sich mit den neurotrophen Eigenschaften von CDNF in vitro. Nach erfolgreicher Protektion sollte durch molekulare und biochemische Methoden der unbekannte Mechanismus der neuroprotektiven Wirkung von CDNF näher untersucht werden. Verschiedene neuronale Zellkulturen wurden dafür mit 6-Hydroxydopamin oder Glutamat geschädigt. Rekombinant hergestelltes CDNF oder dessen SAPLIP-Domäne wurden extern zugeführt und der Einfluss der zugesetzten Proteine analysiert. Im Gegensatz zu den vorherigen Ergebnissen in vivo, konnte in keinem der verwendeten in vitro Systeme eine signifikante Protektion oder Proliferation durch CDNF beobachtet werden.

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