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Titel:Mechanistische Charakterisierung von ISC-Assemblierungsfaktoren bei der Maturierung mitochondrialer [2Fe-2S]- und [4Fe-4S]-Proteine
Autor:Weiler, Benjamin Dennis
Weitere Beteiligte: Lill, Roland (Prof. Dr.)
Veröffentlicht:2018
URI:https://archiv.ub.uni-marburg.de/diss/z2018/0287
URN: urn:nbn:de:hebis:04-z2018-02877
DOI: https://doi.org/10.17192/z2018.0287
DDC: Medizin
Titel (trans.):Mechanistic characterization of ISC assembly factors involved in the maturation of mitochondrial [2Fe-2S] and [4Fe-4S] proteins
Publikationsdatum:2019-01-15
Lizenz:https://creativecommons.org/licenses/by-nc-sa/4.0

Dokument

Schlagwörter:
Fe/S proteins, Fe/S-Proteine, ISC machinery, ISC-Maschinerie,

Zusammenfassung:
Eisen-Schwefel-(Fe/S)-Proteine sind in fast allen Lebensformen zu finden, an einer Vielzahl essentieller zellulärer Prozesse beteiligt und in unterschiedlichen Kompartimenten der eukaryotischen Zelle lokalisiert. In (nicht-grünen) Eukaryoten gibt es zwei Assemblierungs-maschinerien, die für die Maturierung von Fe/S-Proteinen verantwortlich sind. Die zytosolische Fe/S-Protein-Assemblierungsmaschinerie (CIA) mit 11 Proteinfaktoren und die aus 17 Proteinen bestehende mitochondriale Fe/S-Cluster-Assemblierungsmaschinerie (ISC), die an der Biogenese aller zellulären Fe/S-Proteine beteiligt ist. Im ersten Schritt der ISC-Maschinerie wird ein [2Fe-2S]-Cluster de novo auf dem Gerüstprotein ISCU synthetisiert und mit Hilfe eines Chaperonsystems auf das Monothiol-Glutaredoxin GLRX5 transferiert. Der auf GLRX5 transient gebundene [2Fe-2S]-Cluster kann entweder direkt auf [2Fe-2S]-Zielproteine übertragen oder nach in vivo Untersuchungen von den ISCA-IBA57 Proteinen zu einem [4Fe-4S]-Cluster konvertiert werden. Für die Insertion des [4Fe-4S]-Clusters in Apozielproteine werden noch weitere spezialisierte ISC-Proteine benötigt. Hierzu zählt beispielsweise NFU1, das transient einen [4Fe-4S]-Cluster bindet und diesen auf spezielle Zielproteine wie der Aconitase (ACO2) überträgt. Zwei weitere Proteine, BOLA1 und BOLA3, sind vermeintlich zusätzliche spezifische ISC-Proteine. Sowohl die Synthese eines [4Fe-4S]-Clusters als auch die an die [2Fe-2S]- oder [4Fe-4S]-Clustersynthese anschließende Clusterinsertion in entsprechende Zielproteine sind biochemisch weitestgehend unbekannt. Im eukaryotischen System ist bisher weder der [2Fe-2S]- noch der [4Fe-4S]-Clustertransfer unter physiologischen Bedingungen mechanistisch aufgeklärt worden. Alle bisherigen Fe/S-Clustertransfersysteme wurden ausnahmslos in Gegenwart von artifiziellen, thiolspezifischen Reduktionsmitteln wie Dithiothreitol (DTT) durchgeführt. Da DTT in diesen Transfersystemen meist essentiell war, blieb einerseits unklar, welche physiologischen Reduktionsmittel das DTT in der Zelle ersetzen. Andererseits ging aus diesen in vitro Untersuchungen nicht hervor, ob die beobachteten Transferreaktionen überhaupt physiologische Relevanz besitzen und auch in vivo so ablaufen, wie sie in vitro beobachtet wurden. In dieser Arbeit konnte, unter Verwendung des physiologischen Dithiol-Glutaredoxin-Glutathion-Reduktionssystems (Grx1/GSH) anstelle des DTT, ein zielspezifischer [2Fe-2S]- und [4Fe-4S]-Clustertransfer auf Zielproteine in vitro nachgestellt werden. Der [2Fe-2S]-Clustertransfer von GLRX5 auf das natürliche Zielprotein, das mitochondriale Ferredoxin FDX1, erfolgte in weniger als 15 Sekunden ohne Beteiligung weiterer Hilfsproteine. Demgegenüber war der Transfer eines [2Fe-2S]-Clusters von GLRX5 auf ein [4Fe-4S]-Zielprotein nicht spontan ohne weiteres möglich. Auch der [4Fe-4S]-Cluster des späten ISC-Proteins NFU1 konnte in Gegenwart von Grx1/GSH jedoch ohne Hilfsproteine auf [4Fe-4S]-Zielproteine (ACO2, Leu1) übertragen werden. Für die Konversion eines GLRX5-gebundenen [2Fe-2S]- zu einem [4Fe-4S]-Cluster und die anschließende Insertion des [4Fe-4S]-Clusters in ACO2 waren in vitro wie erwartet die späten ISC-Komponenten ISCA1, ISCA2, IBA57, aber überraschenderweise auch die Elektronentransferkette (FDX2/FDXR/NADPH) essentiell notwendig. Grx1/GSH fungierte bei diesen Transferprozessen offensichtlich nur für die Reduktion der Zielproteine, nicht aber für die reduktive Kopplung von [2Fe-2S]2+-Clustern zu einem [4Fe-4S]4+-Cluster, was in vitro auch mit Hilfe von DTT artifiziell erfolgen kann. Zum ersten Mal konnten damit sowohl für den [2Fe-2S]- als auch den [4Fe-4S]-Clustertransfer physiologisch relevante in vitro Ergebnisse erzielt werden, die exakt die in vivo Verhältnisse widerspiegeln. Die mitochondrialen Proteine BOLA1 und BOLA3 der BOLA-Familie wurden als zusätzliche spezifische ISC-Proteine vermutet, da Patienten mit multiplen mitochondrialem Dysfunktionssyndrom 2 (MMDS2) durch den funktionellen Verlust des BOLA3-Proteins ein vergleichbares Krankheitsbild aufwiesen wie Patienten mit Mutationen im NFU1-Gen (MMDS1). In vivo Analysen in Hefe hatten gezeigt, dass Bol1 und Bol3 für die Insertion von [4Fe-4S]-Clustern in spezifische Zielproteine, wie der Lipoatsynthase (LIAS) und der Succinatdehydrogenase (SDH), nicht aber für [2Fe-2S]-Proteine wie Ferredoxine notwendig sind. Beide Hefe Bol-Proteine haben in vivo überlappende Funktionen. In dieser Arbeit konnte mit biochemischen Methoden wie MicroScale Thermophorese, analytischer Gelfiltration, UV/Vis- und CD-Spektroskopie gezeigt werden, dass das humane BOLA1 einen heterodimeren Komplex sowohl mit der Apo- als auch der Holoform des GLRX5 bildet. Der GLRX5-BOLA1-Komplex wies im Vergleich zum homodimeren GLRX5-GLRX5-Komplex unterschiedliche CD-spektroskopische Eigenschaften auf. Der [2Fe-2S]-Cluster war im GLRX5-BOLA1-Komplex stabiler gebunden als im homodimeren GLRX5, gegenüber Reduktion geschützt und konnte nicht auf [2Fe-2S]-Zielproteine transferiert werden. Um zu untersuchen, wie der [2Fe-2S]-Cluster durch BOLA1 koordiniert wird, wurden die konservierten Histidinreste (H102, H67, H58) zu Alanin mutiert. Obwohl in vivo Experimente gezeigt hatten, dass H102 funktionell essentiell ist, war dieser Rest in vitro nicht entscheidend für die Fe/S-Clusterbindung. Der GLRX5-BOLA1H102A-Komplex war reduktionsstabil und zeigte vergleichbare spektroskopische Eigenschaften wie der GLRX5-BOLA1-Komplex. Eine Alaninsubstitution von H67 und H58 und der Dreifachmutante (H102/67/58A) hingegen zeigte veränderte spektroskopische Eigenschaften im GLRX5-BOLA1-Komplex und eine Labilität gegenüber Reduktion. Dennoch bindet der GLRX5-BOLA1-Komplex auch nach Alaninsubstitution aller Histidine einen Fe/S-Cluster, was darauf hindeutet, dass der Fe/S-Cluster auf eine andere Art (GSH) stabilisiert wird. BOLA3 hingegen verhielt sich ganz anders als BOLA1. Es interagierte mit der Apoform von GLRX5, mit der Holoform hingegen nur in Gegenwart von GSH. Der Fe/S-Cluster am GLRX5-BOLA3+GSH-Komplex war labil gebunden, nicht gegen Reduktion geschützt und konnte leicht auf [2Fe-2S]-Zielproteine transferiert werden. Die aus diesen in vitro Daten hervorgehende unterschiedliche biochemische Rolle der humanen GLRX5-BOLA-Komplexe muss physiologisch durch eingehende in vivo Studien aufgeklärt werden.

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