Publikationsserver der Universitätsbibliothek Marburg

Titel: Porins, VDACs and gating : The role of conformational plasticity
Autor: Mertins, Barbara
Weitere Beteiligte: Essen, Lars-Oliver (Prof. Dr.)
Veröffentlicht: 2015
URI: https://archiv.ub.uni-marburg.de/diss/z2015/0074
URN: urn:nbn:de:hebis:04-z2015-00740
DOI: https://doi.org/10.17192/z2015.0074
DDC: Chemie
Titel(trans.): Porine, VDACs und Schaltvorgänge : Funktion der konformationellen Plastizität
Publikationsdatum: 2015-09-10
Lizenz: https://creativecommons.org/licenses/by-nc-sa/4.0

Dokument

Schlagwörter:
Membranprotein, apoptosis, BLM, membraneprotein, VDAC, VDAC, Apoptose, Proteininteraktion, BLM, protein interaction

Summary:
With about 10 000 copies per cell the voltage-dependent anion channels (VDACs) are the most abundant proteins of the mitochondrial outer membrane and are known to be involved in mitochondrial processes such as ATP-, calcium or ROS-transport. Beside this, they were identified as key players of mitochondrial physiology such as being involved in the mitochondrial related apoptotic pathway. Because of their strategic location as well as interaction with pro- and anti-apoptotic proteins, VDACs are involved in various diseases like Alzheimer, Down syndrome, cancer, stroke, and amylotrophic lateral sclerosis. Because of this multifunctionality, VDACs are important targets for medical approaches. After their discovery, VDACs have been extensively studied in terms of their structural organisation and their gating mechanism. The N-terminal region in the pores interior fuelled further debates about the gating mechanism of VDACs. VDACs reply to an applied voltage in a symmetric manner showing one open and several closed states. In this work I classified the closed states into at least three major states. With these quantitative electrophysiological measurements I demonstrated for the first time that a conformational variability of the N-terminus is essential for VDACs function. Through engineering of double-cysteine mVDAC1 variants affixing the N-terminal segment at the bottom and midpoint of the pore I verified that the N-terminus is the major trigger of VDAC´s gating. Additionally, it was shown that channel transitions are not solely dependent on the N-terminus. By analysing EMP as minimal model system for β-barrel gating, I revealed that a loop-independent gating exists, similar to that observed in the double-cysteine mVDAC1 variants. Given VDACs that interact with a plethora of effector proteins, I focused on the pro-apoptotic VDAC-effector Bid/tBid. Recent studies imply that Bid is a key player in neuronal cell death pathways. Accordingly, Bid seems to promote mitochondrial demise by release of death promoting proteins in the cytosol and the acceleration of oxidative stress. Furthermore, Bid-deficient neurons are highly resistant to cell death stimuli including oxygen-glucose deprivation (OGD) and glutamate-induced excitotoxicity in vitro and show reduced damage after cerebral ischemia and brain trauma in vivo. Here I could show a direct interaction between mVDAC1 and Bid/tBid and characterised the electrophysiological influence on VDAC in a quantitative manner. Biophysical analyse by SRCD, SROCD and EPR measurements give first insights into the structure of the VDAC-tBid complex. These data highlight the critical role for VDAC1 as a mitochondrial receptor for Bid thus providing a major control point of neuronal demise.

Zusammenfassung:
Mit rund 10 000 Kopien pro Zelle sind die voltage dependent anion channels (VDACs) die am häufigsten vorkommenden Proteine der äußeren mitochondrialen Membran. VDACs regulieren mitochondriale Prozesse, wie z. B. ATP-, Calcium- und ROS-Transport. Neben dieser klassischen Rolle wurden VDACs als wichtigste Komponente der Mitochondrien Physiologie und der damit verbundenen intrinsischen apoptotischen Signalwege identifiziert. Wegen ihrer strategischen Lage sowie der Interaktion mit pro- und anti-apoptotischen Proteinen, wurden VDACs mit verschiedenen Krankheiten wie Alzheimer, Down Syndrome, Krebs, Schlaganfall, und amylotrophischer lateraler Sklerose in Verbindung gebracht. Aufgrund dieser Multifunktionalität sind VDACs wichtige Ziele für medizinisch-therapeutische Behandlungsansätze. Nach ihrer Entdeckung wurden VDACs in Bezug auf ihre Struktur und den Schaltmechanismus untersucht. Der N-Terminus im Inneren der Pore wurde als das verantwortliche Schalt-Element diskutiert. Durch Anlegen einer externen Spannung, können VDACs in einen offenen und mehrere geschlossene Zustände schalten die ein physiologisches Verhalten imitieren. In dieser Arbeit konnte ich diese geschlossenen Zustände in mindestens drei Haupt-Zustände unterteilen. Mit diesen quantitativen elektrophysiologischen Messungen habe ich zum ersten Mal deutlich gemacht, dass die konformationelle Variabilität des N-Terminus für VDACs Schaltverhalten entscheidend ist. Durch das Erzeugen von Doppel Cystein Varianten, die den N Terminus am unteren Rand und in der Mitte die Pore arretieren, konnte ich zeigen, dass der N-terminus den dominanten Teil beim Schalten von VDACs darstellt. Daneben wurde auch gezeigt, dass zusätzliche Schaltvorgänge in einen geschlossenen Zustand unabhängig vom N-terminus auftreten können. Durch die Analyse von EMP als minimales Modellsystem konnte ich solch ein loop unabhängiges Schalten in Porinen demonstrieren. VDACs interagieren mit pro- und anti-apoptotischen Effektorproteinen. Der Fokus meiner Arbeit lag auf der Aufklärung der VDAC-Bid/tBid Interaktion. Bid als pro-apoptotisches Effektorprotein wurde als wichtiger Akteur im neuronalen Zelltod identifiziert, indem es die Freisetzung von apoptotischen Proteinen in das Zytosol fördert. Darüber hinaus, zeigen Bid defiziente neuronale Zellen eine hohe Resistenz gegen Zelltod-auslösende Reize wie z.B. Glutamattoxizität. Ich konnte die direkte Interaktion von VDAC1 und tBid zeigen und den funktionellen Einfluss mittels elektrophysiologischen Messungen quantifizieren. Biophysikalische Analysen (CD/OCD/EPR) ergaben erste Einblicke in den Protein-Komplex und verdeutlichen die Bedeutung der VDAC1-tBid Interaktion für die Viabilität von Neuronen.

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