Strukturelle Studien des alpha-synuclein, ein Protein impliziert mit der Parkinson-Krankheit

Die Parkinson-Krankheit ist eine der häufigsten degenerativen Erkrankungen des Gehirns mit Steifheit der Muskulatur, Zittern und Verlangsamung der Bewegung als typischen Symptomen. Im Verlauf der Krankheit kommt es zum Verlust von dopaminhaltigen Nervenzellen und proteinhaltigen Ablagerungen in der Substantia Nigra. Eine Hauptkomponente dieser als Lewy- Körper bezeichneten blagerungen ist das presynaptische, 140-Reste umfassende Protein alpha-Synuclein (aS). Bisher wurden drei genetische Mutationen in aS identifiziert, welche in Zusammenhang mit der Parkinson-Krankheit stehen. In vitro, kann die Aggregation von aS über Temperaturerhöhung, pH-Erniedrigung oder durch Zugabe von Polyaminen beschleunigt warden.Wir versuchen Licht in den Ablauf dieser Prozesse über eine NMR-basierte strukturelle Charakterisierung von aS zu bringen. Lösliches, monomers aS besitzt keine wohldefi nierte Sekundär- oder Tertiärstruktur. Aus diesem Grunde war unklar, wie es zu einer spontanen Oligomerisierung von aS kommt und warum hohe Temperaturen oder die Bindung an Polykationen für eine schnelle Aggregation erforderlich sind.Wir entdeckten dass aS trotz seiner hohen Flexibilität im monomeren Zustand eine transiente Tertiärstruktur in Lösung einnimmt, welche durch langreichweitige Wechselwirkungen stabilisiert wird. Hierbei faltet die C-terminale Domäne auf den Kernbereich von aS, der sogenannten non-A-beta component of Alzheimer s disease amyloid (NAC), zurück und verhindert dadurch eine über diesen Bereich vermittelte Oligomerisierung von aS. Die Stabilisierung der autoinhibierten Struktur findet innerhalb von Nano bis Mikrosekunden statt; dies ist die Zeitskala auf welcher Sekundärstrukturelemente während der Faltung von Proteinen entstehen. Polykationen, welche an die C-terminale Domäne binden, sowie erhöhte Temperatur, führen zu einer Destabilisierung der langreichweitigen Wechselwirkungen und induzieren damit eine vollkommene Entfaltung von aS. In diesem entfalteten Zustand ist die hydrophobe NAC Region frei zugänglich, so dass aS ohne Behinderung oligomerisieren kann. Unsere Ergebnisse! deuten an, dass die Destabilisierung der langreichweitigen Wechselwirkungen durch erhöhte Temperatur, Bindung an Polykationen, niedrigen pH oder genetische Mutationen der entscheidende Faktor für die Oligomerisierung von aS ist und damit die aS-haltigen Ablagerungen im Gehirn von Parkinson-Patienten initiiert.In Parkinson s disease (PD), intracellular neuronal inclusions containing amyloid-like aggregates of the protein alpha-synuclein (aS) are deposited in the pigmented nuclei of the brainstem. The mechanisms underlying the structural transition of innocuous, presumably natively unfolded aS to oligomeric neurotoxic forms of the protein are largely unknown.The major aim of this thesis has been the characterization of the ensemble of conformers that aS populates in its monomeric native state and the early transitions that lead to protein oligomerization, by the use of nuclear magnetic resonance (NMR) spectroscopy. The conformational flexibility inherent to this kind of proteins places them beyond the reach of classical structural biology, and a special set of NMR-based experiments had to be implemented in order to study the native soluble state of aS, namely paramagnetic relaxation enhancement (PRE) from nitroxide spin labels and residual dipolar couplings (RDCs).Our results show evidence that monomeric aS assumes conformations that are stabilized by long-range interactions and act to inhibit aggregation. As probed by RDCs, these auto-inhibitory conformations are formed on a ηs to µs timescale that is precisely that in which secondary structure elements form during folding. In addition, PRE-derived distance restraints have been employed to derive a low resolution model for the ensemble of structures of aS compatible with the experimental findings.The conformations populated by aS under disease-state circumstances were further investigated. Missense mutations linked to early onset PD, and environmental conditions that promote aS aggregation were found to release the inherent tertiary structure of the protein. Thus mutant or ligand bound aS overcomes more easily the energetic barrier for self-association, leading to an increased tendency to oligomerize.The homologous protein beta-synuclein (bS), which is proposed to inhibit the toxicity of aS, has been also characterized by means of high resolution NMR. It was found that the conformations populated by this protein do not account for long range interactions as aS does, but a higher degree of residual structure is attained, likely polyproline II extended conformations.Finally, the binding of divalent transition metal cations to aS was studied by a set of spectroscopic techniques. It was found that among several transition metals, Cu(II) is strongly bound by the N-terminus of the protein with an affinity of ~ 100 nM, and the complex is more prone to aggregate that the free protein. Other metals as Fe(II), Mn (II), Ni(II) and Co(II) do not influence protein aggregation since they bind to the C-terminus of the protein with 104 lower affinities. Our results suggest that impairment of Cu(II) homeostasis links the three major amyloid neurodegenerative disorders Alzheimer s, Prion and Parkinson s disease.From the therapeutic point of view, it is foreseen that the reinforcement of these native, auto-inhibitory, long-range interactions in aS may be a key target of new pharmacological agents designed to impede or even reverse aggregate formation in Parkinson s disease. Conformationally altered aS may also constitute a general molecular mechanism underlying the induction of PD by both environmental and genetic conditions. Thus, agents specifically designed to stabilize the native state of aS may also prove useful in impeding or reversing its pathologic aggregation in familial forms of PD