Parkin-mediated ubiquitination and Parkinson's disease: Role of ubiquitin-conjugating enzymes (E2s) and ubiquitin-interacting motif (UIM)-containing proteins

Project: DFG ProjectsDFG Scholarships: Research Fellowships

Project Details

Description

Mutationen im Parkin-Protein verursachen eine Form der Parkinson-Krankheit. Parkinfungiert als eine E3-Ligase in der Ubiquitinierung, wodurch Ubiquitin (ub) an Lysin-Restedes Zielproteins konjugiert wird. Die Ubiquitinierung hat in Abhängigkeit von denVerbindungen der ubs untereinander unterschiedliche physiologische Auswirkungen auf dasjeweilige Substrat (Degradation durch das Proteasom, Endozytose, Modulation derTranskription, etc.). Die Art der Ubiquitinierung durch Parkin ist abhängig von dessen Partner- ub-konjugierender (E2) Enzyme und anderer Interaktoren. Im beantragten Projekt führen wireine systematische Evaluierung aller Gehirn-exprimierter E2-Enzyme auf deren möglicheInteraktion mit Parkin durch und bestimmen die jeweilige Ubiquitinierungsart. Wir werdendie Rolle von UIM (ub-Interaktionsmotive enthaltenden) Proteinen in der Verhinderung derFormierung uneinheitlich verbundener Ubiquitinketten durch bestimmte E2-Parkin-Paareuntersuchen, die den Abbau durch das Proteasom verhindern könnten. Die Ergebnisse unsererUntersuchungen werden es ermöglichen festzustellen, wie spezifische nicht-synonymeMutationen in funktionellen Domänen von Parkin seine Ligase-Aktivität verändern. Durch dieIdentifizierung der Art der ub-Kette, die durch spezifische E2-Parkin-Paare zusammen mitUIM-Proteinen an die Substrate konjugiert wird, wird es gelingen, die diesen Interaktionenzugrunde liegenden zellulären Reaktionswege zu prognostizieren und die Rolle von Parkin inseiner normalen und pathologischen Funktion besser zu verstehen.

Key findings

Mutations in the Parkin protein cause Parkinson's disease (PD). Parkin is an E3 ubiquitin ligase in ubiquitination, the process that conjugates the small modifier protein ubiquitin, to lysine residues of target proteins. Ubiquitination is an important cellular mechanism that can result in various physiological consequences (proteasomal degradation, transcription modulation, etc.). Parkin is a versatile E3 ligase capable of self-ubiquitination and ubiquitination of various other proteins. We have identified a functional and direct interaction between Ataxin-3 and Paritin. Ataxin 3 is an enzyme mutations in which are causing SCA3, the most common dominantly inherited ataxia worldwide, that can clinically present with features of parkinsonism. In our experiments, the interaction between Ataxin-3 and Parkin was greatly enhanced by Parkin selfubiquitination. However, Parkin polyubiquitination, but not monoubiquitination (or multimonoubiquitination) enhanced the binding of Ataxin-3. Remarkably, mutant Ataxin-3 had deubiquitinated Parkin more efficiently than the wild-type enzyme, and mutant but not wild-type Ataxin-3 promoted the clearance of Parkin via the autophagy pathway. This finding is consistent with the reduction in Parkin levels observed in the brains of SCA3 transgenic mice. Thus, our results implicate increased turnover of Parkin in the pathogenesis of SCA3. In addition, we have investigated the role of Parkin as autophagic degradation of mitochondria. Parkin is selectively recruited to dysfunctional mitochondria where it subsequently becomes enzymatically active and induces polyubiquitination of the mitochondrial fusion proteins - Mitofusins (Mfn1 and Mfn2) and other outer mitochondrial membrane proteins. In this way, Parkin initiates the autophagic clearance of damaged mitochondria by lysosomes, i.e. mitophagy. We have studied mitochondria-associated activity of fluorescently tagged wild-type Parkin and four PD-associated mutants. All examined forms of Parkin were located in the cytoplasm under basal conditions. However, after treatment with a mitochondrial membrane depolarizing agent, live-cell imaging showed that the speed and the extent of Parkin recruitment to mitochondria differed significantly among its variants. The immunoblotting experiments indicated that the time frame, extent, and the type of Parkin ubiquitination vary in a similar manner. We have also investigated involvement of Ataxin-3 in the dysfunctional mitochondrial clearance pathway and no signs of relocalization of Ataxin-3 were obvious. The ubiquitination of Mitofusins seems to be an important step preceding mitophagy. In the further experiments we determined, by mass spectrometry, that a single lysine molecule was ubiquitinated and we identified the position of this residue within the Mfn2 sequence. In the next step, we mutated the lysine residue ubiquitinated by Parkin to a non-ubiquitinable amino acid (arginine). After overexpression of wild-type Mfn2 and Mfn2 mutant in fibroblasts we noticed that in the absence of the specific lysine residue, Parkin does not ubiquitinate another lysine on Mfn2. Thus, we determined that the ubiquitination is highly specific and that this lysine is indeed crucial for the ubiquitination. The position of this lysine residue seem to be either structurally best accessible to Parkin or functionally important since in its absence no other lysine is ubiquitinated by Parkin. In in-vitro ubiquitination experiments we determined that Parkin alone is sufficient to ubiquinate Mitofusins.

Statusfinished
Effective start/end date01.01.1031.12.11

UN Sustainable Development Goals

In 2015, UN member states agreed to 17 global Sustainable Development Goals (SDGs) to end poverty, protect the planet and ensure prosperity for all. This project contributes towards the following SDG(s):

  • SDG 3 - Good Health and Well-being

Research Areas and Centers

  • Academic Focus: Center for Brain, Behavior and Metabolism (CBBM)

DFG Research Classification Scheme

  • 206-06 Molecular and Cellular Neurology and Neuropathology

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