Schlehe, Julia (2008): Folding, function and subcellular localization of parkin. Dissertation, LMU München: Faculty of Chemistry and Pharmacy |
Preview |
PDF
Schlehe_Julia.pdf 2MB |
Abstract
Idiopathic Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimers disease. The specific molecular events that provoke neurodegeneration in PD are still unknown, which is an impediment to the development of neuroprotective drugs. Only recently, genes linked to hereditary forms of PD have been identified. Idiopathic and hereditary variants of PD share important pathological features, most notably the demise of dopaminergic neurons in the substantia nigra. Functional characterization of PD-associated gene products might help to understand the molecular mechanisms underlying the pathogenesis and maybe, in the future, to find preventive and curative treatments for PD. Among the mutated genes is the parkin gene (PARK2), encoding a E3 ubiquitin ligase. Mutations in the parkin gene are responsible for the majority of autosomal recessive parkinsonism. Previous work of our group revealed that misfolding and aggregation of parkin is a major mechanism of parkin inactivation, accounting for the loss-of-function phenotype of various pathogenic parkin mutants, including C-terminal deletion mutants and some missense mutants [1,2]. Remarkably, also wildtype parkin is prone to misfolding under certain cellular conditions, suggesting a more general role of parkin in the pathogenesis of PD. One aim of this thesis was to study the folding characteristics of parkin. To this end, I cloned several parkin mutants and analyzed them in cell-culture based assays to determine their folding properties. Folding analysis of these mutants revealed that pathogenic mutations can lead to aberrant parkin conformers with two distinct phenotypes. One class of mutations destabilized the native conformation of parkin, leading to its proteasomal degradation immediately after synthesis. Another class of mutants first adopted a detergent-soluble conformation, similarly to wildtype parkin. However, within hours these mutants formed relatively stable detergent-insoluble aggregates. A comparative analysis of HHARI, an E3 ubiquitin ligase with a similar modular signature, revealed that folding of parkin is specifically dependent on the integrity of the C-terminal domain, but not on the presence of a putative PDZ binding motif at the extreme C-terminus. This study provided new insight into the propensity of parkin to misfold and suggested that pathogenic mutations can induce the formation of non-native conformers at distinct steps in the folding pathway of parkin. Another focus of this thesis was the functional characterization of parkin. We and others observed that parkin protects neurons against diverse cellular insults in different model systems, indicating that it may play a role in maintaining neuronal integrity. To address the underlying mechanism, we analyzed the effect of parkin on different signaling pathways. Our results revealed that parkin has a permissive effect on NFkappaB signaling by ubiquitylating two components of the signaling cascade in a non-degradative manner. Notably, parkin lost its neuroprotective capacity in the presence of a dominant negative inhibitor of NFkappaB. In addition, we could show that parkin expression is significantly up-regulated in neurons under stress conditions, indicating that parkin is a stress-responsive protein.
Item Type: | Theses (Dissertation, LMU Munich) |
---|---|
Keywords: | Parkin; E3 Ubiquitin Ligase; Parkinson's Disease; Folding |
Subjects: | 500 Natural sciences and mathematics 500 Natural sciences and mathematics > 540 Chemistry and allied sciences |
Faculties: | Faculty of Chemistry and Pharmacy |
Language: | English |
Date of oral examination: | 10. November 2008 |
1. Referee: | Winklhofer, Konstanze F. |
MD5 Checksum of the PDF-file: | 08bd27205a174f59d07cc84478c2b770 |
Signature of the printed copy: | 0001/UMC 17400 |
ID Code: | 9266 |
Deposited On: | 26. Nov 2008 12:55 |
Last Modified: | 24. Oct 2020 06:50 |