Robinson, Alexis Anne; (2007) Molecular mechanisms of DJ-1 mutations in Parkinson's disease. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Mutations in PARK7 - the human chromosome 1p36 locus which harbours the DJ-1 gene - have been shown to be responsible for the onset of autosomal recessive Parkinson's disease. The exact function of DJ-1 is unknown due to its diverse role in numerous biological processes including oncogenesis, transcriptional regulation and oxidative stress. This study focuses on the reported missense mutations of DJ-1 in an attempt to elucidate their pathogenic mechanisms. Effects on DJ-1 dimerisation and DJ-1 interaction with known protein partners were initially assessed using the yeast two-hybrid system. Results were confirmed in a mammalian cell system using affinity purification methods. This study demonstrated that dimerisation of DJ-1 is required for all DJ-1 binding protein interactions, and only mutation L166P had an effect on protein dimerisation. DJ-1 mutations were found to have a specific disruptive effect on DJ-1 interaction with DJ-1 binding proteins. Mutation M26I abolished interactions with SUMO-1, a small ubiquitin-like modifier, by an unknown mechanism. Of particular interest was the finding that three distinct DJ-1 missense mutations (A104T, D149A and E163K) selectively abolished interaction with the so-called DJ-1 binding protein (DJBP). Further investigation of DJBP, involving PCR amplification from human brain cDNA, revealed the existence of multiple isoforms of DJBP, generated by alternative splicing. Sequence analysis indicated the potential of DJBP to function as a mitochondrially- located calcium-binding protein, due to the identification of EF-hand motifs and an N-terminal mitochondrial targeting sequence. Disruption of the DJ-1/DJBP interaction may provide a molecular explanation for the underlying cause of DJ-1-related Parkinson's disease. The predicted role of DJBP as a mitochondrially-located calcium-binding protein supports the involvement of DJ-1 and DJBP in a pathway which exerts a protective effect on the cell under conditions of oxidative stress. Mutations which abolish DJ-1/DJBP interaction may abrogate the protective effect, resulting in increased susceptibility to cellular stress, providing a link between mitochondria and the pathogenesis of Parkinson's disease. These results also suggest that the gene encoding DJBP on human chromosome 22q13 is a suitable candidate for genetic analysis in Parkinson's disease.

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