Abstract:
The work described in this thesis has focused on the study of the molecular mechanism that links deregulated mRNA translation to Parkinson's disease (PD). The work included a review about the nascent literature on the pathological role of protein products encoded by key genes that are mutated in PD, and their role(s) in affecting mRNA translation. In Chapter 3, the recombinant production of human ribosomal proteins using Escherichia coli as expression system was explored. An improved method for the production of human ribosomal proteins was developed. The use of a polyhistidine-tagged thioredoxin as a fusion protein was proved crucial for the expression, solubilisation and recovery of pure proteins for biochemical studies. In Chapter 4, the human gene of RioK2 was used as a model system to study the effect of rare codons and consecutive rare codons AGG-AGA on the expression of human genes in E. coli. Expression studies revealed a relationship between the number of rare codons and problems in translation. The presence of consecutive codons AGG-AGA in a sequence with a high number of rare codons, led to the production of a truncated protein. In contrast, AGG-AGA in a codon harmonised sequence did not lead to truncation, thus indicating translational problems caused by AGG-AGA as sequence dependent. In Chapter 5, the role of human ribosomal protein S15 phosphorylation in PD was studied using different techniques. Pulldown binding assays and interaction studies revealed no changes in protein-protein affinity, in particular with ribosomal protein S18. In contrast, a peptide model was used to simulate the structure of S15 and S18 C-terminal tails in solution. NMR studies revealed a change in interactions and dynamicity of the tail of S18, as a consequence of S15 phosphorylation. Overall, this study provides insights on the molecular mechanism that affect mRNA translation in PD.