Parkin is an E3-protein
ubiquitin ligase, which plays an important role as a scavenger in cell metabolism. Since the discovery of the link between Parkin and
Parkinson's disease, Parkin was placed in the center of
Parkinson's disease research. Previously, we isolated a mutant form of the
Parkin protein (Q311R and A371T) from a
Parkinson's disease patient. In this study, we aimed at characterizing this mutant
Parkin protein by using biochemical and proteomic approaches. We used
neuroblastoma cells (SH-SY5Y) as our model and created two inducible cell lines that expressed the wild type and the mutant Parkin
proteins. We first investigated the effect of expressing both the wild type and the mutant Parkin
proteins on the overall
proteome by using 2D-DIGE approach. The experiments yielded the identification of 22 differentially regulated
proteins, of which 13 were regulated in the mutant Parkin expressing cells. Classification of the identified
proteins based on biological process and molecular function revealed that the majority of the regulated
proteins belonged to protein folding and energy metabolism. Ingenuity Pathway Analysis predicted the presence of a link between the regulated
proteins of the mutant Parkin expressing cells and
Parkinson's disease. We also performed biochemical characterization studies on the wild type and the mutant Parkin
proteins to make sense out of the differences observed at the
proteome level. Both
proteins displayed
biological activity, had similar stabilities and localized similarly to the cytoplasm and the nucleus in SH-SY5Y cells. The
mutant protein, however, was cut by a
protease and subjected to a post-translational modification. The observed differences at the
proteome level might be due to the differences in processing of the mutant
Parkin protein. Overall, we were able to create a possible link between a pair of Parkin mutations to its pertinent disease by using 2D-DIGE in combination with biochemical and molecular approaches.