| The research herein demonstrates that parkin, an E3 ubiquitin ligase enzyme, binds the human septin, CDC-rel2, and the regions that mediate this binding include the N-terminus of parkin and the N-terminus of CDC-rel2. In addition to the association between parkin and CDC-rel2, it is shown that parkin interacts with another major protein associated with familial PD, α-synuclein. Parkin-α-synuclein binding was demonstrated with the use of co-immunoprecipitation experiments, and parkin-α-synuclein co-localization was demonstrated with immunohistologic techniques.; Binding between parkin and CDC-rel2/α-synuclein was further demonstrated by the co-immunoprecipitation of parkin and CDC-rel2/α-synuclein both within transfected human neuroblastoma cells as well as from lysates of human brain. Finally, to map more precisely the regions of parkin that mediate its binding with CDC-rel2, various myc-tagged deletion constructs of parkin were utilized for co-immunoprecipitations of transfected human neuroblastoma cells. Regions of parkin able to bind CDC-rel2 include the N-terminus, the N-proximal RING finger, and the N-proximal RING finger with the IBR domain.; In addition, it is demonstrated that parkin mediates its own ubiquitination, thereby controlling its own metabolism. In a cell culture ubiquitination assay, it is shown that parkin is able to ubiquitinate CDC-rel2 and enhance CDC-rel2 catabolism. Subsequent pulse-chase assays demonstrate that parkin increases CDC-rel2 turnover in transfected human cell culture. Finally, brain lysates from patients with ARJP were found to contain significantly higher levels of CDC-rel2 than those of age-matched controls. These data suggest that parkin-regulated turnover of CDC-rel2 is important in normal brain physiology, and that absence of parkin function results in accumulation of CDC-rel2, perhaps to toxic levels. Further studies of CDC-rel2's role in normal brain physiology, and examination of CDC-rel2 toxicity in neurons, are warranted to elucidate further the mechanism of neurodegeneration in ARJP.; In conclusion, the parkin protein is a ubiquitin ligase enzyme, responsible for the ubiquitination and subsequent proteasomal degradation of substrate proteins. CDC-rel2 is a protein substrate of parkin-mediated ubiquitination, and proteasomal degradation of CDC-rel2 is enhanced by the presence of parkin. This dissertation demonstrates that parkin is an important mediator of CDC-rel2 metabolism, and that parkin dysfunction leads to accumulation of CDC-rel2 in neurons. |
