Study On Inhibition Of P53 Transcriptional Activity By DJ-1, Parkinson's Disease Related Protein

Parkinson's disease (PD), the second common neurodegenerative diseases following Alzheimer's disease in the world, affects 1% people over sixty years old. In China, PD has been one of the most popular neurodegenerative disorders, destroying the patients' lives and bringing a heavy burden to the family as well as the society. PD is a progressive movement disorder that results primarily from the death of dopaminergic neurons in the substantia nigra and the presence of some kind of protein aggregates termed Lewy Body, a hallmark of PD, in surviving neurons. The loss of dopamine production in the brain causes the primary symptoms of Parkinson disease including tremor, slowness of movement, rigidity and difficulty with balance. Up to now, the etiology of PD has still been a myth. Environmental factors and genetic factors are believed to contribute to the onset of the PD. From the genetic point of view, PD can be either sporadic form or familial form. In familial PD, the inheritable mutations in the PD-related gene produce the mutant protein which alteres the normal function of its wild type. Although most cases of PD are sporadic, the study on the rare familial PD promotes our understanding of the pathogenic pathway. These studies suggest that oxidative stress, defect in mitochondria, dysfunction in proteasome system and apoptosis play a vital role in the pathology of PD.DJ-1 was originally identified as an oncogene product. Recently, loss of DJ-1 function due to the mutations and deletion in DJ-1 gene is found to be linked with autosomal recessive early onset PD in populations of various ethnic origins. Although increasing studies reveal that DJ-1 has multiple roles in various biological processes, the exact function of DJ-1 remains unclear.Sumoylation has emerged as an important posttranslational modification, which is also involved in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and polyglutamine diseases. Sumoylation regulates the substrate stability, biological activities and its subcellular localization. DJ-1 is able to be sumoylated at K130 but the effect of this modification on the normal function of DJ-1 remains to be elucidated.As most of the oncogene products are able to inhibit apoptosis, in this thesis, we tried to determine the role of DJ-1 in anti-apoptosis as well as the mechanisms in which DJ-1 protects cell from death. In addition, we also focus on the regulation of DJ-1 by sumoylation and the effect of sumoylated DJ-1 in the pathogenic pathway of PD. The main findings of our study are listed as below.1. Under stimulus, overexpression of DJ-1 is able to protect cell from death by inhibiting the cleavage of caspase-9 and caspase-3. Furthermore, Bax, an important factor in the apoptosis pathway, is down-regulated in the cells expressing DJ-1. This result is reproducible in several cell lines harboring p53 but not in p53 null cell line. In addition, knock-down of DJ-1 increase Bax level in a p53 dependent manner. These results imply that the regulation of Bax by DJ-1 is associated with p53 status. Using pull-down and immunoprecipitation experiments, we show that DJ-1 is able to bind to p53 through its DNA-binding domain and C-terminus. Moreover, the results of the report gene assay imply that DJ-1 is able to inhibit p53 transcriptional activity on Bax promoter. In summary, we demonstrate here that DJ-1 exerts its cytoprotection through a p53-Bax-caspase pathway. DJ-1 decreases the Bax protein level and therefore blocks the caspase activation by possibly repressing p53 transcriptional activity.2. In our experimental system, we also observed that DJ-1 is able to be sumoylated at K130. However, a non-sumoylatable mutant of DJ-1, DJ-1(K130R), which shifts from nucleus to cytoplasm, loses its ability to repress p53 transcriptional activity on Bax promoter as well as that of rescuing cell death. In this regard, proper sumoylation of DJ-1 is suggested to be important for its targeting nucleus to regulate p53 activity.Taken together, our findings suggest that DJ-1 rescues cell death through p53-Bax-caspase pathway. DJ-1 binds to p53 physically in the nucleus, decreases Bax expression by attenuating p53 transcriptional activities, inhibits subsequent caspase activation and finally rescues cell death. Whereas, the non-sumoylatable K.130R mutant of DJ-1, loses its cytoprotective effect due to the sequestration in cytoplasm. As loss of DJ-1 function is responsible for the onset of PD, the findings here will be of help to expand our knowledge of DJ-1 functions, as well as its possible role in PD.