A Study On Inhibition Of TRAIL Signaling Pathway By DJ-1, A Parkinson's Disease And Cancer Related Protein

Parkinson's disease is one of the most common neurodegenerative disease linkedto age, second only to Alzheimer's disease. It is characterized clinically byparkinsonism, including slowness of movement, tremor and rigidity; andpathologically by a selective loss of the midbrain dopamine neurons in the substantianigra. Although most PD cases are sporadic and late onset, several proteins accountfor familial PD have been identified in recent years, including DJ-1. There are strongevidences that mitochondrial dysfunction, proteasome system inhibition, increasedoxidative stress and apoptosis play a causal role in the pathology of PD.DJ-1 was originally identified as a novel oncogene product in conjunction withthe small G-protein Ras in NIH3T3 cells. DJ-1, a conserved protein with 189 aminoacids, is ubiquitously expressed but is found at particularly high levels in the testis,brain and kidney. Many studies have shown that DJ-1 distributes in both cytoplasmand nucleus, and partially in mitochondria. It functions in multiple pathways thataffect cell survival. DJ-1 is involved in the pathogenesis of several types of cancer,such as ovarian carcinoma, melanoma and breast, lung and prostate cance. In contrastto its functions in cell survival, deletions or loss of function point mutations in DJ-1are reported to be responsible for recessive early-onset Parkinson's disease in 2003.The most commonly studied PD-associated mutant, L166P, is reported to be unstableand to mislocalize to the mitochondria, leading to a loss of the cytoplasmic function ofDJ-1. However, the mechanism by which DJ-1 is involved in Parkinson's disease andtumorigenesis is still largely unknown.I performed experiments using both HCT116 p53 wild type cells (HCT116+/+)and HCT p53 null cells (HCT116-/-) to explore the role of DJ-1 in theTRAIL-induced apoptosis pathway. The results suggest that DJ-1 attenuates thepro-caspase-8 activation and TRAIL-induced apoptosis in a p53-independent manner.When active caspase-8 was induced independent of TRAIL treatment, overexpressionof resistant mutants of DJ-1 or L166P in DJ-1 knockdown cells did not influence thelevels of cleaved PARP, morphological changes or apoptotic cell numbers. These dataindicate that DJ-1 cannot survive cells after caspase-8 activated and that DJ-1 mayfunction upstream of active caspase-8. Moreover, the results of western blot andRT-PCR indicate that knockdown or overexpression of DJ-1 had no effect on the levels of the 4 TRAIL receptors. I proved DJ-1 interacts with the adapter proteinFADD by both GST-pulldown and immunoprecipitation experiments. However, theinteraction between L166P and FADD is much weaker than DJ-1 due to itsmislocalization to the mitochondria. Furthermore, the results of GST-pulldown implythat DJ-1 expressed by E. coli directly interacted with the N-terminal death effectordomain (DED) of FADD, which recruits pro-caspase-8. Next, in vitro competitivebinding assays showed that the binding of the N-terminal of caspase-8 to FADD wassignificantly decreased with increased amounts of DJ-1. Immunoblot showed thatDJ-1 knockdown resulted in increased interactions between endogenous FADD andpro-caspase-8 on TRAIL treatment.In summary, the results of our study showed that DJ-1 inhibits TRAIL-inducedapoptosis by blocking pro-caspase-8 recruitment to FADD. Our study provides amechanistic explanation for explain the role of DJ-1 plays in Parkinson's disease andtumorigenesis.