The Investigation Of The Interaction Between DJ-1 And Mitochondrial Stress Protein Mortalin

Background and Objective:The DJ-1 gene is associated with early-onset Parkinson's disease (PD). It expresses a 189 amino acids long protein and is a member of ThiJ/ PfpI family. DJ-1 has a wide range of distribution in human tissue and brain. The present studies suggest that DJ-1 function as an oxidative stress sensor and chaperone and protect neurons from protein misfolding and mitochondrial dysfunction, which are two critical mechanisms in PD pathogenesis. Functional loss of DJ-1 will lead to neurodegenerative disorders. Our previous study used a proteomic approach to identify the proteins associated with DJ-1 in dopaminergic MES cells exposed to rotenone versus controls. Mortalin is identified as one of the DJ-1-associated proteins. Mortalin is a 679 amino acids long protein and belonging to of the heat shock protein 70 family. Like the other heat shock protein 70 family members, Mortalin has two functional domains:N-terminal ATPase-binding domain and C-terminal substrate-binding domain. Mortalin is an essential mitochondrial chaperone and believed to perform multiple cellular functions ranging from cell survival, cell proliferation, stress response, mitochondrial biogenesis, and intracellular trafficking. In our previous study we have detected the decrease of Mortalin level in mitochondrial-enriched fraction from Parkinson's disease patient brains as compared with age-matched controls. It suggests that Mortalin might participate in PD pathogenesis. The objective of this project is to confirm the physical interaction between DJ-1 and Mortalin as well as to investigate whether the interaction will affect the function of DJ-1 as an oxidative stress sensor and chaperone.Methods:1. Cell cultures and transfection;2. Expression and purification of GST fusion protein;3. Interaction between DJ-1 and Mortalin is investigated by GST Pull-down assay;4. Interaction between DJ-1 and Mortalin is investigated by Co-immunoprecipitation;5. Subcellular locations of DJ-land Mortalin are investigated by immunocytochemistry;6. Cell viability is investigated by MTT assay;7. Protein refolding ability is investigated by luciferase refolding assay.Results:1. Using C57 mice brain tissue lysate, we did not detect Mortalin in DJ-1 protein complex by GST Pull-down assay.2. Using C57 mice, PC12 cells and SH-SY5Y cells lysate, we did not detect Mortalin in DJ-1 protein complex by Co-immunoprecipitation;3. Immunocytochemistry assay shows that Mortalin level is decreased in mitochondria after rotenone treatment;4. Western blotting assay shows that Mortalin level is increased in cytoplasm after rotenone treatment;5. Immunocytochemistry assay shows that co-localization level of DJ-1 and Mortalin is increased;6. DJ-1 increases cell viability after rotenone exposure as compared with controls, and DJ-1 increases luciferase refolding after heat shock denature as compared with controls.Conclusion:1. We did not detect the interaction between DJ-1 and mortalin in our experimental system.2. Mortalin redistributes after rotenone treatment;3. DJ-1 functions as anti-oxidative stress sensor and the chaperone.