Parkinson's Disease Related Protein Parkin. Of Pink1 And Mortalin Interaction On Mitochondrial Homeostasis

Mutations in Parkin are the most common reason of autosomal recessive PD. As an E3-ubiquitin ligase, Parkin is associated with mitochondrial dynamics and mitophagy. Mutations in PTEN-induced putative kinase 1 (PINK1) is also associated with PD. PINK1 through its transmembrane domain anchors in outer mitochondrial membrane. Recent studies support the close link between PINK1 and Parkin:PINK1 functions upstream of Parkin and both of them act in a common genetic pathway that maintains mitochondrial morphology and function.Mortalin, as a molecular chaperone, mainly locates in mitochondrial matrix and functions in mitochondrial homeostasis. The reduced expression level of mortalin in the affected brain regions of PD patients is demonstrated. It also interacts with a series of PD-related proteins and plays an indispensible role in helping naive proteins folding and importing proteins to mitochondrial matrix.So in this issue the major aims are to detect the interactions among Parkin and PINK1, Parkin and mortalin, and how may they affect mitochondrial function. Methods and results are indicated below:1) The relevant Parkin RING1 domain mutations were generated by Over-lap PCR method with wild type Parkin as template and confirmed by sequencing.2) Interactions between Parkin and PINK1:our immunofluorescence results show that without co-transfection with PINK1, Parkin is distributed throughout the cytoplasm and nucleus, and its RING1 related point mutations and deletion does not affect their subcellular localization. When PINK1 cotransfection with Parkin, the percentage of WT or MT Parkin co-localized with mitochondrial is significantly changed, the former reached 40% while the later did not exceed 15%.Immunoprecipitation results indicate that WT Parkin interacts with PINK1, and Parkin RING1 domain mutation R275W impairs its interaction with PINK1.3) Interactions between Parkin and mortalin:HeLa cells were transfected with pEGFP-N2 vectors encoding wild-type and mutant Parkin and treated with CCCP 1h. We found that mortalin mainly located in mitochondria, and Parkin co-localized with mitochondria after CCCP treatment. The interactions of endogenous mortalin with WT or MT Parkin in vivo were tested by CO-IP. We found that the R275W and R1 domain deletion (but not the T240R mutation) impaired the ability of Parkin to interact with endogenous mortalin4) Knockdown of mortalin in HeLa cells using lentivirus expressing shRNA targeting the sequence of mortalin. Real-time PCR, western blot analysis were performed to measure the effect. We investigated Ientivirus-mediated RNA interference substantially suppressed the mortalin expression.5) The effects of knockdown of mortalin on mitochondrial morphology, apoptosis and Parkin-mitochondrial translocation. Our results show that obvious truncated and fragmented mitochondria and a higher rate of apoptosis are found in mortalin knockdown HeLa cells under H2O2-induced stress conditions, and knockdown of mortalin do not abrogate the CCCP-induced mitochondrial translocation of Parkin.6) Parkin overexpression rescued the mitochondrial dysfunction induced by mortalin knockdown. Our results show that knockdown of mortalin induces an unexpected collapse of MMP, abnormal accumulation of ROS and low copy number of mtDNA and Parkin overexpression could rescue the mitochondrial dysfunction.