Study On The Interaction Of BAG5, PINK1 And Parkin In The Pathogenesis Of Parkinson's Disease

Background:Parkinson's disease (PD) is a common neurodegenerative disorder which is characterized by resting tremor, muscle rigidity, balance and posture to reduce obstacles to movement. Prevalence rate of PD over 65 years old is up to 1.7 percent, and the people over 85 years old are reach to 4.8 percent, and PD is a serious threat to the health of the elderly. The molecular genetic studies have led to the identification of different monogenic forms of PD. Genetic factors in the pathogenesis of Parkinson's disease is becoming increasingly important. From 1999,16 loci for autosomal recessive Parkinsonism have been mapped, and 11 genes have been cloned, they are parkin,PINK1,DJ-1,ATP13A2,PLA2G6 and FBXO7, which are related to autosomal recessive early-onset parkinsonism.Parkin was a protein which in humans was encoded by the PARK2 gene in 1998, and expressed 465 amino acid product. The precise function of this protein is unknown; however, the protein is a component of a multiprotein E3 ubiquitin ligase complex which in turn is part of the ubiquitin-proteasome system that mediates the targeting of substrate proteins for proteasomal degradation, also play an important role in protecting the mitochondria's function.In 2004, Valente found that PINK1 gene (PTEN-induced putative kinase 1) was the second common virulence gene besides Parkin. The PINK1 gene comprises eight exons, which encode a ubiquitously expressed 581 amino acid product. The protein contains an N-terminal mitochondrial targeting domain and a highly conserved kinase domain that sequence comparisons suggest is a serine/threonine protein kinase structurally belonging to the calmodulin-dependent kinase class of protein kinases. To date, functional studies have shown that PINK1 protein may play an important role in protecting the mitochondria's function, wild-type PINK1 protects cells against proteasomal inhibition as well as staurosporine-induced apoptosis. The study suggested that the interaction between PINK1 protein and other protein many play an important role in the pathogenesis of PD.Bcl-2-associated athanogene (BAG)-family proteins were originally identified by their ability to associate with the anti-apoptotic protein, Bcl-2. BAG-family proteins contain a single BAG domain, except for BAG-5 which has four BAG repeats. BAG-5 expresses in the brain cortex, hippocampus, red nucleus and substantia nigra pars compacta, and mainly gathered in the substantia nigra pars compacta. Little is known about the role of BAG-5 in cells other than its ability to bind Hsp70, and to inhibit both parkin E3 ligase and Hsp70 chaperone activity enhancing dopaminergic neuron degeneration.We have used yeast two-hybrid system to identify PINKI interacting protein and aquired the protein BAG5. We confirmed that PINKI directly interacts with BAG5. We confirmed the interaction in vivo by co-immunoprecipitation and in vitro by pull-down, These data not only establish the basis for further investigating PINK1 protein function, but also suggest the interaction between BAG5 protein, PINK1 protein and Parkin protein many play an important role in the pathogenesis of PD.Objective:To research the possible mechanism of the interaction between the PINK1 protein, Parkin protein and BAG protein in the Parkinson's disease.Methods:1) By using the electrophysiological method, we studied Intracellular distribution of wild-type PINK1 protein and wild-type BAG5 protein,subcellular distribution of PINK1 and BAG5 after cotransformation.2) We generated different deletion of BAG5 and PINK1 to identificated of the domains in BAG5 interaction with PNK1 and the domains in PINK1 interaction with BAG5 by GST pull-down assay.3) To further identify whether BAG5 can be phosphorylated by PINK1 by using autoradiography and phosphorylation in vitro. 4) Chase-time experiment was undertaken to investigate the effect of BAG5 protein on the half-lives of wild-type PINK1 and R492X mutation of PINK1.5) Plasmid transfection, RNA interference technique and co-immunoprecipitation were utilized to analyze co-regulation of BAG5 and PINK1 on the ubiquitination of wild-type PINK1.6) Plasmid transfection, RNA interference technique and co-immunoprecipitation were utilized to analyze co-regulation of BAG5 and PINK1 on the ubiquitination of wild-type PINK1.7) Plasmid transfection and co-immunoprecipitation were utilized to analyze the effects of wild-type Parkin and the mutations of Parkin on the ubiquitination of wild-type PINK1.8) Immunohistochemistry and western-blot were utilized to analyze the level of BAG5,PINK1 and Parkin in the MPTP-induced PD model of dopaminergic neuron in substantia nigra.Results:1) The subcellular co-localization of PINK1 protein and BAG5 protein were detected by immunocytochemistry labeling, and after cotransformating in HEK 293 cell the subcellular localization of BAG5 protein changed and showed aggregation in mitochondrion.2) Using GST pull-down assay to detect the 5 domains:BAG5(9-86) (the first BAG domain),BAG5(182-260) (the second BAG domain),BAG5(365-442) (the forth BAG domain) and BAG5(87-181) (the domain between the first BAG domain and the second BAG domain) all can directly interact with PINK1. PINK1(156-509)(the serine/ threonine protein kinase domain) can directly interact with BAG5.3) Using GST pull-down assay and autoradiography we found BAG5 can not be phosphorylated by PINK1 in vitro.4) We found BAG5 protein prolonged the half-lives of wild-type PINK1, but shortened the half-lives of R492X mutation of PINK1. BAG5 protein stabilized the wild-type PINK1 and promoted the degradation of R492X mutation of PINK1.5) We found BAG5 protein can regulate the ubiquitination of wild-type PINK1 and the R492X mutation of PINK1 by using Plasmid transfection, RNA interference technique and co-immunoprecipitation. BAG5 protein can inhibit the ubiquitination of wild-type PINK1, and promote the ubiquitination of of R492X mutation of PINK1.6) By using Plasmid transfection, RNA interference technique and co-immunoprecipitation, we found BAG5 protein and Parkin protein through different pathway to regulate the ubiquitination of wild-type PINK1 and they regulate the ubiquitination of wild-type PINK1 independently.7) We found wild-type Parkin inhibit the ubiquitination of wild-type PINK1, and promote the ubiquitination of R492X mutation of PINK 1, by using Plasmid transfection, RNA interference technique and co-immunoprecipitation.8) By using immunohistochemistry and western-blot technique, we found the protein expression level of BAG5 and Parkin increased, however, the level of PINK1 protein decreased in the MPTP-induced PD model of dopaminergic neuron in substantia nigra.Conclusion:1) There are co-localization of PINK 1 protein and BAG5 protein in the cell.2) The first BAG domain, the second BAG domain, the forth BAG domain and the domain between the first BAG domain and the second BAG domain all can directly interact with PINK1. The serine /threonine protein kinase domain of PINK1 can directly interact with BAG5.3) BAG5 can not be directly phosphorylated by PINK1 in vitro.4) BAG5 protein can regulate the degradation of the wild-type PINK1 and the R492X mutation of PINK1; BAG5 protein can inhibit the ubiquitination of wild-type PINK1, and promote the ubiquitination of R492X mutation of PINK1.5) BAG5 protein and Parkin protein through different pathway to regulate the ubiquitination of wild-type PINK1 and they regulate the ubiquitination of wild-type PINK1 independently.6) The E3 ligase of PINK1 is the substrate of Parkin, Parkin promote the degradation of E3 ligase of PINK1, and inhibits the ubiquitination of wild-type PINK1.7) BAG5,PINK1 and Parkin proteins involved in the pathological process of MPTP-induced PD.