Screening And Confirmation Of Proteins Interacting With PINK1

Parkinson's disease(PD) is a common neurodegenerative disorder. The cause of the disease is still unknown,although substantial evidence suggests that mitochondrial dysfunction is a major contributor:Several mitochondrial toxins induce PD-like symptoms in humans and animal models;systemic mitochondrial dysfunction appears to be a feature of a large proportion of PD sufferers;and several genes involved in rare heritable forms of Parkinsonism have been implicated in mitochondrial biology,including the PTEN-induced kinase 1(PINK1).PINK1 protein has been shown to localize in the mitochondria.The mechanism by which PINK1 protects cells remains unknown.Studying on the function of PINK1 is important to understand the neuropathology of PD.Many,if not all,essential biological processes require selective interactions between proteins.Therefore,an important step in the analysis of PINK1 function is identification of the interaction partners of PINK1. The objectives of the present study are to identify proteins which may interact with PINK1 on the following five aspects:1.The full-length code domain sequence of PINK1 was amplified from pcDNA3.1-P1NK1 which was constructed in our previous experiments and was cloned to pGBKT7 vector.The integrity of the constructs was confirmed by sequencing.Yeast strain AH109 cells were transiently transformed with pGBKT7-PINK1.We prepared Western blots from the transformants and confirmed that PINK1 protein could be normally expressed in Saccharomyces cerevisiae(strain AH109). 2.In order to obtain enough plasmids for library screening in yeast, we must amplify the premaded MATCHMAKER Human Fetal Brain GAL4 cDNA & genomic libraries.Plate 27μl library directly on 300 150-mm plates,and incubate at 30℃for 24-36 hours,the resulting colonies were nearly confluent.Then we scraped colonies,and obtained 1000 ml yields.Then prepare eDNA plasmid with QIAGEN Tip2500 kit. We obtained 2.593mg library plasmid in total.3.By using large-scale transformation,AH109/pGBKT7-PINK1 cells were transformed with library plasmid.According to the manual of the library,we screened for MATCHMAKER Human Fetal Brain GAL4 cDNA & genomie libraries four times.We screened 6.65×10~6 independent clones in total,and acquired 77 His~+ clones.After retesting Ade~+/Mel1~+ phenotypes,61 Ade~+/His~+/Mel1~+ clones were acquired.4.By restreaking Ade~+/His~+/Mel1~+ clones,amplifying library inserts by PCR and characterizing PCR products by digesting with HaeⅢ,we sorted clones to eliminate duplicates.We acquired 49 Ade~+/His~+/Mel1~+ clones.By eliminating self-activating clones,we acquired 2 true positive clones.By sequencing and BLAST in NCBI,we know the two positive inserts are part of KIF1A(kinesin family member 1A) and BAG5 (BCL2-associated athanogene 5).PINK1 protein may interact with KIF1A and BAG5 respectively.5.KIF1A is not supposed to be a candidate protein interacting with PINK1 because the acquired sequence is located on untranslated region of KIF1A.The acquired sequence of BAG5 is located on its N-terminal coding region,so we continued experiments on confirming interaction between PINK1 and BAG5.Incubate GST-BAG5 fusion protein from JM109 and HA-PINK1 fusion protein from HEK293A cells together with G4B(Glutathione 4B),which were then collected and washed.Collected complexes were resolved by SDS-PAGE and processed for further analysis by Western blotting,63kDa and 52kDa PINK1 protein bands in both input and HA-PINK1+GST-BAG5 were found.Collect cell extracts from HEK293A cells cotransfected with PKH3-HA-PINK1 and pEGFP-N3-BAGS,immunoprecipitate the cell extracts with anti-GFP antibody,then perform Western blotting by using anti-HA antibody, 63kDa and 52kDa PINK1 protein bands in cotransfected cells were found, both the full-length and the processed forms of endogenous PINK1 co-immunoprecipitated with BAGS.These results suggest that PINK1 interacts with BAG5 in vitro and in vivo.The following in vitro binding assay showed us that PINK1 directly interacts with BAG5.For the first time,we used yeast two-hybrid system to identify PINK1 interacting proteins and acquired two proteins,which were BAG5 and KIF1A,either of them may interact with PINK1.And we confirmed that PINK1 directly interacts with BAG5.These data not only establish the basis for further investigating PINK1 gene function,but also suggest a novel PINK1 related mitochondrial pathway in PD pathogenesis,even in understanding the relationship between mitochondrial and aging.