Protective Effect Of ANKIB1 On Paraquat-Induced Apoptosis In SH-SY5Y Cell And Functional Analysis Of CnAα4

BackgroundNeurodegenerative diseases featured with neuronal cell apoptosis, seriously threat human health. However, there is no effective treatment clinically. It is necessary to identify novel molecular targets against neurodegenerative diseases. It is well-known that CN is the only Ca2+-CaM protein phosphatase consisting of one catalytic subtype CnA and a regulatory subunit CnB. Moreover, CN could promote NFAT to translocate into nucleus from cytoplasm, it is closely associated with cell apoptosis. ANKIB1 contains one CN binding motif, unfortunately, the relationship between ANKIB1 and neuronal cell apoptosis remain largely unknown. CnAa4 is a new human CnA1 transcript variant, while, the functions of CnAa4 are still a secret to us.Objectives1. To explore the role of ANKIB1 in paraquat-induced SH-SY5Y cells apoptosis and the possible mechanisms;2. To reveal the functions of CnAa4.Methods1. Exploring the role of ANKIB 1 in paraquat-induced SH-SY5Y cells apoptosisSH-SY5Y cells, transfected with pCMV-Myc-ANKIBl, were treated with paraquat for 48 h. western blot was used to analyse the cell lysis with Cleaved caspase3, cleaved PARP and p-IκB antibody.2. ANKIB 1 and CaMPull down and co-immunoprecipitation (Co-IP) were adapted to study the interactions between ANKIB1 and CaM. Then purified GST-ANKIB1(1-480) was pulled down by CaM-agarose in the presence of varying concentrations of Ca2+. The bound proteins were probed with anti-GST monoclonal antibody.Immunoblot and qPCR were adapted to verify whether CaM regulates ANKIB1 gene expression or not. Protein stability experiment was employed to know the stability of ANKIB1 with or without CaM.3. ANKIB1 and the transcriptional activity of NFATHEK293T cells were transfected with the plasmid pCMV-Myc-ANKIB1, then the cells were treated with ionomycin or cyclosporin A for 6 h. Luciferase reporter assay were employed to analyze the role of ANKIB1 on the transcriptional activity of NFAT.4. Revealling the functions of CnAa4Purified GST, GST-CnA a 1 or GST-CnA a 4 were pulled down by CaM-agarose. Translocation of NFATcl mediated by CnA a 1 or CnA a 4 were imaged using a fluorescence microscopy, luciferase reporter assay were used to assess the ability of CnAα4 in activating the transcriptional activity of NFAT.Results1. ANKIB1 protects SH-SY5Y cells against paraquat-induced apoptosis;2. The interactions between ANKIB1 and CaM change in the presence of varying concentrations of Ca2+. CaM regulates ANKIB1 gene expression via improving its stability;3. ANKIB1 inhibits the transcriptional activity of NFAT;4. CnA a 4 binds to CaM, promotes NFATc1 to translocate into nucleus from cytoplasm, and is more effective in activating NFATc1 than CnAal.Conclusions1. ANKIB1 interacts with CaM, inhibits the transcriptional activity of NFAT, and protectes SH-SY5Y cells against paraquat-induced apoptosis;2. CnAa4 binds to CaM, and owns higher phosphatase catalytic activity than CnAal.