Analysis Of Fmr1 Ko Mice Cortex Proteome And Phosphoproteome To Understand The Mechanisms Of Fragile X Mental Retardation Syndrome

Fragile X mental retardation Syndrome(FXS)was the most common cause of inherited intellectual disability.FXS occured in about 1 in 4,000 males and 1 in 8,000females.The main characteristics of the syndrome was intellectual disability,elongated face,large or protruding ears,flat feet,larger testes.Besides,some individuals with FXS will show autism.The reason for FXS was that the 5’UTR region of Fmr1(Fragile X mental retardation gene 1,located on the X chromosome at position q27.3,occurred more than200 CGG trinucleotide repeats and nearby CpG islands became methylated.Fmr1 gene and FMRP protein expresssion decreased.The most classical pathologic features were brain cortex neuron long dendritic spines of increased density and unmatured structure.FXS brings great paint,not only to patient,but also to the family and the entire society.Until now,there were only some drugs which can just partially improve the symptoms.Many small compounds have been developed and entered clinical trial.These results seems to be unsatisfied,there were still no drugs for the key symptoms.It has been reported that in the peripheral tissue of FXS patients and Fmr1 KO mice neuron,kinases and phosphatase induced signal transduction were abnormal.Lose of FMRP lead to the abnormality of signal transduction,and the main feature was phosphorylation levels increased.Besides,loss of FMRP may promote DNA damage in meiotic cells and trigger apoptosis in spermatocytes.FMRP was a mRNA binding protein and mainly play a role as translation repressor.There were many reports about FMRP binding with which mRNA,regulating which mRNA and maintaining stability of which mRNA.FMRP bind nearly 900 mNRAs,those including presynaptic,postsynaptic and autism related genes.This indicated FXS were strongly associated with synapse protein translation.Studying on the protein translation(newly protein synthesis)may shed a light on the mechanism of FXS.Based on LC-MS,proteomic technology were involved in digestion of complex tissues and cell,and then loading the peptides mixtures on LC/MS/MS to get the MS2spectra,comparing with those spectra with database to analysis of amino acid sequence of protein and peptides,Thus leading to identification and quantification of large scales of proteome data.This work collected wild type and Fmr1 KO mice cortex,with filter-aided sample preparation(FASP)method,TMT labeling quantification technology,high pH RP fraction and LC/MS to get large scales of proteome data.Besides,with the TiO~2enrichment method to get the phosphoproteome data.With antibody and target mass spectrometry to validate results.And more,we generated a mimic phosphorylation and mimic loss of phosphorylation of PRKRA,transfected them into Hela and SH-5Y cell lines,explored the localization of basal level and stress induced level.As FMRP bind with mRNA and regulate protein synthesis,we took L-azidohomoalaine(AHA)as a tag of newly synthesized proteins,and click chemistry method to enrich,LC-MS to detect newly synthesized proteins.We have identified 4927 proteins in brain cortex,among those,upregulated were56,downregulated were 36.these differently expressed proteins were enriched mainly on the pathway of synapse,localization and response to stimulus.On the phosphoproteome level,we have identified 7696 phosphosites,among those,there were upregulated 139,downregulated 2.Those changed phosphosites were mainly enriched in cytoskeletal protein binding,actin binding and so on.With motif-X analysis,we classified the upregulated sites with…SP…,…TP…,..RxxS..motif.We developed target MS method and confirmed its reliability and repeatability,and validated some of candidates from proteome data.We generated PRKRA WT,S18A and S18D constructs.Its phosphorylation didn’t affect itself localization.PRKRA was co-localized with FMRP.Under stress,The PRKRA S18A decreased cell apoptosis and S18D increased cell apoptosis.These results offered a way to explain that loss of FMRP trigger apoptosis.We have set up the AHA labeling method to enrich newly synthesized proteins and identified them with LC/MS.Those differently new synthesied proteins may be the FMRP target proteins.This work drew a large scales of proteome and phosphoproteome landscape of Fmr1KO cortex,validated some of changed protein and phosphosites.We have generated phospho-mimicking mutant and dephospho-mimicking mutant,PRKRA S18D increased cell apoptosis and PRKRA S18A decreased apoptosis.As FMRP can regulate protein synthsis,we developed AHA labelling method to enrich and quantify new synthesized proteins.All of these results expanded the limits of understanding the FXS,and more importantly,showed some potential molecular targets and shed a light on the war against to FXS.