Screening And Identification Of FXR1P Interacting Proteins

Screening and Identification of FXR1P interacting proteins Fragile X syndrome is one of the most common form of inherited mental retardation, which have a diversity of clinical manifestation. The main is mental retardation range from moderate to severe, and associated with giant testis, particular face, minimal brain dysfunction, epileptic attack.The check of electroencephalogram present typical and diffused moment slow wave releasing and some other clinical pathological features. Research shows that more than 99% of the FraX are caused by the expression decrease or absence of FMRP(fragile X mental retardation protein), the coding products of FMR1(fragile X mental retardation 1) located at Xq27.3,which is caused by the abnormal amplification of the unstable CGG repeat sequence in the 5'untranslated region of FMR1 and the subsequent methylation of its CpG island. FXR1/2 (fragile X related gene 1/2) located in 3q28 and 17p13.1 respectively were found have considerable similarity with FMR1 in sequence and structure, so they were called fragile X gene family. Any two of the three genes can exist in the form of homodimers or heterodimers. The screening and identification of the series of interaction proteins of FXR1P will be helpful for the clarification of the function of FXR1,understand the regulatory mechanism of FXR1P,and then to comprehend the relationship between FXR1P and FraX.The FXR1P interacting proteins were researched by the yeast two hybrid system. Firstly ,we constructed bait plasmid pGBKT7-FXR1, and mated the transformed bait plasmid pGBKT7-FXR1 in yeast AH109 with the yeast Y187 transformed of Matchmaker human fetal brain pGADT7-cDNA library plasmid. Screened positive clones through detection the express of a series of report genes, and identified the positive AD/library plasmids by enzymatic digestion and then validated the protein-protein interaction by retransformed the AD/library plasmids to yeast Y187 and applied one-to-one small scale mating with yeast AH109 transformed bait plasmid pGBKT7-FXR1. seven proteins which may interact with FXR1P were acquired, namely Btf,SAFB,CMAS,FTH1,CSH1,GOLGA4,and HSD17B1; Later, the interaction of two screend proteins associated with apoptosis with FXR1P was checked with co-immunopricipitation(Co-IP) experiment. Four eukaryon expression vectors, pCMV-FXR1-Myc, pCMV-Flag-FXR1, pCMV-Btf-Myc, pCMV-SAFB-Myc were constructed, and the interaction of FXR1P and Btf, SAFB were validated by Co-IP in the mammals cells. We found FXR1P can interact with Btf, but the interaction between FXR1P and SAFB was not checked. Thus we concluded that Btf is a new member of the FXR1P-related regulation network and play a role in the development process of nervous and muscle system.FXR1P may involeved in the progress of cell apoptosis.