CCDC152 Regulates The Mechanism Of GPCR Signal Transduction

CCDC152(Coiled-coil Domain Containing 152)bears four coiled-coil domains and belongs to CCDC family.It is conserved in mammals,but the function is unknown.The bioinformatics structure study predicted that CCDC152 is strucaturally similar to the GCN4 of yeast.GCN4 acts as a homodimer to bind and activate certain gene transcription,which can initiate the expression of multiple amino acid synthases in yeast under the depletion of amino acid(s).The most basic role of histone modifications is to remodel the chromation.Histone acetylation can affect the chromatin structure,participates in the regulation of gene transcription at a particular site,and thus plays an important role in cell growth anddifferentiation.G protein-coupled receptor(GPCR)is the largest signal transduction superfamily in the human body.It interacts with small molecules such as neurotransmitters,hormones,light,and odor molecules and so on,and plays an important role in cell signal transduction.The occurrence of major human diseases is often associated with GPCR dysfunction.Therefore,GPCR becomes an important target in the drug discovery.This subject is guided by the analysis of bioinformatics structure,combined with the functional characteristics of GCN4 and the results of transcriptome sequencing,studies the relationship between CCDC152 and GPCR signal system,explores the function of CCDC 152 and the relationship with histone remodeling,the main research methods and results are as follows:1 Factors that affect the expression of CCDC 1521.1 Glucose supplement or amino acid deficiency induced changes of the expression of CCDC 152(1)The effect of high concentration of glucose on the expression of CCDC 152.The expression of CCDC 152 protein was detected by WB at different time points post treatment with 1.5g/L or 2.25g/L glucose of 293T or HepG2 cells,respectively.The results show that CCDC 152 expression was significantly reduced in 293T cells treated with 1.5g/L or 2.25g/L of glucose,whereas significantly increased in HepG2 cells treated with 1.5g/L of glucose.(2)The effect of amino acid deficiency on the expression of CCDC152.The expression of CCDC152 protein was detected by WB in 293T and HT22 cells cultured in amino acid deficient medium at different time.The results show that the expression of CCDC152 was significantly increased in 293T cells cultured in amino acid depletion medium for lh,whereas significantly increased in HT22 cells for 1,2 and 6h.1.2 The regulation of the expression of CCDC152 by different GPCR agonists,Mela,GnRH,DOI and LPA(1)In 293T cells,luciferase reporter assay and WB were used to detect the effect of Mela on the expression of CCDC152.The results show that in 293T cells,long-term Mela treatment resulted in a significant reduction in CCDC152,via Gai signaling.(2)The effects of GnRH on the expression of CCDC152 in Panc-1 and Ovcar-3 cells were detected by WB.The results show that GnRH influenced the expression of CCDC152 through Gai and Gaq in Panc-1 and Ovcar-3 cells.(3)The effect of GnRH and DOI on the expression of CCDC152 in HT22 cells were detected by WB.The results show that GnRH and DOI influenced CCDC152 expression via activated Gaq in HT22 cells.(4)In 293T cells,luciferase reporter assay and WB were used to assay the effect of LPA on the expression of CCDC152.The results show that 0.1uM or luM LPA treatment 293T cells for 1h or 2h caused a significant increase in CCDC152 expression.(5)F-actin staining was used to detect the effect of LPA on stress fiber formation and filament stabilization in 293T cells.The results show that LPA treatment of 293T cells,stress fiber formation was enhanced,over-expression of CCDC152 disrupted the stress fiber formation and induced significant pseudopodium.1.3 The study of CCDC152 promoter activity.(1)To study the subcellular localization of CCDC152 in different cells,HepG2 and 293T cells were transfected with CCDC 152-Red,and the localization of CCDC 152-Red in HepG2 and 293T cells was observed.WB was used to detect CCDC152 expression in nuclear and cytoplasm in 293T and Panc-1 cells.The results show that in HepG2 and 293T cells,CCDC 152-Red localization in the nucleus.CCDC152 expression in nucleus was significantly higher than in cytoplasm in both 293T and Panc-1 cells.(2)Luciferase reporter assay was used to analyze CCDC 152 binds to the promoter sequence and the effect of Ca2+,Mela on activity of CCDC 152 promoter.The results show that in 293T cells,CCDC152 was bound the AP-1,GCN4 and CRE sequence ATGACTCAT,CCDC152 promoter activity was significantly inhibited with the treatm-ent of 10mM Mela for 30min whereas activated with 2mM Ca2+ for 5min.2.Effects of CCDC152 on GPCR and downstream key gene expressionWB was used to detect the effect of CCDC152 overexpression on GPCR and downstream key genes expression.The results showed that the expression of CTGF,EGR1,DKK1,TEAD1 and VCL were decreased.3.The possible mechanism of CCDC152 affecting gene expression-histone modificationWB and fluorescence microscopy identified HepG2 stable screening of cell lines,WB and Coomassie brilliant blue G-250 were used to detect the expression of CCDC152 and histone H3S10p and H3K14ac modification.The results showed that the expression of CCDC152 in HepG2 cells was positively correlated with the acetylation of histone H3K14 and negatively correlated with the phosphorylation of histone H3S10.In summary:CCDC152 mainly located in the nucleus,can respond to glucose and the lack of amino acids in the environment,GPCR agonists Mela,GnRH,DOI and LPA may regulate the expression of CCDC152 through different G protein pathway.CCDC152 affects the expression of GPCR and downstream key genes by affecting the modification of histones.