Bioinformatic Analysis And Experimental Study On Homologous Proteins Of TBX5 And GLI1

IntroductionTBX5,as a member of the T-box-containing transcription factor family,encodes a protein of 518 amino acids and is expressed in the embryonic heart and developing limb tissues and plays an important role in normal heart and limb development.TBX5 mutations could cause Holt-Oram syndrome,an autosomal dominant condition characterized by congenital cardiac malformations and upper limb anomalies.The molecular mechanism by which most TBX5 mutations at the C-terminal region cause Holt-Oram syndrome remains unclear.Moreover,the mechanism underlying the transactivation by C-terminus of TBX5 is largely unknown.Thus,the sequence-based study of C-terminus of TBX5 is needed for a better understanding of the molecular mechanism underlying TBX5 mutation-related Holt-Oram syndrome,and of TBX5 function as a transcriptional activator.It has been shown that TBX5 interacts with GATA4 and NKx2-5 which are transcriptional factors related to heart development. G80R,R237Q and R237W mutations reduce the TBX5 DNA-binding ability with target genes,leading to decreased transcriptional activation of TBX5 target genes and a loss of synergistic transcriptional activation with NKx2-5.G80R and R237W missense mutations in TBX5 are also found to disrupt the TBX5 physical interaction with GATA4,a cardiac transcriptional factor implicated in human cardiac septal defects, whereas Q49K,I54T,G169R and S252I mutations are not.Thus,to further explore any other TBX5-interacting proteins would shed light on the mechanism for human heart development and congenital heart diseases.Also playing a role as a transcriptional factor like TBX5,GLI1 mediates hedgehog (Hh)pathway and plays a crucial role during development of embryos in Drosophila and vertebrate.GLI1 protein contains two known domains,that is,Zn-finger region and putative transcriptional repressor SFP1 region.A serine-rich region conserved through the GLI/Ci family in the NH₂-terminal region was recently termed as N-terminal Regulatory region,the function of which remains elusive.Nevertheless,the sequence-function study for the NHE-terminal region of GLI1 is rare.The contribution of NHE-terminal region to the transcriptional activity of GLI1 is unclear.About one third of lethal cancers are associated with mutations of the components in Hh pathway or the super activation of Hh pathway by excessive response to its ligands,with GLI1 mediating this process and playing an important role.GLI1 overexpression is related to a variety of neoplasms,for instance,glioma,melanomas,prostate cancer,esophageal squamous cell carcinoma and gastric cancer.So far,many antitumor drugs target the crucial kinases in the signaling pathway.MEK and PI3K inhibitors are promising for their application in clinical cancer therapy.Due to their specific inhibition of MEK and PI3K pathways,however,these two inhibitors may have certain unforeseeable side-effects and the comprehensive clinical application is restricted.Thus,to study the impact of MEK and PI3K inhibitors on the tumor-related protein expression and tumor cell invasiveness would offer theory foundation for their application in clinical tumor therapy.MethodsExperimental materials:1.The protein database of all species http://www.ncbi.nlm.nih.gov/sites/entrez?db=protein&cmd=search&term=2.The non-redundant database of protein http://www.ncbi.nlm.nih.gov/RefSeq/3.PSI-BLAST(Position-Specific Iterated BLAST)website PSI-BLAST:http://www.ncbi.nlm.nih.gov/blast/Blast.cgi4.FFAS03(Fold & Function Assignment System)websites FFAS03:http://ffas.ljcrf.edu/ffas-cgi/egi/ffas.pl5.HHpred(Homology detection & structure prediction by HMM-HMM)websites HHpred:http://toolkit.tuebingen.mpg.de/hhpred6.M-coffee for alignment: http//www.ch.embnet.org/software/TCoffee.html7.Phosphorylation sites prediction: http://pred.ngri.re.kr/PredPhospho.htm8.The kunming mice were provided by China Medical University.9.Gastric cancer SGC7901 cells were cultured in 1640 medium supplemented with 10%(v/v)calf serum,penicillin and streptomycin in humidified 5%(v/v)CO₂ atmosphere at 37℃.10.MEK,PI3K inhibitor concentration:12.5μM and 25μM in DMSO, respectively.Experimental Methods1.Non-redundant database search for TBX5 and GLI1 homologous proteins by bioinformatic tools(PSI-BLAST,FFAS03 and HHpred)2.Multiple alignments of TBX5 and GLI1 proteins from different species by M-Coffee3.Phosphorylation sites prediction for C-terminal region of TBX5 and NH₂-terminal region of GLI1 by PredPhospho4.Immunoprecipitation study of nuclear protein from mouse heart tissue using Tbx5 antibody and the band of interest is subject to mass spectrometry analysis.Pierce nuclear protein extraction protocol and immunoprecipitation protocol(#23600)were used.Experimental group:active gel with 15μl Tbx5 polyclonal antibody,control group:inactive gel with 15μl Tbx5 polyclonal antibody,IgG group:active gel with 5μl IgG.20μl mouse heart tissue nuclear protein were used for immunoprecipitation.5.After the verification of the protein of interest by mass spectrometry,the interaction of the two interacting proteins was confirmed by western blotting.6.The grouping of gastric cancer SGC7901 cells and treatment:The five groups of gastric cancer SGC7901 cells:control group(normal gastric cancer SGC7901 cells), DMSO group(2μl DMSO in 4ml medium),MEK inhibitor group(concentration: 12.5μM),PI3K inhibitor group(concentration:25μM)and co-acting group(MEK inhibitor and PI3K inhibitor added together in the medium,12.5μM and 25μM in concentration,respectively).7.The GLI1 gene and GLI1 protein expression level in gastric cancer SGC7901 cells detected by RT-PCR and western blotting at the 0,12^th,24^th,48^thand 72^ndhour of MEK and PI3K inhibitors treatment.8.Detection of CD14,CD44 and TLR2 expression by flow cytometry in gastric cancer SGC7901 cells at the 12^th,24^th,48^thand 72^ndhour of MEK and PI3K inhibitors treatment.9.Transwell test to measure in vitro invasiveness of gastric cancer SGC7901 cells at the 12^th,24^th,48^thand 72^ndhour of MEK and PI3K inhibitors treatment.Results1.C-terminus of TBX5 and N-terminus of GLI1 are highly homologous to the C-terminal domain of yeast DNA-direeted RNA polymeraseâ…¡largest subunit.2.Tyr291 and Tyr342 at C-terminus of TBX5,and Ser84 and Ser102 at N-terminus of GLI1 are identified as potential phosphorylation sites.3.Tbx5 protein in mouse heart tissue interacts with Myh6.4.Western blotting confirmed the interaction between mouse Tbx5 and Myh6.5.At the 48^thand 72^ndhour of MEK and PI3K inhibitors treatment,the GLI1 gene and GLI1 protein expression in gastric cancer SGC7901 cells were downregulated.6.At the 72^ndhour of MEK and PI3K inhibitors treatment,CD44 protein expression in gastric cancer SGC7901 cells were downregulated.MEK and PI3K inhibitors had synergistic effect.7.At the 72^ndhour of MEK and PI3K inhibitors treatment,CD14 protein expression in gastric cancer SGC7901 cells were downregulated.MEK and PI3K inhibitors had synergistic effect.8.MEK and PI3K inhibitors,alone or in combination,do not affect the TLR2 expression level significantly in gastric cancer SGC7901 cells from 12^thto 72^ndof treatment.9.MEK and PI3K inhibitors,alone or in combination,make the gastric cancer SGC7901 cells less invasive at the 72nd hour of treatment.Conclusion1.Residues 267-448 at C-terminus of TBX5 and residues 2-175 at N-terminus of GLI1 both are highly homologous to the C-terminal domain of yeast DNA-directed RNA polymeraseâ…¡largest subunit.2.The partial function of the C-terminus of TBX5 might be to interact with or recruit other proteins for its transcriptional activity.GLI1 Ser84 and Ser102 residues were identified as potential PKA and/or PKC phosphorylation sites. 3.The Tbx5 protein in the mouse heart tissue interacts with Myh6 protein.4.At the 48^thand 72^ndhour of MEK and PI3K inhibitors treatment,the GLI1 gene and GLI1 protein expression in gastric cancer SGC7901 cells were downregulated.5.At the 72^ndhour of MEK and PI3K inhibitors treatment,CD44 and CD14 protein expression in gastric cancer SGC7901 cells were significantly downregulated. MEK and PI3K inhibitors had synergistic effect.MEK and PI3K inhibitors,alone or in combination,do not affect the TLR2 expression level in gastric cancer SGC7901 cells.6.MEK and PI3K inhibitors,alone or in combination,made the gastric cancer SGC7901 cells less invasive at the 72^ndhour of treatment.