The Roles Of Transmembrane-4 Protein, A Novel Member Of Ubiquitin-associated Domain Protein Family, In Diabetic Vasculopathy

Partâ… The cloning and bioinformatics analyses of TM4 geneObjective:To clone transmembrane-4(TM4) gene from human aorta tissues, and analyze the structure and function of TM4 gene and protein by bioinformatics for next studies.Methods:Full-length ORF of TM4 gene was cloned into pDrive cloning vector and identified by enzyme-cutting and sequencing.The structure and function of TM4 gene and protein were further analyzed by bioinformatics technology.Results:Two alternatively spliced variants of TM4 gene exist in aorta tissue, which ORF is 1035bp and 915bp respectively.Aorta tissue mainly expresses the longer spliced variant.The longer spliced variant of TM4 gene had been cloned into pDrive cloning vector and pDrive-TM4 vector had been successfully constructed.Results of bioinformatics analyses are as follows: TM4 gene coding 344 amino acids is located on chromosome 13q32.3 and generally expressed in many tissues.There is no the same sequence with TM4 gene and protein in database,but TM4 gene and protein have high homology with ubiquitin-associated domain containing 2(UBAC2) gene and protein in other species.TM4 protein is a membrane protein having four transmembrane domain,which contains a PKC phosphorylation site of serine residue in the 89th amimo acid and a conserved ubiquitin associated domain (UBA) in protein carboxyl terminal.UBA domain is high conserved in spices. TM4 protein may involve in the transportation of one or more substances.Conclusions:TM4 gene is generally expressed in many tissues and there are two spliced variants in tissues.TM4 protein is a membrane protein containing four transmembrane domain.UBA domain may play an important role in function of TM4 protein.TM4 protein is probably involved in substance transportation. Partâ…¡The construction and verification of TM4 gene recombinant eukaryotic and adenovirus expression vectorObjective:To construct recombinant eukaryotic and adenovirus expression vector of TM4 gene.Methods:The full-length ORF of TM4 gene was amplified from pDrive-TM4 re- combinant plasmid and was further subcloned into eukaryotic expression vector PDC315 and PDC315-GFP.Sequences of TM4 gene in PDC315-GFP-TM4 and PDC315-TM4 recombinant plasmids were confirmed by sequencing,enzyme- cutting,and further cell transfection.The recombinant adenovirus vector expressing TM4 protein was successfully produced by the recombination of PDC315-TM4 and PDC315-GFP-TM4 shuttle plasmids and adenovirus genomic plasmid pBHGIoxE1,3Cre,which were identified by PCR and cell infection.Results:PDC315-TM4 and PDC315-GFP-TM4 recombinant eukaryotic expression vectors were successfully constructed,and had been confirmed by enzyme-cutting and sequencing.PDC315-GFP-TM4 recombinant plasmid can exactly express GFP protein.The recombinant adenovirus vector expressing TM4 gene was produced successfully and identified by PCR and cell infection.Conclusions:The recombinant eukaryotic and adenovirus expression vector which can express TM4 protein had been successfully constructed.Partâ…¢Screening of candidate interaction partners of TM4 protein using yeast two- hybrid systemObjective:To screen the candidate interacting partners of TM4 protein using yeast two-hybrid system.Methods:The coding region of TM4 gene was amplified from pDrive-TM4 recombinant plasmid and was further cloned into pGB vector to construct pGB-TM4 recombinant yeast plasmid,pGB-TM4 recombinant plasmid was used as a bait to test if it can self-activate the expression of report-gene.The yeast strain Y190 was transfected by pGB-TM4 recombinant plasmid to test if bait gene could activate the expression of report-gene.Then,yeast cDNA library was screened and positive clone was identified and yeast plasmid was extracted.Prey and bait plasmids were cotransfected into yeast Y190 strain to further examine the interaction between prey and bait protein.Finally,the positive plasmids were sequenced,and the Blastn program was used to characterize the sequence.Results:We had successfully constructed the recombinant pGB-TM4 plasmid which can't self-activate the expression of report-gone.We identified five positive clones that showed specific activation of reporter gene expression.All positive plasmids were identified as NR4A1 gene by sequencing and blastn program.Conclusions:NR4A1 protein is probably a candidate partner of TM4 protein based on the results of yeast two-hybrid system.Partâ…£Generation of mouse model of TM4 gene mutation by gene trappingObjective:To generate the mouse model of TM4 gene mutation by gene trapping.Methods:The mouse model of TM4 gene mutation was produced by gene-trap approach.ES cell line RRQ018 which TM4 gene had been inactivated was obtained from The International Gene Trap Consortium(IGTC). Chimeric mice about 50%coat color chimerism were generated by microinjection of recombinant ES cells into C57BL/6J blastocysts and transferred into foster mothers.Heterozygous mice were obtained by mating between male chimeric mice and female C57BL/6J mouse.Their gone types were identified by PCR analysis of tail DNA.Finally,homozygous mice were generated by intercross of heterozygous mice.Whether TM4 gene had been mutated or not was identified by PCR technology.Results:9 male chimeric mice were generated and number 5 and 8 chimeric mice could generate offspring with gray fur.The gene-trap vector has been successfully inserted into intron of TM4 gene,which leads to hypomorphic mutation in F2 mouse.Conclusions:The mouse model of hypomorphic mutation of TM4 gene had been generated,but those of null mutation hadn't been produced. Partâ…¤Preliminary study of roles of TM4 protein in diabetic vascular injuryObjective:To investigate the tissue and vascular cell expression profiles of TM4 protein and its subcellular localization;To research the effect of high glucose,AGEs,pioglitazone,TPA on expression of TM4 protein;To study the influence of TM4 protein on cell proliferation;To investigate the influence of hypomorphic mutation of TM4 gene on expression of nur77 protein.Methods:RT-PCR and immunohistochemistry were applied to investigate the expression of TM4 gene in tissues and vascular cell;Laser confocal microscopy was employed to reveal the localization of TM4 protein in HEK293 cell line;Western blotting was used to observe the effect of high glucose, AGEs pioglitazone,TPA on expression of TM4 protein;XTT method was applied to study the influence of TM4 protein on cell proliferation;The influence of hypomorphic mutation of TM4 gene on expression of nur77 protein was tested by Western blot.Results:TM4 gene is generally expressed in many tissues.The expression of TM4 gene can be detected in vascular endothelial cell,smooth muscle cell and macrophages.TM4 protein is detected in tunica intima,media and adventitia by immunohistochemistry.The subcellular location of TM4 protein is in nuclear membrane and cellular membrane.High glucose,AGEs and protein kinase C activator upregulate the expression of TM4 protein,but pioglitazone downregulates that of TM4 protein.Overexpression of TM4 protein promotes cell proliferation.The expression of nur77 protein is upregulated becase of hypomorphic mutation of TM4 gene.Conclusions:TM4 protein is generally expressed in many tissues,and in vascular endothelial cell,smooth muscle cell and macrophages.The expression of TM4 protein is regulated by high glucose,AGEs and so on. Overexpression of TM4 protein promotes cell proliferation.TM4 protein may play an important role in diabetic vascular injury.The expression of nur77 protein is upregulated becase of hypomorphic mutation of TM4 gene.