cDNA cloning and mRNA expression of the translationally controlled tumor protein(TCTP)gene from Japanese sea perch(Lateolabrax japonicus)

A homologue of the lower vertebrates translationally controlled tumor protein (TCTP) was cloned from the marine fish Japanese sea perch (Lateolabrax japonicus) by the technology of homology cloning. The full-length cDNA sequence of the sea perch TCTP gene contained a 5' untranslated region (UTR) of 47 bp, a 3' UTR of 433 bp, and a putative open reading frame (ORF) of 510 bp encoding a polypeptide of 170 amino acids. The deduced amino acid sequence of the sea perch TCTP gene showed a high similarity to that of zebrafish, rohu, rabbit, chicken and human. Sequence analysis revealed there were a signature sequence of TCTP family, an N-glycosylation site, and five Casein kinase phosphorylation sites in the sea perch TCTP. The temporal expression of TCTP genes in healthy and lipopolysaccharide (LPS) challenged fishes was measured by semi-quantitative reverse transcription-PCR (RT-PCR). The results indicated that LPS could up-regulate the expression of sea perch TCTP in the examined tissues, including head-kidney, spleen and liver.

Cloning, Expression and Characterization of Translationally Controlled Tumor Protein (TCTP) Gene from Flatfish Turbot (Scophthalmus maximus)

A full-length cDNA encoding translationally controlled tumor protein of marine flatfish turbot （Scophthalmus maximus）, SmTCTP, was isolated with rapid amplification of cDNA Ends （RACE）. SmTCTP consisted of a 5＇ untranslated region （UTR） of 84 bp, a 3＇ UTR of 451 bp and an open reading frame （ORF） of 513 bp, encoding a protein of 170 amino acid residues, which contained two signature sequences of TCTP family. The 5＇UTR of SmTCTP started with a 5＇-terminal oligopyrimidine tract （5＇-TOP）, a typical feature for translationally controlled mRNAs. The deduced amino acid sequence of SmTCTP was similar to the other known vertebrate TCTPs in a range of 58.8% to 64.1%. The length of fish TCTPs was diverse among species, e.g., TCTP of turbot and sea perch （Lateolabraxjaponicus） is 170 aa in length, while that of zebrafish （Danio rerio） and rohu （Labeo rohita） is 171 aa in length. Northern blot analysis revealed that SmTCTP has only one type of mRNA. Its expression level in albino skin was slightly higher than that in normal skin. We constructed the pET3Oa-SmTCTP expression plasmid. The recombinant protein of His-tag SmTCTP was over-expressed in E. coli, purified and identified with peptide mass fingerprinting. These results may pave the way of further investigation of the biological function of TCTP in fish.

Translationally controlled tumor protein exerts a proin?ammatory role in acute rejection after liver transplantation

Background:Translationally controlled tumor protein(TCTP),which has been verified to have a proinflammatory activity,plays an important role in allergy.However,it remains unclear whether TCTP has an impact on the acute rejection(AR)after liver transplantation.Methods:Three protocols were used to delineate the role of TCTP in AR after liver transplantation.First,in rat orthotopic liver transplantation(OLT),the expression of TCTP was measured by enzyme-linked immunosorbent assay(ELISA),real-time PCR,Western blot and immunofluorescence assays.Second,in mixed lymphocyte reaction(MLR),the role of TCTP in lymphocyte proliferation was measured by carboxyfluorescein succinimidyl ester(CFSE)labeling and the impact of TCTP on inflammatory factor release was detected by cytokine arrays.Third,in human OLT,the level of serum TCTP was detected by ELISA,and the relationship between TCTP and model for early allograft function(MEAF)score was assessed by Spearman's correlation.Results:In rat OLT,AR resulted in great harm to allografts,manifesting as deterioration of liver function,increasing inflammatory factors and infiltrating lymphocytes.Meanwhile,TCTP was overexpressed in serum and allografts.Higher level of TCTP was associated with higher rejection activity index(RAI).In an MLR protocol,TCTP knockdown inhibited the proliferation of mixed inflammatory cells and significantly suppressed the release of 15 cytokines and chemokines.In human OLT,the serum TCTP was up-regulated within a week after operation.Additionally,the increasing speed of serum TCTP positively correlated with MEAF scores(r=0.449;P=0.0088).

Proteomic analysis identifies translationally controlled tumor protein as a mediator of phosphatase of regenerating liver-3-promoted proliferation, migration and invasion in human colon cancer cells

Background Considerable evidence suggests that phosphatase of regenerating liver-3 （PRL-3） plays multiple roles in cancer metastasis; however, the molecular mechanisms remain largely unknown. The aim of this study was to identify proteins associated with PRL-3-promoted colon cancer metastasis, by comparative proteomic analysis. Methods Proteomes of human colon cancer LoVo cells transfected with PRL-3 gene （LoVo-PRL-3） or empty vector PAcGFP-C3 （LoVo-control） were compared using 2D gel electrophoresis. Proteins that varied significantly in concentration were selected and identified using mass spectrometry. Expression of translationally controlled tumor protein （TCTP） mRNA and protein in LoVo-PRL-3 and LoVo-control cells was detected by real-time PCR and Western blotting. Small interfering RNA （siRNA） targeting TCTP was used for silencing TCTP expression in LoVo-PRL-3 cells. Functional significance of TCTP in PRL-3-promoted colon cancer cell proliferation, migration and invasion was investigated by Cell Counting Kit-8 assay and transwell chamber. Results Seventeen proteins displaying significant and reproducible differences between LoVo-PRL-3 and LoVo-control cells were identified. Ten proteins were upregulated and seven were downregulated in LoVo-PRL-3 cells when compared with LoVo-control cells. Eight identified proteins are associated with distinct steps of tumor metastasis： ubiquitin-like protein ISG15, interleukin-18, TCTP, serpin B5, annexin A3, macrophage-capping protein, ATP-dependent RNA helicase DDX3X, and cathepsin D. Real-time PCR and Western blotting results showed that both TCTP mRNA and protein were significantly increased in LoVo-PRL-3 cells compared to LoVo-control cells. Transfection with TCTP siRNA significantly reduced the expression of both mRNA and protein levels of TCTP in LoVo-PRL-3 cells. Knockdown of TCTP by siRNA inhibited PRL-3-promoted proliferation, migration and invasion of LoVo-PRL-3 cells. Conclusion Our results imply that TCTP might be a mediator of PRL-3-promoted proliferation, migration and invasion of human colon cancer cells.

Translationally controlled tumor protein: the mediator promoting cancer invasion and migration and its potential clinical prospects

Translationally controlled tumor protein(TCTP) is a highly conserved multifunctional protein localized in the cytoplasm and nucleus of eukaryotic cells. It is secreted through exosomes and its degradation is associated with the ubiquitin-proteasome system(UPS), heat shock protein 27(Hsp27), and chaperone-mediated autophagy(CMA). Its structure contains three α-helices and eleven β-strands, and features a helical hairpin as its hallmark. TCTP shows a remarkable similarity to the methionine-R-sulfoxide reductase B(Msr B) and mammalian suppressor of Sec4(Mss4/Dss4) protein families, which exerts guanine nucleotide exchange factor(GEF) activity on small guanosine triphosphatase(GTPase) proteins, suggesting that some functions of TCTP may at least depend on its GEF action. Indeed, TCTP exerts GEF activity on Ras homolog enriched in brain(Rheb) to boost the growth and proliferation of Drosophila cells. TCTP also enhances the expression of cell division control protein 42 homolog(Cdc42) to promote cancer cell invasion and migration. Moreover, TCTP regulates cytoskeleton organization by interacting with actin microfilament(MF) and microtubule(MT) proteins and inducing the epithelial-mesenchymal transition(EMT) process. In essence, TCTP promotes cancer cell movement. It is usually highly expressed in cancerous tissues and thus reduces patient survival;meanwhile, drugs can target TCTP to reduce this effect. In this review, we summarize the mechanisms of TCTP in promoting cancer invasion and migration, and describe the current inhibitory strategy to target TCTP in cancerous diseases.

Pathophysiological roles of Pim-3 kinase in pancreatic cancer development and progression

Pim-3 is a member of the provirus integration site for Moloney murine leukemia virus(Pim)family proteins that exhibit serine/threonine kinase activity.Similar to the other Pim kinases(Pim-1 and Pim-2),Pim-3 is involved in many cellular processes,including cell proliferation,survival,and protein synthesis.Although Pim-3is expressed in normal vital organs,it is overexpressed particularly in tumor tissues of endoderm-derived organs,including the liver,pancreas,and colon.Silencing of Pim-3 expression can retard in vitro cell proliferation of hepatocellular,pancreatic,and colon carcinoma cell lines by promoting cell apoptosis.Pim-3 lacks the regulatory domains similarly as Pim-1 and Pim-2 lack,and therefore,Pim-3 can exhibit its kinase activity once it is expressed.Pim-3 expression is regulated at transcriptional and post-transcriptional levels by transcription factors(e.g.,Ets-1)and post-translational modifiers(e.g.,translationally-controlled tumor protein),respectively.Pim-3 could promote growth and angiogenesis of human pancreatic cancer cells in vivo in an orthotopic nude mouse model.Furthermore,a Pim-3 kinase inhibitor inhibited cell proliferation when human pancreatic cancer cells were injected into nude mice,without inducing any major adverse effects.Thus,Pim-3 kinase may serve as a novel molecular target for developing targeting drugs against pancreatic and other types of cancer.