BackgroundTranslationally Controlled Tumor Protein (TCTP) found in nasal lavage fluids of allergic patients was named IgE-dependent histamine-releasing factor (HRF). Human recombinant HRF (HrHRF) has been recently reported to be much less effective than HRF produced from activated mononuclear cells (HRFmn).Methods and FindingsWe found that only NH2-terminal truncated, but not C-terminal truncated, TCTP shows cytokine releasing activity compared to full-length TCTP. Interestingly, only NH2-terminal truncated TCTP, unlike full-length TCTP, forms dimers through intermolecular disulfide bonds. We tested the activity of dimerized full-length TCTP generated by fusing it to rabbit Fc region. The untruncated-full length protein (Fc-HrTCTP) was more active than HrTCTP in BEAS-2B cells, suggesting that dimerization of TCTP, rather than truncation, is essential for the activation of TCTP in allergic responses. We used confocal microscopy to evaluate the affinity of TCTPs to its putative receptor. We detected stronger fluorescence in the plasma membrane of BEAS-2B cells incubated with Del-N11TCTP than those incubated with rat recombinant TCTP (RrTCTP). Allergenic activity of Del-N11TCTP prompted us to see whether the NH2-terminal truncated TCTP can induce allergic airway inflammation in vivo. While RrTCTP had no influence on airway inflammation, Del-N11TCTP increased goblet cell hyperplasia in both lung and rhinal cavity. The dimerized protein was found in sera from allergic patients, and bronchoalveolar lavage fluids from airway inflamed mice.ConclusionsDimerization of TCTP seems to be essential for its cytokine-like activity. Our study has potential to enhance the understanding of pathogenesis of allergic disease and provide a target for allergic drug development.

Translationally controlled tumor protein (TCTP) is a growth-related protein under transcriptional as well as translational control. We screened a rat skeletal muscle cDNA library using yeast two-hybrid system and found that TCTP interacts with the third large cytoplasmic domain of ␣1 as well as ␣2 isoforms of Na,K-ATPase, believed involved in the regulation of Na,K-ATPase activity. Interaction between TCTP and Na,K-ATPase was confirmed by coimmunoprecipitation in yeast and mammalian cells. We also showed, using 86 Rb ؉ uptake assay, that overexpression of TCTP inhibited Na,K-ATPase activity in HeLa cells. Northern and Western blotting studies of HeLa cells transiently transfected with GFPtagged TCTP showed that overexpression of TCTP did not change mRNA and protein levels of Na,K-ATPase. Recombinant TCTP protein purified from an Escherichia coli expression system inhibited purified HeLa cell plasma membrane Na,K-ATPase in a dose-dependent manner. Using deletion analysis, we also found that the C-terminal 102-172-amino-acid region of rat TCTP that contains the TCTP homology region 2 is essential for its association with, and inhibition of, Na,K-ATPase. Na,K-ATPase, a multimembrane-spanning enzyme, is essential for maintaining transmembrane gradients of Na ϩ and K ϩ ions and thus for cell homeostasis (1). These ionic gradients serve to control essential cellular processes such as cell volume, membrane potential, and nutrient transport (2). In addition, Na,K-ATPase is involved in cell proliferation and differentiation, heart and vascular muscle contraction, and neurotransmitter and hormone secretion (3). Thus dysfunction of this enzyme can profoundly affect cell function. Na,K-ATPase is composed of a catalytic 110-kDa ␣ subunit and a glycosylated 40 -60-kDa ␤ subunit. The ␣ subunit contains binding sites for cations, ATP, and cardiac glycosides. It has been suggested that there might exist a diffusible cytoplasmic regulator of Na,K-ATPase activity, possibly modulated by protein kinases and hormones (4, 5). The third large cytoplasmic domain (CD3) 1 of Na,K-ATPase was proposed to be one of the domains involved in the regulation of its activity by insulin, thereby playing an important role in the catalytic function and regulation of this enzyme (6). Interactions between the Nterminal region of the Na,K-ATPase ␣ subunit with phosphoinositide-3 kinase (7), cytoplasmic domain 2 (CD2) and CD3 with ankyrin (8, 9), CD3 with cofilin (10), and purified Na,KATPase with actin (11) and adducin (12) have also been demonstrated.We looked for other cytoplasmic agents that might interact with the CD3 of Na,K-ATPase ␣ subunit and regulate its activity and found that translationally controlled tumor protein (TCTP) acts as a cytoplasmic repressor of Na,K-ATPase. TCTP is a growth-related protein, under tight transcriptional as well as translational control (13,14). It occurs as a 23-kDa protein in humans and has a 21-kDa homologue in mice but shows no significant homology with any other family of proteins. Based on structural studies o...

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Translationally controlled tumor protein (TCTP), also known as IgE-dependent histamine-releasing factor, is a growth-related tumor protein. Although the primary sequence of rat TCTP does not reveal any recognizable Ca2+ -binding motif, previous studies have demonstrated that rat TCTP consisting of 172 amino acids is a Ca2+ -binding protein. However, the region of TCTP required for Ca2+ interaction has not been mapped to the molecule. Here, we reported that the Ca2+ binding region of TCTP, which was mapped by using a combination of deletion constructs of rat TCTP and 45Ca2+ -overlay assay, was confined to amino acid residues 81-112. This binding domain did not show any peculiar loop of calcium-binding motif such as CaLB domain and EF hand motif and it seems to be constituted of random coil regions neighboring the a helix. Thus, our data confirm that TCTP is a novel family of Ca2+ -binding protein.

Translationally controlled tumor protein (TCTP), is a highly conserved protein involved in fundamental processes, such as cell proliferation and growth, tumorigenesis, apoptosis, pluripotency, and cell cycle regulation. TCTP also inhibits Na,K-ATPase whose subunits have been suggested as a marker of epithelial-to-mesenchymal transition (EMT), a crucial step during tumor invasiveness, metastasis and fibrosis. We hypothesized that, TCTP might also serve as an EMT inducer. This study attempts to verify this hypothesis. We found that overexpression of TCTP in a porcine renal proximal tubule cell line, LLC-PK1, induced EMT-like phenotypes with the expected morphological changes and appearance of EMT related markers. Conversely, depletion of TCTP reversed the induction of these EMT phenotypes. TCTP overexpression also enhanced cell migration via activation of mTORC2/Akt/GSK3β/β-catenin, and invasiveness by activating MMP-9. Moreover, TCTP depletion in melanoma cells significantly reduced pulmonary metastasis by inhibiting the development of mesenchymal-like phenotypes. Overall, these findings support our hypothesis that TCTP is a positive regulator of EMT and suggest that modulation of TCTP expression is a potential approach to inhibit the invasiveness and migration of cancer cells and the attendant pathologic processes including metastasis.