Dimerization of Translationally Controlled Tumor Protein Is Essential For Its Cytokine-Like Activity

Background

Translationally 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 Findings

We 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.

Conclusions

Dimerization 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.     

Molecular cloning, heterologous expression and functional characterization of a novel translationally-controlled tumor protein (TCTP) family member from Loxosceles intermedia (brown spider) venom

Envenoming with brown spiders (Loxosceles genus) is common throughout the world. Cutaneous symptoms following spider bite accidents include dermonecrosis, erythema, itching and pain. In some cases, accidents can cause hypersensibility or even allergic reactions. These responses could be associated with histaminergic events, such as an increase in vascular permeability and vasodilatation. A protein that may be related to the effects of spider venom was identified from a previously obtained cDNA library of the L. intermedia venom gland. The amino acid sequence of this protein is homologous to proteins from the TCTP (translationally-controlled tumor protein) family, which are extracellular histamine-releasing factors (HRF) that are associated with the allergic reactions to parasites. Herein, we described the cloning, heterologous expression, purification and functional characterization of a novel member of the TCTP family from the Loxosceles intermedia venom gland. This recombinant protein, named LiRecTCTP, causes edema, enhances vascular permeability and is likely related to the inflammatory activity of the venom. Moreover, LiRecTCTP presents an immunological relationship with mammalian TCTPs.     

TSAP6 facilitates the secretion of translationally controlled tumor protein/histamine-releasing factor via a nonclassical pathway

Translationally controlled tumor protein (TCTP) is cytoplasmic and structurally related to guanine-nucleotide free chaperones. TCTP (also called histamine-releasing factor) has been described previously as a secreted protein that participates in inflammatory responses by promoting the release of histamine. How TCTP is eventually exported out of the cell to promote such activities is unknown. Here we show that TCTP secretion was insensitive to either brefeldin A or monensin, suggesting that it proceeds via an endoplasmic reticulum/Golgi-independent or nonclassical pathway. Moreover, our analyses also suggest that secreted TCTP originates from pre-existing pools. TSAP6, a p53-inducible 5-6 transmembrane protein, was found to interact with TCTP in a yeast two-hybrid hunt. GST pull down assays confirmed their direct interaction, and immunofluorescence analysis revealed their partial co-distribution to vesicular-like structures at the plasma membrane and around the nucleus. Functionally, the overexpression of TSAP6 consistently leads to enhanced secretion of both endogenously and exogenously expressed TCTP. Finally, we found TCTP in preparations of small secreted vesicles called exosomes, which have been suggested as a possible pathway for nonclassical secretion. Overexpression of TSAP6 also increased TCTP levels in exosome preparations. Altogether, these data identify a novel role for TSAP6 in the export of TCTP and indicate that this multipass membrane protein could have a general role in the regulation of vesicular trafficking and secretion.     

A knockout mouse approach reveals that TCTP functions as an essential factor for cell proliferation and survival in a tissue- or cell type-specific manner

Translationally controlled Tumor Protein (TCTP) is an evolutionally highly conserved protein which has been implicated in many cellular functions that are related to cell growth, death, and even the allergic response of the host. To address the physiological roles of TCTP, we generated TCTP knockout mice by targeted gene disruption. Heterozygous mutants appeared to be developmentally normal. However, homozygous mutants (TCTP(-/-)) were embryonic lethal. TCTP(-/-) embryos were smaller in size than the control littermates at all postimplantation stages examined. Although TCTP is widely expressed in both extraembryonic and embryonic tissues, the most prominent defect of the TCTP(-/-) embryo at embryonic stage day 5.5 (E5.5) was in its epiblast, which had a reduced number of cells compared with wild-type controls. The knockout embryos also suffered a higher incidence of apoptosis in epiblast starting about E6.5 and subsequently died around E9.5-10.5 with a severely disorganized structure. Last, we demonstrated that TCTP(-/-) and control mouse embryonic fibroblasts manifested similar proliferation activities and apoptotic sensitivities to various death stimuli. Taken together, our results suggest that despite that TCTP is widely expressed in many tissues or cell types, it appears to regulate cell proliferation and survival in a tissue- or cell type-specific manner.     

Identification of the self-interaction of rat TCTP/IgE-dependent histamine-releasing factor using yeast two-hybrid system

To further understand the biological function of translationally controlled tumor protein (TCTP), also known as IgE-dependent histamine-releasing factor (HRF), the yeast two-hybrid system was used to screen interacting molecules. We isolated cDNA clones coding for TCTP/HRF, suggesting that it may have a self-interacting property. Domain mapping of the interaction revealed that the C-terminal region of residue 126-172 is involved in self-interaction. The self-interacting property of TCTP/HRF was further supported by FPLC gel-filtration chromatography and coimmunoprecipitation analysis from transfected COS-7 cells. Our data suggests that TCTP/HRF may have a potential to self-interact through the C-terminal region, and the self-interaction property may be related to its biological function.     

Ligand Binding Reveals a Role for Heme in Translationally-Controlled Tumor Protein Dimerization

The translationally-controlled tumor protein (TCTP) is a highly conserved, ubiquitously expressed, abundant protein that is broadly distributed among eukaryotes. Its biological function spans numerous cellular processes ranging from regulation of the cell cycle and microtubule stabilization to cell growth, transformation, and death processes. In this work, we propose a new function for TCTP as a “buffer protein” controlling cellular homeostasis. We demonstrate that binding of hemin to TCTP is mediated by a conserved His-containing motif (His⁷⁶His⁷⁷) followed by dimerization, an event that involves ligand-mediated conformational changes and that is necessary to trigger TCTP''s cytokine-like activity. Mutation in both His residues to Ala prevents hemin from binding and abrogates oligomerization, suggesting that the ligand site localizes at the interface of the oligomer. Unlike heme, binding of Ca²⁺ ligand to TCTP does not alter its monomeric state; although, Ca²⁺ is able to destabilize an existing TCTP dimer created by hemin addition. In agreement with TCTP''s proposed buffer function, ligand binding occurs at high concentration, allowing the “buffer” condition to be dissociated from TCTP''s role as a component of signal transduction mechanisms.     

Binding of Translationally Controlled Tumour Protein to the N-Terminal Domain of HDM2 Is Inhibited by Nutlin-3

Translationally Controlled Tumour Protein (TCTP), a highly conserved protein present in all eukaryotic organisms, has a number of intracellular and extracellular functions including an anti-apoptotic role. TCTP was recently shown to interact with both p53 and HDM2, inhibiting auto-ubiquitination of the latter and thereby promoting p53 degradation. In this study, we further investigated the interaction between TCTP and HDM2, mapping the reciprocal binding sites of TCTP and HDM2. TCTP primarily interacts with the N-terminal, p53-binding region of HDM2 through its highly basic domain 2. Furthermore, we discovered that Nutlin-3, a small molecule known to promote apoptosis and cell cycle arrest by blocking binding between HDM2 and p53, has a similar inhibitory effect on the interaction of HDM2 and TCTP. This result may provide an additional explanation of how Nutlin-derived compounds currently in clinical trials function to promote apoptosis in cancer cells.     

TCTP is a critical survival factor that protects cancer cells from oxidative stress-induced cell-death

The translationally controlled tumor protein (TCTP) displays growth-promoting and antiapoptotic properties. To gain information on the role of TCTP in cancer disease, we studied the modulation of TCTP and cell survival under stress conditions on tumor cell lines of different origins. When cancer cells were exposed to a mild oxidative stress, such low doses of Arsenic trioxide (ATO) or hydrogen peroxide (H₂O₂), up-regulation of TCTP was observed in cells survived to the treatment. Differently, a strong oxidative hit provided by ATO combined with glutathione (GSH) depletion or condition of glucose deprivation caused a down-modulation of TCTP followed by cell death.Clones with a forced expression of TCTP or with silenced TCTP were obtained from the breast cancer cell line MDA-MB-231. The sensitivity to oxidative stress was strongly enhanced in down-modulated TCTP cells while decreasing in cells with high levels of TCTP.Together these results indicate that TCTP is a survival factor that protects cancer cells from oxidative stress-induced cell-death. We propose TCTP as a “stress hallmark” that may be exploited as a therapeutic target to decrease the resistance of cancer cells to anticancer therapy.     

TCTP Is an Androgen-Regulated Gene Implicated in Prostate Cancer

TCTP has been implicated in a plethora of important cellular processes related to cell growth, cell cycle progression, malignant transformation and inhibition of apoptosis. In addition to these intracellular functions, TCTP has extracellular functions and plays an important role in immune cells. TCTP expression was previously shown to be deregulated in prostate cancer, but its function in prostate cancer cells is largely unknown. Here we show that TCTP expression is regulated by androgens in LNCaP prostate cancer cells in vitro as well as human prostate cancer xenografts in vivo. Knockdown of TCTP reduced colony formation and increased apoptosis in LNCaP cells, implicating it as an important factor for prostate cancer cell growth. Global gene expression profiling in TCTP knockdown LNCaP cells showed that several interferon regulated genes are regulated by TCTP, suggesting that it may have a role in regulating immune function in prostate cancer. In addition, recombinant TCTP treatment increased colony formation in LNCaP cells suggesting that secreted TCTP may function as a proliferative factor in prostate cancer. These results suggest that TCTP may have a role in prostate cancer development.     

Inhibition of Na,K-ATPase-suppressive activity of translationally controlled tumor protein by sorting nexin 6

Translationally controlled tumor protein (TCTP) has both extra- and intracellular functions. Our group recently reported that TCTP interacts with Na,K-ATPase and suppresses its activity. Our studies led to the identification of sorting nexin 6 (SNX6) which binds with TCTP as a potential negative regulator of TCTP. SNX6 does not interact directly with any cytoplasmic domains of Na,K-ATPase. However, when overexpressed, it restores the Na,K-ATPase activity suppressed by TCTP. This was confirmed by measurements of purified plasma membrane Na,K-ATPase activity after incubation with recombinant TCTP and SNX6. SNX6 alone has no effect on Na,K-ATPase activity, but activates Na,K-ATPase via inhibition of TCTP. Inhibition of endogenous TCTP by the overexpression of SNX6 or knockdown of TCTP expression by siTCTP increased Na,K-ATPase activity above the basal level. The interaction between SNX6 and TCTP thus appears to regulate Na,K-ATPase activity.     

Molecular cloning,expression analysis and chromosome localization of the <Emphasis Type="Italic">Tpt1</Emphasis> gene coding for the pig translationally controlled tumor protein (TCTP)

This work describes the cloning and structural analysis of a Tpt1 cDNA coding for the porcine translationally controlled tumor protein (TCTP) molecule and its expression in porcine cells and tissues. Pig Tpt1 cDNA is 842-pb long that displays typical features of translationally controlled mRNAs, including a 5′-UTR containing a 5′-terminal oligopyrimidine tract (5′-TOP), and a 3′-UTR with a high CG-content and one AU rich element (ARE). Both 5′-UTR and 3′-UTR are highly conserved when they are compared with those of other mammals. The pig Tpt1 cDNA contains a 516-b open reading frame that encodes a predicted TCTP protein composed of 172 amino acids that exhibits extensive conservation compared with TCTP sequences from other species and a common structural feature with all the other TCTP proteins analyzed in mammals. Expression analysis demonstrated that Tpt1 mRNA is ubiquitously expressed in normal porcine tissues and cells, showing a higher expression in spleen, lymph nodes and lung, and a lower one in skin and heart. The pig Tpt1 gene localizes on the porcine chromosome 11, region p11.     

Osteoclastogenic activity of translationally-controlled tumor protein (TCTP) with reciprocal repression of p21

Translationally-controlled tumor protein (TCTP) plays a role in a number of cellular processes, but there is limited information about its function in cell differentiation. Previous observations of a twofold induction of TCTP mRNA during osteoclast differentiation prompted us to investigate its involvement in osteoclast differentiation. The osteoclastogenicity of TCTP gradually expressed during osteoclast differentiation was confirmed in mouse and human cells using loss-of-function studies and TCTP heterogeneous mice and transgenic mice. Higher expression ratios of TCTP to p21 could represent TCTP-mediated phenotypic induction of osteoclast differentiation accompanied by p21 down-regulation, attenuating the proliferation of osteoclast precursor cells.     

Proton Pump Inhibitors Exert Anti-Allergic Effects by Reducing TCTP Secretion

Background

Extracellular translationally controlled tumor protein (TCTP) is known to play a role in human allergic responses. TCTP has been identified outside of macrophages, in activated mononuclear cells, and in biological fluids from allergic patients. Even TCTP devoid of signal sequences, is secreted to extracellular environment by an yet undefined mechanism. This study is aimed at understanding the mechanism of TCTP release and its regulation. A secondary goal is to see if inhibitors of TCTP release can serve as potential anti-allergic asthmatic drugs.

Methodology/Principal Findings

Using Western blotting assay in HEK293 and U937 cells, we found that TCTP secretion is reduced by omeprazole and pantoprazole, both of which are proton pump inhibitors. We then transfected HEK293 cells with proton pump expression vectors to search for the effects of exogeneously overexpressed H⁺/K⁺-ATPase on the TCTP secretion. Based on these in vitro data we checked the in vivo effects of pantoprazole in a murine model of ovalbumin-induced allergy. Omeprazole and pantoprazole reduced TCTP secretion from HEK293 and U937 cells in a concentration-dependent fashion and the secretion of TCTP from HEK293 cells increased when they over-expressed H⁺/K⁺-ATPase. In a murine model of ovalbumin-induced allergy, pretreatment with pantoprazole reduced infiltration of inflammatory cells, increased goblet cells, and increased TCTP secretion induced by OVA challenge.

Conclusion

Since Omeprazole and pantoprazole decrease the secretion of TCTP which is associated with the development of allergic reaction, they may have the potential to serve as anti-allergic (asthmatic) drugs.     

Identification of anti-allergic effect of Clonorchis sinensis-derived protein venom allergen-like proteins (CsVAL)

Previous studies demonstrated that Clonochis sinensis-derived crude antigens suppress development of allergic responses. We investigated the effects of C. sinensis venom allergen-like (CsVAL) proteins on immune-modulating activities in allergic inflammatory response. Using RBL-2H3 rat mast cells, we demonstrated that CsVAL inhibits antigen-induced β-hexosaminidase release from immunoglobulin E-sensitized RBL-2H3 cells, and this inhibitory activity occurs by suppressing Lyn phosphorylation and intracellular reactive oxygen species production. In addition, CsVAL peptide treatment inhibits activation of protein kinase C-α and extracellular signal-regulated kinase 1/2, which are involved in degranulation of immunoglobulin E-sensitized mast cells. Furthermore, immunization with CsVAL suppressed development of skin inflammation by assessing ear thickness and cutaneous infiltration by eosinophils and mast cells in oxazolone-induced contact hypersensitivity in vivo mouse model. These results suggest that CsVAL is a promising candidate as an effective mast cell inhibitor for allergic and inflammatory diseases.     

Novel translationally controlled tumor protein homologue in the buccal gland secretion of Lampetra japonica

We have cloned a homologue of the translationally controlled tumor protein (TCTP) from the buccal gland of Lampetra japonica according to information from a cDNA library and primary analysis of expressed sequence tags. Sequence analysis of L. japonica TCTP showed that it had two signature regions of high sequence homology termed TCTP-1 and TCTP-2, respectively. TCTP is highly conserved in evolution. It showed more than 40% identification similarities with parasite TCTPs that had effect on immune responses of host. Phylogeny of 31 TCTP sequences showed that lamprey was closer to jawed vertebrates than to Amphioxus and was a sister group of gnathostomes. TCTP gene from L. japonica was expressed in a pET23b vector and purified by using His Bind affinity chromatography. Polyclonal antibody to recombinant protein was generated in New Zealand Rabbit. Immunoblot analysis to localize the recombinant protein in buccal gland secretion proves that recombinant TCTP is a secretion protein, which may be secreted through a non-classical secretion pathway. A characterization study shows that recombinant TCTP has histamine-releasing function in vitro. It mediated histamine release from rat basophilic leukemia (RBL-2H3) cells. TCTP links both the innate and the adaptive immune responses by modulating the secretion of cytokines from mast cells, basophils, eosinophils, and T and B lymphocytes. These may indicate a potential role of TCTP in the inflammatory process and immune regulation between L. japonica and host.     

Quantitative N-terminal analysis of fibrinogen-fibrin-related antigen [FR antigen] from human plasma.

Structure-activity studies have been performed on a series of naturally occurring and 'tailor-made' polypeptides, by measurement of ability to induce selective histamine release from normal rat peritoneal mast cells in vitro. Compounds investigated include corticotropin and melittin derivatives, mast-cell-degranulating peptide from bee venom, polymyxin B, bradykinin and various synthetic poly(amino acids) and short-chain peptides. It was confirmed that a cluster of four basic residues (lysine or arginine) was optimal for histamine release by corticotropin and melittin polypeptides, provided that the C-terminal carboxyl group was substituted (by, for instance, amidation). In contrast, the presence of a free C-terminal carboxyl group or nearby dicarboxylic acid residues led to a considerable diminution in histamine-releasing activity. Likewise, polypeptides comprised essentially of acidic amino acids were inactive. On the basis of these observations it has been possible to predict that synthetic peptides comprising a particular sequence within the Fc region of human immunoglobulin E, the immunoglobulin class particularly involved in mediation of allergic reactions of the immediate type, would possess potent histamine-releasing activity when similarly made to react with normal rat mast cells. The further study of such a structure should throw new light on the molecular basis of allergen-antibody triggering of mast cells.