Jeehye Maeng

A protein transduction domain located at the NH2-terminus of human translationally controlled tumor protein for delivery of active molecules to cells.

Protein transduction domains (PTDs) are small peptides, able to penetrate biological membranes and deliver various types of cargo both in vitro and in vivo. Because use of PTDs originating from viral origins resulted in undesired effects, PTDs originating from non-viral origins are needed. Here, we report that a 10-amino acid peptide (MIIYRDLISH) derived from the NH(2)-terminus of human translationally controlled tumor protein (TCTP) functions as a PTD. This peptide was internalized through lipid raft-dependent endocytosis and partial macropinocytosis, and did not enter lysosome and nucleus. Beta-galactosidase fused to TCTP-PTD, when injected into mice, was efficiently delivered to liver, kidney, spleen, heart, and lungs of the animals. Preincubation of TCTP-PTD with adenovirus increased adenoviral mediated-gene expression in cells and also improved immune response to intranasally administered adenovirus expressing the triple repeat of G glycoprotein of respiratory syncytial virus (RSV), rAd/3×G. These findings suggest that TCTP-PTD might overcome the limitations of polycation-mediated transduction and serve as an efficient vehicle for drug delivery.

Interaction of translationally controlled tumor protein with Apaf-1 is involved in the development of chemoresistance in HeLa cells.

BackgroundTranslationally controlled tumor protein (TCTP), alternatively called fortilin, is believed to be involved in the development of the chemoresistance of tumor cells against anticancer drugs such as etoposide, taxol, and oxaliplatin, the underlying mechanisms of which still remain elusive.MethodsCell death analysis of TCTP-overexpressing HeLa cells was performed following etoposide treatment to assess the mitochondria-dependent apoptosis. Apoptotic pathway was analyzed through measuring the cleavage of epidermal growth factor receptor (EGFR) and phospholipase C-γ (PLC-γ), caspase activation, mitochondrial membrane perturbation, and cytochrome c release by flow cytometry and western blotting. To clarify the role of TCTP in the inhibition of apoptosome, in vitro apoptosome reconstitution and immunoprecipitation was used. Pull-down assay and silver staining using the variants of Apaf-1 protein was applied to identify the domain that is responsible for its interaction with TCTP.ResultsIn the present study, we confirmed that adenoviral overexpression of TCTP protects HeLa cells from cell death induced by cytotoxic drugs such as taxol and etoposide. TCTP antagonized the mitochondria-dependent apoptotic pathway following etoposide treatment, including mitochondrial membrane damage and resultant cytochrome c release, activation of caspase-9, and -3, and eventually, the cleavage of EGFR and PLC-γ. More importantly, TCTP interacts with the caspase recruitment domain (CARD) of Apaf-1 and is incorporated into the heptameric Apaf-1 complex, and that C-terminal cleaved TCTP specifically associates with Apaf-1 of apoptosome in apoptosome-forming condition thereby inhibiting the amplification of caspase cascade.ConclusionsTCTP protects the cancer cells from etoposide-induced cell death by inhibiting the mitochondria-mediated apoptotic pathway. Interaction of TCTP with Apaf-1 in apoptosome is involved in the molecular mechanism of TCTP-induced chemoresistance. These findings suggest that TCTP may serve as a therapeutic target for chemoresistance in cancer treatment.

Dimerization of TCTP and its clinical implications for allergy

Following the detection of histamine-releasing activity (HRA) in the supernatants of peripheral blood mononuclear cell cultures, research efforts were directed at characterizing the source of this activity, mostly focusing, on IgE-dependent histamine-releasing factors (HRFs). HRF is now variously called translationally controlled tumor protein (TCTP), p21, p23, and fortilin. TCTP exhibits cytokine-like functions including release of histamine, induction of TH2 cytokines and chemoattractants, augmentation of B cell proliferation, and immunoglobulin production during late phase allergic inflammation. Because of its association with the allergic status of patients, TCTP emerged as a potential key agent in the modulation of allergic diseases. Several lines of evidence suggest that TCTP exhibits its cytokine-like functions only after it is modified by the proteases, altered oxidant-antioxidant balance and immunoglobulin E, present in the inflamed sites. This review will try to show that dimerization is the critical modification of TCTP if not the only modification, responsible for its cytokine-like activity causing allergic diseases.

Design and evaluation of variants of the protein transduction domain originated from translationally controlled tumor protein

Protein transduction domains (PTDs) have been successfully employed to deliver therapeutic cargos both in vitro and in vivo because of their cellular penetrating ability. We previously reported that a 10-amino acid peptide (MIIYRDLISH) derived from the NH(2)-terminus of human translationally controlled tumor protein (TCTP) functions as a PTD. TCTP-PTD is quite different from other well-known PTDs in its hydrophobic composition and structural character, and the sequence requirements for transduction remain unknown. To identify the role of each residue, we compared the cellular uptake of various deletion mutants and Ala substituents of TCTP-PTD. The results showed that the amino terminal residues and the hydrophobic nature of the peptide, with a minimal length of nine residues, were necessary for transduction. Based on the elucidated sequence requirements, we designed and evaluated variants to improve the efficiency and solubility through sequential modification of TCTP-PTD. During the optimization process, we also delineated the contribution of residues and the advantageous composition of sequences for cellular uptake.

Cellular uptake mechanism of TCTP-PTD in human lung carcinoma cells.

We reported previously that human translationally controlled tumor protein (TCTP) contains, at its NH2-terminus, a protein transduction domain (PTD), which we called TCTP-PTD, with the amino acid sequence MIIYRDLISH. In this report we describe how TCTP-PTD penetrates A549 human lung cancer cell membranes and promotes protein internalization. Cellular uptake of fluorescent TCTP-PTD and a recombinant fusion protein consisting of TCTP-PTD and GFP (green fluorescent protein) was analyzed by confocal fluorescence microscopy and flow cytometry. Inhibitor assays using several agents that perturb the internalization process revealed that TCTP-PTD transduces the cells partly via lipid-raft/caveola-dependent endocytosis and partly by macropinocytosis in a dynamin/actin/microtubule-dependent pathway. To trace the pathway followed by the penetration of TCTP-PTD, the localization of PTDs was investigated in the lipid-raft, subcellular, and ER fractions. We found that, after entry, TCTP-PTD is localized in the cytoplasm and cytoskeleton, but not in the nucleus, and is transported into endoplasmic reticulum (ER). Expression levels of caveolin-1 in A549 and HeLa cells are different, and these differences appear to contribute to the sensitivity of TCTP-PTD uptake inhibition, against lipid-raft depleter, nystatin. This elucidation of the underlying mechanism of TCTP-PTD translocation may help the design of approaches that employ TCTP-PTD in the cellular delivery of bioactive molecules.

Insulin Induces Phosphorylation of Serine Residues of Translationally Controlled Tumor Protein in 293T Cells

Insulin induces the activation of Na,K-ATPase while translationally controlled tumor protein (TCTP) inhibits this enzyme and the associated pump activity. Because binding of insulin with its membrane receptor is known to mediate the phosphorylation of multiple intracellular proteins, phosphorylation of TCTP by insulin might be related to the sodium pump regulation. We therefore examined whether insulin induces TCTP phosphorylation in embryonic kidney 293T cells. Using immunoprecipitation and Western blotting, we found that insulin phosphorylates serine (Ser) residues of TCTP. Following fractionation of the insulin-treated cells into cytosol and membrane fractions, phosphorylated TCTP at its Ser residue (p-Ser-TCTP) was detected exclusively in the cytosolic part and not in the membrane fraction. Phosphorylation of TCTP reached maximum in about 10 min after insulin treatment in 293T cells. In studies of cell-type specificity of insulin-mediated phosphorylation of TCTP, insulin did not phosphorylate TCTP in HeLa cells. Computational prediction and immunoprecipitation using several constructs having Ser to Ala mutation at potential p-Ser sites of TCTP revealed that insulin phosphorylated the serine-9 and -15 residues of TCTP. Elucidations of how insulin-mediated TCTP phosphorylation promotes Na,K-ATPase activation, may offer potential therapeutic approaches to diseases associated with vascular activity and sodium pump dysregulation.

Transduction of translationally controlled tumor protein employing TCTP-derived protein transduction domain

Protein transduction domains (PTDs), which are cell-penetrating peptides, have been employed for delivery of various cargos. We previously showed that the N-terminal fragment of translationally controlled tumor protein functions as a PTD (TCTP-PTD) by as yet poorly understood mechanisms. In this study, we generated several green fluorescent protein (GFP)-tagged TCTP fusion proteins by conjugating a single PTD or tandem PTDs at the N-terminus, the C-terminus, and both termini and compared their transduction efficiencies in human lung adenocarcinoma A549 cells to determine whether the protein transducing function of TCTP depends on the location or the number of PTD moieties in the TCTP molecule. Fluorimetric analysis and Western blotting assays revealed that TCTP-GFP fusion protein containing one or two TCTP-PTDs at its N-terminus showed more efficient cellular entry than either the C-terminal TCTP-PTD or TCTP-PTD with PTDs located at both the N- and C-terminals. This study demonstrates the feasibility of transduction of TCTP target cells employing its TCTP-PTD by simple co-incubation with purified proteins.

Up-Regulation of Rhoa/Rho Kinase Pathway by Translationally Controlled Tumor Protein in Vascular Smooth Muscle Cells

Translationally controlled tumor protein (TCTP), a repressor for Na,K-ATPase has been implicated in the development of systemic hypertension, as proved by TCTP-over-expressing transgenic (TCTP-TG) mice. Aorta of TCTP-TG exhibited hypercontractile response compared to that of non-transgenic mice (NTG) suggesting dys-regulation of signaling pathways involved in the vascular contractility by TCTP. Because dys-regulation of RhoA/Rho kinase pathway is implicated in increased vascular contractility, we examined whether TCTP induces alterations in RhoA pathway in vascular smooth muscle cells (VSMCs). We found that TCTP over-expression by adenovirus infection up-regulated RhoA pathway including the expression of RhoA, and its downstream signalings, phosphorylation of myosin phosphatase target protein (MYPT-1), and myosin light chain (MLC). Conversely, lentiviral silencing of TCTP reduced the RhoA expression and Rho kinase signalings. Using immunohistochemical and Western blotting studies on aortas from TCTP-TG confirmed the elevated expression of RhoA and increase in p-MLC (phosphorylated MLC). In contrast, down-regulation of RhoA and p-MLC were found in aortas from heterozygous mice with deleted allele of TCTP (TCTP+/−). We conclude that up-regulation of TCTP induces RhoA-mediated pathway, and that TCTP-induced RhoA plays a role in the regulation in vasculature. Modulation of TCTP may offer a therapeutic target for hypertension and in vascular contractility dysfunction.

On the Mechanisms Underlying the Secretion and Export of TranslationallyControlled Tumor Protein/Histamine Releasing Factor (TCTP/HRF)

Numerous studies have demonstrated that TCTP/HRF is a unique cytokine, modulating the release of inflammatory mediators from various cell types including cells involved in allergic phenomena. Despite the absence of a leader sequence in its NH2-terminus, TCTP/HRF is regarded as a secreted protein found outside of cells as well as in fluids from allergic patients and parasitic organisms. Recent studies clarified several potential mechanisms leading to its secretion. For example, these studies showed that TCTP/HRF is exported from cells via a non-classical, endoplasmic reticulum (ER)/Golgi-independent mechanism associated with exosomal transport. TSAP6, a p53-inducible transmembrane protein, has been shown to enhance exosome production, and facilitate the secretion of TCTP/HRF into extracellular milieu. Additionally, H,K-ATPase also appears to play a role in the transport of TCTP/HRF, since inhibitors of H,K-ATPase also inhibit TCTP/HRF exit. The exact mechanisms involved in TCTP/HRF secretion has not yet emerged. Here we attempted to collate the available information in the current understanding of the mechanisms underlying the release of TCTP/HRF and of the factors that seem to influence these mechanisms.

Dimerized Translationally Controlled Tumor Protein-Binding Peptide Ameliorates Atopic Dermatitis in NC/Nga Mice

Our previous study showed that dimerized translationally controlled tumor protein (dTCTP) plays a role in the pathogenesis of allergic diseases, such as asthma and allergic rhinitis. A 7-mer peptide, called dTCTP-binding peptide 2 (dTBP2), binds to dTCTP and inhibits its cytokine-like effects. We therefore examined the protective effects of dTBP2 in house dust mite-induced atopic dermatitis (AD)-like skin lesions in Nishiki-nezumi Cinnamon/Nagoya (NC/Nga) mice. We found that topical administration of dTBP2 significantly reduced the AD-like skin lesions formation and mast cell infiltration in NC/Nga mice, similarly to the response seen in the Protopic (tacrolimus)-treated group. Treatment with dTBP2 also decreased the serum levels of IgE and reduced IL-17A content in skin lesions and inhibited the expression of mRNAs of interleukin IL-4, IL-5, IL-6, IL-13, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC) and thymic stromal lymphopoietin (TSLP). These findings indicate that dTBP2 not only inhibits the release of Th2 cytokine but also suppresses the production of proinflammatory cytokines in AD-like skin lesions in NC/Nga mice, by inhibiting TCTP dimer, in allergic responses. Therefore, dTCTP is a therapeutic target for AD and dTBP2 appears to have a potential role in the treatment of AD.