受翻译调节的肿瘤蛋白的结构与功能

受翻译调节的肿瘤蛋白（translationally controlled tumor protein, TCTP）是一种普遍存在并且大量表达的蛋白,在进化上高度保守,与其它任何蛋白家族均未显示出明显的序列同源性.该家族的蛋白基本上都具有TCTP1和TCTP2两个特征结构区.TCTP与Mss4/Dss4(mammalian suppressor of Sec4)蛋白家族结构相似,二者构成结构超家族.TCTP的合成受到钙、真核翻译起始因子eIF4E(eukaryotic translation initiation factor 4E)和双链RNA依赖的蛋白激酶(dsRNA dependent protein kinase,PKR)的调节.具有与钙结合,与微管蛋白结合,抗细胞凋亡,抑制翻译,促进组胺释放等生物学活性.另外,它还可作为肿瘤逆转的靶标.系统发育分析提示,在真核细胞进化中, TCTP的直向同源基因起源于1.0×109年前.本文对TCTP的分子特点, 生物学功能及其研究现状进行了综述.     

猪TCTP基因的表达规律及其对脂肪细胞分化的影响

翻译控制肿瘤蛋白（TCTP, translationally controlled tumor protein）是一类广泛存在各种生物、序列高度保守的蛋白,最初认为TCTP是一类生长相关蛋白,近年研究发现TCTP可能具有非常重要的生物学功能.本研究通过高通量测序(Solexa)技术、实时定量PCR (RT qPCR)对瘦肉型和脂肪型猪不同生长阶段脂肪组织、脂肪细胞中TCTP的表达规律进行了研究,采用siRNA技术,沉默TCTP,研究了其对脂肪细胞分化的影响.结果表明：TCTP在瘦肉型猪脂肪组织中的mRNA表达量显著高于脂肪型猪（P＜0.01）|在不同日龄猪脂肪组织中,TCTP的mRNA表达量随着日龄增长而降低|在不同组织中的检测结果发现,TCTP在心、肝、肾、肌肉和脂肪中有较高的表达,肺和脾中表达量较低|TCTP的mRNA表达量在猪前体脂肪细胞增殖过程中逐渐增高,在分化阶段逐渐下降|沉默TCTP促进了脂肪细胞的分化,引起了PPARγ、C/EBPα、SREBP 1c的显著升高（P ＜0.01）.以上研究发现,TCTP在脂肪沉积过程中可能具有抑制作用,为进一步研究肥胖关键基因调控机制提供科学依据.     

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.     

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.     

Biochemical characterisation of TCTP questions its function as a guanine nucleotide exchange factor for Rheb

Translationally controlled tumour protein (TCTP) is involved in malignant transformation and regulation of apoptosis. It has been postulated to serve as a guanine nucleotide exchange factor for the small G-protein Rheb. Rheb functions in the PI3 kinase/mTOR pathway. The study presented here was initiated to characterise the interaction between TCTP and Rheb biochemically. Since (i) no exchange activity of TCTP towards Rheb could be detected in vitro, (ii) no interaction between TCTP and Rheb could be detected by NMR spectroscopy, and (iii) no effect of TCTP depletion in cells on the direct downstream targets of Rheb could be observed in vivo, this study shows that TCTP is unlikely to be a guanine nucleotide exchange factor for Rheb.     

Overexpression of Arabidopsis translationally controlled tumor protein gene AtTCTP enhances drought tolerance with rapid ABA-induced stomatal closure

Translationally controlled tumor protein (TCTP), also termed P23 in human, belongs to a family of calcium- and tubulin-binding proteins, and it is generally regarded as a growth-regulating protein. Recently, Arabidopsis TCTP (AtTCTP) has been reported to function as an important growth regulator in plants. On the other hand, plant TCTP has been suggested to be involved in abiotic stress signaling such as aluminum, salt, and water deficit by a number of microarray or proteomic analyses. In this study, the biological functions of AtTCTP were investigated by using transgenic Arabidopsis plants overexpressing AtTCTP. Interestingly, AtTCTP overexpression enhanced drought tolerance in plants. The expression analysis showed that AtTCTP was expressed in guard cells as well as in actively growing tissues. Physiological studies of the overexpression lines showed increased ABA- and calcium-induced stomatal closure ratios and faster stomatal closing responses to ABA. Furthermore, in vitro protein-protein interaction analysis confirmed the interaction between AtTCTP and microtubules, and microtubule cosedimentation assays revealed that the microtubule binding of AtTCTP increased after calcium treatment. These results demonstrate that the overexpression of AtTCTP confers drought tolerance to plants by rapid ABA-mediated stomatal closure via the interaction with microtubules in which calcium binding enhances the interaction. Collectively, the present results suggest that the plant TCTP has molecular properties similar to animal TCTPs, such as tubulin- and calcium-binding, and that it functions in ABA-mediated stomatal movement, in addition to regulating the growth of plants.     

TCTP protects from apoptotic cell death by antagonizing bax function

Translationally controlled tumor protein (TCTP) is a potential target for cancer therapy. It functions as a growth regulating protein implicated in the TSC1-TSC2 -mTOR pathway or a guanine nucleotide dissociation inhibitor for the elongation factors EF1A and EF1Bbeta. Accumulating evidence indicates that TCTP also functions as an antiapoptotic protein, through a hitherto unknown mechanism. In keeping with this, we show here that loss of tctp expression in mice leads to increased spontaneous apoptosis during embryogenesis and causes lethality between E6.5 and E9.5. To gain further mechanistic insights into this apoptotic function, we solved and refined the crystal structure of human TCTP at 2.0 A resolution. We found a structural similarity between the H2-H3 helices of TCTP and the H5-H6 helices of Bax, which have been previously implicated in regulating the mitochondrial membrane permeability during apoptosis. By site-directed mutagenesis we establish the relevance of the H2-H3 helices in TCTP's antiapoptotic function. Finally, we show that TCTP antagonizes apoptosis by inserting into the mitochondrial membrane and inhibiting Bax dimerization. Together, these data therefore further confirm the antiapoptotic role of TCTP in vivo and provide new mechanistic insights into this key function of TCTP.     

Structure of TCTP reveals unexpected relationship with guanine nucleotide-free chaperones

The translationally controlled tumor-associated proteins (TCTPs) are a highly conserved and abundantly expressed family of eukaryotic proteins that are implicated in both cell growth and the human acute allergic response but whose intracellular biochemical function has remained elusive. We report here the solution structure of the TCTP from Schizosaccharomyces pombe, which, on the basis of sequence homology, defines the fold of the entire family. We show that TCTPs form a structural superfamily with the Mss4/Dss4 family of proteins, which bind to the GDP/GTP free form of Rab proteins (members of the Ras superfamily) and have been termed guanine nucleotide-free chaperones (GFCs). Mss4 also acts as a relatively inefficient guanine nucleotide exchange factor (GEF). We further show that the Rab protein binding site on Mss4 coincides with the region of highest sequence conservation in the TCTP family. This is the first link to any other family of proteins that has been established for the TCTP family and suggests the presence of a GFC/GEF at extremely high abundance in eukaryotic cells.     

Identification of the interaction between the human recombinant histamine releasing factor/translationally controlled tumor protein and elongation factor-1 delta (also known as eElongation factor-1B beta)

The human recombinant histamine releasing factor (HrHRF), also known as translationally controlled tumor protein (TCTP), p23 and fortilin, has been described to have both extra- and intracellular functions. To elucidate an extra- or intracellular role for HrHRF, we used the yeast two-hybrid system with HrHRF as the bait and a Jurkat T cell library. We isolated a partial cDNA clone of the human elongation factor-1 delta (EF-1delta) encoding for amino acids 12 to 281. This interaction was confirmed by co-immunoprecipitation experiments. Previously, both HrHRF and EF-1delta have been isolated and identified in association with malignancy in numerous studies. EF-1delta is part of the EF-1 complex responsible for kinetic proofreading in protein synthesis. Additionally, DNA microarray data classifies TCTP (HrHRF) as co-regulated with ribosomal proteins and recent structural analysis of TCTP (HrHRF) relates it to a guanine nucleotide-free chaperone. Our findings of an interaction between HrHRF and EF-1delta taken with some of the recently published information concerning the TCTP (HrHRF) mentioned above suggest a possible intracellular role for TCTP/HrHRF.     

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.     

Solution structure and mapping of a very weak calcium-binding site of human translationally controlled tumor protein by NMR

Human translationally controlled tumor protein (TCTP) is a growth-related, calcium-binding protein. We determined the solution structure and backbone dynamics of human TCTP, and identified the calcium-binding site of human TCTP using multi-dimensional NMR spectroscopy. The overall structure of human TCTP has a rather rigid well-folded core and a very flexible long loop connected by a short two-strand β-sheet, which shows a conserved fold in the TCTP family. The C-terminal portions of loop L_α3β8 and strand β9 and the N-terminal region of strand β8 may form a calcium-binding site in the human TCTP structure, which is largely conserved in the sequence alignment of TCTPs. The K_d value for the calcium binding is 0.022-0.025 M indicating a very weak calcium-binding site.     

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.     

Conformational Ensemble and Biological Role of the TCTP Intrinsically Disordered Region: Influence of Calcium and Phosphorylation

The translationally controlled tumor protein (TCTP) is a multifunctional protein that may interact with many other biomolecules, including itself. The experimental determinations of TCTP structure revealed a folded core domain and an intrinsically disordered region, which includes the first highly conserved TCTP signature, but whose role in the protein functions remains to be elucidated. In this work, we combined NMR experiments and MD simulations to characterize the conformational ensemble of the TCTP intrinsically disordered loop, in the presence or not of calcium ions and with or without the phosphorylation of Ser46 and Ser64. Our results show that these changes in the TCTP electrostatic conditions induce significant shifts of its conformational ensemble toward structures more or less extended in which the disordered loop is pulled away or folded against the core domain. Particularly, these conditions impact the transient contacts between the two highly conserved signatures of the protein. Moreover, both experimental and theoretical data show that the interface of the non-covalent TCTP dimerization involves its second signature which suggests that this region might be involved in protein–protein interaction. We also show that calcium hampers the formation of TCTP dimers, likely by favoring the competitive binding of the disordered loop to the dimerization interface. All together, we propose that the TCTP intrinsically disordered region is involved in remodeling the core domain surface to modulate its accessibility to its partners in response to a variety of cellular conditions.     

Growth performance of two Indian major carps, catla (Catla catla) and rohu (Labeo rohita) fed diets containing different levels of Spirulina platensis

The influence of Spirulina platensis meal on the growth and carcass composition of two Indian major carps, catla, Catla catla and rohu, Labeo rohita was investigated in a 90-day culture trial. Four experimental diets were used and Spirulina replaced fish meal protein from the standard diet at 25%, 50%, 75% and 100% levels. There was no significant difference in the final weight attained by catla at all levels of Spirulina incorporation as compared to the fish-meal-based control diet. However, the replacement of fish meal by more than 25% Spirulina resulted in significantly superior growth of rohu. The specific growth rate and protein efficiency ratio recorded in rohu improved with higher levels of Spirulina inclusion, while in catla they did not differ significantly from the control treatment. In both the species, the digestibility of dry matter, protein and fat was found to improve marginally with increasing levels of Spirulina incorporation. The carcass composition showed an inverse relationship between protein and fat deposition. In general, fish fed with Spirulina diets had a significantly higher percentage of fat. The study demonstrated the usefulness of Spirulina for partial or complete replacement of fish meal in the diets of catla and rohu.     

Characterization of TCTP, the Translationally Controlled Tumor Protein, from Arabidopsis thaliana

The translationally controlled tumor protein (TCTP) is an important component of the TOR (target of rapamycin) signaling pathway, the major regulator of cell growth in animals and fungi. TCTP acts as the guanine nucleotide exchange factor of the Ras GTPase Rheb that controls TOR activity in Drosophila melanogaster. We therefore examined the role of Arabidopsis thaliana TCTP in planta. Plant TCTPs exhibit distinct sequence differences from nonplant homologs but share the key GTPase binding surface. Green fluorescent protein reporter lines show that Arabidopsis TCTP is expressed throughout plant tissues and developmental stages with increased expression in meristematic and expanding cells. Knockout of TCTP leads to a male gametophytic phenotype with normal pollen formation and germination but impaired pollen tube growth. Silencing of TCTP by RNA interference slows vegetative growth; leaf expansion is reduced because of smaller cell size, lateral root formation is reduced, and root hair development is impaired. Furthermore, these lines show decreased sensitivity to an exogenously applied auxin analog and have elevated levels of endogenous auxin. These results identify TCTP as an important regulator of growth in plants and imply a function of plant TCTP as a mediator of TOR activity similar to that known in nonplant systems.     

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.     

小麦TCTP基因的克隆及白粉菌诱导下的表达

翻译控制肿瘤蛋白(translationally controlled tumor protein,TCTP)最初在鼠肿瘤细胞中被发现,研究表明TCTP广泛存在于动植物细胞中,并具有多种生物学功能。本研究用RT PCR和RACE技术在抗白粉病栽培小麦Brock中克隆了一个TCTP基因,该基因全长766 bp,推测编码一个168个氨基酸的多肽。ScanProsite分析表明,该多肽链具有2个TCTP特征结构区（TCTP1和TCTP2）和7个可能的功能位点。表达半定量分析发现,该基因受华北地区流行的优势小种15号白粉菌诱导,且随着诱导时间的增加其表达量增加。本研究将可能在小麦白粉病抗性研究领域开辟新的研究思路。