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.     

翻译调控肿瘤蛋白的结构、功能以及在肿瘤发展中的作用

翻译调控肿瘤蛋白(translational controlled tumor protein,TCTP),又称p23、组胺释放因子(histamine releasing factor,HRF)等,是一类在动植物中具有高度保守性和同源性的蛋白,主要介导细胞凋亡、细胞增殖与分化、细胞骨架重排、炎症反应等重要事件,与肿瘤的发生发展进程密切相关.针对TCTP的相关研究不仅有助于进一步了解各种肿瘤的生理病理周期,同时也提示其在寻找治愈肿瘤的方法中有望成为新的靶点.本文将对TCTP的结构、生物学功能以及在各种肿瘤中的作用进行综述.     

Translationally Controlled Tumor Protein Is a Novel Biological Target for Neurofibromatosis Type 1-associated Tumors

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease that predisposes individuals to develop benign neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs). Due to the lack of information on the molecular mechanism of NF1-associated tumor pathogenesis or biomarkers/therapeutic targets, an effective treatment for NF1 tumors has not been established. In this study, the novel NF1-associated protein, translationally controlled tumor protein (TCTP), was identified by integrated proteomics and found to be up-regulated via activated MAPK/PI3K-AKT signaling in response to growth factors in NF1-deficient Schwann cells. Immunohistochemical analysis of NF1-associated tumors revealed that the TCTP expression level correlated with tumorigenicity. In NF1-deficient MPNST cells, TCTP protein but not mRNA was down-regulated by NF1 GTPase-activating protein-related domain or MAPK/PI3K inhibitors, and this correlated with suppression of mammalian target of rapamycin (mTOR) signaling. mTOR inhibition by rapamycin also down-regulated TCTP protein expression, whereas knockdown or overexpression of TCTP suppressed or activated mTOR signaling, respectively, and affected cell viability. These results suggest that a positive feedback loop between TCTP and mTOR contributes to NF1-associated tumor formation. Last, the anti-tumor effect of artesunate, which binds to and degrades TCTP, was evaluated. Artesunate significantly suppressed the viability of MPNST cells but not normal Schwann cells, and the TCTP level inversely correlated with artesunate sensitivity. Moreover, combinational use of artesunate and rapamycin enhanced the cytotoxic effect on MPNST cells. These findings suggest that TCTP is functionally implicated in the progression of NF1-associated tumors and could serve as a biological target for their therapy.     

Dimerization of VirD2 Binding Protein Is Essential for Agrobacterium Induced Tumor Formation in Plants

The Type IV Secretion System (T4SS) is the only bacterial secretion system known to translocate both DNA and protein substrates. The VirB/D4 system from Agrobacterium tumefaciens is a typical T4SS. It facilitates the bacteria to translocate the VirD2-T-DNA complex to the host cell cytoplasm. In addition to protein-DNA complexes, the VirB/D4 system is also involved in the translocation of several effector proteins, including VirE2, VirE3 and VirF into the host cell cytoplasm. These effector proteins aid in the proper integration of the translocated DNA into the host genome. The VirD2-binding protein (VBP) is a key cytoplasmic protein that recruits the VirD2–T-DNA complex to the VirD4-coupling protein (VirD4 CP) of the VirB/D4 T4SS apparatus. Here, we report the crystal structure and associated functional studies of the C-terminal domain of VBP. This domain mainly consists of α-helices, and the two monomers of the asymmetric unit form a tight dimer. The structural analysis of this domain confirms the presence of a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) fold. Biophysical studies show that VBP is a dimer in solution and that the HEPN domain is the dimerization domain. Based on structural and mutagenesis analyses, we show that substitution of key residues at the interface disrupts the dimerization of both the HEPN domain and full-length VBP. In addition, pull-down analyses show that only dimeric VBP can interact with VirD2 and VirD4 CP. Finally, we show that only Agrobacterium harboring dimeric full-length VBP can induce tumors in plants. This study sheds light on the structural basis of the substrate recruiting function of VBP in the T4SS pathway of A. tumefaciens and in other pathogenic bacteria employing similar systems.     

The Plasmodium falciparum Translationally Controlled Tumor Protein (TCTP) Is Incorporated More Efficiently into B Cells than Its Human Homologue

Plasmodium falciparum secretes a homologue of the translationally controlled tumor protein (TCTP) into serum of infected individuals, although its role in pathogenesis or virulence is unknown. To determine the effect of P. falciparum TCTP on B cells as compared to human TCTP, fluorescently labeled proteins were incubated on primary cultures of mouse splenic B cells and analyzed by flow cytometry and confocal microscopy. Our results indicate that both recombinant proteins are incorporated into B cells, but differ significantly in their rate and percentage of incorporation, being significantly higher for P. falciparum TCTP. Furthermore, P. falciparum TCTP showed a lower B cell proliferative effect than human TCTP, suggesting a mechanism through which the former could interfere in the host''s immune response.     

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.     

Dimerization of Tetherin Is Not Essential for Its Antiviral Activity against Lassa and Marburg Viruses

Tetherin (also known as BST2, CD317 or HM1.24) has recently been reported to inhibit a wide range of viruses. However, the antiviral mechanism of action of tetherin has not been determined. Both ends of the tetherin molecule are associated with the plasma membrane and it forms a homodimer. Therefore, a model in which progeny virions are retained on the cell surface by dimer formation between tetherin molecules on the viral envelope and plasma membrane has been proposed as the antiviral mechanism of action of this molecule. To investigate this possibility, we examined the correlation between dimerization and antiviral activity of tetherin in Lassa and Marburg virus-like particle production systems using tetherin mutants deficient in dimer formation. However, the tetherin mutant with complete loss of dimerization activity still showed apparent antiviral activity, indicating that dimerization of tetherin is not essential for its antiviral activity. This suggests that tetherin retains progeny virions on the cell surface by a mechanism other than dimerization.     

Evolutionarily Conserved Binding of Translationally Controlled Tumor Protein to Eukaryotic Elongation Factor 1B

Translationally controlled tumor protein (TCTP) is an abundant protein that is highly conserved in eukaryotes. However, its primary function is still not clear. Human TCTP interacts with the metazoan-specific eukaryotic elongation factor 1Bδ (eEF1Bδ) and inhibits its guanine nucleotide exchange factor (GEF) activity, but the structural mechanism remains unknown. The interaction between TCTP and eEF1Bδ was investigated by NMR titration, structure determination, paramagnetic relaxation enhancement, site-directed mutagenesis, isothermal titration calorimetry, and HADDOCK docking. We first demonstrated that the catalytic GEF domain of eEF1Bδ is not responsible for binding to TCTP but rather a previously unnoticed central acidic region (CAR) domain in eEF1Bδ. The mutagenesis data and the structural model of the TCTP-eEF1Bδ CAR domain complex revealed the key binding residues. These residues are highly conserved in eukaryotic TCTPs and in eEF1B GEFs, including the eukaryotically conserved eEF1Bα, implying the interaction may be conserved in all eukaryotes. Interactions were confirmed between TCTP and the eEF1Bα CAR domain for human, fission yeast, and unicellular photosynthetic microalgal proteins, suggesting that involvement in protein translation through the conserved interaction with eEF1B represents a primary function of TCTP.     

Phylogenetic and Structural Analysis of Translationally Controlled Tumor Proteins

The translationally controlled tumor protein (TCTP) is conserved in all eukaryotes studied thus far. Recent evidence points to an important role for TCTP in the induction of cell proliferation in animals through an interaction with G proteins. TCTP may also constitute an intercellular secreted signal that modulates the immune response in the vertebrates. Because of its sequence conservation and ubiquity, the analysis of its amino acid sequence divergence between different taxa may provide insight into the structural constraints on the evolution of this protein. In the present study, we analyzed the phylogeny of TCTP sequences from a wide range of organisms and found that, with some exceptions, the groupings formed were consistent with the evolutionary history. Indeed, at the level of lower-order taxa, the groupings are in agreement with their established phylogeny, thus indicating that the substitution rates of the TCTP residues varied evenly between members of the same clade. Predicted three-dimensional structures of representative TCTPs, based on the reported 3D structure of Schizosaccharomyces pombe, indicated that these proteins are highly conserved among diverse taxonomic groups. However, analysis of the primary structure indicated subtle differences in the domain-forming pocket that potentially interacts with G proteins, particularly among Diplomonadidae, Apicomplexa, and other parasites of vertebrates. These differences support the notion that these specific TCTPs could block the normal immune response by acting as dominant negative mutants. Structural differences were also observed in a reported sequence of TCTP from Plasmodium knowlesi, in which the presence of an extra alpha-helix could also interfere in the interaction with G proteins.     

植物翻译控制肿瘤蛋白的分子结构特征与功能预测分析

翻译控制肿瘤蛋白(The translationally controlled tumor protein,TCTP)是一类广泛存在于动物、植物及酵母中的,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的蛋白.采用生物信息学的方法和工具对已在GenBank上注册的拟南芥(Arabidopsis thaliana)、牵牛(Ipomoea nil)、草莓(Fragaria x ananassa)、橡胶树(Heveabrasiliensis)、南瓜(Cucurbita maxima)、卷心菜(Brassica oleracea)的翻译控制肿瘤蛋白的核苷酸及氨基酸序列进行分析,并对其组成成分、转运肽、跨膜结构域、疏水性/亲水性、蛋白质的二级及三级结构、分子系统进化关系等进行预测和分析.结果 表明,该基因的全长包括5'、3'非翻译区和一个开放读码框,其编码的蛋白是一个无跨膜结构域,不具转运肽的亲水性蛋白,包括一个β-折叠核心区和一个主要的α-螺旋区,不规则卷曲散布于整个蛋白质中,具有TCTP-1和TCTP-2两个特征结构域.     

麻疯树Jc-Tctp1基因的同源性分析及时空表达模式鉴定

从麻疯树胚乳cDNA文库中获得了翻译调节肿瘤蛋白(translationally controlled tumor protein,TCTP)cDNA 序列,命名为 Jc-Tctp 1 (GenBank 登录号为EF091818).该序列全长1410 bp,开放阅读框由507个碱基组成.Jc-Tctp 1 编码的推测蛋白产物含有168个氨基酸残基,该蛋白具有典型的TCTP蛋白特点:由3个α螺旋组成α螺旋核心、4个β片层结构构成β片层核心,及微管结合结构域(MTB)和钙结合结构域(CaB).其推测氨基酸序列与巴西橡胶树(Hevea brasiliensis)、油棕(Elaeis guineensis)、大豆(Glycine max)、水稻(Oryza sativa,japonica cultivar-group)、黑杨(Populus trichocarpa)、番茄(Lycopersicon esculentum)、西加云杉(Picea sitchensis)、拟南芥(Arabidopsis thaliana)以及玉米(Zea may)的TCTP同源性依次为93%、90%、85%、84%、85%、84%、77%、80%和77%.经构建含Jc-TCTP1在内的植物TCTP蛋白分子进化树分析,发现单子叶植物并未按照其生物学分类的地位出现聚合.用半定量RT-PCR研究Jc-Tctp1 基因的表达模式,结果显示,其表达在转录水平上具有一定的组织和时间特异性,茎、I期胚乳、胚中最为丰富,而在Ⅱ期胚乳和花中表达最弱.     

TCTP在肝癌细胞增殖过程中的作用及机制研究

目的:研究翻译控制肿瘤蛋白(TCTP)在肝癌细胞增殖过程中的作用及相关机制。方法:通过western blot技术检测14对肝癌与癌旁组织中TCTP的蛋白表达水平。通过siRNA(small interference RNA)技术在肝癌细胞系SMMC-7721和BEL-7404中下调TCTP的表达,然后通过CCK-8实验、克隆形成实验和EdU实验观察下调TCTP对肝癌细胞增殖的影响。通过western blot技术分析TCTP促进肝癌发生这一过程中可能涉及的分子通路。结果:相比于对应的癌旁组织,TCTP在肝癌组织中显著高表达。用siRNA技术下调TCTP水平后能够明显抑制肝癌细胞的增殖能力。下调TCTP的表达之后,AKT和ERK蛋白的磷酸化水平也随之降低。结论:TCTP在肝癌组织中显著高表达,并且在肝癌细胞的增殖过程中发挥着极其重要作用,其作用机制可能与AKT和ERK通路的磷酸化激活有关。     

Dark-Induced Accumulation of mRNA for a Homolog of Translationally Controlled Tumor Protein (TCTP) in Pharbitis

A cDNA encoding a homolog of human translationally controlledtumor protein (TCTP) was isolated from cotyledons of the short-dayplant Pharbitis nil cv. Violet. The level of the correspondingmRNA increased gradually during darkness. This increase wasinhibited by end-of-day exposure to far-red light. (Received October 30, 1997; Accepted January 7, 1998)     

TCTP在辐射诱导胶质瘤细胞旁效应中的作用及机制研究

目的:研究肿瘤翻译控制蛋白(TCTP)在辐射诱导胶质瘤细胞旁效应中的作用及机制。方法:给予不同剂量的X射线照射U87、SHG44两种胶质瘤细胞,观察U87以及SHG44细胞的克隆形成率,并在给予最佳照射剂量后,通过Western Blot检测TCTP蛋白表达水平。将经过最佳X射线照射剂量的U87以及SHG44两种胶质瘤细胞与未经过辐射照射的细胞放在一起共培养,通过MTT实验检测胶质瘤细胞的增殖率,Western Blot检测共培养的胶质瘤细胞与经过辐射的胶质瘤细胞中Caspase3蛋白表达水平。结果:U87以及SHG44两种胶质瘤细胞的克隆形成率随着X射线照射剂量增加而显著性降低(P0.05),给予最佳X射线照射剂量后,与未经过X射辐射照射后的细胞相比,其TCTP蛋白表达水平明显升高(P0.05)。经过辐射照射与未经过辐射照射的胶质瘤细胞经过共培养后,与经过辐射的胶质瘤细胞相比,细胞的增殖率明显升高,同时共培养的胶质瘤细胞与经过辐射的胶质瘤细胞相比,Caspase3的蛋白表达明显降低(P0.05)。结论:TCTP的表达增高能够诱导未经过辐射的U87以及SHG44两种胶质瘤细胞的抗凋亡作用增强,其作用机制可能与Caspase3的表达降低有关。     

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.     

Translationally Controlled Tumor Protein,a Dual Functional Protein Involved in the Immune Response of the Silkworm,Bombyx mori

Insect gut immunity is the first line of defense against oral infection. Although a few immune-related molecules in insect intestine has been identified by genomics or proteomics approach with comparison to well-studied tissues, such as hemolymph or fat body, our knowledge about the molecular mechanism underlying the gut immunity which would involve a variety of unidentified molecules is still limited. To uncover additional molecules that might take part in pathogen recognition, signal transduction or immune regulation in insect intestine, a T7 phage display cDNA library of the silkworm midgut is constructed. By use of different ligands for biopanning, Translationally Controlled Tumor Protein (TCTP) has been selected. BmTCTP is produced in intestinal epithelial cells and released into the gut lumen. The protein level of BmTCTP increases at the early time points during oral microbial infection and declines afterwards. In vitro binding assay confirms its activity as a multi-ligand binding molecule and it can further function as an opsonin that promotes the phagocytosis of microorganisms. Moreover, it can induce the production of anti-microbial peptide via a signaling pathway in which ERK is required and a dynamic tyrosine phosphorylation of certain cytoplasmic membrane protein. Taken together, our results characterize BmTCTP as a dual-functional protein involved in both the cellular and the humoral immune response of the silkworm, Bombyx mori.     

Dimerization of Bacterial Diaminopimelate Epimerase Is Essential for Catalysis

Diaminopimelate (DAP) epimerase is involved in the biosynthesis of meso-DAP and lysine, which are important precursors for the synthesis of peptidoglycan, housekeeping proteins, and virulence factors in bacteria. Accordingly, DAP epimerase is a promising antimicrobial target. Previous studies report that DAP epimerase exists as a monomeric enzyme. However, we show using analytical ultracentrifugation, X-ray crystallography, and enzyme kinetic analyses that DAP epimerase from Escherichia coli exists as a functional dimer in solution and the crystal state. Furthermore, the 2.0-Å X-ray crystal structure of the E. coli DAP epimerase dimer shows for the first time that the enzyme exists in an open, active conformation. The importance of dimerization was subsequently probed by using site-directed mutagenesis to generate a monomeric mutant (Y268A). Our studies show that Y268A is catalytically inactive, thus demonstrating that dimerization of DAP epimerase is essential for catalysis. Molecular dynamics simulations indicate that the DAP epimerase monomer is inherently more flexible than the dimer, suggesting that dimerization optimizes protein dynamics to support function. Our findings offer insight into the development of novel antimicrobial agents targeting the dimeric antibiotic target DAP epimerase.     

A Leucine Zipper-Like Domain Is Essential for Dimerization and Encapsidation of Bluetongue Virus Nucleocapsid Protein VP4

The bluetongue virus (BTV) minor protein VP4, with molecular mass of 76 kDa, is one of the seven structural proteins and is located within the inner capsid of the virion. The protein has a putative leucine zipper near the carboxy terminus of the protein. In this study, we have investigated the functional activity of this putative leucine zipper by a number of approaches. The putative leucine zipper region (amino acids [aa] 523 to 551) was expressed initially as a fusion protein by using the pMAL vector of Escherichia coli, which expresses a maltose binding monomeric protein. The expressed fusion protein was purified by affinity chromatography, and its size was determined by gel filtration chromatography. Proteins of two sizes, 51 and 110 kDa, were recovered, one equivalent to the monomeric form and the other equivalent to the dimeric form of the fusion protein. To prove that the VP4-derived sequence was responsible for dimerization of this protein, a mutated fusion protein was created in which a VP4 leucine residue (at aa 537) within the zipper was replaced by a proline residue. Analyses of the mutated protein demonstrated that the single mutation indeed prevented dimerisation of the protein. The dimeric nature of VP4 was further confirmed by using purified full-length BTV-10 VP4 recovered from recombinant baculovirus-expressing BTV-10 VP4-infected insect cells. Using chemical cross-linking and gel filtration chromatography, we documented that the native VP4 indeed exists as a dimer in solution. Subsequently, Leu537 was replaced by either a proline or an alanine residue and the full-length mutated VP4 was expressed in the baculovirus system. By sucrose density gradient centrifugation and gel filtration chromatography, these mutant forms of VP4 were shown to lack the ability to form dimers. The biological significance of the dimeric forms of VP4 was examined by using a functional assay system, in which the encapsidation activity of VP4 into core-like particles (CLPs) was studied (H. LeBlois, T. French, P. P. C. Mertens, J. N. Burroughs, and P. Roy, Virology 189:757–761, 1992). We demonstrated conclusively that dimerization of VP4 was essential for encapsidation by CLPs.