Translationally Controlled Tumor Protein Genes Research Articles

Molecular Characterization and Activity Analysis of Promoters from Two Cucumber Translationally Controlled Tumor Protein Genes (CsTCTPs)

Aims: The aim of the paper was to isolate and characterize the promoters of two cucumber TCTP genes (CsTCTP1 and CsTCTP2) and evaluate their active domains.
 Study Design: CsTCTP1 and CsTCTP2 promoter activity were analyzed under treatments with different exogenous hormones.
 Place and Duration of Study: In 2017, these experiments were conducted in College of Bioscience and Biotechnology of Shenyang Agricultural University (Lab 240).
 Methodology: CsTCTPpro::GUS constructs were generated by using double digests method. Transient expression was mediated by Agrobacterium tumefaciens GV3101. Histochemical and Fluorometric GUS Assays were follow by the biochemical method.
 Results: Bioinformatics analysis revealed some hormone- and defense-related response elements. Histochemical and fluorometric GUS assays demonstrated that the 0.7-kb CsTCTP1 promoter (proT1-0.7kb) and 0.7-kb and 1.3-kb CsTCTP2 promoters (proT2-0.7kb and proT2-1.3kb) had strong transcriptional activity. In addition, we used exogenous hormones (abscisic acid [ABA], salicylic acid [SA], and ethylene [ETH]) for treatment. The results showed that proT1-0.7kb and proT2-1.3kb activity were upregulated in the ABA treatment group, suggesting that these promoter sequences may contain ABA-related response elements. However, in the SA and ETH treatment groups, the activity of all the promoter fragments of CsTCTP1 and CsTCTP2 declined to different degrees, suggesting that SA and ETH may have negative regulatory effects on CsTCTP1 and CsTCTP2 promoters.
 Conclusion: Taken together, these results suggest that the proT1-0.7kb promoter of CsTCTP1 and proT2-1.3kb promoter of CsTCTP2 may contain ABA-related response elements, and SA and ETH may have negative regulatory effects on the CsTCTP1 and CsTCTP2 promoters. This study will help to further understand the expression patterns and the regulatory mechanism of gene transcriptional regulation.

Comprehensive analysis of Translationally Controlled Tumor Protein (TCTP) provides insights for lineage-specific evolution and functional divergence.

BackgroundTranslationally controlled tumor protein (TCTP) is a conserved, multifunctional protein involved in numerous cellular processes in eukaryotes. Although the functions of TCTP have been investigated sporadically in animals, invertebrates, and plants, few lineage-specific activities of this molecule, have been reported. An exception is in Arabidopsis thaliana, in which TCTP (AtTCTP1) functions in stomatal closuer by regulating microtubule stability. Further, although the development of next-generation sequencing technologies has facilitated the analysis of many eukaryotic genomes in public databases, inter-kingdom comparative analyses using available genome information are comparatively scarce.MethodologyTo carry out inter-kingdom comparative analysis of TCTP, TCTP genes were identified from 377 species. Then phylogenetic analysis, prediction of protein structure, molecular docking simulation and molecular dynamics analysis were performed to investigate the evolution of TCTP genes and their binding proteins.ResultsA total of 533 TCTP genes were identified from 377 eukaryotic species, including protozoa, fungi, invertebrates, vertebrates, and plants. Phylogenetic and secondary structure analyses reveal lineage-specific evolution of TCTP, and inter-kingdom comparisons highlight the lineage-specific emergence of, or changes in, secondary structure elements in TCTP proteins from different kingdoms. Furthermore, secondary structure comparisons between TCTP proteins within each kingdom, combined with measurements of the degree of sequence conservation, suggest that TCTP genes have evolved to conserve protein secondary structures in a lineage-specific manner. Additional tertiary structure analysis of TCTP-binding proteins and their interacting partners and docking simulations between these proteins further imply that TCTP gene variation may influence the tertiary structures of TCTP-binding proteins in a lineage-specific manner.ConclusionsOur analysis suggests that TCTP has undergone lineage-specific evolution and that structural changes in TCTP proteins may correlate with the tertiary structure of TCTP-binding proteins and their binding partners in a lineage-specific manner.

Bioassay-Guided Fractionation of Emilia sonchifolia Extract on the Induction of Ovarian Maturation in Fenneropenaeus merguiensis

This study was carried out to identify the active compounds of Emilia sonchifolia on induction of ovarian maturation in Fenneropenaeus merguiensis. The crude extracts from dichloromethane and acetone were investigated in vitro. The crude extract from acetone induced up-regulation of shrimp ovarian peritrophin (SOP) and translationally controlled tumor protein (TCTP) genes, which were highly expressed during early phase of ovarian development higher than dichloromethane extract. Furthermore, fraction 14 (F14) from acetone extract up-regulated both of the SOP and the TCTP genes to the greatest extent. Leading to in vivo study, the effect of ribosomal protein L10a (RPL10a), which acts as shrimp ovarian stimulator plus with F14 on shrimp ovarian maturation was investigated by injection. The best result was observed in the group that received RPL10a plus with F14 at 0.8 μg/g body weight of shrimp. The RPL10a plus F14 enhanced the shrimp ovaries from undeveloped stage to 57% of early developing stage within 15 days. Meanwhile, the control group remained 100% of the undeveloped stage. Hence, F14 seems to play a positive role in the induction of shrimp ovarian maturation. The component of F14 was identified using mass spectroscopy and presented as 1,2-benzenedicarboxylic acid; 2,4-di-tert-butyl phenol; 2,4,5-trimethoybenzylidene;palmitic acid; 1-heneicosyl formate; 1-heptadecanol; ethyl-4-ethoxybenzoate and stearic acid.

Molecular characterization, expression analysis and heterologous expression of two translationally controlled tumor protein genes from Cucumis sativus.

The translationally controlled tumor protein (TCTP) is a family of abundant and ubiquitous proteins involved in several important primary functions. Cucumbers harbor two TCTP genes, CsTCTP1 and CsTCTP2; however, their functional roles remain unclear. In this study, we isolated CsTCTP1 and CsTCTP2 (XP-004134215 and XP-004135602, respectively) promoters, full-length cDNA and genomic sequences from Cucumis sativus. Bioinformatics analysis, containing cis-acting elements, structural domains, phylogenetic tree and conserved motifs, suggested the conservation and divergence of CsTCTP1 and CsTCTP2, thus providing knowledge regarding their functions. Expression analysis indicated that CsTCTP1 and CsTCTP2 exhibited tissue-specific expression and were regulated by biotic or abiotic stresses in C. sativus. Furthermore, CsTCTP1 and CsTCTP2 were regulated by ABA and may be associated with the TOR (target of rapamycin) signaling pathway. In a prokaryotic expression analysis, CsTCTP1 and CsTCTP2 showed positive responses to salt and heat stresses and a negative response to drought and HgCl2 stresses. TCTP may exert multiple functions in various cellular processes.

Expression Profiles, Characterization and Function of HbTCTP in Rubber Tree (Hevea brasiliensis).

As a highly conserved protein, the translationally controlled tumor protein (TCTP) carries out vital roles in various life processes. In rubber tree, two TCTP genes, HbTCTP and HbTCTP1, were cloned, but only HbTCTP1 was studied in details. In this study, cis-acting regulatory elements, expression patterns, subcellular localization, interacting proteins, and antioxidant activity of HbTCTP were systematically analyzed. Besides the common cis-acting regulatory elements, HbTCTP promoter also harbored various known cis-elements that respond to hormone/stresses. Being consistent with the aforementioned results, HbTCTP was regulated by drought, low temperature, high salt, ethylene (ET), wounding, H2O2, and methyl jasmonate (MeJA) treatments. HbTCTP was expressed throughout different tissues and developmental stages of leaves. In addition, HbTCTP was associated with tapping panel dryness (TPD). HbTCTP was localized in the membrane, cytoplasm and the nucleus, and interacted with four proteins rubber elongation factor (REF), 17.5 kDa heat shock family protein, annexin, and REF-like stress related protein 1. Being similar to HbTCTP1, HbTCTP also indicated antioxidant activity in metal-catalyzed oxidation (MCO) system. Our results are useful for further understanding the molecular characterization and expression profiles of HbTCTP, but also lay a solid foundation for elucidating the function of HbTCTP in rubber tree.

AtTCTP2, an Arabidopsis thaliana homolog of Translationally Controlled Tumor Protein, enhances in vitro plant regeneration.

The Translationally Controlled Tumor Protein (TCTP) is a central regulator of cell proliferation and differentiation in animals, and probably also in plants. Arabidopsis harbors two TCTP genes, AtTCTP1 (At3g16640), which is an important mitotic regulator, and AtTCTP2 (At3g05540), which is considered a pseudogene. Nevertheless, we have obtained evidence suggesting that this gene is functional. Indeed, a T-DNA insertion mutant, SALK_045146, displays a lethal phenotype during early rosette stage. Also, both the AtTCTP2 promoter and structural gene are functional, and heterozygous plants show delayed development. AtTCTP1 cannot compensate for the loss of AtTCTP2, since the accumulation levels of the AtTCTP1 transcript are even higher in heterozygous plants than in wild-type plants. Leaf explants transformed with Agrobacterium rhizogenes harboring AtTCTP2, but not AtTCTP1, led to whole plant regeneration with a high frequency. Insertion of a sequence present in AtTCTP1 but absent in AtTCTP2 demonstrates that it suppresses the capacity for plant regeneration; also, this phenomenon is enhanced by the presence of TCTP (AtTCTP1 or 2) in the nuclei of root cells. This confirms that AtTCTP2 is not a pseudogene and suggests the involvement of certain TCTP isoforms in vegetative reproduction in some plant species.

Structural divergence of plant TCTPs.

The Translationally Controlled Tumor Protein (TCTP) is a highly conserved protein at the level of sequence, considered to play an essential role in the regulation of growth and development in eukaryotes. However, this function has been inferred from studies in a few model systems, such as mice and mammalian cell lines, Drosophila and Arabidopsis. Thus, the knowledge regarding this protein is far from complete. In the present study bioinformatic analysis showed the presence of one or more TCTP genes per genome in plants with highly conserved signatures and subtle variations at the level of primary structure but with more noticeable differences at the level of predicted three-dimensional structures. These structures show differences in the “pocket” region close to the center of the protein and in its flexible loop domain. In fact, all predictive TCTP structures can be divided into two groups: (1) AtTCTP1-like and (2) CmTCTP-like, based on the predicted structures of an Arabidopsis TCTP and a Cucurbita maxima TCTP; according to this classification we propose that their probable function in plants may be inferred in principle. Thus, different TCTP genes in a single organism may have different functions; additionally, in those species harboring a single TCTP gene this could carry multiple functions. On the other hand, in silico analysis of AtTCTP1-like and CmTCTP-like promoters suggest that these share common motifs but with different abundance, which may underscore differences in their gene expression patterns. Finally, the absence of TCTP genes in most chlorophytes with the exception of Coccomyxa subellipsoidea, indicates that other proteins perform the roles played by TCTP or the pathways regulated by TCTP occur through alternative routes. These findings provide insight into the evolution of this gene family in plants.

CDNA cloning, genomic organization and expression analysis during somatic embryogenesis of the translationally controlled tumor protein (TCTP) gene from Japanese larch (Larix leptolepis)

A full-length cDNA and genomic sequences of a translationally controlled tumor protein (TCTP) gene were isolated from Japanese larch (Larix leptolepis) and designated LaTCTP. The length of the cDNA was 1043bp and contained a 504bp open reading frame that encodes a predicted protein of 167 amino acids, characterized by two signature sequences of the TCTP protein family. Analysis of the LaTCTP gene structure indicated four introns and five exons, and it is the largest of all currently known TCTP genes in plants. The 5′-flanking promoter region of LaTCTP was cloned using an improved TAIL-PCR technique. In this region we identified many important potential cis-acting elements, such as a Box-W1 (fungal elicitor responsive element), a CAT-box (cis-acting regulatory element related to meristem expression), a CGTCA-motif (cis-acting regulatory element involved in MeJA-responsiveness), a GT1-motif (light responsive element), a Skn-1-motif (cis-acting regulatory element required for endosperm expression) and a TGA-element (auxin-responsive element), suggesting that expression of LaTCTP is highly regulated. Expression analysis demonstrated ubiquitous localization of LaTCTP mRNA in the roots, stems and needles, high mRNA levels in the embryonal-suspensor mass (ESM), browning embryogenic cultures and mature somatic embryos, and low levels of mRNA at day five during somatic embryogenesis. We suggest that LaTCTP might participate in the regulation of somatic embryo development. These results provide a theoretical basis for understanding the molecular regulatory mechanism of LaTCTP and lay the foundation for artificial regulation of somatic embryogenesis.

Identification of upregulated immune-related genes in Vibrio harveyi challenged Penaeus monodon postlarvae

A subtracted cDNA library was constructed and analyzed to elucidate the response of Penaeus monodon postlarvae challenged with Vibrio harveyi. As many as 960 randomly selected cDNA fragments generated through suppression subtractive hybridization were single pass sequenced. Forty five genes and 20 hypothetical proteins were identified, a few being first reports from shrimps. The most abundant immune relevant genes were ferritin, hemocyanin, and TCTP (translationally controlled tumor protein) indicating their upregulation as also confirmed through qPCR. Post-infection qPCR analyses confirmed 2.04, 2.09, 3.28, 5.49, 6.47, and 11.63 fold rise respectively in ferritin, penaeidin, MnSOD, lysozyme, TCTP, and hemocyanin genes. These genes may be involved in the regulation of the host defense against V. harveyi.

Characterization and Biological Activity of the Ribosomal Protein L10a of the White Shrimp: Fenneropenaeus merguiensis De Man During Vitellogenesis

The molecular events in the ovaries of Fenneropenaeus merguiensis De Man during vitellogenesis were investigated. The ribosomal protein L10a (RPL10a) was characterized and cloned. It consisted of 669 bp and the deduced polypeptide had 217 amino acids (GeneBank/EBI accession number FJ623402). The calculated molecular mass and pI were 25.7 kDa and 10.06, respectively. An immunohistochemical technique showed that RPL10a was located in the cytoplasm and nucleus of developing oocytes and follicle cells. Treatment of undeveloped ovarian explant cultures with recombinant histidine (His)-RPL10a stimulated the expression of translationally controlled tumor protein (TCTP), heat shock protein 70 (HSP70), and shrimp ovarian peritrophin (SOP) genes, previously shown to be involved in ovarian maturation. The transcripts of all three genes in the ovarian explants showed their highest expression after 4 h incubation with the His-RPL10a at 37 degrees C. The TCTP and HSP70 transcripts declined after 12 h, while the transcript of SOP remained high until 24 h. The His-RPL10a did not stimulate the expression of the TCTP, SOP, and HSP70 genes in shrimp muscle tissue. The information on the molecular behavior of the RPL10a in this study may, in the future, lead to new methods to stimulate ovarian development in shrimp.

Molecular characterization of a calcium binding translationally controlled tumor protein homologue from the filarial parasites Brugia malayi and Wuchereria bancrofti

We have cloned homologues of the mammalian translationally controlled tumor protein (TCTP) from the human filarial parasites Wuchereria bancrofti and Brugia malayi. TCTP genes from B. malayi and W. bancrofti were expressed in a T7 promoter vector as histidine tagged fusion proteins. Both the recombinant B. malayi TCTP (rBm-TCTP) and recombinant W. bancrofti TCTP (rWb-TCTP) have a molecular mass of approximately 28 kDa with the histidine tag. Sequence analyses showed that there is a 98% similarity between the two filarial TCTPs at amino acid levels and are immunologically cross-reactive. Analysis of soluble proteins from various lifecycle stages of B. malayi suggested that the expression of Bm-TCTP might be differentially regulated and occurs in multimeric form. Recombinant TCTP were found to form multimers in solution under non-reducing conditions. The tendency for filarial TCTPs to become multimers was predicted by the presence of the Lupas coiled coil structure in their sequence. Despite the absence of a signal sequence, Bm-TCTP is present abundantly in the excretory/secretions (ES) of microfilariae. Characterization studies showed that both Bm- and Wb-TCTPs are calcium-binding proteins and have histamine-releasing function in vitro. When injected intraperitoneally both the filarial TCTPs induced inflammatory infiltration of eosinophils into the peritoneal cavity of mice suggesting that the filarial TCTPs may have a role in the allergic inflammatory responses associated with filarial infections.