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1.
14-3-3蛋白家族是由多个高度保守的成员构成的调节性蛋白质家族,它们主要以磷酸化的形式与伴侣蛋白相互作用,并能够以多种方式来影响靶蛋白。通过构建14-3-3蛋白原核表达载体,纯化重组蛋白获得14-3-3蛋白抗体。为了验证14-3-3蛋白基因在耐铝中的作用,构建14-3-3酵母表达载体,得到14-3-3过表达酵母菌株。在5mmol/L铝浓度下,转基因酵母比对照酵母长势好,这表明14-3-3蛋白通过促进生长赋予酵母对铝胁迫的耐受性。 相似文献
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Sun Am KIM Mi Young NOH Yong Hun JO Seung Han OH Iksoo KIM Yong Seok LEE Heung Cheol KIM Sook Jae SEO In Seok BANG Yeon Soo HAN 《Entomological Research》2009,39(2):129-134
The 14-3-3 proteins are known to play an important regulatory role in apoptosis, and various cell signaling cascades. However, no investigation on mosquito 14-3-3 has been reported. To investigate the role of 14-3-3 proteins in mosquito midgut cells undergoing apoptosis, we decided to take advantage of Anopheles gambiae genome data, and were able to find Ag14-3-3ζ cDNA and protein sequences from Ensembl ( http://www.ensembl.org ). Further in silico analysis using BLAST search revealed that Ag14-3-3ζ protein is a polypeptide of 248 amino acids, and shares high identity with 14-3-3ζ homologues from Aedes aegypti (100%), Drosophila melanogaster (96%) and Bombyx mori (93%). Due to the perfect match and high homology, we hypothesized that Ag14-3-3ζ peptide antibody may recognize 14-3-3ζ homologs from other anopheline mosquitoes and insects. We thus generated 14-3-3ζ polyclonal antibody against a unique region located in the C-terminal end of Ag14-3-3ζ after in silico epitope analysis. As expected, zoo-western blot analysis of 14-3-3 proteins revealed that a polyclonal antibody against Ag14-3-3ζ peptide recognizes 14-3-3 homologs from dipteran and lepidopteran insects. To our knowledge, this is the first report on polyclonal antibody production against mosquito 14-3-3ζ. The mosquito-based 14-3-3ζ antibody will be very useful for studying the functional characterization of 14-3-3ζ in the context of host–pathogen interactions in midgut and other immune cells. 相似文献
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The 14-3-3 family of phosphoserine/phosphothreonine-binding proteins dynamically regulates the activity of client proteins in various signaling pathways that control diverse physiological and pathological processes. In response to environmental cues, 14-3-3 proteins orchestrate the highly regulated flow of signals through complex networks of molecular interactions to achieve well-controlled physiological outputs, such as cell proliferation or differentiation. Accumulating evidence now supports the concept that either an abnormal state of 14-3-3 protein expression, or dysregulation of 14-3-3/client protein interactions, contributes to the development of a large number of human diseases. In particular, clinical investigations in the field of oncology have demonstrated a correlation between upregulated 14-3-3 levels and poor survival of cancer patients. These studies highlight the rapid emergence of 14-3-3 proteins as a novel class of molecular target for potential therapeutic intervention. The current status of 14-3-3 modulator discovery is discussed. 相似文献
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The 14-3-3 protein family interacts with more than 2000 different proteins in mammals, as a result of its specific phospho-serine/phospho-threonine binding activity. Seven paralogs are strictly conserved in mammalian species. Here, we show that during adipogenic differentiation of 3T3-L1 preadipocytes, the level of each 14-3-3 protein paralog is regulated independently. For instance 14-3-3β, γ, and η protein levels are increased compared to untreated cells. In contrast, 14-3-3ε protein levels decreased after differentiation while others remained constant. In silico analysis of the promoter region of each gene showed differences that explain the results obtained at mRNA and protein levels. 相似文献
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hPFTAIRE1 (PFTK1), a Cdc2-related protein kinase, is highly expressed in human brain. It exhibits cytoplasmic distribution in Hela cells, although it contains two nuclear localization signals (NLSs) in its N-terminus. To search for its substrates and regulatory components, we screened a two-hybrid library by using the full-length hPFTAIRE1 as a bait. Four 14-3-3 isoforms (β,ε,η,τ) were identified interacting with the hPFTAIRE1. We found a putative 14-3-3 binding consensus motif(RHSSPSS) in the hPFTAIRE 1, which overlapped with its second NLS. Deletion of the RHSSPSS motif or substitution of Ser^119 gwithAla in the conserved binding motif abolished the specific interaction between the hPFTAIRE 1 and the 14-3 -3 proteins. The mutant S 120A hPFTAIRE1 also showed a weak interaction to the 14-3-3 proteins. The results suggested that the Ser^119 is crucial for the interaction between hPFTAIREI and the 14-3-3 proteins. All the hPFTAIRE1 mutants distributed in cytoplasm of Hela cells and human neuroblastoma cells (SH-SY5Y) when fused to the C-terminus of a green fluorescent protein (GFP), indicating that binding with the 14-3-3 proteins does not contribute to the subcellular localization of the hPFTAIRE1, although the binding may be involved in its signaling regulation. 相似文献
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14-3-3蛋白家族是一组高度保守的可溶性酸性蛋白质,分子量在28~33kD之间,广泛分布于各种真核生物之中。该蛋白能够特异地结合含有磷酸化丝氨酸或苏氨酸的肽段,参与多种信号转导途径。14-3-3蛋白调节着许多重要细胞生命活动,如:新陈代谢、细胞周期、细胞生长发育、细胞的存活和凋亡以及基因转录,该蛋白家族异常与疾病的发生密切相关,尤其是14-3-3蛋白在脑脊液中的分布与一些神经系统疾病密切相关。14-3-3蛋白已成为一些疾病的临床诊断指标,其作为疾病治疗的靶点也在研究之中。主要阐述了14-3-3蛋白的结构、功能、及其在疾病治疗中的应用。 相似文献
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Akira Ichikawa Sun-Yup Shim Yoshinori Katakura Kiichiro Teruya Shuichi Hashizume Sanetaka Shirahata 《Cytotechnology》2001,36(1-3):101-107
We reported previously that adenocarcinoma-reactive human monoclonal antibody AE6F4, which had been generated by in vitro immunization method, recognizes both 14-3-3protein and cytokeratin 8 (CK8). In this study, to analyze the cross-reactivity
of AE6F4 antibody, epitopes of AE6F4 antibody on 14-3-3 proteins and CK8 were studied by using synthetic linear peptide scanning
technology. To determine the locations of B cell epitope, 48 and 95 of decapeptides covering the entire 14-3-3 proteins and
CK8, respectively,were synthesized and binding to AE6F4 antibody was examined by ELISA. The AE6F4 antibody was strongly reactive
to peptides containing amino acid sequences TLWTSDTQGD in 14-3-3 proteins and INFLRQLYEE in CK8. These results indicate that
AE6F4 antibody can recognize the different peptide sequences in 14-3-3 proteins and CK8.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
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棉花143-3L基因的分子鉴定及其在纤维发育中优势表达分析 总被引:1,自引:0,他引:1
14-3-3蛋白以二聚体形式存在于所有真核生物中,是一种高度保守的调节蛋白,在细胞生长、增殖、凋亡、信号转导等生命活动中发挥着重要调控作用。我们在棉纤维cDNA文库中分离克隆到1个基因(cDNA),编码14-3-3蛋白类似物,命名为Gh14-3-3L(Gossypiumhirsutum14-3-3-like)。该cDNA长度为1,029bp,包含762bp开放阅读框,其编码蛋白由253个氨基酸组成。Gh14-3-3L与其他真核生物的14-3-3蛋白具有较高的同源性,并具有14-3-3蛋白的基本结构:二聚体结构域、磷酸化丝氨酸富集识别序列、4个CC结构和1个EFHand结构。Northern杂交分析显示Gh14-3-3L在棉纤维发育早期优势表达,且在10DPA棉纤维细胞中表达量最高,这表明Gh14-3-3L基因可能涉及棉纤维细胞伸长过程的调节。研究还表明,该基因在胚珠和花瓣组织中也有较强的表达,但在其他组织中表达较弱或不表达。 相似文献
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Plants and protozoa contain a unique family of calcium-dependent protein kinases (CDPKs) which are defined by the presence of a carboxyl-terminal calmodulin-like regulatory domain. We present biochemical evidence indicating that at least one member of this kinase family can be stimulated by 14-3-3 proteins. Isoform CPK-1 from the model plant Arabidopsis thaliana was expressed as a fusion protein in E. coli and purified. The calcium-dependent activity of this recombinant CPK-1 was shown to be stimulated almost twofold by three different 14-3-3 isoforms with 50% activation around 200 nM. 14-3-3 proteins bound to the purified CPK-1, as shown by binding assays in which either the 14-3-3 or CPK-1 were immobilized on a matrix. Both the 14-3-3 binding and activation of CPK-1 were specifically disrupted by a known 14-3-3 binding peptide LSQRQRSTpSTPNVHMV (IC50=30 μM). These results raise the question of whether 14-3-3 can modulate the activity of CDPK signal transduction pathways in plants. 相似文献
11.
Sluchanko NN Chernik IS Seit-Nebi AS Pivovarova AV Levitsky DI Gusev NB 《Archives of biochemistry and biophysics》2008,477(2):305-312
Effect of mutations mimicking phosphorylation on the structure of human 14-3-3ζ protein was analyzed by different methods. Mutation S58E increased intrinsic Trp fluorescence and binding of bis-ANS to 14-3-3. At low protein concentration mutation S58E increased the probability of dissociation of dimeric 14-3-3 and its susceptibility to proteolysis. Mutation S184E slightly increased Stokes radius and thermal stability of 14-3-3. Mutation T232E induced only small increase of Stokes radius and sedimentation coefficient that probably reflect the changes in the size or shape of 14-3-3. At low protein concentration the triple mutant S58E/S184E/T232E tended to dissociate, whereas at high concentration its properties were comparable with those of the wild type protein. The triple mutant was highly susceptible to proteolysis. Thus, mutation mimicking phosphorylation of Ser58 destabilized, whereas mutation of Ser184 induced stabilization of 14-3-3ζ structure. 相似文献
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Monroy FP 《Experimental parasitology》2008,118(1):134-138
14-3-3 Proteins are expressed in most eukaryotes organisms and play varied and crucial roles in a wide range of regulatory processes. In mammalian cells, seven 14-3-3 isoforms have been identified. However, it is not known what effect infection has on 14-3-3 isoform expression. In this study human colonic carcinoma cell lines were infected with Toxoplasma gondii for 24h and expression of 14-3-3 proteins was determined by RT-PCR. HT-29 cells only expressed 3 out of the 7 isoforms while 5 and all 7 isoforms were found in HCT-116 and Caco-2 cells, respectively. Infection had little or no effect in the expression of 14-3-3gamma, epsilon, sigma, and xi; but in HCT-116 cells induced expression of 14-3-3eta and sigma, while 14-3-3beta, eta, and xi were induced in HT-29 cells. If 14-3-3 proteins are involved in cell survival and/or prevention of parasite replication, longer incubation times may be required as no differences in percentage of infection were found among the cell lines at 24h post-infection. 相似文献
15.
Marco Lalle Carlo Bavassano Federica Fratini Prisca Boisguerin Edoardo Pozio 《International journal for parasitology》2010,40(2):201-4484
14-3-3s are a family of phosphoserine/phosphothreonine binding proteins directly affecting many protein functions by regulating enzyme activity, intracellular localisation or mediating protein-protein interaction. The single 14-3-3 (g14-3-3) of the flagellated parasite Giardia duodenalis is phosphorylated at residue threonine 214 (T214) and polyglycylated at the extreme C-terminus in a stage-specific manner. To define the role of each post-translational modification, Giardia transgenic lines expressing a N-terminally FLAG-tagged g14-3-3, or the single point mutant T214A, or the E246A and the E247A mutants of the putative polyglycylation sites, were generated in this study. By affinity chromatography and MALDI-MS analysis, Glu246 was identified as the only site of polyglycylation. The absence of a polyglycine chain results in the nuclear localisation of the protein at any parasite life-stage, suggesting a role for polyglycylation in 14-3-3 nucleo/cytoplasm shuttling. Moreover, cyst formation was strongly induced in parasites expressing the E246A mutant and delayed in those harbouring the T214A mutant. Finally, in vitro overlay assays with a GST_T214E mutant indicated that phosphorylation can alter in vitro the binding properties of 14-3-3. The present data suggest that g14-3-3 post-translational modifications act in combination to affect encystation efficiency in Giardia. 相似文献
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Jaubert S Laffaire JB Ledger TN Escoubas P Amri EZ Abad P Rosso MN 《International journal for parasitology》2004,34(7):873-880
14-3-3 proteins are highly conserved ubiquitous proteins found in all eukaryotic organisms. They are involved in various cellular processes including signal transduction, cell-cycle control, apoptosis, stress response and cytoskeleton organisation. We report here the cloning of two genes encoding 14-3-3 isoforms from the plant parasitic root-knot nematode Meloidogyne incognita, together with an analysis of their expression. Both genes were shown to be transcribed in unhatched second stage larvae, infective second stage larvae, adult males and females. The Mi-14-3-3-a gene was shown to be specifically transcribed in the germinal primordium of infective larvae, whereas Mi-14-3-3-b was transcribed in the dorsal oesophageal gland in larvae of this stage. The MI-14-3-3-B protein was identified by mass spectrometry in in vitro-induced stylet secretions from infective larvae. The stability and distribution of MI-14-3-3 proteins in host plant cells was assessed after stable expression of the corresponding genes in tobacco BY2 cells. 相似文献
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Purification, Properties, and Immunohistochemical Localisation of Human Brain 14-3-3 Protein 总被引:9,自引:1,他引:8
Paul F. Boston Peter Jackson Pamela A. M. Kynoch R. J. Thompson 《Journal of neurochemistry》1982,38(5):1466-1474
Abstract: A protein has been purified from human brain that appears to be the human equivalent of bovine 14-3-3 protein. On polyacrylamide gel electrophoresis the protein migrates as a faster major component, termed 14-3-3-2 protein, and a slower minor component, termed 14-3-3-1 protein, which consists of approximately 12% of the total protein. Both 14-3-3-1 and 14-3-3-2 have a native molecular weight of approximately 67,000. 14-3-3-2 appears to have the subunit composition (αβ; 14-3-3-1 has the composition ββ. Peptide mapping with Stuphvlococcus aureus V8 proteinase shows that α and β subunits are unrelated but the β and β' subunits show some common peptides. Immunoperoxidase labelling shows that 14-3-3 is localised in neurones in the human cerebral cortex. 14-3-3 shows no enolase, creatine kinase, triose phosphate isomerase, ATPase, cyclic nucleotide-dependent protein kinase, or purine nucleoside phosphorylase activity. 14-3-3 does not bind calcium and does not appear to be related to calmodulin, calcineurin, tubulin, neurofilament proteins, clathrin-associated proteins, or tropomyosin. The functional significance of this neuronal protein remains obscure. 相似文献