中间纤维与细胞信号通路的研究进展

中间纤维家族由约70个中间纤维蛋白组成,在真核细胞内组成横跨核膜和胞质的网状骨架.中间纤维最初仅仅被当做是细胞骨架的一种,主要起机械支撑作用.这个观点正发生快速的改变,因为越来越多的研究发现中间纤维蛋白参与各种主要的细胞信号通路,如细胞应激、细胞凋亡和14-3-3信号通路等.     

14-3-3 Binding to Cyclin Y contributes to cyclin Y/CDK14 association

Cyclin Y is a highly conserved cyclin among eumetazoans, yet its function and regulation are poorly understood. To search for Cyclin Y-interacting proteins, we screened a yeast two-hybrid library using human Cyclin Y （CCNY） as a bait and identified the following interactors： CDK14 and four members of the 14-3-3 family （ε,β,η,τ）. The interaction between CCNY and 14-3-3 proteins was confirmed both in vitro and in vivo. The results showed that Ser-100 and Ser-326 residues in CCNY were crucial for 14-3-3 binding. Interestingly, binding of CCNY to 14-3-3 significantly enhanced the association between CCNY and CDK14. Our findings may add a new layer of regulation of CCNY binding to its kinase partner.     

A Cdc2-related protein kinase hPFTAIRE1 from human brain interacting with 14-3-3 proteins

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.     

14-3-3 proteins as potential therapeutic targets

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.     

PRAS40与14-3-3蛋白各亚型相互作用的酵母双杂交系统检测

PRAS40是近几年新发现的Akt作用底物,14-3-3结合蛋白。为确定PRAS40与14-3-3蛋白7种亚基间相互作用关系,利用gateway方法构建用于酵母双杂交系统的诱饵质粒pEG-PRAS40及转录激活质粒pJG-PRAS40,将PRAS40和14-3-3各亚型质粒分别作为诱饵蛋白质粒及转录激活质粒共转化酵母细胞EGY48,通过氨基酸营养缺陷生长实验及β-半乳糖苷酶显色反应分析两种蛋白相互作用程度。酶切鉴定证实成功地构建了pEG-PRAS40和pJG-PRAS40质粒,酵母双杂交实验结果显示PRAS40可以和14-3-3亚型tau,beta,zeta及epsilon相结合,epsilon较强,beta和zeta次之,tau较弱。此结果将为深入研究PRAS40与14-3-3蛋白生物学功能及发现药物靶标奠定基础。     

The down-regulated expression of 14-3-3σ may activate the other six 14-3-3 isoforms and they may take over the role of 14-3-3σ in the tumor genesis

Seven isoforms of 14-3-3 protein family have different functions in the cancer genesis and progress. It is found that six isoforms were up-regulated expression and inclined to sustain the cancer survival. Conversely, 14-3-3σ strongly promotes cancer apoptosis. Its down-regulated expression was found in many cancer tissues and thought to be an early event in the tumor genesis. Interestingly, no suggestions are made about the possible effect that the down-regulated expression of 14-3-3σ activated the other six 14-3-3 isoforms and they take over the role of 14-3-3σ in the tumor genesis. The inactivation of 14-3-3σ in the early stage of tumor genesis is a clue to trigger the other six 14-3-3 isoforms activation.     

拟南芥IQM3基因的表达分析及其突变体的鉴定

对拟南芥(Arabidopsis thaliana)IQM3基因的功能进行了分析.结果表明,推定IQM3的启动子中存在多种光、非生物胁迫和植物激素反应的顺式作用元件,可能参与植物对环境变化的反应.RT-PCR分析表明,IQM3在拟南芥莲座叶、花序叶、茎、花和根中的表达较强,但在荚果中的表达很弱;IQM3基因的T-DNA插入突变体iqm3-1和iqm3-2分别是功能缺失和超表达突变体,对这些突变体的表型分析表明,IQM3基因与种子萌发及幼苗子叶膨大有密切关系.     

PrP106-126多肽诱导凋亡细胞中14-3-3β的降解

本研究将PrP106-126多肽和HeLa细胞共孵育4h和8h,采用Hoechst染色分析发现PrP106-126诱导凋亡细胞细胞核出现不同程度的染色质浓集,固缩及碎裂的细胞凋亡征象。Western blotting检测发现PrP106-126诱导细胞中的多聚ADP核糖聚合酶(poly ADP-ribose polymerase,PARP)降解,提示PrP106-126通过caspase3途径引起细胞凋亡现象。PrP106-126诱导的细胞中14-3-3β在不同孵育时间也出现降解,而Real-time PCR检测14-3-3β mRNA未发生变化。本研究证明PrP106-126通过caspase3诱导HeLa细胞凋亡,并可导致抗凋亡蛋白14-3-3β的降解而加速凋亡的形成。     

Induction kinetics of delayed light emission in spinach chloroplasts

Induction curves of the delayed light emission in spinach chloroplasts were studied by measuring the decay kinetics after each flash of light. This study differs from previous measurements of the induction curves where only the intensities at one set time after each flash of light were recorded. From the decay kinetics after each flash of light, the induction curves of the delayed light emission measured 2 ms after a flash of light were separated into two components: one component due to the last flash only and one component due to all previous flashes before the last one. On comparing the delayed light induction curves of the two components with the fluorescence induction curves in chloroplasts treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and in chloroplasts treated with hydroxylamine and 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the component due to the last flash only is found to be dependent on the concentration of open reaction centers and the component due to all previous flashes except the last is dependent on the concentration of closed reaction centers. This implies that the yield of the fast decaying component of the delayed light emission is dependent on the concentration of open reaction centers and the yield of the slow decaying component is dependent on the concentration of closed reaction centers.     

Expression and methylation status of 14-3-3 sigma gene can characterize the different histological features of ovarian cancer

We hypothesize that 14-3-3 sigma gene expression and its regulation by methylation can characterize histological types of primary human epithelial ovarian cancer. To test this hypothesis, ovarian cancer cell lines and 54 ovarian cancer tissue samples were analyzed for expression and methylation of 14-3-3 sigma gene using methylation specific PCR. The results of our experiments demonstrate that 14-3-3 sigma gene was methylated and inactivated in ES-2 ovarian cell line, which was derived from clear cell adenocarcinoma. Treatment of this cell line with demethylating agent 5-aza-2'-deoxycytidine restored the expression of 14-3-3 sigma gene. In human ovarian cancer tissues, the expression of 14-3-3 sigma protein was inactivated in most of the ovarian clear cell carcinoma tissues. Interestingly, 14-3-3 sigma protein expression was positive in significantly higher percentages of serous (89.5%), endometrioid (90%), and mucinous (81.8%) ovarian adenocarcinoma tissues. The ovarian clear cell carcinoma samples with inactivated 14-3-3 sigma protein were highly methylated, suggesting that inactivation of 14-3-3 sigma gene is through DNA methylation. Using direct DNA sequencing, 14-3-3 sigma gene methylation on all the 17 CpG sites was significantly higher in ovarian clear cell carcinoma as compared to other histological types of ovarian cancer (serous, endometrioid, and mucinous). This is the first report suggesting that 14-3-3 sigma gene expression and methylation status can characterize histological features of different types of ovarian cancer.     

Differential expression and accumulation of 14-3-3 paralogs in 3T3-L1 preadipocytes and differentiated cells

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.     

Subcellular localization of 14-3-3 proteins in Toxoplasma gondii tachyzoites and evidence for a lipid raft-associated form

A polyclonal antibody was raised against a Toxoplasma gondii 14-3-3-gluthatione S-transferase fusion protein obtained by cloning a 14-3-3 cDNA sequence determined from the T. gondii database. This antibody specifically recognized T. gondii 14-3-3 without any cross-reaction with mammalian proteins. Immunofluorescence microscopy studies of the tachyzoites or the T. gondii-infected cells suggested cytosolic and membranous localizations of 14-3-3 protein. Different subcellular fractions were prepared for electrophoresis analysis and immunodetection. 14-3-3 proteins were found in the cytosol, the membrane fraction and Triton X-100-resistant membranes. Two 14-3-3 isoforms were detected. The major one was mainly cytoplasmic and to a lesser extent membrane-associated, whereas the minor isoform was associated with the detergent-resistant lipid rafts.     

The mutational spectrum of procarbazine in Drosophila melanogaster.

The antineoplastic agent Procarbazine was tested for the induction of genetic damage in Drosophila melanogaster. The compound was administered to adult males by oral application. The following types of genetic damage were measured: (1) sex-linked recessive lethals; (2) dominant lethals; (3) total and partial sex-chromosome loss; and (4) translocations. Procarbazine is highly mutagenic in causing recessive lethal mutations in all stages of spermatogenesis. In sperm a clear-cut concentration-effect relationship is not apparent, but in spermatids such a relationship is obtained for mutation induction at low levels of procarbazine exposure, while at high concentrations the induction of recessive lethals is not a function of concentration. A low induction of total sex-chromosome loss (X,Y) and dominant lethals was observed in metabolically active germ cells (spermatids), but procarbazine failed to produce well-defined breakage events, such as partial sex-chromosome loss (YL,YS) and II-III translocations. The results obtained in Drosophila melanogaster are discussed and compared with the mutational pattern reported in the mouse after procarbazine treatment.     

14-3-3蛋白参与植物应答非生物胁迫的研究进展

14-3-3蛋白是一种在真核生物细胞中普遍存在且高度保守的蛋白。该蛋白在大多数物种中由一个基因家族编码,并以同源或异源二聚体的形式存在。不同的14-3-3蛋白同工型具有不同的细胞特异性,可通过识别特异的磷酸化或非磷酸化序列与靶蛋白相互作用。14-3-3蛋白在植物生长和发育的各个方面都起重要作用。本文主要围绕植物14-3-3蛋白的种类、结构、磷酸化或非磷酸化识别序列及其响应干旱、冷冻、盐碱、营养和机械胁迫等的分子机制研究进展进行综述。     

Subcellular Localisation of 14-3-3 Isoforms in Rat Brain Using Specific Antibodies

Abstract: The 14-3-3 protein family, which is present at particularly high concentrations in mammalian brain, is known to be involved in various cellular functions, including protein kinase C regulation and exocytosis. Despite the fact that most of the 14-3-3 proteins are cytosolic, a small but significant proportion of 14-3-3 in brain is tightly and selectively associated with some membranes. Using a panel of isoform-specific antisera we find that the ε, η, γ, β, and ζ isoforms are all present in purified synaptic membranes but absent from mitochondrial and myelin membranes. In addition, the η, ε, and γ isoforms but not the β and ζ isoforms are associated with isolated synaptic junctions. When different populations of synaptosomes were fractionated by a nonequilibrium Percoll gradient procedure, the ε and γ isoforms were present and the β and ζ isoforms were absent from the membranes of synaptosomes sedimenting in the more dense parts of the gradient. The finding that these proteins are associated with different populations of synaptic membranes suggests that they are selectively expressed in different classes of neurones and raises the possibility that some or all of them may influence neurotransmission by regulating exocytosis and/or phosphorylation.     

Molecular characterization of a 14-3-3 zeta gene from Plodia interpunctella: A potential marker for phylogenetic inference

The 14-3-3 proteins are a large family of approximately 30 kDa acidic proteins and acting in the regulation of many biological processes. In this study, a 14-3-3 zeta (Pi14-3-3z) gene from the Indian meal moth, Plodia interpunctella (Lepidoptera, Pyralidae) was isolated and characterized. The full-length cDNA of Pi14-3-3z is 1382 bp, including a 5'-untranslated region (UTR) of 141 bp, 3′-UTR of 497 bp and an open reading frame (ORF) of 744 bp encoding a polypeptide of 247 amino acids which contains a 14-3-3 homologues domain (PF00244). The deduced Pi14-3-3z protein sequence has 81%–100% identity with the homologues in comparison to with other individuals. qPCR analysis revealed that Pi14-3-3z was expressed at the four developmental stages and in all tissues tested. Based on the amino acid of 14-3-3z, phylogenetic analysis demonstrated a similar topology with the traditional classification, suggesting 14-3-3z protein has the potential value in phylogenetic inference.     

柞蚕14-3-3基因的鉴定及表达分析

14-3-3蛋白是一种可以改变其结合蛋白构象的酸性蛋白质.柞蚕14-3-3 cDNA序列全长1 220 bp,包括一个126 bp的5'非编码区和一个350 bp的3'非编码区.该基因的开放读码框长度为744 bp,编码247个氨基酸.序列比对结果表明,柞蚕14-3-3蛋白与家蚕的14-3-3蛋白具有高度同源性.此外对柞蚕14-3-3基因进行了原核表达和重组蛋白纯化.SDS-PAGE和免疫印迹结果表明,分子量大小约32 kD的重组蛋白在大肠杆菌中得到了成功表达.     

14-3-3ζ/τ heterodimers regulate Slingshot activity in migrating keratinocytes

Defining the pathways required for keratinocyte cell migration is important for understanding mechanisms of wound healing and tumor cell metastasis. We have recently identified an α6β4 integrin-Rac1 signaling pathway via which the phosphatase Slingshot (SSH) activates/dephosphorylates cofilin, thereby determining keratinocyte migration behavior. Here, we assayed the role of 14-3-3 isoforms in regulating the activity of SSH1. Using amino or carboxy terminal domains of 14-3-3ζ, we demonstrate that in keratinocytes 14-3-3ζ/τ heterodimers bind SSH1, in the absence of Rac1 signaling. This interaction leads to an inhibition of SSH1 activity, as measured by an increase in phosphorylated cofilin levels. Overexpression of the carboxy terminal domain of 14-3-3ζ acts as a dominant negative and inhibits the interaction between 14-3-3τ and SSH1. These results implicate 14-3-3ζ/τ heterodimers as key regulators of SSH1 activity in keratinocytes and suggest they play a role in cytoskeleton remodeling during cell migration.     

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

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。