植物14⁃3⁃3蛋白结构与功能的研究进展

14⁃3⁃3蛋白是一类进化上高度保守的磷酸化识别蛋白,以同源或异源二聚体的形式发挥功能。植物14⁃3⁃3蛋白不但参与调控植物开花、叶绿体发育和移动、气孔开放、种子发育、细胞伸长和分裂等过程,还参与调控植物对环境胁迫的响应,如干旱、盐碱和温度等胁迫响应。目前,已鉴定与植物14⁃3⁃3蛋白互作的蛋白超过300个,说明14⁃3⁃3蛋白在植物生长发育过程中具有重要作用。本文主要总结了近年来植物14⁃3⁃3蛋白的结构及功能方面的研究进展。

14⁃3⁃3蛋白影响肿瘤放射敏感性的机制研究进展

14⁃3⁃3蛋白是一组常表达于真核生物中的小分子蛋白质,随着近年来对其功能研究不断加深,相关研究发现其在肿瘤的发生发展及放射敏感性中具有重要意义,其机制可能与14⁃3⁃3蛋白对DNA损伤后修复、细胞周期调控、信号转导等多种途径有关。本文就14⁃3⁃3蛋白对不同肿瘤放射敏感性的影响进行综述。

14⁃3⁃3ζ蛋白在乳腺癌发生发展中的作用研究进展

14⁃3⁃3蛋白家族(14⁃3⁃3 protein family)是一类酸性且高度保守的小分子蛋白,广泛存在真核生物细胞内。14⁃3⁃3蛋白自身无明显的催化活性,主要与其他蛋白相互作用而发挥调控靶蛋白的作用。14⁃3⁃3ζ是14⁃3⁃3蛋白家族的重要成员之一,近几年受到学者的广泛关注,且越来越多的学者开展14⁃3⁃3ζ与癌症的研究。本文综述了近几年研究者对14⁃3⁃3ζ与癌症的研究,尤其与乳腺癌耐药、复发、转移等相关研究,为乳腺癌的诊疗提供新思路、寻找新靶标。

Molecular Characterization of Cotton 14-3-3L Gene Preferentially Expressed During Fiber Elongation

The 14-3-3 protein, highly conserved in all eukaryotic cells, is an important regulatory protein. It plays an important role in the growth, amplification, apoptosis, signal transduction, and other crucial life activities of cells. A eDNA encoding a putative 14-3-3 protein was isolated from cotton fiber eDNA library. The eDNA, designated as Gh14-3-3L （Gossypium hirsutum 14-3-3-like）, is 1,029 bp in length （including a 762 bp long open reading frame and 5＇-/3＇-untranslated regions） and deduced a protein with 253 amino acids. The GhI4-3-3L shares higher homology with the known plant 14-3-3 proteins, and possesses the basic structure of 14-3-3 proteins： one dimeric domain, one phosphoralated-serine rich motif, four CC domains, and one EF Hand motif. Northern blotting analysis showed that Gh14-3-3L was predominantly expressed during early fiber development, and reached to the peak of expression in 10 days post anthers （DPA） fiber cells, suggesting that the gene may be involved in regulating fiber elongation. The gene is also expressed at higher level in both ovule and petal, but displays lower or undeteetable level of activity in other tissues of cotton.

Overexpression of 14-3-3 protein protects pheochromocytoma cells against 1-methyl-4-phenylpyridinium toxicity

Objective To investigate the effects of 14-3-3 protein overexpression on the 1-methyl-4-phenylpyridinium （MPP^＋） induced pheochromocytoma （PC12） cell death and the potential mechanisms. Methods pcDNA3.1（＋）-14-3-3 plasmids, which could be expressed in mammalian cell, were constructed and transfected into PC 12 cells with Lipofectamine 2000. The expression of 14-3-3 protein, Bcl-2 protein, and BAD protein were determined by western blot. 3-（4,5- dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay, microplate reader, and flow cytometric analysis were used to measure cell viability, the caspase activity, and apoptotic ratio respectively. Results （1） The expression of 14-3-3 protein increased significantly three weeks after pcDNA3.1（＋）-14-3-3 plasmids transfected into PC 12 cells. （2） MPP^＋ caused a decrease of cell viability in a dose-dependent manner. At 100μmol/L MPP^＋, cell viability reduced approximately 50%. （3） The caspase activity increased along with the MPP^＋ concentrations rising and reached its maximum value （0.34 μmol/mg protein） at 100 μmol/L MPP＊. However caspase activity decreased significantly when the MPP^＋ concentration exceeded 100 μmol/L. （4） Overexpression of 14-3-3 protein decreased the apoptosis ratio of PC 12 cells treated with 100μmol/L MPP^＋ from 26.5% to 8.6%. （5） Bcl-2 protein tended to decrease but BAD protein tended to increase after treatment of PC 12 cells with 100 μmol/L MPP^＋. Overexpression of 14-3-3 protein significantly increased the cellular level of Bcl-2 protein and decreased that of BAD protein. Conclusion Overexpression of 14-3-3 protein may reduce MPP^＋-induced apoptotic cell death in PC12 cells by up-regulating the Bcl-2 expression and down-regulating the BAD expression. These results may provide a promising target for treatment of Parkinson＇s disease.

Involvement of ERK1/2 and p38 MAPK in up-regulation of 14-3-3 protein induced by hydrogen peroxide preconditioning in PC12 cells

Objective To investigate the protective effects of hydrogen peroxide preconditioning （HPP） on the pheochromocytoma （PC12） cells treated with 1-methyl-4-phenylpyridinium （MPP^＋） and to explore the potential mechanisms. Methods The viability and apoptosis of PC 12 cells were determinded by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay and 4′,6′-diamidino-2-phenylindole （DAPI） staining, respectively. The expressions of 14-3-3 protein and phospholylated p38 mitogen-activated protein kinase （MAPK） were determined by Western blot. Enzyme-linked immunosorbent assay （ELISA） was used to measure the activity of extracellular signal-regulated protein kinase 1/2 （ERK1/2）. Results The cell viability decreased and the number of apoptotic cells increased dramatically in MPP^＋ group compared with that in Control group. HPP induced a significant increase in cell viability and a marked decrease in population of apoptotic cells of the MPP^＋- treated PC 12 cells, accompanied with up-regulation of 14-3-3 protein and increase of ERK 1/2 and p38 MAPK activities. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in PC 12 cells induced by HPP. Conclusion HPP protects PC 12 cells against MPP＋ toxicity by up-regulating 14-3-3 protein expression through the ERK1/2 and p38 MAPK signaling pathways.

14-3-3 proteins—an update

14-3-3 is a highly conserved acidic protein family, composed of seven isoforms in mammals. 14-3-3 protein caninteract with over 200 target proteins by phosphoserine-dependent and phosphoserine-independent manners. Little isknown about the consequences of these interactions, and thus are the subjects of ongoing studies. 14-3-3 controls cellcycle, cell growth, differentiation, survival, apoptosis, migration and spreading. Recent studies have revealed newmechanisms and new functions of 14-3-3, giving us more insights on this fascinating and complex family of proteins.Of all the seven isoforms, 14-3-3σ seems to be directly involved in human cancer. 14-3-3σ itself is subject to regulationby p53 upon DNA damage and by epigenetic deregulation. Gene silencing of 14-3-3σ by CpG methylation has beenfound in many human cancer types. This suggests that therapy-targeting 14-3-3σ may be beneficial for future cancertreatment.

The role of epigenetic inactivation of 14-3-3σ in human cancer

Cancer cells show characteristic alterations in DNA methylation patterns. Aberrant CpG methylation of specificpromoters results in inactivation of tumor suppressor genes and therefore plays an important role in carcinogenesis. Thep53-regulated gene 14-3-3σ undergoes frequent epigenetic silencing in several types of cancer, including carcinoma ofthe breast, prostate, and skin, suggesting that the loss of 14-3-3σ expression may be causally involved in tumor progression.Functional studies demonstrated that 14-3-3σ is involved in cell-cycle control and prevents the accumulation of chro-mosomal damage. The recent identification of novel 14-3-3σ-associated proteins by a targeted proteomics approachimplies that 14-3-3σ regulates diverse cellular processes, which may become deregulated after silencing of 14-3-3σexpression in cancer cells.

Clinical implication of 14-3-3 epsilon expression in gastric cancer

AIM:To evaluate for the first time the protein and mRNA expression of 14-3-3εin gastric carcinogenesis.METHODS:14-3-3εprotein expression was determined by western blotting,and mRNA expression was examined by real-time quantitative RT-PCR in gastric tumors and their matched non-neoplastic gastric tissue samples.RESULTS:Authors observed a significant reduction of 14-3-3εprotein expression in gastric cancer(GC)samples compared to their matched non-neoplastic tissue.Reduced levels of 14-3-3εwere also associated with diffuse-type GC and early-onset of this pathology.Our data suggest that reduced 14-3-3εmay have a role in gastric carcinogenesis process.CONCLUSION:Our results reveal that the reduced 14-3-3εexpression in GC and investigation of 14-3-3ε interaction partners may help to elucidate the carcino-genesis process.

14-3-3 λ protein interacts with ADF1 to regulate actin cytoskeleton dynamics in Arabidopsis

Actin cytoskeleton dynamics is critical for variety of cellular events including cell elongation, division and morphogenesis, and is tightly regulated by numerous groups of actin binding proteins. However it is not well understood how these actin binding proteins are modulated in a physiological condition by their interaction proteins. In this study, we describe that Arabidopsis 14-3-3 λ protein interacted with actin depolymerizing factor 1(ADF1) in plant to regulate F-actin stability and dynamics. Loss of 14-3-3 λin Arabidopsis resulted in longer etiolated hypocotyls in dark and changed actin cytoskeleton architecture in hypocotyl cells. Overexpression of ADF1 repressed 14-3-3 λ mutant hypocotyl elongation and actin dynamic phenotype. In addition, the phosphorylation level of ADF1 was increased and the subcellular localization of ADF1 was altered in 14-3-3 λ mutant. Consistent with these observations, the actin filaments were more stable in 14-3-3 λ mutant. Our results indicate that 14-3-3 λ protein mediates F-actin dynamics possibly through inhibiting ADF1 phosphorylation in vivo.

Molecular cloning of partial 14-3-3 genes in the marine sponge Hymeniacidon perleve and its role in differentiating infectious and non-infectious bacteria

Sponges （phylum Porifera） are the phylogenetically oldest metazoa and highly efficient filter feeders. In the marine ecosystem, they are unconditionally exposed to environmental stresses. Understanding the sponge-bacteria interaction is hence of both eco- logical and biological significance. This study investigated the specific interaction between the sponge Hymeniacidon perleve and the non-infectious bacteria, Escherichia coli and infectious bacteria, Vibrio spp. by measuring the 14-3-3 mRNA expression of H. perleve. Three partial cDNAs of 14-3-3 proteins and partial 18S RNA in H. perleve were cloned and sequenced. Using Re- verse-transcription real-time PCR, the 14-3-3 mRNA expression of H. perleve was examined when exposed to three common bacteria in aquatic water--E, coli and two Vibrio spp. for different time and dosages. H. perleve could efficiently remove E. coli from the water column without self-infection; however Vibrio at higher dosages infected H. perleve. When H. perleve was ex- posed to E. coli （1.1×10^7 CFU mL^-1）, V. anguillarum II （1.2×10^6 CFU mL^-1） and V. alginolyticus （3.6×10^5 CFU mL-1） for 6 h, the 14-3-3 mRNA expression in the V. anguillarum II and V. alginolyticus groups was down-regulated by 2.67- and 2.36-fold, respectively. The 14-3-3 mRNA expression in the E. coli group was not significantly different. However, no clear trend was ob- served on the 14-3-3 transcript levels of H. perleve in response to different doses of V. anguillarum II for different time. The re- sults demonstrated that infectious bacteria can be discriminated by 14-3-3 mRNA expression of sponge H. perleve.