14-3-3蛋白的研究进展

14-3-3是一个在真核生物中广泛表达的酸性蛋白家族,主要以同源/异源二聚体形式存在,通过磷酸化丝/苏氨酸作用与靶蛋白或靶蛋白的2个结构域结合。7种不同亚型的高度保守的14-3-3蛋白与人类细胞中多种不同的信号通路有密切的关系。14-3-3通过调节靶蛋白间的相互作用在信号转导途径中发挥调控作用。     

与14-3-3ζ相互作用的蛋白Polo样激酶1的筛选与鉴定

目的:应用酵母双杂交系统筛选与14-3-3ζ相互作用的蛋白,进一步鉴定其与Polo样激酶1(Plk1)相互作用。方法:构建pGBKT7-14-3-3ζ诱饵表达载体,筛选HeLa细胞cDNA文库中与14-3-3ζ相互作用蛋白,进一步通过共转酵母、免疫荧光以及外源性和内源性的细胞免疫共沉淀实验验证两者的相互作用。结果:通过酵母双杂交系统筛选出的阳性相互作用蛋白中包括Plk1,进一步通过共转酵母,外源性和内源性的细胞免疫共沉淀实验证实两者的相互作用,免疫荧光实验证实两者共定位于有丝分裂过程中胞质分裂期的中体。结论:Plk1是高度保守的丝氨酸/苏氨酸蛋白激酶,在中体的成熟,有丝分裂期染色体的分离,胞质分裂以及DNA的损伤应答等环节发挥重要作用,其与14-3-3ζ的相互作用为14-3-3蛋白家族参与有丝分裂(M期)的调控提供了直接证据。     

14-3-3蛋白与凋亡

14 3 3蛋白是分布十分广泛的真核生物蛋白家族 ,在体内发挥着诸多的生物学功能。 14 3 3蛋白能够在多个通路和环节与一些调节因子相互作用 ,抑制凋亡的发生 ;14 3 3蛋白主要是通过以下几种作用方式参与凋亡的调节 :控制靶蛋白 (如Bad ,Bax ,Fkhrl 1)的亚细胞定位 ,作为接头蛋白 (如A2 0与c Raf 1)促进蛋白质 蛋白质相互作用以及调节关键酶 (如ASK1)的活性 ,但具体作用机制有待进一步研究     

14－3－3蛋白的新认识

14-3-3蛋白是一个在真核生物中广泛表达的高度保守的酸性蛋白家族,主要以同源/异源二聚体形式存在,在哺乳动物中由7种亚型组成。7种不同亚型的14-3-3蛋白在人类细胞中发挥着重要作用。本文就14-3-3蛋白的序列保守性和独特性、结构特征、结合靶点、调控机制以及作为潜在药物治疗靶点的相关研究进展做一综述。     

蛋白14-3-3蛋白亚型与癌症

14-3-3蛋白家族在真核细胞中广泛表达且高度保守,据报道其与心血管疾病、神经元损伤、帕金森病以及支气管哮喘等疾病有关联,近年来研究发现14-3-3蛋白与癌症的发生和发展也密切相关,而涉及的癌症包括胆管癌、肝癌、肺癌、鼻咽癌、口腔癌、食管癌、胃癌、乳腺癌、卡波济肉瘤、脑膜癌、直肠癌、星形细胞瘤等几乎所有常见癌症。14-3-3蛋白对癌症增殖、转移、凋亡以及耐药等影响作用已经得到证实,其调控机制也得到部分揭示,并且已合成了影响14-3-3蛋白与其他蛋白相互作用的特异小分子阻断药物。人体内的14-3-3蛋白家族包含β,ε,γ,η,θ(τ),ζ和σ7种亚型,不同亚型有不同的组织定位和功能。阐明各亚型在不同肿瘤中的分布、作用及其调控机制,对我们进一步认识和治疗癌症均有一定的指导意义。     

14-3-3蛋白各亚型在大鼠黑质和PC12细胞中的分布

本实验采用免疫组织化学、免疫荧光组织化学和免疫印迹技术对14-3-3蛋白各亚型在大鼠黑质(SN)和PC12细胞中的分布情况进行了研究。在大鼠黑质内,免疫组化和免疫荧光结果显示14-3-3蛋白的γ、ε、θ、ζ四个亚型呈阳性反应,而β和η亚型未见明显阳性反应;在PC12细胞中,6个亚型均呈免疫荧光阳性反应;在黑质中,Westernblot结果显示14-3-3各亚型蛋白表达的相对水平为ε>γ>ζ>θ;而在PC12细胞中各亚型的表达水平为γ>ε>β>ζ>η>θ。本实验的结果提示ε、γ、ζ和θ亚型可能在黑质中发挥主要作用。本研究的结果为研究14-3-3蛋白在Parkinson病中的作用提供了形态学依据。     

大鼠脂肪细胞中14-3-3蛋白与葡萄糖转运子4的相互作用

目的研究在大鼠脂肪细胞中,14-3-3蛋白与葡萄糖转运子4(GLUT4)之间是否存在相互作用。方法用胶原酶I消化雄性SD大鼠附睾上的脂肪垫,获得分离的脂肪细胞。在纯化的细胞中,采用低渗裂解和甘油梯度速率沉降技术获得3个含有GLUT4的组分:T、H和L。用交联了1F8(GLUT4特异性单抗)的琼脂糖微珠对脂肪细胞总提取物以及上述3个组分分别进行免疫吸附实验,经吸附后在上清液和微珠的洗脱液中分别进行14-3-3蛋白和GLUT4的免疫印迹分析。结果在脂肪细胞的总提取物以及上述GLUT4的3个组分T、H和L中,14-3-3蛋白都能够与GLUT4发生免疫共沉淀。共沉淀下来的14-3-3蛋白在免疫印迹分析时显示为2个条带,其相对分子质量分别约为29 ku和60 ku,提示14-3-3蛋白可能以二聚体的形式与GLUT4发生相互作用。结论在生理条件下,14-3-3蛋白与GLUT4在大鼠脂肪细胞中存在相互作用。     

窖蛋白-1与肿瘤

窖蛋白是胞膜窖结构中的特征性结构蛋白。窖蛋白在细胞内吞、胆固醇运输、细胞膜组装、调节信号转导和肿瘤生成、转移等生命活动中扮演了重要角色。窖蛋白基因家族中的窖蛋白-1(Cav-1)可能参与肿瘤生成和转移的病理机制,以及与多药耐药等密切相关。窖蛋白-1参与调节细胞周期与信号转导,在细胞凋亡程序中发挥重要作用,具有抗血管新生,负性调节生长因子,参与肿瘤细胞侵袭和转移的作用。     

信号蛋白14-3-3 β及其缺失突变体与朊蛋白体外相互作用的初步研究

目的 确定PrP蛋白与14-3-3蛋白是否发生分子间的相互作用,以及相互作用区域.方法 表达纯化PrP和人14-3-3 β及其缺失突变体.通过Pull-down和免疫共沉淀方法研究全长人14-3-3 β及缺失突变体与PrP蛋白是否发生分子间相互作用以及相互作用的区域.结果 14-3-3 β能与PrP23-231发生体外的相互作用,且与PrP相互作用的区域位于14-3-3 β N端第1位至38位氨基酸.结论 首次证实人14-3-3 β与PrP相互作用的区域位于14-3-3 β N端第1位至38位氨基酸.     

细胞周期蛋白D与细胞周期调控研究进展

细胞周期是细胞生命活动中一个最重要的过程,在细胞周期的4个时相中,G1期是启动细胞周期循环的关键。细胞周期蛋白(cyclin)、细胞周期蛋白依赖性激酶(CDKs)和CDK抑制蛋白(CDKIs)是参与细胞周期调控的主要因子。而cyclinD作为细胞周期的启动因子,作用于G1期,在细胞增殖中发挥着重要作用。综述了cyclinD的结构、功能,cyclinD与CKI蛋白的相互作用及其表达失调与某些疾病的关系。     

Association of Chk1 with 14-3-3 proteins is stimulated by DNA damage

The protein kinase Chk1 is required for cell cycle arrest in response to DNA damage. We have found that the 14-3-3 proteins Rad24 and Rad25 physically interact with Chk1 in fission yeast. Association of Chk1 with 14-3-3 proteins is stimulated in response to DNA damage. DNA damage results in phosphorylation of Chk1 and the 14-3-3 proteins bind preferentially to the phosphorylated form. Genetic analysis has independently implicated both Rad24 and Rad25 in the DNA-damage checkpoint pathway. We suggest that DNA damage-dependent association of phosphorylated Chk1 with 14-3-3 proteins mediates an important step along the DNA-damage checkpoint pathway, perhaps by directing Chk1 to a particular substrate or to a particular location within the cell. An additional role for 14-3-3 proteins in the DNA-damage checkpoint has been suggested based on the observation that human Chk1 can phosphorylate Cdc25C in vitro creating a 14-3-3 binding site. Our results suggest that in fission yeast the interaction between the 14-3-3 proteins and Cdc25 does not require Chk1 function and is unaffected by DNA damage, in sharp contrast to the interaction between the 14-3-3 proteins and Chk1.     

14-3-3zeta is indispensable for aggregate formation of polyglutamine-expanded huntingtin protein

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder caused by polyglutamine (polyQ) expansions in the huntingtin (Htt) protein. A hallmark of HD is the presence of aggregates-predominantly composed of NH(2)-terminal fragments of polyQ-expanded Htt-in the nucleus and cytoplasm of affected neurons. We previously proposed that 14-3-3zeta might act as a sweeper of misfolded proteins by facilitating the formation of aggregates possibly for neuroprotection; these aggregates are referred to as inclusion bodies. However, evidence available in this regard is indirect and circumstantial. In this study, analysis of the aggregation-prone protein Htt encoded by HD gene exon 1 containing polyglutamine expansions (Htt86Q) revealed that 17 residues in the NH(2)-terminal of this protein are indispensable for its aggregate formation. Immunoprecipitation assays revealed that 14-3-3beta, gamma, eta, and zeta interact with Htt86Q transfected in N2a cells. Interestingly, the small interfering ribonucleic acid (siRNA) suppression of 14-3-3zeta exclusively abolished Htt86Q aggregate formation, whereas 14-3-3beta or eta siRNA suppression did not. This indicates that 14-3-3zeta participates in aggregate formation under nonnative conditions. Our data support a novel role for 14-3-3zeta in the aggregate formation of nonnative, aggregation-prone proteins.     

Intranuclear immunolocalization of 14-3-3 protein isoforms in brains with spinocerebellar ataxia type 1

Immunolocalization of 14-3-3 protein isoforms, one of the interacters with ataxin 1, was investigated in spinocerebellar ataxia type 1 (SCA 1) brains using isoform-specific antibodies. Samples from the pons and from the cerebellum of four SCA1 cases and three controls were studied. The intensity of the immunoreactivity (IR) and its subcellular topography were analyzed. In control subjects, granular immunoreactivity for an epitope common to all known isoforms of 14-3-3 proteins (14-3-3 COM) found in the cytoplasm of some pontine and dentate nucleus neurons was weak. It was observed in some Purkinje cells, while its intensity varied. Many nuclei of those neurons and Purkinje cells of SCA1 were intensely immunopositive for 14-3-3 COM, while it was less in their cytoplasm. Expanded polyglutamine epitope was colocalized to 14-3-3 COM epitope in some pontine neurons, sometimes accumulated in intranuclear inclusion-like structures. This findings support previous reports that 14-3-3 proteins stabilize mutant ataxin 1 in nucleus and possibly lead to neurodegeneration. However, nuclear localization of 14-3-3 proteins in SCA1 brains was dependent on its isoforms, i.e. pontine neurons intensely positive for beta, Purkinje cells for tau and dentate nucleus neurons for both, while all of those neurons were consistently positive for zeta isoform, although sigma isoform tended to be located in the cytoplasm. Nuclear accumulation and isoform- and region-dependent subcellular localizations of 14-3-3 proteins may be related to SCA1 pathology, which exhibits marked regional variability.     

Interaction with 14-3-3 proteins promotes functional expression of the potassium channels TASK-1 and TASK-3

The two-pore-domain potassium channels TASK-1, TASK-3 and TASK-5 possess a conserved C-terminal motif of five amino acids. Truncation of the C-terminus of TASK-1 strongly reduced the currents measured after heterologous expression in Xenopus oocytes or HEK293 cells and decreased surface membrane expression of GFP-tagged channel proteins. Two-hybrid analysis showed that the C-terminal domain of TASK-1, TASK-3 and TASK-5, but not TASK-4, interacts with isoforms of the adapter protein 14-3-3. A pentapeptide motif at the extreme C-terminus of TASK-1, RRx(S/T)x, was found to be sufficient for weak but significant interaction with 14-3-3, whereas the last 40 amino acids of TASK-1 were required for strong binding. Deletion of a single amino acid at the C-terminal end of TASK-1 or TASK-3 abolished binding of 14-3-3 and strongly reduced the macroscopic currents observed in Xenopus oocytes. TASK-1 mutants that failed to interact with 14-3-3 isoforms (V411*, S410A, S410D) also produced only very weak macroscopic currents. In contrast, the mutant TASK-1 S409A, which interacts with 14-3-3-like wild-type channels, displayed normal macroscopic currents. Co-injection of 14-3-3ζ cRNA increased TASK-1 current in Xenopus oocytes by about 70 %. After co-transfection in HEK293 cells, TASK-1 and 14-3-3ζ (but not TASK-1ΔC5 and 14-3-3ζ) could be co-immunoprecipitated. Furthermore, TASK-1 and 14-3-3 could be co-immunoprecipitated in synaptic membrane extracts and postsynaptic density membranes. Our findings suggest that interaction of 14-3-3 with TASK-1 or TASK-3 may promote the trafficking of the channels to the surface membrane.     

Immunolocalization of 14-3-3 isoforms in brains with Pick body disease

Immunolocalization of 14-3-3 protein isoforms in relation to Pick bodies in Pick body disease (PBD) brains was investigated. Weakly granular immunoreactivity of 14-3-3 proteins was found in neurons in control subjects and in Pick body disease brains. In addition to this granular immunoreactivity, many Pick bodies were immunopositive for 14-3-3 proteins as confirmed with double-immunofluorescence with an anti-PHF tau (AT8) and anti-14-3-3 that recognizes all its isoforms (common). When probed with isoform-specific antibodies, Pick bodies were positive for beta, gamma, epsilon, eta, tau, and zeta isoform and exhibited immunostaining pattern similar to that observed with the anti-14-3-3 proteins (common). In addition, immunoreactivity of sigma isoform, so far considered to be exclusively extraneuronal, was unexpectedly found in Pick bodies, normal hippocampal neurons and brain homogenate from age-matched controls. Although localization of 14-3-3 proteins in Pick bodies suggests their involvement in Pick body formation, their role may be variable dependent on the isoforms differently expressed in different area in the brain.     

Increased expression of 14-3-3varepsilon protein in intrinsically aged and photoaged human skin in vivo

Skin aging is a complicated process associated with the passage of time and environmental exposure, especially to UV light. This aging phenomenon is related to alterations in various cellular mechanisms, such as changes in apoptosis, perturbations to cellular signaling, and an increased genetic instability. In this study, we investigated changes of proteins involved in intrinsic aging by the proteomic analysis of human sun-protected (upper inner arm) young and aged dermis. One of the proteins upregulated in aged dermis was identified as 14-3-3epsilon. This protein is an isoform of 14-3-3 protein, which is involved in cellular processes like signal transduction, cell cycle arrest, and apoptosis. 14-3-3epsilon is consistently found to be upregulated in the sun-protected dermis of aged skin, by Western blotting and immunohistochemical staining. In addition, we demonstrate that the expression of 14-3-3epsilon is further upregulated in the sun-exposed (photodamaged) dermis, and that the UV irradiation of young skin significantly upregulates 14-3-3epsilon in vivo. Our results suggest the possibility that the cellular processes related to 14-3-3epsilon protein play an important role in the photoaging and intrinsic aging of human skin.