水稻锚蛋白基因家族分析和锚定膜蛋白的表达模式研究

锚蛋白重复序列模体是生物体内最普遍的蛋白质序列模体之一,在多种细胞活动中主要介导蛋白质与蛋白质的相互作用。采用生物信息学的方法,在基因组水平上搜索和鉴定了水稻锚定重复序列蛋白,通过分析蛋白质二级结构,进行水稻锚蛋白基因家族的聚类分析,并在此基础上,用RT-PCR的方法分析水稻锚定膜蛋白的表达模式,为水稻锚蛋白基因的研究提供了理论依据。     

锚蛋白重复序列介导的蛋白质-蛋白质相互作用

锚蛋白重复序列(ANK)是生物体中广泛利用的一种序列模体.ANK模体在ANK结构域中折叠成β₂α₂结构,在空间上则形成L型结构.数目不等的ANK串联起来, 依靠氢键和疏水相互作用,组成紧密、稳定的结构域,并且形成了种类众多但功能各异的ANK蛋白质分子.ANK结构域介导蛋白质与蛋白质的相互作用,它能够和多种配体结合,实现纷繁复杂的生物功能.着重介绍几类结构已知的ANK家族蛋白质分子及复合物的结构特征、生理功能及与疾病的关系.     

菜豆ANK基因家族鉴定及ANK25的表达模式分析

锚蛋白重复序列模体是生物体内最普遍的蛋白质序列模体之一,在多种细胞活动中主要介导蛋白质-蛋白质的相互作用。本研究利用菜豆基因组数据库,通过生物信息学手段对菜豆ANK家族成员及分子生物学特性进行了鉴定。结果显示,菜豆基因组中含有30个ANK家族基因,分布于9条染色体上,其中第5条染色体上含有的ANK基因最多,包含13个基因。蛋白结构域分析发现,ANK25除了含有ANK结构域外还含有RING结构域。亚细胞定位结果显示,ANK25主要分布在细胞膜上。表达模式分析发现,ANK25对干旱、盐和ABA胁迫有响应。本研究为进一步研究菜豆ANK的分类及功能提供了有利的依据。     

细菌外毒素序列中特有模体的识别及其基因本体注释分析

【目的】识别细菌外毒素序列中特有模体,进一步理解外毒素的致病机制。【方法】构建非致病性细菌蛋白质数据库,利用InterProScan对数据库中非致病菌蛋白质序列以及收集的经实验确认的89条细菌外毒素蛋白质序列进行模体搜索。【结果】在89条细菌外毒素序列中,分析得到了39个细菌外毒素特有模体。【结论】得到的外毒素特有模体与外毒素功能密切相关,为在致病性细菌基因组内搜索外毒素序列奠定了基础;同时通过对外毒素特有模体的基因本体(Gene ontology,GO)注释分析,进一步阐明了细菌外毒素的致病机制。     

应用蛋白组方法筛选血浆中乙肝病毒PreS1结合蛋白

目的筛选血浆中乙型肝炎病毒PreS1结合蛋白。方法表达纯化了PreS1-谷胱甘肽-S-转移酶（glutathione—S-transferase,GST）融合蛋白,利用该蛋白与血浆进行Pull—down实验,并设立GST与血浆Pull—down,GST、PreS1-GST与PBS Pull—down对照,Pull-down产物进行双向电泳分离（2-DE）,差异蛋白点通过质谱鉴定。结果成功表达纯化出PreS1-GST融合蛋白,通过双向电泳分析发现一个PreS1特异结合蛋白,经质谱鉴定为含锚蛋白重复序列的蛋白57（ANKRD57）。结论锚蛋白重复序列的主要功能是介导蛋白质与蛋白质之间的相互作用,ANKRD57与PreS1特异结合后的生理功能值得深入研究。     

2个银叶真藓HSP70序列的生物信息学相关分析

从银叶真藓(Bryum argenteum)转录组数据库出发,使用Pfam数据库提供的HMM模型共得到33条长度大于200aa,注释的热休克蛋白Ba HSP70;其中2条(Ba HSP70-1,Ba HSP70-2)具有完整ORF,NCBI核酸数据库登录号为KP087877和KP087878。使用生物信息学在线分析工具和软件,对真藓HSP70的两条蛋白质序列从氨基酸组成、保守结构域、理化性质、疏水性/亲水性、信号肽、蛋白质结构、模体的识别及同源性分析等方面进行了预测和分析。结果表明:2条Ba HSP70s基因序列ORF全长分别为2 396 bp和2 356 bp,分别编码649aa和650aa。序列模体分析表明Ba HSP70s和其它报道的植物HSP70均含有4个相同的模体,并且各模体在蛋白质序列上顺序一致。通过对2条Ba HSP70s进行氨基酸多序列比对及基因树分析,发现Ba HSP70-1和Ba HSP70-2雪莲相似度最高,分别是91.2%和86.6%。本研究为进一步研究HSP70基因的克隆和功能验证奠定了基础。     

酵母转录因子PHO81锚蛋白重复序列表达和功能分析

酵母转录因子ＰＨＯ８１蛋白含有６个锚蛋白重复序列。将ＰＨＯ８１锚蛋白重复序列与谷胱甘肽转移酶（ＧＳＴ）融合表达（ＧＳＴ－ＡＮＫ）,表达产物以包含体形式存在。利用氧化型和还原型谷城肽系统对包含体进行复性,得到了可溶性蛋白,亲和纯化后,进行了活性分析。通过免疫共沉淀、分别制备了ＰＨＯ８５－ＰＨＯ８０和ＰＨＯ８５－ＰＡＰ１激酶复合物,用重组的ＰＨＯ４蛋白作底物,在激酶反应体系中加入ＧＳＴ－ＡＮＫ,发现它     

线性短模体：介导蛋白质相互作用的新模块

线性短模体是天然无序蛋白实现生物学功能的重要组件.线性短模体具有柔性结构和短小的序列,可以介导瞬时、可逆的蛋白质相互作用,并在发生相互作用时表现出杂泛性.随着实验技术的更新和预测手段的发展,越来越多的线性短模体被发现和重新定义,例如BH3线性短模体.本文重点总结了线性短模体在结构、生物学功能以及进化等方面的特点.对线性短模体功能的研究将为解析细胞信号转导网络、疾病靶标确认、新药发现等领域带来新的思路.     

富脯氨酸模体研究进展

综述了近年来富脯氨酸模体的研究进展状况,并将其分为PxxP核心序列模体、聚脯氨酸模体、PPxY核心序列模体以及其他类型的模体4类,论述了富脯氨酸膜体的生物学功能及其常出现的原因.     

锌指蛋白基因ZNF191

锌指是Ｍｉｌｌｅｒ等人 (1 985年 )最初在爪蟾转录因子ＴＦ ＩＩＩＡ中发现的一种特殊结构 ,即在蛋白质一级结构上 ,存在着Ｃ Ｘ2 4 Ｃ Ｘ12 Ｈ Ｘ3 Ｈ的序列单元 (Ｃ :半胱氨酸 ;Ｈ :组氨酸 ;Ｘ :任意氨基酸 ) ,其中的 2个半胱氨酸和 2个组氨酸通过配位键与一个锌离子结合 ,成手指状结构 ,这类序列单元被称为锌指模体 ,含有锌指模体的蛋白质称为锌指蛋白。研究表明 ,锌指蛋白作为一类转录因子而广泛地参与基因表达的调控。这种调控作用主要是通过它与特定的ＤＮＡ序列 (如启动子 )和蛋白质的结合而实现的。锌指蛋白与胚胎发育和细胞分化…     

Inhibition of cofactor activity of protein S by a complex of protein S and C4b-binding protein. Evidence for inactive ternary complex formation between protein S, C4b-binding protein, and activated protein C

To elucidate the mechanism by which C4b-binding protein inhibits the cofactor activity of protein S for anticoagulant-activated protein C, the interactions between protein S, activated protein C, and C4b-binding protein were studied using solid-phase enzyme immunoassays. Both activated protein C and C4b-binding protein bound to protein S fixed to microplate wells. C4b-binding protein did not inhibit the binding of activated protein C to protein S, nor did activated protein C inhibit the binding of C4b-binding protein to protein S. Activated protein C bound to a protein S-C4b-binding protein complex which was cross-linked with a chemical reagent as well as it bound to free protein S. Protein S-C4b-binding protein complex competitively inhibited activated protein C-binding to free protein S and also the cofactor activity of free protein S. Immunoblotting analysis showed ternary complex formation with protein S, C4b-binding protein, and activated protein C in the liquid phase by treatment with the cross-linking reagent. These findings suggest that the protein S-C4b-binding protein complex inhibits the cofactor activity of free protein S probably by inhibition of functionally active protein S-activated protein C complex formation by the apparent competitive formation of an inactive ternary complex with protein S, C4b-binding protein, and activated protein C.     

Biochemical basis of the constitutive repressor cleavage activity of recA730 protein. A comparison to recA441 and recA803 proteins.

The recA730 mutation results in constitutive SOS and prophage induction. We examined biochemical properties of recA730 protein in an effort to explain the constitutive activity observed in recA730 strains. We find that recA730 protein is more proficient than the wild-type recA protein in the competition with single-stranded DNA binding protein (SSB protein) for single-stranded DNA (ssDNA) binding sites. Because an increased aptitude in the competition with SSB protein has been previously reported for recA441 protein and recA803 protein, we directly compared their in vitro activities with those of recA730 protein. At low magnesium ion concentration, both ATP hydrolysis and lexA protein cleavage experiments demonstrate that these recA proteins displace SSB protein from ssDNA in a manner consistent with their in vivo repressor cleavage activity, i.e. recA730 protein > recA441 protein > recA803 protein > recAwt protein. Additionally, a correlation exists between the proficiency of the recA proteins in SSB protein displacement and their rate of association with ssDNA. We propose that an increased rate of association with ssDNA allows recA730 protein to displace SSB protein from the ssDNA that occurs naturally in Escherichia coli and thereby to become activated for the repressor cleavage that leads to SOS induction. RecA441 protein is similarly activated for repressor cleavage; however, in this case, significant SSB protein displacement occurs only at elevated temperature. At physiological magnesium ion concentration, we argue that recA803 protein and wild-type recA protein do not displace sufficient SSB protein from ssDNA to constitutively induce the SOS response.     

Severe acute respiratory syndrome coronavirus 7a accessory protein is a viral structural protein

Severe acute respiratory syndrome coronavirus (SCoV) 7a protein is one of the viral accessory proteins. In expressing cells, 7a protein exhibits a variety of biological activities, including induction of apoptosis, activation of the mitogen-activated protein kinase signaling pathway, inhibition of host protein translation, and suppression of cell growth progression. Analysis of SCoV particles that were purified by either sucrose gradient equilibrium centrifugation or a virus capture assay, in which intact SCoV particles were specifically immunoprecipitated by anti-S protein monoclonal antibody, demonstrated that 7a protein was associated with purified SCoV particles. Coexpression of 7a protein with SCoV S, M, N, and E proteins resulted in production of virus-like particles (VLPs) carrying 7a protein, while 7a protein was not released from cells expressing 7a protein alone. Although interaction between 7a protein and another SCoV accessory protein, 3a, has been reported, 3a protein was dispensable for assembly of 7a protein into VLPs. S protein was not required for the 7a protein incorporation into VLPs, and yet 7a protein interacted with S protein in coexpressing cells. These data established that, in addition to 3a protein, 7a protein was a SCoV accessory protein identified as a SCoV structural protein.     

Inhibition of protein Ca cofactor function of human and bovine protein S by C4b-binding protein

Vitamin K-dependent protein S exists in two forms in plasma, as free protein and in a bimolecular, noncovalent complex with the regulatory complement protein C4b-binding protein (C4BP). The effects of C4BP on the protein Ca cofactor activity of protein S were studied in a plasma system and in a system using purified components from both human and bovine origin. Bovine protein S was found to interact with human C4BP with a 5-fold higher affinity than that observed for the interaction between human protein S and human C4BP. The binding of protein S, from either species, to human C4BP results in the loss of the protein Ca cofactor function. In bovine plasma, protein S could be totally complexed by the addition of human C4BP, with a concomitant total loss of protein Ca cofactor activity. The addition of purified human C4BP to human plasma resulted in only partial loss of protein Ca cofactor activity and the plasma protein S was not completely complexed. Human protein S functioned as a cofactor to human protein Ca, but not to bovine protein Ca, whereas bovine protein S demonstrated very little species specificity and functioned as a cofactor both with human and bovine protein Ca. The species specificity of the protein Ca-protein S interaction was useful in elucidating the effect of C4BP in the plasma system. In the system with purified bovine components, protein S was required for the degradation of factor Va by low concentrations of protein Ca, whereas in the system with human components protein Ca alone, even when added at very low concentrations, exhibited potential to degrade factor Va, and the presence of protein S only enhanced the reaction rate approximately 5-fold. In both these systems, the stimulating effect of protein S on factor Va degradation by protein Ca was completely lost when protein S bound to C4BP.     

Ultrastructure of Endosperm Protein Bodies in Developing Rice Grains Differing in Protein Content

Ultrastructural aspects of the development of protein bodies(aleurone grains) in endosperm of grains of two rices differingin protein content are described. Formation of rough endoplasmicreticulum complexes prior to protein deposition was observedonly in the higher protein grain. From 8 days after floweringthree types of protein body were observed, one of which wasrestricted to the peripheral endosperm (sub-aleurone) layers.The higher protein grain had a greater number of protein bodiesand rough endoplasmic reticulum in the endosperm cells thanthe lower protein grain. Increase in total protein with maturitywas the result of increased number of protein bodies ratherthan increase in size; the protein bodies were concentratedin the peripheral endosperm layers.     

Biochemical properties of the Escherichia coli recA430 protein. Analysis of a mutation that affects the interaction of the ATP-recA protein complex with single-stranded DNA

The biochemical properties of the recA430 protein have been examined and compared to those of wild-type recA protein. We find that, while the recA430 protein possesses ssDNA-dependent rATP activity, this activity is inhibited by the Escherichia coli single-stranded DNA binding protein (SSB protein) under many conditions that enhance wild-type recA protein rATPase hydrolysis. Stimulation of rATPase activity by SSB protein is observed only at high concentrations of both rATP (greater than 1 mM) and recA430 protein (greater than 5 microM). In contrast, stimulation of ssDNA-dependent dATPase activity by SSB protein is less sensitive to protein and nucleotide concentration. Consistent with the nucleotide hydrolysis data, recA430 protein can carry out DNA strand exchange in the presence of either rATP or dATP. However, in the presence of rATP, both the rate and the extent of DNA strand exchange by recA430 protein are greatly reduced compared to wild-type recA protein and are sensitive to recA430 protein concentration. This reduction is presumably due to the inability of recA430 protein to compete with SSB protein for ssDNA binding sites under these conditions. The cleavage of lexA repressor protein by recA430 protein is also sensitive to the nucleotide cofactor present and is completely inhibited by SSB protein when rATP is the cofactor but not when dATP is used. Finally, the steady-state affinity and the rate of association of the recA430 protein-ssDNA complex are reduced, suggesting that the mutation affects the interaction of the ATP-bound form of recA protein with ssDNA. This alteration is the likely molecular defect responsible for inhibition of recA430 protein rATP-dependent function by SSB protein. The biochemical properties observed in the presence of dATP and SSB protein, i.e. the reduced levels of both DNA strand exchange activity and cleavage of lexA repressor protein, are consistent with the phenotypic behavior of recA430 mutations.     

UvsY protein of bacteriophage T4 is an accessory protein for in vitro catalysis of strand exchange

The uvsX and uvsY genes are essential to genetic recombination, recombination-dependent DNA synthesis and to the repair of DNA damage in bacteriophage T4. Purified UvsX protein has been shown to catalyze strand exchange and D-loop formation in vitro, but the role of UvsY protein has been unclear. We report that UvsY protein enhances strand exchange by UvsX protein by interacting specifically with UvsX protein: gene 32 protein (gp32) is not necessary for this effect and UvsY protein has no similar effect on the RecA protein of E. coli. UvsY protein, like UvsX protein, protects single-stranded DNA from digestion by nucleases, but, unlike UvsX protein, shows no ability to protect double-stranded DNA. UvsY protein enhances the rate of single-stranded-DNA-dependent ATP hydrolysis by UvsX protein, particularly in the presence of gp32 or high concentrations of salt, factors that otherwise reduce the ATPase activity of UvsX protein. The enhancement of ATP hydrolysis by UvsY protein is shown to result from the ability of UvsY protein to increase the affinity of UvsX protein for single-stranded DNA.     

Regulation of dnaB function in DNA replication in Escherichia coli by dnaC and lambda P gene products

The dnaB protein of Escherichia coli, a multifunctional DNA-dependent ribonucleotide triphosphatase and dATPase, cross-links to ATP on ultraviolet irradiation under conditions that support rNTPase and dATPase activities of dnaB protein. The covalent cross-linking to ATP is specifically inhibited by ribonucleotides and dATP. Tryptic peptide mapping demonstrates that ATP cross-links to only the 33-kDa tryptic fragment (Fragment II) of dnaB protein. The presence of single-stranded DNA alters the covalent labeling of dnaB protein by ATP, suggesting a possible role of DNA on the mode of nucleotide binding by dnaB protein. Present studies demonstrate that the dnaC gene product binds ribonucleotides independent of dnaB protein. On dnaB-dnaC protein complex formation, covalent incorporation of ATP to dnaB protein decreases approximately 70% with a concomitant increase of ATP incorporation to dnaC protein by approximately 3-fold. The mechanism of this phenomenon has been analyzed in detail by titrating dnaB protein with increasing amounts of dnaC protein. The binding of dnaC protein to dnaB protein appears to be a noncooperative process. The lambda P protein, which interacts with dnaB protein in the bacteriophage lambda DNA replication, does not bind ATP in the presence or absence of dnaB protein. However, lambda P protein enhances the covalent incorporation of ATP to dnaB protein approximately 4-fold, suggesting a direct physical interaction between lambda P and dnaB proteins with a probable change in the modes of nucleotide binding to dnaB protein. The lambda P protein likely forms a lambda P-dnaB-ATP dead-end ternary complex. The implications of these results in the E. coli and bacteriophage lambda chromosomal DNA replication are discussed.     

Identification of a new protein involved in the regulation of the anticoagulant activity of activated protein C. Protein S-binding protein

The apparent molecular weight of functional protein S in citrated plasma was observed to be between 115,000 and 130,000 as measured by sedimentation equilibrium in the air-driven ultracentrifuge. The molecular weight of the functional protein decreased to approximately 62,000 when copper ions were added to the plasma. This suggested the presence of a protein S-binding protein in plasma, which was confirmed by gel filtration experiments. Frontal analysis of plasma indicated that functional protein S could exist in as many as three forms. Addition of copper ions to plasma reduced the number of forms to one. In order to isolate the binding protein, plasma was fractionated first on a column of immobilized iminodiacetic acid that had been equilibrated with copper ions. The proteins that eluted in a 0.6 M NaCl wash were passed over a column of protein S immobilized on agarose beads. A protein, eluted in the 0.6 M NaCl wash, was observed to bind to protein S in gel filtration experiments. When added to plasma depleted of both protein S and the binding protein, the binding protein was observed to enhance the anticoagulant activity of activated protein C only in the presence of protein S. Protein S-binding protein was also observed to enhance the rate of factor Va inactivation by activated protein C and protein S.     

6-Hydroxydopamine upregulates iron regulatory protein 1 by activating certain protein kinase C isoforms in the dopaminergic MES23.5 cell line

Iron-induced oxidative stress is thought to play a crucial role in the pathogenesis of Parkinson's disease. Our previous studies demonstrated that decreased expression of ferroportin 1 contributes to 6-hydroxydopamine induced intracellular iron accumulation and that decreased ferroportin 1 expression is caused by increased expression of iron regulatory protein 1. Iron regulatory protein 1 is a central regulator of iron homeostasis and is a likely target of extracellular agents to program changes in cellular iron metabolism. Therefore, the mechanism of iron regulatory protein 1 upregulation induced by 6-hydroxydopamine has become a significant focus of research. Iron regulatory protein 1 is regulated by protein kinase C, although this regulation is tissue specific. Therefore, in the present study, we aimed to determine whether alteration of protein kinase C activity modified iron regulatory protein 1 expression in the dopaminergic MES23.5 cell line, Furthermore, we investigated whether 6-hydroxydopamine induced iron regulatory protein 1 upregulation is mediated by protein kinase C, thus achieving regulation of cellular iron levels. The results showed that iron regulatory protein 1 was upregulated by phorbol 12-myristate-13-acetate, the PKC activator in dopaminergic MES23.5 cells, and ferroportin 1 expression and iron efflux were decreased as a result of iron regulatory protein 1 upregulation. The protein kinase C inhibitor bisindolylmaleimide I hydrochloride abolished the effect of phorbol 12-myristate-13-acetate. Protein kinase C-δ and protein kinase C-ζ, but not protein kinase C-? were activated by 6-hydroxydopamine. The protein kinase C-δ inhibitor rottlerin inhibited protein kinase C-δ phosphorylation and abolished iron regulatory protein 1 upregulation induced by 6-hydroxydopamine. The protein kinase C-ζ pseudo-substrate inhibitor inhibited protein kinase C-ζ phosphorylation and abolished iron regulatory protein 1 upregulation induced by 6-hydroxydopamine. These data indicate that iron regulatory protein 1 is regulated by protein kinase C in dopaminergic MES23.5 cells and that protein kinase C activated by 6-hydroxydopamine regulates iron regulatory protein 1 expression, thus achieving regulation of cellular iron levels.