Proteins with haemagglutinin activity in larvae of the Colorado beetle, Leptinotarsa decemlineata

The haemagglutinating activity of larval haemolymph of Leptinotarsa decemlineata against red blood cells of various origins has been examined. This activity appeared to be unspecific, since all the different types of erythrocytes were agglutinated by a haemolymph dilution of $\text{1}\text{128}$ to $\text{1}\text{512}$. Only horse erythrocytes were agglutinated to a greater degree ($\text{1}\text{3200}$. Red blood cells became much more sensitive after treatment with trypsin, while formol fixation also resulted in a better agglutinability. Sulphated polysacchrides (heparin, mucin, dextran sulphate) were good inhibitors of the haemagglutination reaction. A weaker inhibition was obtained with hexosamines. As demonstrated by immunoelectrophoresis, two haemagglutinins occur in larval haemolymph. One is specific for larvae and pupae, and is therefore called the larval-pupal haemagglutinin. It is absent in adults. The second haemagglutinin is the well-known chromoprotein 2, which is present in all developmental stages, including the egg, where it constitutes an important element of yolk proteins. The affinity of chromoprotein 2 toward dextran sulphate was confirmed by precipitation tests in agarose.     

Tau融合蛋白及其缺失突变体与朊蛋白的体外作用分析

在部分朊病毒病（prion diseases）中,高度磷酸化的微管相关蛋白tau与朊蛋白(prion protein,PrP)发生共定位,tau蛋白可能在朊病毒病的病理机制中有重要作用. 本室已经证明二者可以发生分子间相互作用,本文进一步分析了tau蛋白与prion的体外相互作用及作用位点. 利用RT-PCR方法从人源细胞系SHSY5Y cDNA中扩增出微管相关蛋白tau全长cDNA序列,克隆至质粒pGEX-2T载体,在大肠杆菌中诱导表达融合蛋白GST-tau. 利用GST pull-down及免疫共沉淀方法检测全长tau蛋白与PrP23-231的分子间相互作用. 进一步表达tau 蛋白的各种缺失突变体,确定tau蛋白与PrP蛋白的相互作用位点. 结果表明,所表达的全长tau蛋白及各种缺失突变体均为可溶性蛋白,Western印迹结果显示,各种蛋白均能很好的被tau蛋白单抗识别. GST pull-down和免疫共沉淀实验均显示,原核表达的全长tau蛋白可与全长的PrP蛋白在体外发生相互作用,并确定相互作用位点位于tau蛋白的N端序列及中段的重复区. 上述结果为研究tau蛋白与PrP的相互作用在朊病毒病的发病机制中的意义提供了一定的理论基础.     

Interactions Between Late-Acting Proteins Required for Peptidoglycan Synthesis during Sporulation

The requirement of peptidoglycan synthesis for growth complicates the analysis of interactions between proteins involved in this pathway. In particular, the latter steps that involve membrane-linked substrates have proven largely recalcitrant to in vivo analysis. Here, we have taken advantage of the peptidoglycan synthesis that occurs during sporulation in Bacillus subtilis to examine the interactions between SpoVE, a nonessential, sporulation-specific homolog of the well-conserved and essential SEDS (shape elongation, division, and sporulation) proteins, and SpoVD, a nonessential class B penicillin binding protein. We found that localization of SpoVD is dependent on SpoVE and that SpoVD protects SpoVE from in vivo proteolysis. Co-immunoprecipitations and fluorescence resonance energy transfer experiments indicated that SpoVE and SpoVD interact, and co-affinity purification in Escherichia coli demonstrated that this interaction is direct. Finally, we generated a functional protein consisting of an SpoVE-SpoVD fusion and found that a loss-of-function point mutation in either part of the fusion resulted in loss of function of the entire fusion that was not complemented by a wild-type protein. Thus, SpoVE has a direct and functional interaction with SpoVD, and this conclusion will facilitate understanding the essential function that SpoVE and related SEDS proteins, such as FtsW and RodA, play in bacterial growth and division.     

The Site Specificity of Two Species of Gregarina in Tenebrio molitor Larvae

The site specificity of the apicomplexans Gregarina cuneata and Gregarina sleini , in larval Tenebrio molitor was investigated. Gregarina cuneata was found to inhabit the anteriormost region of the larval midgut, while G. steini was restricted to the posterior portion of the intestine. The site specificity of the pair was conserved in single and concurrent infections. Interspecific interactions do not seem to be presently responsible for the resource partitioning by the 2 gregarine species. Key words. Gregarina, site specificity, Tenebrio molitor.     

Specificity of Collybistin-Phosphoinositide Interactions: IMPACT OF THE INDIVIDUAL PROTEIN DOMAINS*

The regulatory protein collybistin (CB) recruits the receptor-scaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We developed an in vitro assay based on solid-supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes doped with different phosphoinositides on silicon/silicon dioxide substrates to quantify the binding of various CB2 constructs using reflectometric interference spectroscopy. Based on adsorption isotherms, we obtained dissociation constants and binding capacities of the membranes. Our results show that full-length CB2 harboring the N-terminal Src homology 3 (SH3) domain (CB2_SH3+) adopts a closed and autoinhibited conformation that largely prevents membrane binding. This autoinhibition is relieved upon introduction of the W24A/E262A mutation, which conformationally “opens” CB2_SH3+ and allows the pleckstrin homology domain to properly bind lipids depending on the phosphoinositide species with a preference for phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate. This type of membrane tethering under the control of the release of the SH3 domain of CB is essential for regulating gephyrin clustering.     

Characterization of Storage Proteins in Daucus carota L.: Two Novel Proteins Display Zygotic Embryo Specificity

Although maturation-related proteins are well known in the endospermof albuminous seeds, an important question is whether the zygoticembryo possesses its own maturation proteins. We report on theisolation and partial characterization of storage proteins ofcarrot (Daucus carota L. var Nandor) dry achenes and isolatedzygotic embryos, using one- and two-dimensional electrophoresistechniques, HPLC and amino acid sequencing. The presence ofa series of abundant polypeptides showing charge heterogeneity,that are rapidly degraded upon germination, was revealed inthe endosperm. These proteins consisted of glycoproteins, themost abundant of which displayed a molecular mass (M_r) of 58,000,albumins of M_r 42,000 comprising at least one rß-1,3-glucanase,and two globulins of M_r 90,000 and 50,000–55,000 respectively,the second being an oligomer composed of three subunits of M_r13,000, 20,000 and 30,000. None of these storage proteins identifiedin the endosperm were detected in zygotic embryos. In contrast,two novel proteins were isolated from zygotic embryos, namelya globulin family of M_r 50,000 and pI 6.3–6.8, which wasnamed "daucin", and a late embry-ogenesis abundant (LEA) proteinfamily of M_r 25,000 and pI6.3–6.6, named "RAB25". Sincethe latter proteins are apparently absent of the endosperm,these results suggest that the maturation of carrot zygoticembryos requires its own specific set of storage and LEA proteins. (Received July 15, 1997; Accepted October 28, 1997)     

Molecular Dynamics Simulations of Proteins in Water Without the Truncation of Long-range Coulomb Interactions

A new program package (COSMOS90) for molecular dynamics simulations was developed to simulate large molecular systems consisting of more than tens of thousands of atoms without the truncation of long-range coulomb interactions. This program package was based on a new approximation scheme (PPPC) for calculating efficiently the coulomb interactions without sacrificing accuracy. In this approximation scheme, the group of charges at a long distance from each atom was represented by a total charge and total dipole moment of the group. In order to assess the accuracy of PPPC and the ability of COSMOS90, molecular dynamics simulations were carried out for a large system consisting of 16108 atoms (human lysozyme in water) for 50 ps using this program package. The coulomb energy per solute atom was calculated with only five percent of the error found in the 10 Å cut-off approximation (about 0.9 kcal/mol versus 18 kcal/mol, respectively). The molecular dynamics simulations using COSMOS90 require no more CPU time than the simulations based on the 10 Å cut-off approximation of the conventional programs for macromolecular simulations.     

人Calpain1与心脏结构蛋白的相互作用

为探讨人calpain1与心脏结构蛋白之间的相互作用 ,采用酵母双杂交体系筛选与人calpain1大亚基相互作用的蛋白质 ,通过DNA序列测定及BLAST分析同源性 ,获得了阳性克隆 12 (pACT2 12 ) ,16 (pACT2 16 ) ,2 2 (pACT2 2 2 )和 37(pACT2 37)等 .12和 2 2分别与人心脏α肌动蛋白 (humancardiacmusclealpha actin ,ACTC)有 99%和 10 0 %同源性 .16和 37分别与人心脏的肌球蛋白结合蛋白C(humancardiacmyosin bindingproteinC ,MYBPC3)和人α2 辅肌动蛋白 (humancardiacalpha 2actinin ,ACTN2 )有 10 0 %同源性 .这些阳性克隆均属于心脏中心肌细胞的结构蛋白 ,参与心肌细胞的收缩运动 .为鉴定calpain1大亚基的哪些结构域可能参与这些相互作用 ,阳性克隆再分别与含有人calpain1大亚基结构域Ⅱ、Ⅲ、Ⅳ的诱饵质粒共同转化AH10 9进行酵母双杂交 ,发现pACT2 12(ACTC)和pACT2 16 (MYBPC3)均能与全长人calpain1大亚基的结构域Ⅱ和Ⅲ相互作用 ;pACT2 16(MYBPC3)还能与大亚基的结构域Ⅳ作用 ;而pACT2 37(ACTN)虽能与全长人calpain1大亚基作用 ,却不与其单独的结构域Ⅱ、Ⅲ或Ⅳ发生作用 .定量分析阳性克隆与人calpain1大亚基作用的强弱的结果显示 ,pACT2 12、16或 37分别与诱饵质粒pGBKT7 CANP共同转化AH10 9后     

Purification and Substrate Specificity of Two Cysteine Proteinases of Giardia lamblia

The proteinase activity present in homogenates of trophozoites of Giardia lamblia , active on azocasein and urea-denaturated hemoglobin, was separated into two different enzymes by a series of purification procedures. These procedures included gel filtration on Fractogel TSK HW-55 (F), organomercurial agarose affinity chromatography, and ion exchange chromatography on DEAE-cellulose. By chromatography on Sephadex G-100, two purified enzymes exhibited relative molecular weights of M_r= 95,000 and 35,000 ± 10%, respectively. On the basis of inhibition by thiol reagents and abrogation of this effect by dithiothreitol and cysteine, they were identified as cysteine proteinases. Proteinase I (M_r= 95,000) and proteinase II (M_r= 35,000) were active against the β-chain of insulin releasing characteristic fragments. However, differences in substrate specificities of the two enzymes could be observed by using synthetic peptides that represent sequences 1–6, 8–18, and 20–30 of the insulin β-chain. Furthermore, the synthetic tetrapeptides Arg-Gly-Phe-Phe, Arg-Gly-Leu-Hyp, and Arg-Arg-Phe-Phe were hydrolyzed by the two proteinases releasing Phe-Phe and Leu-Hyp, respectively. Compared with Arg-Gly-Phe-Phe, the rates of hydrolysis of Arg-Gly-Leu-Hyp and Arg-Arg-Phe-Phe at substrate concentrations of 1 mM were 91% and 63% (proteinase I) and 80% and 57% (proteinase II), respectively.     

酵母双杂交技术研究与人孕酮受体B相互作用的蛋白质

应用酵母双杂交技术研究与人孕酮受体B（hPRB）发生相互作用的蛋白质,有助于进一步阐明其在乳腺癌的发生发展中发挥重要作用的调控机制。应用酵母双杂交系统3,以hPRB不同结构域为诱饵,筛选人乳腺cDNA文库,寻找能与之相互作用的蛋白质,并运用X—α—Gal等实验提供的信息,筛除假阳性克隆。最终以AF1-DBD结构域作为诱饵最终筛出了1个阳性克隆,经测序及生物信息学分析,这个克隆所编码的蛋白为PIAS3（活化的STAT3的蛋白抑制剂）。结果表明,孕激素受体可以和PIAS3发生相互作用,它们的相互作用有可能参与乳腺癌的生长调控。     

The Interplay Between Disordered Regions in RNAs and Proteins Modulates Interactions Within Stress Granules and Processing Bodies

Condensation, or liquid-like phase separation, is a phenomenon indispensable for the spatiotemporal regulation of molecules within the cell. Recent studies indicate that the composition and molecular organization of phase-separated organelles such as Stress Granules (SGs) and Processing Bodies (PBs) are highly variable and dynamic. A dense contact network involving both RNAs and proteins controls the formation of SGs and PBs and an intricate molecular architecture, at present poorly understood, guarantees that these assemblies sense and adapt to different stresses and environmental changes. Here, we investigated the physico-chemical properties of SGs and PBs components and studied the architecture of their interaction networks. We found that proteins and RNAs establishing the largest amount of contacts in SGs and PBs have distinct properties and intrinsic disorder is enriched in all protein-RNA, protein-protein and RNA-RNA interaction networks. The increase of disorder in proteins is accompanied by an enrichment in single-stranded regions of RNA binding partners. Our results suggest that SGs and PBs quickly assemble and disassemble through dynamic contacts modulated by unfolded domains of their components.     

水稻胚胎发育时期的特异性蛋白质

对不同发育时期的水稻胚蛋白质,进行了单向SDS聚丙烯酰胺凝胶电泳和双向凝胶电泳分析。单向电泳图中存在20kD左右、胚分化完成期以后特有的蛋白质。根据双向电泳图谱,稻胚总蛋白按其在胚胎发育过程中的动态可分为7类,其中组成性的蛋白质约占85％,在不同发育时期基本不变;具有发育时期特异性的蛋白质约占15％。以分化期胚蛋白质的抗体,对各时期的胚蛋白质进行交叉免疫电泳,所得图谱总体上是相似的,但不同时期的图谱之间出现沉淀峰的峰形、位置和数目的差异。交叉免疫电泳图谱的这种变化可能反映了稻胚发育过程中某些分化期蛋白质发生的变化。     

Vital for Viruses: Intrinsically Disordered Proteins

Viruses infect all kingdoms of life; their genomes vary from DNA to RNA and in size from 2kB to 1 MB or more. Viruses frequently employ disordered proteins, that is, protein products of virus genes that do not themselves fold into independent three-dimensional structures, but rather, constitute a versatile molecular toolkit to accomplish a range of functions necessary for viral infection, assembly, and proliferation. Interestingly, disordered proteins have been discovered in almost all viruses so far studied, whether the viral genome consists of DNA or RNA, and whatever the configuration of the viral capsid or other outer covering. In this review, I present a wide-ranging set of stories illustrating the range of functions of IDPs in viruses. The field is rapidly expanding, and I have not tried to include everything. What is included is meant to be a survey of the variety of tasks that viruses accomplish using disordered proteins.     

Linker Dependence of Avidity in Multivalent Interactions Between Disordered Proteins

Multidomain proteins often interact through several independent binding sites connected by disordered linkers. The architecture of such linkers affects avidity by modulating the effective concentration of intramolecular binding. The linker dependence of avidity has been estimated theoretically using simple physical models, but such models have not been tested experimentally because the effective concentrations could not be measured directly. We have developed a model system for bivalent protein interactions connected by disordered linkers, where the effective concentration can be measured using a competition experiment. We characterized the bivalent protein interactions kinetically and thermodynamically for a variety of linker lengths and interaction strengths. In total, this allowed us to critically assess the existing theoretical models of avidity in disordered, multivalent interactions. As expected, the onset of avidity occurs when the effective concentration reached the dissociation constant of the weakest interaction. Avidity decreased monotonously with linker length, but only by a third of what is predicted by theoretical models. We suggest that the length dependence of avidity is attenuated by compensating mechanisms such as linker interactions or entanglement. The direct role of linkers in avidity suggests they provide a generic mechanism for allosteric regulation of disordered, multivalent proteins.     

DNA错配修复蛋白MutS和MutL的相互作用研究

MutL 和 MutS 是DNA错配修复系统中起关键作用的修复蛋白. 利用基因融合技术高效表达了MutL 和 MutS融合蛋白,并利用它们发展了一种研究二者相互作用的简便方法. 融合蛋白MutL-GFP (Trx-His6-GFP-(Ser-Gly)6-MutL),MutL-Strep tagⅡ (Trx-His6-(Ser-Gly)6-Strep tagⅡ-(Ser-Gly)6-MutL) 和 MutS (Trx-His6-(Ser-Gly)6-MutS) 被构建并在大肠杆菌中高效表达. 收集菌体细胞、超声波破碎后离心取上清进行SDS-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 分析,结果表明有与预期分子质量相应的诱导表达条带出现,其表达量约占全细胞蛋白的30％且以可溶形式存在. 利用固定化金属离子配体亲和层析柱分别纯化融合蛋白,其纯度达到90％. 通过将MutS蛋白固定的方法研究两种MutL融合蛋白分别与MutS之间的相互作用. 结果表明：只有MutS蛋白与含有错配碱基DNA分子结合后才与MutL蛋白发生相互作用. 通过检测MutL融合蛋白标记的绿色荧光信号或酶学显色信号来鉴定相互作用的发生. 建立的融合分子系统方法也为研究其他的蛋白质或生物大分子之间的相互作用提供了一个技术平台.     

蛋白磷酸化修饰在植物-病原微生物互作中的作用研究进展

蛋白磷酸化修饰是植物细胞信号调控的普遍机制。植物-病原微生物互作过程中, 关键调控蛋白的磷酸化状态影响免疫信号的激活。多种病原微生物通过干扰宿主蛋白的磷酸化状态攻击免疫系统, 以提高致病性。该文对植物免疫调控过程中关键元件的磷酸化修饰及其在免疫信号中的调控作用进行了综述。研究植物-病原菌互作过程中关键蛋白的磷酸化修饰, 有助于深入探讨植物-病原微生物互作的分子机理。该文将为寻找广谱抗病的新途径提供理论依据。     

水稻类Tubby蛋白质在叶片生长和白叶枯病抗性反应中的表达

类Tubby蛋白质（Tubby-like protein,TLP）在动植物中广泛存在,暗示其在生命过程中发挥重要的作用。水稻（Oryza sativa）基因组中有14个TLP家族成员,首先制备了这些蛋白质的抗体,用免疫印迹方法检测了它们在水稻叶片不同生长时期的表达情况,揭示其表达模式;然后对Xa21介导的水稻白叶枯病抗性反应不同时间点进行检测,发现OsTLP2、OsTLP7、OsTLP8和OsTLP9等4个蛋白质的表达发生了变化;进一步比较它们在抗病、感病反应和对照处理中的表达情况,发现不同反应间的表达也有区别。该研究结果为阐释水稻TLP在叶片生长过程中的功能,尤其是在水稻-白叶枯病菌互作过程中的作用提供了重要线索。     

不同红麻种子耐老化性差异及热稳定蛋白的研究

利用10份红麻品种为材料,研究红麻种子在人工老化过程中耐老化差异及其与热稳定蛋白的关系。结果表明:(1)随着老化处理程度加深,10份红麻品种种子的发芽率、发芽指数及活力指数均逐渐下降,但品种之间有显著差异;(2)热稳定蛋白含量随着老化处理的加深呈现上升的趋势,通过SDS-PAGE电泳显示,辽55在140h老化处理中出现一条差异带,分子量为62.8kD,其他9个品种各个处理间未出现差异带。10份红麻品种在老化过程中,辽55最耐老化,在辽55中发现的热稳定蛋白可能与红麻品种的耐老化性及种子活力的丧失有关。