Genetic and pharmacological suppression of oncogenic mutations in ras genes of yeast and humans

The activity of an oncoprotein and the secretion of a pheromone can be affected by an unusual protein modification. Specifically, posttranslational modification of yeast a-factor and Ras protein requires an intermediate of the cholesterol biosynthetic pathway. This modification is apparently essential for biological activity. Studies of yeast mutants blocked in sterol biosynthesis demonstrated that the membrane association and biological activation of the yeast Ras2 protein require mevalonate, a precursor of sterols and other isoprenes such as farnesyl pyrophosphate. Furthermore, drugs that inhibit mevalonate biosynthesis blocked the in vivo action of oncogenic derivatives of human Ras protein in the Xenopus oocyte assay. The same drugs and mutations also prevented the posttranslational processing and secretion of yeast a-factor, a peptide that is farnesylated. Thus, the mevalonate requirement for Ras activation may indicate that attachment of a mevalonate-derived (isoprenoid) moiety to Ras proteins is necessary for membrane association and biological function. These observations establish a connection between the cholesterol biosynthetic pathway and transformation by the ras oncogene and offer a novel pharmacological approach to investigating, and possibly controlling, ras-mediated malignant transformations.     

A seven-transmembrane RGS protein that modulates plant cell proliferation

G protein-coupled receptors (GPCRs) at the cell surface activate heterotrimeric G proteins by inducing the G protein alpha (Galpha) subunit to exchange guanosine diphosphate for guanosine triphosphate. Regulators of G protein signaling (RGS) proteins accelerate the deactivation of Galpha subunits to reduce GPCR signaling. Here we identified an RGS protein (AtRGS1) in Arabidopsis that has a predicted structure similar to a GPCR as well as an RGS box with GTPase accelerating activity. Expression of AtRGS1 complemented the pheromone supersensitivity phenotype of a yeast RGS mutant, sst2Delta. Loss of AtRGS1 increased the activity of the Arabidopsis Galpha subunit, resulting in increased cell elongation in hypocotyls in darkness and increased cell production in roots grown in light. These findings suggest that AtRGS1 is a critical modulator of plant cell proliferation.     

Protein insertion into the mitochondrial inner membrane by a twin-pore translocase

The mitochondrial inner membrane imports numerous proteins that span it multiple times using the membrane potential Deltapsi as the only external energy source. We purified the protein insertion complex (TIM22 complex), a twin-pore translocase that mediated the insertion of precursor proteins in a three-step process. After the precursor is tethered to the translocase without losing energy from the Deltapsi, two energy-requiring steps were needed. First, Deltapsi acted on the precursor protein and promoted its docking in the translocase complex. Then, Deltapsi and an internal signal peptide together induced rapid gating transitions in one pore and closing of the other pore and drove membrane insertion to completion. Thus, protein insertion was driven by the coordinated action of a twin-pore complex in two voltage-dependent steps.     

HTLV-III gag protein is processed in yeast cells by the virus pol-protease

The gag-pol gene of HTLV-III (human T-lymphotropic virus), the virus linked to AIDS (acquired immune deficiency syndrome), was expressed in yeast, and processing of the gag precursor into proteins of the same size as those in the virion was observed. Processing of the gag gene in yeast cells mimics the process that naturally occurs in mammalian cells during maturation of virions. Therefore it was possible to perform mutational analysis of the virus genome to localize the gene that codes for the protease function to the amino terminal coding region of the pol gene. Since this region overlaps the gag gene, it is likely that ribosomal frameshifting occurs from gag to pol. Antibodies in all of the AIDS patients' sera tested recognized the yeast synthesized gag proteins, although the sera showed differences in relative reactivity to the individual gag proteins and the precursor. This yeast system should be valuable not only for production of viral proteins for diagnostic or vaccine purposes but also for analysis of the genetics and biochemistry of viral gene functions--parameters that are difficult to study otherwise with this virus.     

Antiviral Activity of Allokin-Alpha against Herpesvirus in Birds

The article reports the efficacy assessment for the Allokin-alpha (Allokin) preparation by modeling the experimental viral infection in vivo in poultry (day-old chickens). The experimental infection included the following steps: vaccination using the vaccine HVT strain, the intramuscular injection of Allokin, and the preparation of feather follicle extracts. The specific antigen was detected in the feather follicle epithelium using the method of gel diffusion precipitation reaction (DPR). A decrease in the number of positive DPR test results for HVT antigen was observed in the case of Allokin-alpha treatment. It has been demonstrated that the optimal time for the Allokin-alpha administration was on the tenth day after the vaccination with HVT as soon as the number of positive DPR results was 33%, while the number of positive results was 50% when the tested preparation was administered 5 and 15 days after the vaccination (samples were collected on day 28 after the vaccination). Since Allokin-alpha has been successfully used to treat human papillomavirus for 15 years, it may also work well as an antiviral agent in the experimental viral infection simulation by the inoculation of vaccine HVT strain; the preparation is not species specific.     

Pheromone signaling mechanisms in yeast: a prototypical sex machine

The actions of many extracellular stimuli are elicited by complexes of cell surface receptors, heterotrimeric guanine nucleotide-binding proteins (G proteins), and mitogen-activated protein (MAP) kinase complexes. Analysis of haploid yeast cells and their response to peptide mating pheromones has produced important advances in our understanding of G protein and MAP kinase signaling mechanisms. Many of the components, their interrelationships, and their regulators were first identified in yeast. Current analysis of the pheromone response pathway (see the Connections Maps at Science's Signal Transduction Knowledge Environment) will benefit from new and powerful genomic, proteomic, and computational approaches that will likely reveal additional general principles that are applicable to more complex organisms.     

Control of yeast mating signal transduction by a mammalian beta 2-adrenergic receptor and Gs alpha subunit

To facilitate functional and mechanistic studies of receptor-G protein interactions, [corrected] the human beta 2-adrenergic receptor (h beta-AR) has been expressed in Saccharomyces cerevisiae. This was achieved by placing a modified h beta-AR gene under control of the galactose-inducible GAL1 promoter. After induction by galactose, functional h beta-AR was expressed at a concentration several hundred times as great as that found in any human tissue. As determined from competitive ligand binding experiments, h beta-AR expressed in yeast displayed characteristic affinities, specificity, and stereoselectivity. Partial activation of the yeast pheromone response pathway by beta-adrenergic receptor agonists was achieved in cells coexpressing h beta-AR and a mammalian G protein (Gs) alpha subunit-demonstrating that these components can couple to each other and to downstream effectors when expressed in yeast. This in vivo reconstitution system provides a new approach for examining ligand binding and G protein coupling to cell surface receptors.     

Biosynthesis of the Torpedo californica acetylcholine receptor alpha subunit in yeast

Yeast cells were transformed with a plasmid containing complementary DNA encoding the alpha subunit of the Torpedo californica acetylcholine receptor. These cells synthesized a protein that had the expected molecular weight, antigenic specificity, and ligand-binding properties of the alpha subunit. The subunit was inserted into the yeast plasma membrane, demonstrating that yeast has the apparatus to express a membrane-bound receptor protein and to insert such a foreign protein into its plasma membrane. The alpha subunit constituted approximately 1 percent of the total yeast membrane. The alpha subunit constituted approximately 1 percent of the total yeast membrane proteins, and its density was about the same in the plasma membrane of yeast and in the receptor-rich electric organ of Electrophorus electricus. In view of the available technology for obtaining large quantities of yeast proteins, it may now be possible to obtain amplified amounts of interesting membrane-bound proteins for physical and biochemical studies.     

中华蜜蜂化学感受蛋白CSP1的功能模式分析及亚细胞定位

【目的】研究中华蜜蜂(Apis cerana cerana,简称中蜂)重组化学感受蛋白CSP1与不同化学信息素的结合功能、模式及其亚细胞定位,明确触角特异表达的CSP1蛋白功能。【方法】将克隆的中蜂CSP1构建至p ET-32a(+)载体并转入BL21(DE3)感受态细菌中,挑取单克隆菌落接种于LB培养基,培养过夜后按1%(V/V)进行转接,继续培养至OD600≈0.4左右时,加入IPTG至终浓度为1 mmol·L~(-1)后继续诱导5 h。将诱导好的CSP1大肠杆菌菌液离心弃上清,再加入细菌裂解液超声破碎,离心后上清用镍柱对CSP1重组蛋白进行亲和层析纯化,再经PBS透析液透析后,最终获得可溶的具有生物活性的CSP1重组蛋白。设定荧光分光光度计的激发波长为281 nm,测定竞争性荧光探针1-NPN与CSP1的相互作用,用Scatchard方程计算其解离常数,再计算获得CSP1与各种候选化学信息素的亲和力。以CSPMbra A6晶体结构(PDB代码:1n8v)为模板,通过同源建模和分子对接解析CSP1蛋白与化学信息素的结合模式,根据Mol Dock Score选出最佳对接模型进行作用机理分析,获得结合时配基周围的CSP1残基分布以及氢键产生情况,以此获得信息素与CSP1的结合模式。最后将CSP1免疫注射兔子获得多克隆抗体,并对中蜂工蜂触角进行低温固定、脱水和包埋后进行超薄切片,然后对样品切片进行免疫胶体金电镜定位,以解析CSP1在触角感器中的亚细胞分布。【结果】成功诱导获得可溶性的重组中蜂CSP1蛋白,利用荧光光谱分析1-NPN与CSP1的解离常数K1-NPN为2.1μmol·L~(-1),结合位点数n为0.99,表明结合时基本以1:1结合,线性相关系数为0.9933。在9种化学信息物质中,CSP1与两种蜜蜂蜂王信息素成分对羟基苯甲酸甲酯(HOB)和9-羰基-2癸烯酸(9-ODA),和植物挥发物成分3-蒈烯均具有较强的结合能力,其中与CSP1亲和力最强的对羟基苯甲酸甲酯的[IC50]和解离常数KD分别达到10.1和7.68μmol·L~(-1)。分子对接显示不同配基与CSP1的结合分别是通过与CSP1疏水腔中特定氨基酸残基(或借助于氢键)作用结合。典型的如CSP1与HOB相互作用过程中,预测主要由8个氨基酸残基贡献能量,包括4个疏水性残基(Phe30、Phe44、Leu70和Phe85),3个极性中性残基(Tyr26、Tyr27和Ser41)以及1个酸性残基(Asp40),其中Asp40中两个羧基上的氧原子分别与HOB苯环中羟基上的氧原子分别产生一个氢键。免疫电镜定位结果显示CSP1主要表达于板形感器周围附属支持细胞中,少量表达于感器内部,这与气味结合蛋白的定位存在明显区别。【结论】中蜂CSP1与两种蜂王信息素成分和某些植物花香成分具有较强的结合能力,集合了信息素结合蛋白和普通气味结合蛋白的功能和相似的作用模式,但其亚细胞定位与气味结合蛋白存在明显区别,显示化学感受类蛋白生理特征的多样性。     

The LDL receptor gene: a mosaic of exons shared with different proteins

The multifunctional nature of coated pit receptors predicts that these proteins will contain multiple domains. To establish the genetic basis for these domains (LDL) receptor. This gene is more than 45 kilobases in length and contains 18 exons, most of which correlate with functional domains previously defined at the protein level. Thirteen of the 18 exons encode protein sequences that are homologous to sequences in other proteins: five of these exons encode a sequence similar to one in the C9 component of complement; three exons encode a sequence similar to a repeat sequence in the precursor for epidermal growth factor (EGF) and in three proteins of the blood clotting system (factor IX, factor X, and protein C); and five other exons encode nonrepeated sequences that are shared only with the EGF precursor. The LDL receptor appears to be a mosaic protein built up of exons shared with different proteins, and it therefore belongs to several supergene families.     

Expression of the major surface antigen of Plasmodium knowlesi sporozoites in yeast

The circumsporozite protein, a surface antigen of the sporozoite stage of the monkey malarial parasite Plasmodium knowlesi, was expressed in the yeast Saccharomyces cerevisiae by using an expression vector containing the 5' regulatory region of the yeast alcohol dehydrogenase I gene. It was necessary to eliminate the entire 5' upstream region of the parasite DNA to obtain the expression of this protein. Only the circumsporozoite precursor was produced by the yeast transformants, as detected by immunoblotting. About 55 and 20 percent of the circumsporozoite protein produced in yeast was associated wtih the 25,000 g and 150,000 g particulate fractions, respectively. The protein could be solubilized in Triton X-100 and was stable in solubilized extracts.     

Yeast KEX2 protease has the properties of a human proalbumin converting enzyme

Several classes of proteolytic enzymes have been proposed to have a role in the processing of precursor forms of proproteins at paired basic amino acid residues. In higher eukaryotes, a single endopeptidase has yet to fulfill the necessary criteria as the physiologically relevant convertase. The observation of proalbumin circulating in a child with a bleeding disorder caused by an unusual alpha 1-antitrypsin mutation led to speculation that the presence of this alpha 1-antitrypsin mutant was inhibitory to the convertase. This provided an additional means of characterizing the processing enzyme. In this study the yeast KEX2 enzyme, a calcium-dependent thiol protease, was found to have all the properties expected for this processing enzyme. KEX2 correctly recognized and cleaved the prosequence in proalbumin. In addition, KEX2 was specifically inhibited by the mutant alpha 1-antitrypsin but not by other serine protease inhibitors.     

DPR对水稻氮素代谢及籽粒蛋白质产量的影响

为了实现水稻高产、优质、高效，采用盆栽试验，研究了水稻双促调节剂(DPR)在不同土壤上喷施对水稻氮素代谢特性及其籽粒蛋白质产量的影响．结果表明：水稻齐穗后喷施DPR能明显改善水稻功能叶和茎鞘氮素代谢的能力，提高其籽粒和糙米中蛋白氮含量及籽粒的蛋白质产量．DPR处理与对照相比，早、晚稻籽粒蛋白质产量分别提高了12．77％～16．56％和8.39％～13．18％。     

Yeast KEX2 endopeptidase correctly cleaves a neuroendocrine prohormone in mammalian cells

Mammalian cell lines (BSC-40, NG108-15, and GH4C1) that cannot process the murine neuroendocrine peptide precursor prepro-opiomelanocortin (mPOMC) when its synthesis is directed by a vaccinia virus vector were coinfected with a second recombinant vaccinia virus carrying the yeast KEX2 gene, which encodes an endopeptidase that cleaves at pairs of basic amino acid residues. mPOMC was cleaved intracellularly to a set of product peptides normally found in vivo, including mature gamma-lipotropin and beta-endorphin1-31. In GH4C1 cells (a rat pituitary line), product peptides were incorporated into stored secretory granules. These results suggest that the inability of any particular cell line to process a prohormone precursor is due to the absence of a suitable endogenous processing enzyme.     

Regulation of MAPK function by direct interaction with the mating-specific Galpha in yeast

The mating response of the budding yeast Saccharomyces cerevisiae is mediated by a prototypical heterotrimeric GTP-binding protein (G protein) and mitogen-activated protein kinase (MAPK) cascade. Although signal transmission by such pathways has been modeled in detail, postreceptor down-regulation is less well understood. The pheromone-responsive G protein alpha subunit (Galpha) of yeast down-regulates the mating signal, but its targets are unknown. We have found that Galpha binds directly to the mating-specific MAPK in yeast cells responding to pheromone. This interaction contributes both to modulation of the mating signal and to the chemotropic response, and it demonstrates direct communication between the top and bottom of a Galpha-MAPK pathway.     

Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H(+)-adenosine triphosphatase

The TFP1 gene of the yeast Saccharomyces cerevisiae encodes two proteins: the 69-kilodalton (kD) catalytic subunit of the vacuolar proton-translocating adenosine triphosphatase (H(+)-ATPase) and a 50-kD protein. The 69-kD subunit is encoded by the 5' and 3' thirds of the TFP1 coding region, whereas the 50-kD protein is encoded by the central third. Evidence is presented that both the 69-kD and 50-kD proteins are obtained from a single translation product that is cleaved to release the 50-kD protein and spliced to form the 69-kD subunit.     

陌夜蛾性信息素类似物的合成及其生物活性

背景 陌夜蛾（Trachea atriplicis）作为一种重要食叶害虫，近年来，由于其数量以及危害程度的不断增加，已对我国华北、华东等地区的农林业造成重大危害，尤其是对荞麦等作物的产量构成严重威胁。目的 以陌夜蛾性信息素主要组分顺-11-十六碳烯乙酸酯（Z11-16:Ac）为母体结构，合成6种具有不同生物活性的性信息素类似物，为陌夜蛾的综合治理开辟一种新型环境友好的防治策略。方法 选用性信息素组分的前体顺-11-十六碳烯-1-醇（Z11-16:OH）为原料，在缚酸剂和催化剂的作用下，与酸酐或酰氯发生反应，通过硅胶柱层析进行分离纯化后，合成性信息素类似物共6种。包括极性基团为氨基甲酸酯的性信息素类似物M3、M4，极性基团末端引入不饱和键的性信息素类似物M5、M6，以及卤素原子修饰的性信息素类似物M7、M8。通过触角电生理试验（EAG）测定不同剂量性信息素类似物直接刺激下的EAG响应值，并于北京市延庆区进行田间试验对性信息素类似物的生物活性进行验证。结果 EAG试验表明，类似物M1、M2、M6、M7和M8具有显著的剂量-效应关系。其中陌夜蛾雄蛾触角对类似物M7的响应最为强烈，在100 μg剂量下响应值达2.36 mV，是同剂量条件下Z11-16:Ac响应值的77.9%。而M3—M5在10—1 000 μg剂量下，与性信息素组分Z11-16:Ac的相对响应值之间存在显著差异（P<0.05），M5的EAG响应整体活性表现较差，最高相对响应值仅为性信息素组分的21.2%。田间试验表明，类似物M1和M5在添加剂量为1 μg时，平均诱捕量分别为38.00和35.67头/诱捕器/15 d，与性信息素对照相比存在显著差异；类似物M7在添加剂量为100 μg时，平均诱捕量为29.67头/诱捕器/15 d，显著高于性信息素对照；类似物M4具有潜在的抑制活性，随着其添加剂量的增加，平均诱捕量逐渐减少，当添加剂量为1 000 μg时，平均诱捕量仅有3.33头/诱捕器/15 d。 结论经过EAG试验和田间试验验证，类似物M1、M5和M7具有显著的增效活性，可以用作性信息素的增效剂，类似物M4具有显著的抑制活性，可以用作性信息素的抑制剂。研究结果可为使用性信息素类似物绿色防控陌夜蛾提供理论依据。     

利用酵母双杂交系统筛选介体异沙叶蝉中与小麦矮缩病毒外壳蛋白互作的蛋白质

【目的】利用分离泛素酵母双杂交膜系统（split-ubiquitin yeast membrane system）,以小麦矮缩病毒（Wheat dwarf virus,WDV）的外壳蛋白（CP）基因为诱饵对异沙叶蝉（Psammotettix alienus L.）cDNA文库进行筛选,研究异沙叶蝉传播WDV的分子机制。【方法】以笔者实验室饲养的异沙叶蝉为材料,提取其总RNA后取100 ng进行纯化,利用SMART法反转录合成ds cDNA,经过Sfi I酶切纯化,连接到pPR3-N文库载体上,构建得到以pPR3-N为载体的异沙叶蝉分离泛素酵母双杂交膜系统cDNA文库。同时,构建带有Sfi I酶切位点的诱饵载体pDHB1-WDV CP,经功能检测后用诱饵载体初步筛选pPR3-N空文库,寻找适合筛库的条件和确定His基因产物抑制剂3-氨基-1,2,4-三唑（3-AT）的使用浓度。然后用诱饵载体筛选异沙叶蝉cDNA文库,对筛选结果进行分析,再通过共转验证和β-半乳糖苷酶检测进一步验证是否发生互作。利用Uniprot和KEGG在线网站,对筛到的蛋白进行gene ontology（GO）注释和Pathway分析。【结果】初级文库库容量超过2.0×10⁶ cfu,文库实际扩增数量大于1.3×10⁶ cfu,文库重组率大于97%,扩增文库插入片段平均长度大于1 000 bp,表明异沙叶蝉cDNA文库的质量较高。酶切验证显示诱饵载体pDHB1-WDV-CP中CP的插入完整而准确。功能检测表明融合蛋白能够正确表达。在3-AT浓度为5 mmol?L^-1的筛选条件下,诱饵载体筛选异沙叶蝉cDNA文库得到280个克隆,经测序和Blast比对分析最终得到12个可能与WDV的CP发生互作的异沙叶蝉蛋白质。将这12个蛋白质再次进行共转验证和β-半乳糖苷酶检测,最终得到9个蛋白质与WDV CP互作。GO注释显示,9个蛋白参与的生物过程包括蛋白去磷酸化、碳水化合物代谢过程、先天性免疫应答、模式识别受体的信号通路、运输、同向运输和乙醇氧化等;分子功能包括金属离子结合活性、蛋白磷酸酶活性、信号模式识别受体的活性、水解酶活性、磷酸离子载体活性和叶酸运输活性等。参考KEGG数据库,这些蛋白参与的代谢途径有泛素介导的蛋白水解途径、内吞作用、花生四烯酸代谢途径、cAMP信号通路和模式识别受体的信号通路等。【结论】异沙叶蝉分离泛素酵母双杂交膜系统cDNA文库的成功构建与筛选,为研究异沙叶蝉与小麦矮缩病毒的互作机制研究奠定了基础。