基因组水平预测稻瘟菌分泌蛋白组及富集分析

稻瘟菌分泌蛋白在稻瘟菌入侵植物过程中发挥着重要的作用。这些分泌蛋白中有很多是效应蛋白,这些效应蛋白可以干扰寄主的抗性、抑制寄主免疫反应。因此,对稻瘟菌分泌蛋白组的预测及功能分析就显得十分必要,也是目前植物和微生物分子互作研究领域的热点。使用SignalP、TMHMM及SecretomeP等软件,完成稻瘟菌分泌蛋白组的预测。同时,对含信号肽的经典分泌蛋白进行了GO功能富集、KEGG通路分析、结构域统计以及可降解植物成分的经典分泌蛋白预测等分析。结果显示,稻瘟菌含有约789个分泌蛋白,其长度多集中在100-500 aa;GO功能分析发现,这些分泌蛋白多富集在分泌途径及宿主互作中;KEGG分析显示,分泌蛋白在糖代谢途径中发挥着重要作用;大规模筛选预测到156个分泌蛋白具有降解植物细胞壁等成分的功能;同时还发现稻瘟菌中有可能存在大量不含信号肽的非经典分泌蛋白。通过设计的生物信息学流程,实现了稻瘟菌分泌蛋白组的预测;预测出经典分泌蛋白具有可降解植物细胞壁等成分以及参与糖代谢途径的功能;稻瘟菌中存在大量的无信号肽的非经典分泌蛋白。     

内生菌KM-1-2全基因组ORFs信号肽和分泌蛋白预测及功能分析

【目的】内生菌普遍存在于植物中,与宿主在长期的进化中形成了互利共生的关系。目前对内生菌和植物之间的互作机制研究较少,为深入了解银杏叶内生菌KM-1-2与寄主植物作用机制,本研究对其分泌蛋白进行预测,并明确其特征。【方法】组合使用信号肽分析软件Signal P,跨膜螺旋结构分析软件TMHMM 2.0和Phobius,蛋白质细胞定位软件PSORT,亚细胞定位软件Target P和GPI锚定位点分析软件big-PI Predictor,预测KM-1-2基因组范围内所有分泌蛋白,定义为分泌组。【结果】KM-1-2全基因组5299条蛋白序列中发现271个具有典型信号肽的分泌蛋白,占全基因组的2.4%;编码这些蛋白的ORF最短为61 bp,最大为2105 bp,平均为373 bp;引导它们的信号肽长度分布在15–37 aa之间,平均为24 aa。信号肽中出现频率最高的氨基酸依次为丙氨酸、亮氨酸和缬氨酸,信号肽切割类型多属于A-X-A型,即SPI切割类型。共66个蛋白质有功能描述,其中包括26个酶类。这些酶主要包括各种糖苷水解酶、酯酶、蛋白酶、碳氧裂解酶等。【结论】通过上述生物信息学分析方法有效实现了银杏叶内生菌KM-1-2分泌蛋白的预测,这些分泌蛋白功能涉及较多的酶类以及其他未知功能,为进一步研究内生菌和植物的互作提供了基础。     

稻瘟病菌含信号肽小蛋白的计算机分析

结合计算机技术和生物信息学的方法,采用组合的信号肽分析软件SignalPv3.0、TargetPv1.1、Big-PIpredictor、TMHMMv2.0和SecretomeP对已公布的1486个稻瘟菌(magnaporthegrisea)小蛋白基因的N-端氨基酸序列进行信号肽分析,同时系统分析了信号肽的类型及结构。分析结果表明,在1486个稻瘟病菌小蛋白中,119个具有N-端信号肽的典型分泌蛋白。其中116个具有分泌型信号肽,1个具RR-motif型信号肽,2个具信号肽酶II型信号肽。在稻瘟病菌基因组中,分泌型小蛋白的序列是高度趋异的,仅出现少数氨基酸组成完全一致的信号肽,为进一步确认具有相同信号肽的分泌蛋白是否具有同源性,分别用BLAST2SEQUENCES对具有相同信号肽的分泌蛋白进行了序列对比。结果表明,具有相同信号肽的分泌蛋白同源性非常高。同时还采用Sublocv1.0对1486个小蛋白的亚细胞位置进行了预测,结果显示小蛋白的可能功能场所包括细胞质、细胞外、线立体和细胞核,功能场所位于细胞核的小蛋白是最多的。     

信号肽对外源蛋白分泌效率的影响

信号肽在引导外源蛋白分泌过程中具有重要作用,本文从信号肽疏水性结构、构建分泌型载体以及分泌增强子、定位信号等几个方面介绍了信号肽对外源蛋白分泌效率的影响,在大量生产以酵母为表达宿主的治疗性蛋白方面具有广泛的应用前景。     

植物病原细菌Ralstonia solanacearum GMI1000中分泌蛋白信号肽分析

利用SignalP 3.0、TMHMM 2.0、TargetP 1.01、LipoP 1.0和PSORTb蛋白分析软件并结合L值计算,对植物病原细菌Ralstonia solanacearum GMI1000菌株基因组中的全部3 440个ORFs进行了分析预测,确定其中186个ORFs所编码蛋白质的N-端有信号肽序列,且它们的氨基酸残基相对保守.其中134条具有分泌型信号肽,22条具有RR-motif型信号肽,30条具有信号肽酶Ⅱ型信号肽.对各类信号肽及其结构域的长度作了系统的分析.未发现Prepilin-like信号肽和细菌素和信息素信号肽.     

秀丽小杆线虫分泌蛋白组的计算机分析

结合计算机技术和生物信息学的方法,采用组合的信号肽分析软件SignalP v3.0、TargetP v1.01、Big-PI Predictor和TMHMM v2.0,预测了秀丽小杆线虫（Caenorthaditis elegans ws123）的全基因组19855个ORF编码蛋白的信号肽,同时系统分析了信号肽的特征。结果表明,在19855个秀丽小杆线虫的蛋白中,有1990条为带有信号肽的分泌型蛋白,其中,1936条为典型的分泌型信号肽（即信号肽酶Ⅰ型信号肽）,53条为信号肽酶Ⅱ型信号肽,1条为信号肽酶Ⅳ型信号肽;在Ⅰ型信号肽中,有41条为RR-motif亚组型信号肽。在1990条信号肽中,有742条没有典型的N-区,其余1248条包含典型的3个区。比较了秀丽小杆线虫与原核生物分泌蛋白信号肽中20种氨基酸残基在信号肽酶切位点的使用情况,表明：在Ⅰ型信号肽酶切位点,其信号肽中使用的氨基酸总体趋势与原核生物基本相似,但秀丽小杆线虫选用的氨基酸种类更多,变化更大;在Ⅱ型信号肽酶切位点,秀丽小杆线虫脂蛋白信号肽中使用的氨基酸的种类与原核生物有很大的不同。通过与真核单细胞生物比较,作为真核多细胞生物的秀丽小杆线虫,其分泌蛋白信号肽所占比例更高、种类更多,可知线虫信号肽的组成具有很高的多态性,表明该物种的分泌蛋白具有多种功能。此外,分析结果显示,脂蛋白信号肽在结构上比分泌型信号肽更为保守。在秀丽小杆线虫分泌蛋白中出现了少数氨基酸组成完全一致的信号肽,采用BLAST 2 SEQUENECES对具有相同信号肽的分泌蛋白进行了序列比对,结果表明具有相同信号肽的分泌蛋白同源性非常高,它们的存在是生物进化过程中基因倍加（duplication）及环境选择的结果,信号肽特征的详细描述必将对这些蛋白功能的研究提供重要的帮助。      

根癌土壤杆菌C58 Cereon中分泌蛋白信号肽分析

利用SignalP3．0、LipoP1．0、TMHMM2．0和TargetP1．014种蛋白分析软件预测了Agrobacterium tumefaciens C58 Cereon菌株全部基因组的4554个ORF编码的蛋白信号肽,共发现203个信号肽,且它们的氨基酸残基相对保守。其中158条具分泌型信号肽,9条具RR-motif型信号肽,28条具信号肽酶Ⅱ型信号肽,8条具细菌素-信息素型信号肽,但只有分泌蛋白AGR-C-1878p和AGR-C-1880p的信号肽氨基酸残基完全相同,表明信号肽是高度变异的。     

嗜酸氧化亚铁硫杆菌基因组分泌蛋白的初步分析

利用信号肽预测软件SignalP v3.0、跨膜螺旋结构预测软件TMHMM v2.0和非经典分泌蛋白预测软件SecretomeP对嗜酸氧化亚铁硫杆菌全基因组的3 218个氨基酸序列进行预测分析.结果表明在嗜酸氧化亚铁硫杆菌中有507个蛋白为分泌蛋白,其中分泌型信号肽120个(其中有9个为RR-motif亚组型信号肽),脂蛋白信号肽3个,Prepilin-like信号肽4个,非经典分泌蛋白380个.并对分泌型信号肽的长度分布、氨基酸使用频率和酶切位点的氨基酸使用频率作了统计.得分最高的100个非经典分泌蛋白中,有36个具有功能分类,主要是参与细胞壁、能量代谢及转运和结合的蛋白质.嗜酸氧化亚铁硫杆菌的这507个分泌蛋白所参与的生化过程可能发生在膜外的周质空间或是菌体外的场所,为该物种与矿物相互作用,以及对环境做出响应服务.     

地衣芽孢杆菌DSM13分泌蛋白信号肽分析

地衣芽孢杆菌是重要的工业微生物,对于其分泌途径及信号肽进行预测和分析,有助于改善影响蛋白分泌的关键因素,高效生产异源蛋白。本研究首次在全基因范围内,利用SignalPv3.0等方法识别了地衣芽孢杆菌DSM13中各种分泌蛋白的信号肽。DSM13信号肽类型包括分泌型Sec信号肽、双精氨酸Tat信号肽、脂蛋白信号肽、IV型纤毛结构信号肽及生物信息素信号肽。同时分析了分泌途径组成,信号肽长度,氨基酸组成,各分泌信号肽特征,与枯草芽孢杆菌的异同以及重要工业酶制剂的分泌途径。该研究对使DSM13成为更有效分泌表达外源蛋白表达系统,具有重要的理论指导意义。     

苏云金芽胞杆菌分泌蛋白的鉴定及分析

研究苏云金芽胞杆菌(Bacillus thuringiensis,Bt)在生长发育过程中分泌蛋白的类型和含量,尤其是胞外蛋白酶,对于充分了解该菌的杀虫机理、扩大该菌的应用具有重要意义。通过制备Bt_HD73过渡期(transition phase)的分泌蛋白质样品,进行质谱检测并分析其分泌蛋白的种类、含量、功能和信号肽等。结果表明,Bt_HD73在过渡期初期有54种胞外蛋白质,其中酶类最多,占所有分泌蛋白的66.30%,且主要为蛋白水解酶;毒力蛋白次之,占所有分泌蛋白的14.53%。在54种分泌蛋白中,发现27种含有经典Sec型信号肽(Secretory signal peptide,Sec-type SP),信号肽的长度在24-40个氨基酸之间。因此,Bt_HD73产生分泌蛋白的能力较强,在过渡期可分泌大量酶类,尤其是蛋白水解酶。同时还建立了该菌株的信号肽库,包括27种具有引导外源蛋白分泌潜力的信号肽。     

Exo‐ and surface proteomes of the probiotic bacterium Lactobacillus acidophilus NCFM

Lactobacillus acidophilus NCFM is a well‐known probiotic bacterium extensively studied for its beneficial health effects. Exoproteome (proteins exported into culture medium) and surface proteome (proteins attached to S‐layer) of this probiotic were identified by using 2DE followed by MALDI TOF MS to find proteins potentially involved in bacteria–host interactions. The exo‐ and surface proteomes included 43 and 39 different proteins from 72 and 49 successfully identified spots, respectively. Twenty‐two proteins were shared between the two proteomes; both contained the major surface layer protein that participates in host interaction as well as several well‐known and putative moonlighting proteins. The exoproteome contained nine classically‐secreted (containing a signal sequence) and ten nonclassically‐secreted proteins, while the surface proteome contained four classically‐secreted and eight nonclassically secreted proteins. Identification of exo‐ and surface proteomes contributes describing potential protein‐mediated probiotic–host interactions.     

Sorting of proteins to the vacuoles of plant cells.

The secretory system of plant cells sorts a large number of soluble proteins that either are secreted or accumulate in vacuoles. Secretion is a bulk-flow process that requires no information beyond the presence of a signal peptide necessary to enter the endoplasmic reticulum. Many vacuolar proteins are glycoproteins and the glycans are often modified as the proteins pass through the Golgi complex. Vacuolar targeting information is not contained in glycans as it is in animal cells; rather, targeting information is in polypeptide domains as it is in yeast cells. Several such domains have now been identified, but these show little or no amino acid sequence homology. We discuss the possibilities that targeting of protein to plant vacuoles may involve receptors as well as aggregation of protein at low pH.     

Different sequence patterns in signal peptides from mycoplasmas, other gram-positive bacteria, and Escherichia coli: a multivariate data analysis

Signal peptides are essential N-terminal extensions in export proteins, and have a positively charged N-terminus, a hydrophobic central core, and a C-terminal cleavage region. They interact in a consecutive manner with different accessory proteins during the secretion process. Potential patterns or periodicity in the amino acid (aa) sequence were searched, using multivariate techniques, for a large number of signal peptides from mollicutes (mycoplasmas), other Gram-positive bacteria, and Escherichia coli. Mollicutes signal peptides were significantly different from the E. coli and Gram-positive ones by their N-terminal charge, peptide length, and especially, unique periodicities of side chain hydrophobicity and volumes. Their lipoprotein signal peptides were longer than for any other bacteria. Significant differences were also recorded between the other bacterial peptide groups. Specific aa patterns were more related within the signal peptides from several groups of secreted bacillus enzymes, than for all signal peptides from one bacillus species. In E. coli, signal peptides from proteins routed for the various destinations revealed significant and compartment-specific sequence patterns not evident by other methods. This was substantiated from a large number of signal peptide secretion mutants for the E. coli periplasmic space. It is proposed that the differences in aa patterns and side-chain properties are related to the secondary structure sidedness and topology of the signal peptides, and important for specific interactions during the secretion process.     

Trafficking arms: oomycete effectors enter host plant cells

Oomycetes cause devastating plant diseases of global importance, yet little is known about the molecular basis of their pathogenicity. Recently, the first oomycete effector genes with cultivar-specific avirulence (AVR) functions were identified. Evidence of diversifying selection in these genes and their cognate plant host resistance genes suggests a molecular "arms race" as plants and oomycetes attempt to achieve and evade detection, respectively. AVR proteins from Hyaloperonospora parasitica and Phytophthora infestans are detected in the plant host cytoplasm, consistent with the hypothesis that oomycetes, as is the case with bacteria and fungi, actively deliver effectors inside host cells. The RXLR amino acid motif, which is present in these AVR proteins and other secreted oomycete proteins, is similar to a host-cell-targeting signal in virulence proteins of malaria parasites (Plasmodium species), suggesting a conserved role in pathogenicity.     

How rhizobial symbionts invade plants: the Sinorhizobium-Medicago model

Nitrogen-fixing rhizobial bacteria and leguminous plants have evolved complex signal exchange mechanisms that allow a specific bacterial species to induce its host plant to form invasion structures through which the bacteria can enter the plant root. Once the bacteria have been endocytosed within a host-membrane-bound compartment by root cells, the bacteria differentiate into a new form that can convert atmospheric nitrogen into ammonia. Bacterial differentiation and nitrogen fixation are dependent on the microaerobic environment and other support factors provided by the plant. In return, the plant receives nitrogen from the bacteria, which allows it to grow in the absence of an external nitrogen source. Here, we review recent discoveries about the mutual recognition process that allows the model rhizobial symbiont Sinorhizobium meliloti to invade and differentiate inside its host plant alfalfa (Medicago sativa) and the model host plant barrel medic (Medicago truncatula).     

Systematic high-yield production of human secreted proteins in Escherichia coli

Human secreted proteins play a very important role in signal transduction. In order to study all potential secreted proteins identified from the human genome sequence, systematic production of large amounts of biologically active secreted proteins is a prerequisite. We selected 25 novel genes as a trial case for establishing a reliable expression system to produce active human secreted proteins in Escherichia coli. Expression of proteins with or without signal peptides was examined and compared in E. coli strains. The results indicated that deletion of signal peptides, to a certain extent, can improve the expression of these proteins and their solubilities. More importantly, under expression conditions such as induction temperature, N-terminus fusion peptides need to be optimized in order to express adequate amounts of soluble proteins. These recombinant proteins were characterized as well-folded proteins. This system enables us to rapidly obtain soluble and highly purified human secreted proteins for further functional studies.     

Identification of potential host plant mimics of CLAVATA3/ESR (CLE)-like peptides from the plant-parasitic nematode Heterodera schachtii

In this article, we present the cloning of two CLAVATA3/ESR (CLE)-like genes, HsCLE1 and HsCLE2, from the beet cyst nematode Heterodera schachtii, a plant-parasitic cyst nematode with a relatively broad host range that includes the model plant Arabidopsis. CLEs are small secreted peptide ligands that play important roles in plant growth and development. By secreting peptide mimics of plant CLEs, the nematode can developmentally reprogramme root cells for the formation of unique feeding sites within host roots for its own benefit. Both HsCLE1 and HsCLE2 encode small secreted polypeptides with a conserved C-terminal CLE domain sharing highest similarity to Arabidopsis CLEs 1-7. Moreover, HsCLE2 contains a 12-amino-acid CLE motif that is identical to AtCLE5 and AtCLE6. Like all other plant and nematode CLEs identified to date, HsCLEs caused wuschel-like phenotypes when overexpressed in Arabidopsis, and this activity was abolished when the proteins were expressed without the CLE motif. HsCLEs could also function in planta without a signal peptide, highlighting the unique, yet conserved function of nematode CLE variable domains in trafficking CLE peptides for secretion. In a direct comparison of HsCLE2 overexpression phenotypes with those of AtCLE5 and AtCLE6, similar shoot and root phenotypes were observed. Exogenous application of 12-amino-acid synthetic peptides corresponding to the CLE motifs of HsCLEs and AtCLE5/6 suggests that the function of this class of CLEs may be subject to complex endogenous regulation. When seedlings were grown on high concentrations of peptide (10 μm), root growth was suppressed; however, when seedlings were grown on low concentrations of peptide (0.1 μm), root growth was stimulated. Together, these findings indicate that AtCLEs1-7 may be the target peptides mimicked by HsCLEs to promote parasitism.     

Comparative proteomics of extracellular proteins in vitro and in planta from the pathogenic fungus Fusarium graminearum

High-throughput MS/MS was used to identify proteins secreted by Fusarium graminearum (Gibberella zeae) during growth on 13 media in vitro and in planta during infection of wheat heads. In vitro secreted proteins were collected from the culture filtrates, and in planta proteins were collected by vacuum infiltration. A total of 289 proteins (229 in vitro and 120 in planta) were identified with high statistical confidence. Forty-nine of the in planta proteins were not found in any of the in vitro conditions. The majority (91-100%) of the in vitro proteins had predicted signal peptides, but only 56% of the in planta proteins. At least 13 of the nonsecreted proteins found only in planta were single-copy housekeeping enzymes, including enolase, triose phosphate isomerase, phosphoglucomutase, calmodulin, aconitase, and malate dehydrogenase. The presence of these proteins in the in planta but not in vitro secretome might indicate that significant fungal lysis occurs during pathogenesis. On the other hand, several of the proteins lacking signal peptides that were found in planta have been reported to be potent immunogens secreted by animal pathogenic fungi, and therefore could be important in the interaction between F. graminearum and its host plants.