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.     

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。     

粗糙脉孢菌基因组分泌蛋白的初步分析

文章报道利用信号肽预测软件SignalP v3.0和PSORT,跨膜螺旋结构预测软件TMHMMv2.0和THUMBUP,GPI-锚定位点预测软件big-PI Predictor和亚细胞器中蛋白定位分布预测软件TargetP v1.01对粗糙脉孢菌全基因组数据库中已公布的10 082个氨基酸序列进行预测分析。结果表明在粗糙脉孢菌中有437个蛋白为分泌蛋白,编码这些蛋白最小的可读框（open reading frame,ORF）为252 bp,最大为6 604 bp,平均1 433 bp,分泌蛋白信号肽长度介于15~59个氨基酸之间。在437个分泌蛋白中,205个具有功能描述,主要包括各种酶类、细胞能量生成、运转以及自身修复、防卫等多种功能。这些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,为该物种营养的摄取,以及对环境做出响应服务。      

立枯丝核菌AG-3全基因组分泌蛋白的预测分析

[目的]预测、分析立枯丝核菌AG-3全基因组范围内的分泌蛋白，并明确其基本特征，筛选其效应蛋白。[方法]依据已经公布的立枯丝核菌AG-3全基因组数据库中的12 726个蛋白序列，利用信号肽预测软件SignalP-4.1,细胞器定位分析软件ProtComp 9.0,跨膜螺旋结构预测软件TMHMM 2.0, GPI-锚定位点预测软件big-PI Fungal Predictor和亚细胞器中蛋白定位分布预测软件TargetP-1.1进行典型分泌蛋白的预测分析，并用LipoP-1.0进行信号肽切割位点的预测分析，最后对预测得到的分泌蛋白通过EffectorP进行效应蛋白的预测。[结果]在立枯丝核菌AG-3中有401个蛋白被预测为分泌蛋白，其编码蛋白长度集中于100～600 aa。信号肽长度介于11～35 aa之间，-3至-1位置上的氨基酸相对保守，切割位点为A-X-A类型，可被SpⅠ型信号肽酶识别并切割。在预测得到的分泌蛋白中通过EffectorP筛选得到140个效应蛋白。[结论]通过全基因组预测得到401个具有典型分泌蛋白特征的蛋白，从预测的分泌蛋白中筛选得到140个效应蛋白。     

马铃薯晚疫病菌全基因组分泌蛋白的初步分析

利用马铃薯晚疫病菌全基因组测序结果,结合计算机技术和生物信息学的方法,对马铃薯晚疫病菌的蛋白进行分析,为明确该病原菌与寄主互作的分子机制奠定基础。文章应用信号肽预测软件SignalP v3.0和PSORT,跨膜螺旋结构预测软件TMHMM-2.0和THUMBUP,GPI锚定位点预测软件big-PI Predictor,亚细胞器中蛋白定位分布预测软件TargetP v1.01,对已经公布的马铃薯晚疫病菌全基因组22 658个蛋白质氨基酸序列进行分析。结果发现,晚疫病菌全基因组编码蛋白中有671个为潜在的分泌型蛋白,占编码蛋白总数的3.0%。其中有45个分泌蛋白有功能方面的描述,其功能涉及细胞代谢、信号转导等方面;此外,还有一些与激发子类似的分泌蛋白,它们可能与晚疫病菌的毒性有关。     

MAP30蛋白结构功能的生物信息学研究

使用protparam、PHDhtm、PredictProtein等生物信息学在线服务器,对MAP30蛋白进行全面分析预测,研究MAP30蛋白具有的抗HIV活性,为临床应用提供抗科学依据和理论基础。结果表明:MAP30蛋白为稳定的碱性疏水蛋白,序列上存在三段跨膜螺旋结构和一段无序化位区域,肽链上的二硫键可使分子间形成聚集体,是一种分泌蛋白。MAP30蛋白序列包含信号肽、低复杂度区域和RIP样活性区域3个区域,具有细胞外被膜、异构酶、免疫应答三种功能。序列上分布着N-糖基化位点、N-豆蔻酰化位点、Shiga/ricin核糖体失活蛋白活性位点和多段蛋白激酶磷酸化位点。     

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.     

乙型肝炎病毒S蛋白的序列特征分析

乙肝病毒S蛋白是病毒的包膜蛋白,与病毒进入细胞有关,它存在逆转录过程并且具有极强的潜伏性。本论文应用生物信息学分析乙肝病毒S蛋白的序列特征,利用在线分析软件预测乙肝病毒S蛋白的理化性质和亲疏水性、跨膜区域、信号肽特征、磷酸化位点、二级结构以及乙肝病毒S蛋白的最佳抗原表位形成位置等。结果显示了乙肝病毒S蛋白由226个氨基酸组成,理论等电点是8.21,为不稳定蛋白,总平均亲水性为0.649,是疏水蛋白质,并且该蛋白存在信号肽,有4个跨膜区,有30个潜在的磷酸化位点,主要二级结构为α螺旋和无规则卷曲,同时,结合乙型肝炎病毒S蛋白的序列可及性、线性表位、β转角、柔性、抗原性的预测结果,可以找到潜在的抗原表位区域,为乙型肝炎的表位疫苗研制提供重要的参考依据,有利于进一步对乙型肝炎S蛋白的抗原性进行研究。     

三种隐孢子虫钙依赖蛋白激酶的生物信息学分析

对三种隐孢子虫(C.parvum Iowa II、C.hominis TU502和C.muris RN66)钙依赖蛋白激酶(Calcium-dependent protein kinases,CDPKs)进行生物信息学分析,探索该蛋白的结构并预测其功能,为其基因功能的研究提供一定的理论基础。通过隐孢子虫基因组数据库收集数据,获得三种隐孢子虫CDPKs蛋白的序列信息,通过生物信息学软件进行分析,预测该蛋白的理化性质、翻译后修饰位点、功能域、亚细胞定位、二级结构、亲/疏水性、抗原表位等。隐孢子虫CDPKs的蛋白性质不稳定,理论分子量从59.76 k Da到76.63 k Da,p I值为5.33~6.09,CDPKs不具有跨膜区和信号肽,不是跨膜分泌性蛋白,都具有蛋白激酶C磷酸化位点、酪氨酸激酶Ⅱ磷酸化位点、酪氨酸激酶磷酸化位点、c AMP和c GMP依赖蛋白激酶磷酸化位点、N-端糖基化位点、N-端肉豆蔻酰化位点和EF-hand钙结合域,二级结构主要以α螺旋和无规卷曲为主;CDPKs主要存在虫体细胞内,均有20多个潜在的抗原表位。在隐孢子虫中,CDPKs蛋白不仅可单独发挥作用,而且还能通过相互结合发挥其生物学效应;同时,CDPKs有望成为候选疫苗及潜在药物靶点。     

Proteome analysis of Schizosaccharomyces pombe by two-dimensional gel electrophoresis and mass spectrometry

The fission yeast Schizosaccharomyces pombe (S. pombe) is a unicellular eukaryote and contains many genes and regulatory mechanisms that are close to those of mammals. In this study, we performed a global proteomic analysis of the fission yeast S. pombe wild type h(-S) L 972 proteome. More than 1,500 protein spots were visualized on silver stained 2-D gels in the 3-10 pI range with a high resolution and high reproducibility. Protein identification was carried out by MALDI-TOF-MS and/or nanoLC-MS/MS. Advantage of the complementarity of these two MS approaches was used to enhance the identification quality. So far, 364 proteins (representing 157 different proteins) have been identified. We report here the identification of 117 new proteins on our 2-D reference map of this yeast compared to the first reference map. Of these identified proteins, 40.1% were involved in metabolism. The present work provides a very useful tool for all studies relying on S. pombe as a model organism and is a considerable complement to the first reference map of S. pombe published recently by Sun and coworkers (Sun, N., Jang, J., Lee, S., Kim, S. et al.., Proteomics 2005, 5, 1574-1579).     

不同营养方式真菌中分泌蛋白数量及其功能对比研究

【目的】分泌蛋白在植物病原真菌致病过程中发挥着重要的作用,前人多以单种菌株分泌蛋白预测分析,尚未见多种类型真菌中分泌蛋白的预测及对比研究报道。【方法】本研究基于多种不同营养方式真菌的全基因组序列,根据分泌蛋白所具有的基本特征,采用在线分析程序(主要包括SignalP v5.0、ProtComp v9.0等)对模式生物、死体营养型真菌、半活体营养型真菌及活体营养型真菌共19种菌株的分泌蛋白开展预测及功能分析。【结果】在上述真菌约13万条蛋白序列中,分泌蛋白所占比例为0.74%–4.83%,其中,活体营养型真菌所具有的分泌蛋白数量占比最高,平均为3.51%,而死体营养型真菌和模式生物平均比例最低,平均为1.36%。同时,活体营养型真菌具有的功能种类最多,为433种,其次为半活体营养型真菌,为266种,而模式生物具有的功能种类最少,为100种,其中,功能为假设蛋白和非特征蛋白的蛋白数量最多。【结论】该研究为深入解析分泌蛋白在实现不同营养方式真菌的致病机制方面提供理论依据。     

In silico reconstruction of nutrient-sensing signal transduction pathways in Aspergillus nidulans

We report here probable nutrient-sensing signal transduction pathways in Aspergillus nidulans, a model filamentous fungus, based on sequence homology studies with known Saccharomyces cerevisiae and Schizosaccharomyces pombe proteins. Specifically, we identified A. nidulans homologs for yeast proteins involved in (1) filamentation-invasion, (2) cAMP-PKA, (3) pheromone response, (4) cell integrity and (5) TOR signaling pathways. We have also studied autophagy, one of the most important cellular responses regulated by TOR signaling. The Basic Local Alignment Search Tool program "blastp" was used to assess the homology of proteins. We note that by using a highly conservative approach, 70% of the S. cerevisiae signal transduction proteins (107 proteins out of 153 proteins studied) have significant homologs in A. nidulans. Using a slightly less conservative approach, we are able to identify homologs for as high as 91% of the S. cerevisiae signal transduction proteins (139 proteins out of 153 proteins studied). The filamentation-invasion, cell integrity and TOR signaling pathways showed greatest similarity with S. cerevisiae, while the cAMP-PKA and pheromone response pathways showed greater similarity with S. pombe. Based on these results, probable pathways in A. nidulans were constructed using well-established S. cerevisiae and S. pombe models.     

Osteogenic differentiation of adipose derived stem cells promoted by overexpression of osterix

Secreted proteins, which may be involved in the regulation of various biological processes, are the potential targets for diagnosis and treatment of diverse diseases. In this study, to identify the human hepatoma HepG2 cells-derived secreted proteins more extensively, we applied the protein sample preparations using the combinations of denaturation methods and molecular-mass cutoff via ultrafiltration to the two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D LC–MS/MS) analysis. We were able to identify a total of 86 proteins containing widely known secreted proteins of HepG2 such as alpha-fetoprotein, of which 73 proteins including 27 signal peptide-containing proteins have never been reported to be secreted from HepG2 cells in other proteomic studies. Among the identified signal peptide-containing proteins, ten proteins such as growth differentiation factor 15, osteopontin and stanniocalcin 2 were discovered as new secreted proteins of HepG2 cells. These observations suggest that the combinations of different sample preparation methods and 2D LC–MS/MS analysis are useful for identifying a wider range of low-abundance proteins and that the secreted proteins from HepG2 identified in this study may be useful as liver-specific biomarkers for diagnosis and treatment.     

Comparison of alternative extraction methods for secretome profiling in human hepatocellular carcinoma cells

Secreted proteins are important sources for early detection and diagnosis of disease, and as such have received considerable attention. The extraction of low concentration proteins from large volumes of culture media, which are rich in salts and other compounds that interfere with most proteomics techniques, presents a problem for secretome studies. Ultrafiltration, precipitation, and dialysis are three major extraction methods that can be used to overcome this problem. The present study for the first time, compared the merits and shortcomings of these three methods, without bias. Centrifugal ultrafiltration provided the best extraction efficiency, and precipitation provided the highest number of identifiable proteins. The three methods yielded closely related, but different, information on the secretome; thus, they should be considered complementary or, at least, supplementary methods. Three hundred and sixty unique proteins were identified, including 211 potential secreted proteins. Compared with previous studies, this study also identified 42 new secreted proteins. The present study not only offers a reference for the selection of secretome extraction methods, but also expands the secretome database for the investigation of hepatocellular carcinoma.     

Bob1, a Gim5/MM-1/Pfd5 homolog, interacts with the MAP kinase kinase Byr1 to regulate sexual differentiation in the fission yeast, Schizosaccharomyces pombe

The MAPKK Byr1 is an essential component of a Ras-dependent MAPK module required for sexual differentiation in the fission yeast, Schizosaccharomyces pombe. Here we describe the genetic and molecular characterization of a highly conserved protein, Bob1, which was identified from a two-hybrid screen for Byr1-interacting proteins. Byrl and Bobl proteins coprecipitate from S. pombe cell lysates, and both proteins localize to the tips and septa of S. pombe cells. S. pombe bob1 null (bob1delta) mutants lack obvious growth defects but exhibit a significant mating deficiency, which can be suppressed by overexpression of Byrl. Overexpression of Bob1 also leads to inhibition of mating in S. pombe, and this defect is likewise suppressed by Byrl overexpression. Bob1 is highly homologous in structure to the mammalian MM-1/Pfd5 and budding yeast Gim5/Pfd5-Sc proteins, which have been implicated as regulators of actin and tubulins. Similar to budding yeast gim5/pfd5-Sc mutants, S. pombe bob1delta cells have cytoskeletal defects, as judged by hypersensitivity to cytoskeletal disrupting drugs. byr1delta mutants do not share this characteristic with bob1delta mutants, and byr1delta bob1delta mutants are not significantly more sensitive to cytoskeletal disrupting drugs than cells carrying only the bob1delta mutation. Taken together, our results suggest that Bob1 has Byr1-related function(s) required for proper mating response of S. pombe cells and Byrl-independent function(s) required for normal cytoskeletal control. We show that the human MM-1/Pfd5 protein can substitute for its counterpart in fission yeast, providing evidence that the functions of Bob1-related proteins have been highly conserved through evolution. Our results lead us to propose that Bob1-related proteins may play diverse roles in eukaryotic organisms.     

羊布鲁氏菌的分泌蛋白质组分析

分泌蛋白是指那些分泌到细胞外的蛋白质。布鲁氏菌的分泌蛋白可能介导了病原与宿主之间的相互作用, 在布鲁氏菌的毒力方面发挥一定的作用, 但是研究方法的局限性限制了分泌蛋白的研究。本文报道了利用蛋白质组的方法来寻找羊布鲁氏菌的分泌蛋白。首先用TCA-丙酮法提取布鲁氏菌培养上清中的分泌蛋白, 双向电泳进行分离, 然后用质谱来鉴定这些蛋白, 最终鉴定到40种蛋白。通过生物信息学分析, 发现这些蛋白主要是ABC转运系统的底物结合蛋白、外膜蛋白和热休克蛋白。这些蛋白的识别不仅有助于对布鲁氏菌致病机制的理解, 而且也可为     

HIV-1 Vif protein mediates the degradation of APOBEC3G in fission yeast when over-expressed using codon optimization

Interaction between the HIV-1 Vif protein and the cellular host APOBEC3G protein is a promising target for inhibition of HIV-1 replication. Considering that human cells are a very complicated environment for the study of protein interactions, the goal of this study was to check whether fission yeast could be used as a model cell for studying the Vif-APOBEC3G interaction. Vif and APOBEC3G were expressed in fusion with GFP protein in the S. pombe SP223 strain. Subcellular localizations of Vif and APOBEC3G were observed with fluorescent microscopy. Codon optimization was used to over express the Vif protein in S. pombe cells. The degradation of APOBEC3G mediated by Vif was tested through expressing Vif and GFP-APOBEC3G proteins in the same cell. Western Blot analysis was used to measure the corresponding protein levels under different experimental conditions. The results showed that the Vif protein was predominantly localized in the nucleus of S.pombe cells, APOBEC3G was localized in the cytoplasm and concentrated at punctate bodies that were often in close proximity to the nucleus but were not necessarily restricted from other regions in the cytoplasm. Vif protein expression levels were increased significantly by using codon optimization and APOBEC3G was degraded when Vif was over-expressed in the same S. pombe cells. These results indicate that fission yeast is a good model for studying the interaction between the Vif and APOBEC3G proteins.     

Over‐expression of secreted proteins from mammalian cell lines

Secreted mammalian proteins require the development of robust protein over‐expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large‐scale growth of cells in a variety of formats.     

Isolation and characterization of the structural gene for secreted acid phosphatase from Schizosaccharomyces pombe

The Schizosaccharomyces pombe acid phosphatase structural gene (PHO 1) was isolated by complementation of an S. pombe acid phosphatase mutant with a wild type S. pombe DNA recombinant plasmid library. Northern analysis indicates that acid phosphatase is encoded by a 1.4-kilobase mRNA of which approximately 100 bases are 3'-poly(A). The gene contains no introns and the 3' and 5' untranslated regions are short. According to DNA and amino acid sequence data, the S. pombe acid phosphatase has a molecular weight of 50,600. An 18-amino acid sequence at the N terminus was found that is similar to previously identified signal peptides in other eukaryotic secretory proteins. This signal peptide is apparently removed during secretion, since it is absent in the mature secreted acid phosphatase. The gene can be induced 2--3-fold by starvation for phosphate. The signals required for this induction are contained on the isolated DNA clone. Although the gene can be expressed in Saccharomyces cerevisiae, secretion is abnormal.