番茄幼苗盐胁迫响应蛋白质组分析

采用营养液栽培,以盐敏感型番茄品种M82为试材,利用双向电泳(2-DE)研究盐胁迫处理下幼苗叶片蛋白质的表达谱,并采用基质辅助激光解析飞行时间串联质谱(MALDI-TOF/TOF-MS)技术进行差异蛋白质的分离及质谱鉴定。结果表明:(1)盐胁迫处理下,利用2-DE获得差异显著蛋白点20个,其中17个蛋白质点丰度上调表达,3个蛋白质点丰度下调表达。(2)通过质谱分析和蛋白质NCBInr数据库检索,共鉴定出19个差异蛋白,分别为果糖-二磷酸醛缩酶、S-腺苷甲硫氨酸合成酶、甘油醛-3-磷酸脱氢酶等及3个功能未知蛋白;这些鉴定出的差异蛋白质与能量代谢、光合作用、蛋白合成、氧化还原平衡等过程相关,暗示所分离鉴定的蛋白可能参与了番茄的盐胁迫响应,为进一步研究番茄抗逆机制奠定基础。     

大豆种子萌发过程中的差异蛋白质组研究

运用蛋白质组学技术对大豆(Glycinemax)N2899种子萌发0h、8h、36h、60h4个时期蛋白质的差异表达情况进行了研究.结果发现,在考马斯亮蓝染色的双向电泳pH3～10胶上,PDQuest图像分析软件可识别的点约350个,其中表达量变化2.5倍以上的蛋白质点有24个,而绝大部分大豆种子贮藏蛋白在萌发期尚未降解.在萌发的第一阶段,24个差异表达蛋白中有10个蛋白质的丰度发生变化.第二阶段,差异表达蛋白的种类和量增加,其中15个蛋白质是动态变化的,14个蛋白质在胚根突破种皮时表达量达到峰值,表明吸胀后种子内的生命活动越来越强.对这24个蛋白质点进行胶内酶解,用基质辅助激光解析电离飞行时间质谱测定均获得肽质量指纹图谱.搜索大豆的UniGene库初步鉴定出6个蛋白质,分别是核苷二磷酸激酶、热激蛋白、硫氧还蛋白、35ku种子成熟蛋白及种子成熟蛋白PM36.对这些蛋白质在种子萌发过程中可能的作用进行了讨论.     

肾阳虚证候的人血清比较蛋白质组学分析

利用双向电泳（2-DE）优化分离了除去高丰度蛋白（白蛋白和IgG）的老年体虚肾阳虚患者（以健康组为参照）的血清样本,对比分析pH 4～7范围的2-DE谱图,肾阳虚患者和参照组的平均蛋白点分别为(393±32)和(455±19)个. 其中肾阳虚证候表达量上调2倍（P<0.05）以上的蛋白点有26个,下调2倍以上的有33个. 用质谱获得上述差异蛋白的肽质量指纹图谱,并经数据库检索共鉴定出了49种差异蛋白质,其中有10种在肾阳虚血清中特异表达,有6种在健康组血清中特异表达. 蛋白功能分析发现,其中33种蛋白质的差异表达与肾阳虚证密切相关. 蛋白质TCRβ及transthyretin的蛋白印迹实验验证了2-DE 的结果. 该研究结果为阐明中医肾阳虚证的机理提供了一条新途径.     

人肝癌细胞系的糖蛋白质组学研究

糖基化是最重要的蛋白质翻译后形式之一,糖基化蛋白的糖链部分影响着蛋白质的折叠和稳定性以及其生物学功能.许多恶性肿瘤组织与正常组织相比已显示出蛋白质糖基化的差异.采用蛋白质组学分析方法结合先进的糖蛋白荧光染色技术,研究了正常人肝细胞系(ChangLiver)和人肝癌细胞系(Hep3B)糖蛋白糖基化的差异.首先用细胞裂解法提取细胞总蛋白质,进行双向电泳(2-DE),然后用pro-QEmerald488糖蛋白荧光染料进行糖蛋白染色,得到两种细胞系糖基化蛋白表达谱,经2-DE分析软件Dymension分析2-DE图像,比较糖蛋白的糖基化程度,并对糖基化蛋白进行质谱鉴定.结果显示正常人肝细胞表达(74±2)个(n=3),而人肝癌细胞系表达(78±3)个糖蛋白(n=3).两者匹配的糖蛋白质点31个,Hep3B表达而ChangLiver不表达的糖蛋白质点47个,ChangLiver表达而Hep3B不表达的糖蛋白质点43个.两种细胞系糖基化程度存在明显差异,与正常人肝细胞相比,肝癌细胞发生糖基化改变的糖蛋白有25个,其中糖基化水平上调的有10个,下调的有15个,质谱鉴定出12个发生糖基化改变的糖蛋白.这些结果显示蛋白质糖基化改变可能在肝癌的发生和发展中起一定作用.     

胃癌及癌旁组织定量比较蛋白质组学研究

为寻找胃癌特异的肿瘤标记物,用于胃癌临床诊断及药物治疗靶点的选择,本研究采用荧光差异显示凝胶电泳（DIGE）技术分离并筛选 Cy3、Cy5 及 Cy2 荧光素标记的胃癌及对应癌旁组织差异表达蛋白质,用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）或串联质谱技术进行鉴定并分析。结果共筛选出 33 个差异表达蛋白质点,其中 9 个蛋白质点在胃癌组织中上调,24 个蛋白质点下调。对 22 个蛋白质点采用质谱技术成功鉴定,突变结蛋白、锰超氧化物歧化酶、热休克蛋白 60等在胃癌中高表达,热休克蛋白 27、前列腺素 F 合酶、硒结合蛋白 1、锌指蛋白 160、微管蛋白 α6、真核生物翻译延伸因子 1 α1 等在胃癌组织中低表达,并筛选出 5 个未知蛋白。这些差异表达蛋白可望成为胃癌诊断的特异标记物,并与胃癌的发生、发展及预后等有关,为胃癌的诊断、发生机制的研究提供了新的思路。     

小麦种子萌发时期胚差异蛋白表达分析

为了解小麦种子在萌发时期胚蛋白质表达情况,通过双向电泳技术,对"晋麦-47"小麦种子未萌发和萌发3 h、16 h和25 h的胚进行蛋白质分离。结果发现,"晋麦-47"小麦种子未萌发和萌发3 h、16 h和15 h的胚在PDQuest图像分析软件可识别的蛋白点分别有127、131、135和141个,其中表达量变化2.5倍以上的蛋白点有17个。选取表达量在2.5倍以上的9个差异蛋白点进行质谱分析,初步鉴定出4个蛋白质,分别是核苷二磷酸激酶Ⅰ、17.5 kD热休克蛋白、ATP合酶和LEA蛋白27。     

隆线溞孤雌溞和两性雌溞的蛋白质差异表达

本实验提取隆线溞孤雌溞和两性雌溞的可溶性蛋白进行双向电泳和质谱鉴定,分析隆线溞在两种生殖状态下蛋白质组的差异变化。聚丙烯酰胺凝胶SDS-PAGE结果表明:隆线溞在两种生殖状态下存在明显的蛋白质表达差异,孤雌溞的蛋白条带在分子量约50.6kD、36.2kD、32.1kD和25.7kD处表达量较两性雌溞明显;两性雌的蛋白条带在分子量约87.8kD、67.2kD、53.6kD和35.5kD处表达量较孤雌溞明显,其中35.5kD的蛋白条带为两性雌所特有。同时取两个样品的可溶性蛋白进行双向电泳,每个样品重复四次。双向电泳图谱经银染后利用软件分析可知,隆线孤雌平均可检测到约750个蛋白质点,两性雌溞平均可检测到约720个蛋白质点。同时利用软件对凝胶上的蛋白质点进行半定量分析,发现隆线溞从孤雌生殖转化为两性生殖后有18个蛋白质点呈现显著变化,其中14个点表达量明显下降,4个点表达量显著升高。实验结果具有较好的重复性。取4个表达量显著上升的蛋白质点进行质谱分析,得到两个蛋白质点(16号和17号)的测定结果。其中16号点为一类酸性脱氢酶(2I234),它在动物生长发育的各个阶段大量表达,这类蛋白质在隆线溞生殖转化过程中表达量变化尤为显著。本研究结果表明:隆线溞在孤雌生殖和两性生殖状态下存在明显的蛋白质表达差异。     

双向电泳和质谱技术分析樟芝无性孢子萌发相关蛋白

【目的】采用双向电泳(2DE)、质谱技术和实时荧光定量PCR(RT-q PCR)技术分析樟芝无性孢子萌发相关蛋白。【方法】分别提取培养0 h和24 h的樟芝孢子总蛋白并进行双向电泳分离,再用PDQuest软件进行差异蛋白分析,并用MALDI-TOF-MS技术对差异蛋白进行鉴定;其次将鉴定成功的蛋白与孢子萌发相关蛋白的本地数据库进行比对,获得樟芝中的孢子萌发相关蛋白信息,最后用RT-qPCR技术对相关基因的转录水平进行分析。【结果】两组样品共有32个差异蛋白点,其中在24 h表达量上调的蛋白25个,下调的蛋白7个。将32个差异蛋白点挖取鉴定,成功鉴定24个。其中,与孢子萌发相关的蛋白有10个,分别为GerO、Ubc1、Cat-1、Snf1、Cas2、SfaD、Chaperonin、Fad5、Tyrosine-P和ChiA。【结论】该研究结果为进一步解析樟芝无性孢子萌发的分子机制提供了理论依据。     

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。     

炭样小单孢菌中抗生素生物合成相关蛋白的筛选

目的：筛选一株具有广谱抗菌活性的炭样小单孢菌JXNU-1中核苷类抗生素生物合成相关蛋白。方法：通过iTRAQ定量蛋白质组学技术对JXNU-1菌体生长期（36h）和产物合成期（108h）的差异蛋白进行鉴定和功能分析。结果：基于iTRAQ定量蛋白质组学技术共鉴定出炭样小单孢菌总蛋白质2390个,差异表达蛋白172个,在产物合成期（108h）表达上调76个、表达下调96个。通过蛋白GO和COG注释等功能分析,筛选出12个与抗生素合成密切相关蛋白和5个生物合成基因簇。结论：利用iTRAQ技术筛选出炭样小单孢菌JXNU-1的抗生素合成相关蛋白,为阐明该抗生素的生物合成机制奠定实验依据。     

Penicillium griseofulvum F1959, high-production strain of pyripyropene a, specific inhibitor of acyl-CoA: cholesterol acyltransferase 2

Acyl-coenzyme A: cholesterol acyltransferase (ACAT) catalyzes cholesterol esterification and plays an important role in the intestinal absorption of cholesterol, hepatic production of lipoproteins, and accumulation of cholesteryl ester within cells. During the course of screening to find ACAT inhibitors from microbial sources, the present authors isolated pyripyropene A from Penicillium griseofulvum F1959. Pyripyropene A, an ACAT2-specific inhibitor, has already been produced from Aspergillus fumigatus. Yet, Aspergillus fumigatus is a pathogen and only produces a limited amount of pyripyropene A, making the isolation of pyripyropene A troublesome. In contrast, Penicillium griseofulvum F1959 was found to produce approximately 28 times more pyripyropene A than Aspergillus fumigatus, plus this report also describes the ideal conditions for the production of pyripyropene A by Penicillium griseofulvum F1959 and its subsequent purification.     

Comparative Biochemical Studies on F and EDP208 Conjugative Pili

EDP208 pili are encoded by a derepressed derivative of a naturally occurring lac plasmid, F(0)lac (incompatibility group FV), originally isolated from Salmonella typhi. EDP208 pili are serologically unrelated to F pili and do not promote infection by F-specific ribonucleic acid bacteriophages. However, they do confer sensitivity to the F-specific filamentous deoxyribonucleic acid phages. EDP208-containing cells are multi-piliated and contain approximately 20 pili per cell. These pili contain a single polypeptide subunit of 11,500 daltons. EDP208-specific RNA phages were readily isolated from local sewage. These phages were somewhat smaller in diameter than the F-specific ribonucleic acid phages and absorbed relatively weakly to EDP208 pili. Comparing EDP208 pilin to F, it was found that both contain the equivalent of two to three hexose units per subunit as well as blocked N-termini. EDP208 pilin contains one covalently linked phosphate residue per subunit, whereas the F pilin subunit contains two such residues. Although notable differences were found in the case of three or four amino acids, the overall amino acid compositions of F and EDP208 were very similar. Moreover, the tryptic peptide maps of the two proteins contained seven peptides with similar mobilities, suggesting considerable homology in their amino acid sequences. Substantial similarities were also noted in the secondary structures of F and EDP208 pilin on the basis of circular dichroism studies. The alpha-helix content of both proteins was calculated to be 65 to 70%. X-ray fiber diffraction studies have indicated that the arrangements of subunits in F and EDP208 pili are also similar. It was concluded that F and EDP208 pili are closely related structures.     

Effect of the reversal of coenzyme specificity by expression of mutated Pichia stipitis xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

AIMS: To determine the effects on xylitol accumulation and ethanol yield of expression of mutated Pichia stipitis xylitol dehydrogenase (XDH) with reversal of coenzyme specificity in recombinant Saccharomyces cerevisiae. METHODS AND RESULTS: The genes XYL2 (D207A/I208R/F209S) and XYL2 (S96C/S99C/Y102C/D207A/I208R/F209S) were introduced into S. cerevisiae, which already contained the P. stipitis XYL1 gene (encoding xylose reductase, XR) and the endogenously overexpressed XKS1 gene (encoding xylulokinase, XK). The specific activities of mutated XDH in both strains showed a distinct increase in NADP(+)-dependent activity in both strains with mutated XDH, reaching 0.782 and 0.698 U mg(-1). In xylose fermentation, the strain with XDH (D207A/I208R/F209S) had a large decrease in xylitol and glycerol yield, while the xylose consumption and ethanol yield were decreased. In the strain with XDH (S96C/S99C/Y102C/D207A/I208R/F209S), the xylose consumption and ethanol yield were also decreased, and the xylitol yield was increased, because of low XDH activity. CONCLUSIONS: Changing XDH coenzyme specificity was a sufficient method for reducing the production of xylitol, but high activity of XDH was also required for improved ethanol formation. SIGNIFICANCE AND IMPACT OF THE STUDY: The difference in coenzyme specificity was a vital parameter controlling ethanolic xylose fermentation but the XDH/XR ratio was also important.     

Analysis and characterization of the IncFV plasmid pED208 transfer region

pED208 is a transfer-derepressed mutant of the IncFV plasmid, F(0)lac, which has an IS2 element inserted in its traY gene, resulting in constitutive overexpression of its transfer (tra) region. The pED208 transfer region, which encodes proteins responsible for pilus synthesis and conjugative plasmid transfer, was sequenced and found to be very similar to the F tra region in terms of its organization although most pED208 tra proteins share only about 45% amino acid identity. All the essential genes for F transfer had homologs within the pED208 transfer region with the exception of traQ, which encodes the chaperone for stable F-pilin expression. F(0)lac appears to have a fertility inhibition system different than the FinOP system of other F-like plasmids, and its transfer efficiency was increased in the presence of F or R100, suggesting that it could be mobilized by these plasmids. The F-like transfer systems specified by F, R100, and F(0)lac were highly specific for their cognate origins of transfer (oriT) as measured by their abilities to mobilize chimeric oriT-containing plasmids.     

Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc

Pichia stipitis NAD(+)-dependent xylitol dehydrogenase (XDH), a medium-chain dehydrogenase/reductase, is one of the key enzymes in ethanol fermentation from xylose. For the construction of an efficient biomass-ethanol conversion system, we focused on the two areas of XDH, 1) change of coenzyme specificity from NAD(+) to NADP(+) and 2) thermostabilization by introducing an additional zinc atom. Site-directed mutagenesis was used to examine the roles of Asp(207), Ile(208), Phe(209), and Asn(211) in the discrimination between NAD(+) and NADP(+). Single mutants (D207A, I208R, F209S, and N211R) improved 5 approximately 48-fold in catalytic efficiency (k(cat)/K(m)) with NADP(+) compared with the wild type but retained substantial activity with NAD(+). The double mutants (D207A/I208R and D207A/F209S) improved by 3 orders of magnitude in k(cat)/K(m) with NADP(+), but they still preferred NAD(+) to NADP(+). The triple mutant (D207A/I208R/F209S) and quadruple mutant (D207A/I208R/F209S/N211R) showed more than 4500-fold higher values in k(cat)/K(m) with NADP(+) than the wild-type enzyme, reaching values comparable with k(cat)/K(m) with NAD(+) of the wild-type enzyme. Because most NADP(+)-dependent XDH mutants constructed in this study decreased the thermostability compared with the wild-type enzyme, we attempted to improve the thermostability of XDH mutants by the introduction of an additional zinc atom. The introduction of three cysteine residues in wild-type XDH gave an additional zinc-binding site and improved the thermostability. The introduction of this mutation in D207A/I208R/F209S and D207A/I208R/F209S/N211R mutants increased the thermostability and further increased the catalytic activity with NADP(+).     

Nucleotide sequence of the tra YALE region from IncFV plasmid pED208

The pED208 plasmid is a 90-kilobase conjugative plasmid which is the derepressed form of Fo lac plasmid (IncFV). A 3.3-kilobase HindIII-PstI fragment from the pED208 plasmid was cloned and sequenced and was found to contain four open reading frames which were highly homologous to the traA, traL, traE, and traY gene products of the F plasmid. The pED208 traA propilin protein was 119 amino acids in length, consisting of a leader sequence of 55 amino acids and a mature pilin subunit of 64 residues. The leader sequence contained a hydrophobic region followed by a classic signal peptidase cleavage site (Ala-Ser-Ala-55). F and pED208 pilin proteins shared 27 conserved residues and had similar predicted secondary structures. The pED208 traA and traL genes were separated by a single base pair, and no ribosome binding site preceded the traL gene. The pED208 traY gene contained an IS2 insertion element in orientation II 180 nucleotides (60 residues) upstream of the traY stop codon. This insertion of IS2 resulted in a predicted fusion peptide of 69 residues for traY which may provide the observed traY activity. Since IS2 is absent in the wild-type plasmid, Fo lac, derepression and concomitant multipiliation may be due to the insertion of IS2 providing constitutive expression of the pED208 tra operon.     

Ubiquitin-mediated proteasomal degradation of non-synonymous SNP variants of human ABC transporter ABCG2

Clinical relevance is implicated between the genetic polymorphisms of the ABC (ATP-binding cassette) transporter ABCG2 (ABC subfamily G, member 2) and the individual differences in drug response. We expressed a total of seven non-synonymous SNP (single nucleotide polymorphism) variants in Flp-In-293 cells by using the Flp (flippase) recombinase system. Of these, ABCG2 F208S and S441N variants were found to be expressed at markedly low levels, whereas their mRNA levels were equal to those of the other SNP variants and ABCG2 WT (wild-type). Interestingly, protein expression levels of the ABCG2 F208S and S441N variants increased 6- to 12-fold when Flp-In-293 cells were treated with MG132, a proteasome inhibitor. Immunoprecipitation followed by immunoblot analysis showed that the ABCG2 F208S and S441N variant proteins were endogenously ubiquitinated in Flp-In-293 cells, and treatment with MG132 significantly enhanced the level of these ubiquitinated variants. Immunofluorescence microscopy demonstrated that MG132 greatly affected the ABCG2 F208S and S441N variants in terms of both protein levels and intracellular distribution. Immunoblot analysis revealed that those variants were N-glycosylated; however, their oligosaccharides were immature compared with those present on ABCG2 WT. The ABCG2 F208S and S441N variant proteins do not appear to be processed in the Golgi apparatus, but undergo ubiquitin-mediated protein degradation in proteasomes, whereas ABCG2 WT is sorted to the plasma membrane and then degraded via the lysosomal pathway. The present study provides the first evidence that certain genetic polymorphisms can affect the protein stability of ABCG2. Control of proteasomal degradation of ABCG2 would provide a novel approach in cancer chemotherapy to circumvent multidrug resistance of human cancers.     

Protein entrainment during baker's yeast fermentation on a semi-solid substrate in an air-fluidized bed fermentor

Baker's yeast was grown on a semi-solid substrate (homogenized whole potatoes) in an air-fluidized bed fermentor, in which a rapid stream of air simultaneously supplied oxygen and mixed the semi-solid substrate. The potato starch was converted to reducing sugars by -amylase (from Aspergillus).During the course of the batch fermentation, some secreted yeast proteins were trapped by sparging the effluent air into a water chamber. Surprisingly, neither the -amylase nor the potato proteins were the most predominant proteins carried over to the overhead collector during the 24 h run, even though they were the most abundant proteins in the fermentation mash. Fractionation of the yeast-produced proteins during this carry-over process is described, based on gel electrophoresis analyses of both the carried-over proteins and the extracellular proteins in the fermentation bed. Effects of the operating variables on the extracellular protein levels in the fermentation bed and the proteins in the overhead collector are also discussed.     

A mutant strain of Scenedesmus obliquus deficient in ribulose diphosphate carboxylase, cytochrome f and photosystem II activity.

The partial reactions of photosynthesis shown by strain F208, a non-photosynthetic mutant strain of Scenedesmus obliquus, have been compared with those performed by other mutant strains which lacked; Photosystem II activity (strains 11 and F131), cytochrome f (strain 50), P-700 and cytochrome f (strain F 119), and P-700 (strains F139 and 199). In this respect the properties of strain F208 were those that would be expected if Photosystem II activity and cytochrome f were not present in this strain. Examination of the composition of strain F208 has shown the absence of cytochrome f in both the soluble and the membrane-bound form. The considerably lower level of plastoquinone compared to that found in the wild type is characteristic of the strains which lack Photosystem II activities. Fraction 1 protein could not be detected in extracts of strain F208 by sedimentation velocity experiments in the ultracentrifuge, and only 7% of the wild type ribulose diphosphate carboxylase activity was found after chromatography of these extracts on DEAE-cellulose. The properties of strain F208 are compared with those of the ac-20 and cr-1 strains of Chlamydomanas rheinhardi, both of which have a deficiency of ribulose diphosphate carboxylase which is considered to result from a deficiency of chloroplast ribosomes. Strain F208 resembles these strains in its abnormal chloroplast ultrastructure and its decreased levels of the RNA forms derived from the chloroplast ribosomes when compared with the wild type. Chloroplast fragments isolated from strains of S. obliquus which lacked cytochrome f (strains 50 and F208) were able to use diaminodurene and ascorbate as an electron donor to Photosynstem I. Since this reaction was inhibited by mercuric salts it would appear that plastocyanin, but not cytochrome f, was involved in this electron transfer.     

Engineering of the iron site in ribonucleotide reductase to a self-hydroxylating monooxygenase.

Protein R2 of ribonucleotide reductase contains a dinuclear ferric iron center adjacent to a tyrosyl radical in the interior of the protein matrix. A patch of hydrophobic residues surrounds the iron-radical cofactor. Its importance during the oxidative generation of the iron-radical cofactor was investigated by site-directed mutagenesis of Phe-208 to tyrosine. The mutant protein R2 F208Y has prominent absorption bands at 460 and 720 nm reminiscent of those in ferric-catecholate complexes. Resonance Raman spectroscopy shows that the iron center of R2 F208Y contains a bidentate catechol ligand. The mechanism for generation of this protein-derived dihydroxyphenylalanine may be similar to the catalytic cycle of methane monooxygenase.