Construction of a polycystic ovarian syndrome (PCOS) pathway based on the interactions of PCOS-related proteins retrieved from bibliomic data

Polycystic ovary syndrome (PCOS) is a complex but frequently occurring endocrine abnormality. PCOS has become one of the leading causes of oligo-ovulatory infertility among premenopausal women. The definition of PCOS remains unclear because of the heterogeneity of this abnormality, but it is associated with insulin resistance, hyperandrogenism, obesity and dyslipidaemia. The main purpose of this study was to identify possible candidate genes involved in PCOS. Several genomic approaches, including linkage analysis and microarray analysis, have been used to look for candidate PCOS genes. To obtain a clearer view of the mechanism of PCOS, we have compiled data from microarray analyses. An extensive literature search identified seven published microarray analyses that utilized PCOS samples. These were published between the year of 2003 and 2007 and included analyses of ovary tissues as well as whole ovaries and theca cells. Although somewhat different methods were used, all the studies employed cDNA microarrays to compare the gene expression patterns of PCOS patients with those of healthy controls. These analyses identified more than a thousand genes whose expression was altered in PCOS patients. Most of the genes were found to be involved in gene and protein expression, cell signaling and metabolism. We have classified all of the 1081 identified genes as coding for either known or unknown proteins. Cytoscape 2.6.1 was used to build a network of protein and then to analyze it. This protein network consists of 504 protein nodes and 1408 interactions among those proteins. One hypothetical protein in the PCOS network was postulated to be involved in the cell cycle. BiNGO was used to identify the three main ontologies in the protein network: molecular functions, biological processes and cellular components. This gene ontology analysis identified a number of ontologies and genes likely to be involved in the complex mechanism of PCOS. These include the insulin receptor signaling pathway, steroid biosynthesis, and the regulation of gonadotropin secretion among others.     

A global analysis of protein expression profiles in Sinorhizobium meliloti: discovery of new genes for nodule occupancy and stress adaptation

A proteomic examination of Sinorhizobium meliloti strain 1021 was undertaken using a combination of 2-D gel electrophoresis, peptide mass fingerprinting, and bioinformatics. Our goal was to identify (i) putative symbiosis- or nutrient-stress-specific proteins, (ii) the biochemical pathways active under different conditions, (iii) potential new genes, and (iv) the extent of posttranslational modifications of S. meliloti proteins. In total, we identified the protein products of 810 genes (13.1% of the genome's coding capacity). The 810 genes generated 1,180 gene products, with chromosomal genes accounting for 78% of the gene products identified (18.8% of the chromosome's coding capacity). The activity of 53 metabolic pathways was inferred from bioinformatic analysis of proteins with assigned Enzyme Commission numbers. Of the remaining proteins that did not encode enzymes, ABC-type transporters composed 12.7% and regulatory proteins 3.4% of the total. Proteins with up to seven transmembrane domains were identified in membrane preparations. A total of 27 putative nodule-specific proteins and 35 nutrient-stress-specific proteins were identified and used as a basis to define genes and describe processes occurring in S. meliloti cells in nodules and under stress. Several nodule proteins from the plant host were present in the nodule bacteria preparations. We also identified seven potentially novel proteins not predicted from the DNA sequence. Post-translational modifications such as N-terminal processing could be inferred from the data. The posttranslational addition of UMP to the key regulator of nitrogen metabolism, PII, was demonstrated. This work demonstrates the utility of combining mass spectrometry with protein arraying or separation techniques to identify candidate genes involved in important biological processes and niche occupations that may be intransigent to other methods of gene expression profiling.     

Comparative proteomic studies in Rhodospirillum rubrum grown under different nitrogen conditions

Forty-four differentially expressed proteins have been identified in the photosynthetic diazotroph Rhodospirillum rubrum grown anaerobic and photoheterotrophically, with different nitrogen sources, using 2D-PAGE and MALDI-TOF, from gels containing an average of 679 +/- 52 (in N(+)) and 619 +/- 37 (in N(-)) protein spots for each gel. A higher level of expression was found under nitrogen-rich growth, for proteins involved in carbon metabolism (reductive tricarboxylic acid cycle, CO(2) fixation, and poly-beta-hydroxybutyrate metabolism) and amino acid metabolism. The key enzymes RuBisCO and alpha-ketoglutarate synthase were found to be present in higher amounts in nitrogen-rich conditions. Ntr and Nif regulated proteins, such as glutamine synthetase and nitrogenase, were, as expected, induced under nitrogen-fixing conditions and glutamate dehydrogenase was down regulated. A novel 2Fe-2S ferredoxin with unknown function was identified from nitrogen-fixing cultures. In addition to differential expression, two of the identified proteins revealed variable p I values in response to the nitrogen source used.     

Effect of tryptic or peptic digestion or mechanical isolation on the extraction of proteins, antigens, and ribonucleic acids from Sarcocystis muris bradyzoites

Various methods have been used for the isolation of bradyzoites of Sarcocystis species. Using Sarcocystis muris as a model, the effect of 3 methods, trypsin digestion, pepsin digestion, and mechanical isolation, on the subsequent extraction of S. muris proteins, antigens, and RNA was examined. Although some quantitative differences were found among the proteins, antigens, and RNA extracted after the 3 isolation methods, qualitative differences were not evident. The overall isoelectric focusing protein profile showed approximately 30 bands in the pH range of 3-9 and was dominated by 5 bands with pI values of approximately 5.3, 6.3, 6.8, 7.0, and 7.4. In experimentally infected mice, antibodies were recorded from 35 days postinoculation (PI) until the end of examination (130 days PI). The S. muris RNA appeared to consist of at least 10 subunits in the range of 200-4,200 nucleotides. A Toxoplasma gondii DNA fragment specific for small subunit ribosomal RNA (rRNA) hybridized mainly to the S. muris 1,900-nucleotide subunit and a range of smaller subunits, and a probe specific for large subunit rRNA hybridized mainly to the 4,200-nucleotide subunit and a range of smaller subunits. All 3 methods of bradyzoite isolation gave good yields of intact messenger RNAs that showed similar in vitro translation curves.     

Proteomic analysis of schistosoma japonicum schistosomulum proteins that are differentially expressed among hosts differing in their susceptibility to the infection

Schistosomiasis is a tropical, parasitic disease affecting humans and several animal species. The aim of this study was to identify proteins involved in the growth and survival of the parasitic forms inside a host. Schistosomula of Schistosoma japonicum were isolated from three different hosts: the susceptible BALB/c mice; the Wistar rats, which have a considerably lower susceptibility; and the resistant reed vole, Microtus fortis. Soluble proteins of the schistosomula collected from the above three hosts 10 days postinfection were subjected to two-dimensional difference gel electrophoresis. Comparative proteomic analyses revealed that 39, 21, and 25 protein spots were significantly differentially expressed between schistosomula from mice and rats, mice and reed voles, or rats and reed voles, respectively (ANCOVA, p < 0.05). Further, the protein spots were identified by liquid chromatography-tandem MS. Bioinformatics analysis showed that the differentially expressed proteins were essentially those involved in the metabolism of proteins, ribonucleotides, or carbohydrates, or in stress response or cellular movement. This study represents the first attempt at profiling S. japonicum living in different states and provides a basis for a better understanding of the molecular mechanisms in the development and survival of S. japonicum in different host environments.     

Bacterial proteome of streptococcus pneumoniae through multidimensional separations coupled with LC-MS/MS

Streptococcus pneumoniae is a major human respiratory pathogen causing considerable morbidity and mortality worldwide. In order to better understand the pathogenesis of S. pneumoniae, we employed SDS-PAGE combined with LC-MS/MS analysis and in-solution digestion coupled with 2D-LC-MS/MS to obtain the whole-cell proteome of the bacterium. Among the identified 1,210 proteins, 345 proteins were annotated for cellular components, 613 for biological processes, and 421 for molecular functions. Important virulence-associated surface proteins such as Eno, ZmpB, and PrtA were identified. Classification analysis and protein-protein interaction map revealed that these identified proteins are involved in many biological processes including protein biosynthesis, protein folding and proteolysis, cell cycle, or regulation and carbohydrate metabolism. These data represent a comprehensive reference map of S. pneumoniae proteome, providing a useful source for further analysis of the virulence factors and the regulatory network involved in the pathogenesis of the bacterium.     

Insights into fruit function from the proteome of the hypanthium

Apple (Malus×domestica Borkh.) was used as a model to studying essential biological processes occurring in mature fruit hypanthium, commonly referred to as the fruit flesh or pulp, a highly active tissue where numerous metabolic processes such as carbohydrate metabolism and signal transduction occur. To understand the complex biological processes occurring in the hypanthium, a proteomics approach was used to analyze the proteome from freshly harvested ripe apple fruits. A total of 290 well-resolved spots were detected using two-dimensional gel electrophoresis (2-DE). Out of these, 216 proteins were identified representing 116 non-redundant proteins using matrix-assisted laser-desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and either the MASCOT or ProteinProspector engine for peptide mass fingerprinting (PMF) database searching. Identified proteins were classified into 13 major functional categories. Among these, the energy metabolism class was the most represented and included 50% of proteins homologous to Arabidopsis proteins that are involved in the response to biotic and abiotic stresses, suggesting a dual role for these proteins in addition to energy metabolism. We also identified dynein heavy chain in the hypanthium although this protein has been proposed as absent from angiosperms and thus suggest that the lack of dyneins in higher plants studied to date may not be a general characteristic to angiosperm genomic organisation. We therefore conclude that the detection and elucidation of the apple hypanthium proteome is an indispensable step towards the comprehension of fruit metabolism, the integration of genomic, proteomic and metabolomic data to agronomic trait information and thus fruit quality improvements.     

Characterization of Arabidopsis NEET reveals an ancient role for NEET proteins in iron metabolism

The NEET family is a newly discovered group of proteins involved in a diverse array of biological processes, including autophagy, apoptosis, aging, diabetes, and reactive oxygen homeostasis. They form a novel structure, the NEET fold, in which two protomers intertwine to form a two-domain motif, a cap, and a unique redox-active labile 2Fe-2S cluster binding domain. To accelerate the functional study of NEET proteins, as well as to examine whether they have an evolutionarily conserved role, we identified and characterized a plant NEET protein. Here, we show that the Arabidopsis thaliana At5g51720 protein (At-NEET) displays biochemical, structural, and biophysical characteristics of a NEET protein. Phenotypic characterization of At-NEET revealed a key role for this protein in plant development, senescence, reactive oxygen homeostasis, and Fe metabolism. A role in Fe metabolism was further supported by biochemical and cell biology studies of At-NEET in plant and mammalian cells, as well as mutational analysis of its cluster binding domain. Our findings support the hypothesis that NEET proteins have an ancient role in cells associated with Fe metabolism.     

Infection status of dragonflies with Plagiorchis muris metacercariae in Korea.

Plagiorchis muris has been found in both house and field rats as well as in humans. The infection status of the second intermediate hosts of P. muris is prerequisite in understanding their biological features in an ecosystem. Six species of dragonflies were caught in a wide range of areas in Korea; and they were Sympetrum darwinianum, S. eroticum, S. pedomontanum, S. infuscatum, Pantala flavoscens, Calopteryx atrata, and Orthetrum albistylum speciosum. The occurrence of P. muris metacercariae in dragonflies was nationwide with various infection rates. The metacercarial burden of P. muris in the surveyed areas was the highest in S. eroticum followed by S. darwinianum, S. pedomontanum, and C. atrata. The highest infection rate by P. muris metacercariae was found in S. darwinianum followed by S. eroticum. The metacercarial burden was particularly heavy in the dragonflies found in Hamyang-gun and Kosong-gun, Kyongsangnam-do. It is, therefore, likely that dragonflies play a significant role as the second intermediate host in the life cycle of P. muris in Korea.     

Proteomic analysis of the cambial region in juvenile Eucalyptus grandis at three ages

Recent advances in genomics and proteomics have provided an excellent opportunity to understand complex biological processes such as wood formation at the gene and protein levels. The aim of this work was to describe the proteins participating in the processes involved in juvenile wood formation by isolating proteins from the cambial region of Eucalyptus grandis, at three ages of growth (6-month-old seedlings, 3- and 6-year-old trees), and also to identify proteins differentially expressed. Using a 2-D-LC-MS/MS strategy we identified a total of 240 proteins, with 54 corresponding spots being present in at least two ages. Overall, nine proteins classified into the functional categories of metabolism, cellular processes, and macromolecular metabolism showed significant changes in expression. Proteins were classified into seven main functional categories, with metabolism representing 35.2% of the total proteins identified. The comparison of the reference maps showed not only differences in the expression pattern of individual proteins at each age, but also among isoforms. The results described in this paper provide a dynamic view of the proteins involved in the formation of juvenile wood in E. grandis.     

Proteomic analysis of differential protein expression in neuropathic pain and electroacupuncture treatment models

Acupuncture has long been used for pain relief. Although recent studies have shown that acupuncture can reduce neuropathic pain, the mechanism of this effect is not clear and little information is available regarding proteins that are involved in the development of neuropathic pain and the effects of acupuncture. We have developed an animal model for neuropathic pain using young adult male Sprague-Dawley rats. The model was confirmed by behavioral tests. Electroacupuncture (EA) treatment was applied to Zusanli (ST36) of neuropathic pain model to examine the analgesic effect of EA. The protein expression profile of the hypothalamus in both neuropathic pain and EA treatment models was analyzed using two-dimensional electrophoresis-based proteomics. We detected thirty-six proteins that were differentially expressed in the neuropathic pain model compared with normal rats and that restored to normal expression levels after EA treatment. Twenty-one of these proteins were identified in the MS-FiT database and are involved in a number of biological processes, including inflammation, enzyme metabolism and signal transduction. Potential applications of our results include the identification and characterization of signaling pathways involved in EA treatment and further exploration of the role of selected identified proteins in the animal model.     

Oxidative Damage in Rat Brain During Aging: Interplay Between Energy and Metabolic Key Target Proteins

Aging is characterized by a gradual and continuous loss of physiological functions and responses particularly marked in the central nervous system. Reactive oxygen species (ROS) can react with all major biological macromolecules such as carbohydrates, nucleic acids, lipids, and proteins. Since proteins are the major components of biological systems and regulate multiple cellular pathways, oxidative damage of key proteins are considered to be the principal molecular mechanisms leading to age-related deficits. Recent evidences support the notion that a decrease of energy metabolism in the brain contribute to neuronal loss and cognitive decline associated with aging. In the present study we identified selective protein targets which are oxidized in aged rats compared with adult rats. Most of the oxidatively modified proteins we found in the present study are key proteins involved in energy metabolism and ATP production. Oxidative modification of these proteins was associated with decreased enzyme activities. In addition, we also found decreased levels of thiol reducing system. Our study demonstrated that oxidative damage to specific proteins impairs energy metabolism and ATP production thus contributing to shift neuronal cells towards a more oxidized environment which ultimately might compromise multiple neuronal functions. These results further confirm that increased protein oxidation coupled with decreased reducing systems are characteristic hallmarks of aging and aging-related degenerative processes.     

Metabolic pathway analysis of S. pneumoniae: an in silico approach towards drug-design

The emergence of multidrug resistant varieties of Streptococcus pneumoniae (S. pneumoniae) has led to a search for novel drug targets. An in silico comparative analysis of metabolic pathways of the host Homo sapiens (H. sapiens) and the pathogen S. pneumoniae have been performed. Enzymes from the biochemical pathways of S. pneumoniae from the KEGG metabolic pathway database were compared with proteins from the host H. sapiens, by performing a BLASTp search against the non-redundant database restricted to the H. sapiens subset. The e-value threshold cutoff was set to 0.005. Enzymes, which do not show similarity to any of the host proteins, below this threshold, were filtered out as potential drug targets. Five pathways unique to the pathogen S. pneumoniae when compared to the host H. sapiens have been identified. Potential drug targets from these pathways could be useful for the discovery of broad-spectrum drugs. Potential drug targets were also identified from pathways related to lipid metabolism, carbohydrate metabolism, amino acid metabolism, energy metabolism, vitamin and cofactor biosynthetic pathways and nucleotide metabolism. Of the 161 distinct targets identified from these pathways, many are in various stages of progress at the Microbial Genome Database. However, 44 of the targets are new and can be considered for rational drug design. The study was successful in listing out potential drug targets from the S. pneumoniae proteome involved in vital aspects of the pathogen's metabolism, persistence, virulence and cell wall biosynthesis. This systematic evaluation of metabolic pathways of host and pathogen through reliable and conventional bioinformatics approach can be extended to other pathogens of clinical interest.     

LRRC4基因转染U251细胞的比较蛋白质组学分析

LRRC4是我室自主克隆的一个脑组织优势表达基因.前期研究结果表明,外源性LRRC4基因转染至U251细胞,可明显地抑制U251细胞的增殖、黏附、趋化和侵袭等生物学行为. 因此,LRRC4亦是一个脑胶质瘤抑制性基因.为了进一步了解LRRC4在胶质瘤发生发展中的调控作用,本研究采用荧光差异凝胶电泳（2D-DIGE）和质谱分析技术获得了LRRC4转染U251细胞的11个差异表达蛋白质,并用Western 印迹证实了U251细胞在转染LRRC4基因前后热休克蛋白27、stathmin 1和S100钙结合蛋白A11的差异表达变化. 这些差异蛋白质涉及细胞代谢、增殖、转录、信号转导等众多事件,表明LRRC4基因转染U251细胞后可能通过调控这些蛋白质的表达而参与细胞的增殖、黏附、趋化和侵袭等生物学过程.     

Large-scale analysis of phosphorylated proteins in maize leaf

Phosphorylation is an ubiquitous regulatory mechanism governing the activity, subcellular localization, and intermolecular interactions of proteins. To identify a broad range of phosphoproteins from Zea mays, we enriched phosphopeptides from Zea mays leaves using titanium dioxide microcolumns and then extensively fractionated and identified the phosphopeptides by mass spectrometry. A total of 165 unique phosphorylation sites with a putative role in biological processes were identified in 125 phosphoproteins. Most of these proteins are involved in metabolism, including carbohydrate and protein metabolism. We identified novel phosphorylation sites on translation initiation factors, splicing factors, nucleolar RNA helicases, and chromatin-remodeling proteins such as histone deacetylases. Intriguingly, we also identified phosphorylation sites on several proteins associated with photosynthesis, and we speculate that these sites may be involved in carbohydrate metabolism or electron transport. Among these phosphoproteins, phosphoenolpyruvate carboxylase and NADH: nitrate reductase (NR) which catalyzes the rate-limiting and regulated step in the pathway of inorganic nitrogen assimilation were identified. A conserved phosphorylation site was found in the cytochrome b5 heme-binding domain of NADH: nitrate reductase, suggesting that NADH: nitrate reductase is phosphorylated by the same protein kinase or highly related kinases. These data demonstrate that the pathways that regulate diverse processes in plants are major targets of phosphorylation.     

Characterization of protein-binding to the spinach chloroplast psbA mRNA 5' untranslated region.

RNA-binding proteins play a major role in regulating mRNA metabolism in chloroplasts. In this work we characterized two proteins, of 43 and 47 kDa, which bind to the spinach psbA mRNA 5' untranslated region (psbA encoding the D1 protein of photosystem II). The 43 kDa protein, which is present in the stroma and in membranes, co-sediments with a complex of 68S. It was purified, and the N-terminal sequence was determined. Upon homology search it was identified as the chloroplast homologue of the Escherichia coli ribosomal protein S1. The 47 kDa protein, which, in contrast with the 43 kDa protein, sediments with a small sedimentation coefficient, is only detected in the stromal fraction. It is soluble in an uncomplexed form. By deletion analysis, an element within the psbA mRNA 5' untranslated region was identified that is necessary but not sufficient for binding of stromal proteins. The 'central protein binding element' ranges from nucleotide -49 to -9 of the psbA mRNA 5' untranslated region. It comprises the Shine-Dalgarno-like GGAG motif and, 7 nucleotides upstream, an endonucleolytic cleavage site involved in psbA mRNA degradation in vitro . The mechanistic impacts of this region in relation to RNA-binding proteins are discussed.     

Kiwi protein inhibitor of pectin methylesterase amino-acid sequence and structural importance of two disulfide bridges.

A protein acting as a powerful inhibitor of plant pectin methylesterase was isolated from kiwi (Actinidia chinensis) fruit. The complete amino-acid sequence of the pectin methylesterase inhibitor (PMEI) was determined by direct protein analysis. The sequence comprises 152 amino-acid residues, accounting for a molecular mass of 16 277 Da. The far-UV CD spectrum indicated a predominant alpha-helix conformation in the secondary structure. The protein has five cysteine residues but neither tryptophan nor methionine. Analysis of fragments obtained after digestion of the protein alkylated without previous reduction identified two disulfide bridges connecting Cys9 with Cys18, and Cys74 with Cys114; Cys140 bears a free thiol group. A database search pointed out a similarity between PMEI and plant invertase inhibitors. In particular, the four Cys residues, which in PMEI are involved in the disulfide bridges, are conserved. This allows us to infer that also in the homologous proteins, whose primary structure was deduced only by cDNA sequencing, those cysteine residues are engaged in two disulfide bridges, and constitute a common structural motif. The comparison of the sequence of these inhibitors confirms the existence of a novel class of proteins with moderate but significant sequence conservation, comprising plant proteins acting as inhibitors of sugar metabolism enzymes, and probably involved in various steps of plant development.     

Proteomic identification of 3-nitrotyrosine-containing rat cardiac proteins: effects of biological aging

Proteomic techniques were used to identify cardiac proteins from whole heart homogenate and heart mitochondria of Fisher 344/Brown Norway F1 rats, which suffer protein nitration as a consequence of biological aging. Soluble proteins from young (5 mo old) and old (26 mo old) animals were separated by one- and two-dimensional gel electrophoresis. One- and two-dimensional Western blots with an anti-nitrotyrosine antibody show an age-related increase in the immunoresponse of a few specific proteins, which were identified by nanoelectrospray ionization-tandem mass spectrometry (NSI-MS/MS). Complementary proteins were immunoprecipitated with an immobilized anti-nitrotyrosine antibody followed by NSI-MS/MS analysis. A total of 48 proteins were putatively identified. Among the identified proteins were alpha-enolase, alpha-aldolase, desmin, aconitate hydratase, methylmalonate semialdehyde dehydrogenase, 3-ketoacyl-CoA thiolase, acetyl-CoA acetyltransferase, GAPDH, malate dehydrogenase, creatine kinase, electron-transfer flavoprotein, manganese-superoxide dismutase, F1-ATPase, and the voltage-dependent anion channel. Some contaminating blood proteins including transferrin and fibrinogen beta-chain precursor showed increased levels of nitration as well. MS/MS analysis located nitration at Y105 of the electron-transfer flavoprotein. Among the identified proteins, there are important enzymes responsible for energy production and metabolism as well as proteins involved in the structural integrity of the cells. Our results are consistent with age-dependent increased oxidative stress and with free radical-dependent damage of proteins. Possibly the oxidative modifications of the identified proteins contribute to the age-dependent degeneration and functional decline of heart proteins.