Functional elucidation of hypothetical proteins for their indispensable roles toward drug designing targets from Helicobacter pylori strain HPAG1

Helicobacter pylori is a flagellated and slow growing gram-negative bacterium that persistently infects about half of the entire world population. In present study, we examined the proteome of H. pylori strain HPAG1 for identification of key uncharacterized proteins toward their novel regulatory functions. The complete proteome of this strain consists of 1539 proteins, out of which 520 proteins are annotated as hypothetical. Based on the functional motifs in their primary sequences, we were able to classify 254 of these hypothetical proteins into 6 functional categories. Further, KEGG database was used to find the roles of these hypothetical proteins in several pathways and structural prediction was done by homology modeling methods. Thirty-three of these hypothetical proteins were found to have strong association in various pathways including signaling and defense mechanisms. We noted that 27 of these proteins are specific to H. pylori and can be selected for drug designing targets, based on their virulence and regulatory role. We were able to successfully model the 3D structures of three of these proteins: YP_626977.1, YP_626786.1, and YP_628146.1. The stability of these proteins was also validated using molecular dynamics simulations, and their possible role in the regulation of different pathways was explained. These novel annotations may contribute to the understanding of disease mechanism at molecular level and provide novel potential targets for designing new drugs against H. pylori strain HPAG1.     

Towards an understanding of wheat chloroplasts: a methodical investigation of thylakoid proteome

We utilized Percoll density gradient centrifugation to isolate and fractionate chloroplasts of Korean winter wheat cultivar cv. Kumgang (Triticum aestivum L.). The resulting protein fractions were separated by one dimensional polyacrylamide gel electrophoresis (1D-PAGE) coupled with LTQ-FTICR mass spectrometry. This enabled us to detect and identify 767 unique proteins. Our findings represent the most comprehensive exploration of a proteome to date. Based on annotation information from the UniProtKB/Swiss-Prot database and our analyses via WoLF PSORT and PSORT, these proteins are localized in the chloroplast (607 proteins), chloroplast stroma (145), thylakoid membrane (342), lumens (163), and integral membranes (166). In all, 67% were confirmed as chloroplast thylakoid proteins. Although nearly complete protein coverage (89% proteins) has been accomplished for the key chloroplast pathways in wheat, such as for photosynthesis, many other proteins are involved in regulating carbon metabolism. The identified proteins were assigned to 103 functional categories according to a classification system developed by the iProClass database and provided through Protein Information Resources. Those functions include electron transport, energy, cellular organization and biogenesis, transport, stress responses, and other metabolic processes. Whereas most of these proteins are associated with known complexes and metabolic pathways, about 13% of the proteins have unknown functions. The chloroplast proteome contains many proteins that are localized to the thylakoids but as yet have no known function. We propose that some of these familiar proteins participate in the photosynthetic pathway. Thus, our new and comprehensive protein profile may provide clues for better understanding that photosynthetic process in wheat.     

小柴旦盐湖脱硫棒状菌属的环境适应性机制分析

【目的】青藏高原小柴旦盐湖富含硫酸盐卤水,宏基因组学分析揭示该生境蕴藏着丰富的具有耐盐、固碳和脱硫功能的微生物。本研究拟通过生物信息学分析揭示潜在的固碳脱硫微生物脱硫棒状菌(Desulfotignum)的代谢多样性和环境适应性机制。【方法】利用宏基因组分箱分析和公共数据库下载获得小柴旦盐湖脱硫棒状菌属的基因组,通过文献跟踪和16S rRNA基因数据库检索揭示脱硫棒状菌的全球生境分布,基于基因组分类数据库(genome taxonomy database, GTDB)中120个细菌标记蛋白的系统发育树对脱硫棒状菌属的亚群进行分类,通过重构脱硫棒状菌属不同亚群的生理代谢潜能以及基因组比较分析来解析其环境适应机制和代谢多样性。【结果】脱硫棒状菌属全球分布广泛且主要栖息在高盐生境。小柴旦盐湖沉积物中共得到了9个脱硫棒状菌基因组,结合公共数据库中2个基因组,根据基因组系统发育分析、平均核苷酸一致性(average nucleotide identity,ANI)和平均氨基酸一致性(average amino acid identity, AAI)分析将这11个脱硫棒状菌基因组分为了2个亚群(G1...     

Multiple Horizontal Gene Transfer Events and Domain Fusions Have Created Novel Regulatory and Metabolic Networks in the Oomycete Genome

Complex enzymes with multiple catalytic activities are hypothesized to have evolved from more primitive precursors. Global analysis of the Phytophthora sojae genome using conservative criteria for evaluation of complex proteins identified 273 novel multifunctional proteins that were also conserved in P. ramorum. Each of these proteins contains combinations of protein motifs that are not present in bacterial, plant, animal, or fungal genomes. A subset of these proteins were also identified in the two diatom genomes, but the majority of these proteins have formed after the split between diatoms and oomycetes. Documentation of multiple cases of domain fusions that are common to both oomycetes and diatom genomes lends additional support for the hypothesis that oomycetes and diatoms are monophyletic. Bifunctional proteins that catalyze two steps in a metabolic pathway can be used to infer the interaction of orthologous proteins that exist as separate entities in other genomes. We postulated that the novel multifunctional proteins of oomycetes could function as potential Rosetta Stones to identify interacting proteins of conserved metabolic and regulatory networks in other eukaryotic genomes. However ortholog analysis of each domain within our set of 273 multifunctional proteins against 39 sequenced bacterial and eukaryotic genomes, identified only 18 candidate Rosetta Stone proteins. Thus the majority of multifunctional proteins are not Rosetta Stones, but they may nonetheless be useful in identifying novel metabolic and regulatory networks in oomycetes. Phylogenetic analysis of all the enzymes in three pathways with one or more novel multifunctional proteins was conducted to determine the probable origins of individual enzymes. These analyses revealed multiple examples of horizontal transfer from both bacterial genomes and the photosynthetic endosymbiont in the ancestral genome of Stramenopiles. The complexity of the phylogenetic origins of these metabolic pathways and the paucity of Rosetta Stones relative to the total number of multifunctional proteins suggests that the proteome of oomycetes has few features in common with other Kingdoms.     

Structural characterization of CA1462, the <Emphasis Type="Italic">Candida albicans</Emphasis> thiamine pyrophosphokinase

Background  

In search of new antifungal targets of potential interest for pharmaceutical companies, we initiated a comparative genomics study to identify the most promising protein-coding genes in fungal genomes. One criterion was the protein sequence conservation between reference pathogenic genomes. A second criterion was that the corresponding gene in Saccharomyces cerevisiae should be essential. Since thiamine pyrophosphate is an essential product involved in a variety of metabolic pathways, proteins responsible for its production satisfied these two criteria.     

Reconstruction of metabolic pathways for the cattle genome

Background  

Metabolic reconstruction of microbial, plant and animal genomes is a necessary step toward understanding the evolutionary origins of metabolism and species-specific adaptive traits. The aims of this study were to reconstruct conserved metabolic pathways in the cattle genome and to identify metabolic pathways with missing genes and proteins. The MetaCyc database and PathwayTools software suite were chosen for this work because they are widely used and easy to implement.     

In silico quest for putative drug targets in <Emphasis Type="Italic">Helicobacter pylori</Emphasis> HPAG1: molecular modeling of candidate enzymes from lipopolysaccharide biosynthesis pathway

Aimed at identification and structural characterization of novel putative therapeutic targets in H. pylori, the etiological agent of numerous gastrointestinal diseases including peptic ulcer and gastric cancer, the present study comprised of three phases. First, through subtractive analysis of metabolic pathways of Helicobacter pylori HPAG1 and human, as documented in the KEGG database, 11 pathogen-specific pathways were identified. Next, all proteins involved in these pathogen-specific pathways were scrutinized in search of promising targets and the study yielded 25 candidate target proteins that are likely to be essential for the pathogen viability, but have no homolog in human. The lipopolysaccharide (LPS) biosynthesis pathway was found to be the largest contributor (nine proteins) to this list of candidate proteins. Considering the importance of LPS in H. pylori virulence, 3D structural models of three predicted target enzymes of this pathway, namely 2-dehydro-3-deoxy-phosphooctonate aldolase, UDP-3-O-[3-hydroxymyristoyl] N-acetylglucosamine deacetylase and Phosphoheptose isomerase, were then built up using the homology modeling approaches. Binding site analysis and docking of the known biological substrate PEP to 2-dehydro-3-deoxyphosphooctonate aldolase revealed the potential binding pocket present in the single monomeric form of the enzyme and identified 11 amino acid residues that might play the key roles in this protein-ligand interaction.     

Evolution of the 12 kDa FK506-binding protein gene

Background information. The FKBPs (FK506‐binding proteins) belong to a ubiquitous family of proteins that are found in a wide range of taxonomic groups. These proteins participate in a variety of pathways, including protein folding, down‐regulation of T‐cell activation and inhibition of cell‐cycle progression. Results. A cDNA encoding the 12 kDa FKBP gene orthologue (FKBP12) in Bombyx mori was been isolated from both Bm‐5 cultured cells and silk‐gland tissue. Using the FKBP12 cDNA in combination with the B. mori 6× whole‐genome shotgun database, we were able to identify the FKBP12 gene, as well as the positions of its intron—exon junctions. Conclusions. FKBP12 exon sizes and intronic positions are highly conserved among FKBP12 orthologues in 24 diverse genomes. Comparison of 41 FKBP12 genes revealed several intronic insertion and deletion events throughout evolution. In addition, paralogous FKBP12 isoforms were identified in all 12 vertebrate genomes. Both structural and phylogenetics analyses suggest that the isoforms may be evolving independently, possibly due to the distinct functional roles played by each paralogue.     

Genomic insights into versatile lifestyle of three new bacterial candidate phyla

Metagenomic explorations of the Earth’s biosphere enable the discovery of previously unknown bacterial lineages of phylogenetic and ecological significance. Here, we retrieved 11 metagenomic-assembled genomes (MAGs) affiliated to three new monophyletic bacterial lineages from the seawater of the Yap Trench. Phylogenomic analysis revealed that each lineage is a new bacterial candidate phylum, subsequently named Candidatus Qinglongiota, Candidatus Heilongiota, and Candidatus Canglongiota. Metabolic reconstruction of genomes from the three phyla suggested that they adopt a versatile lifestyle, with the potential to utilize various types of sugars, proteins, and/or short-chain fatty acids through anaerobic pathways. This was further confirmed by a global distribution map of the three phyla, indicating a preference for oxygen-limited or particle-attached niches, such as anoxic sedimentary environments. Of note, Candidatus Canglongiota genomes harbor genes for the complete Wood- Ljungdahl pathway and sulfate reduction that are similar to those identified in some sulfate-reducing bacteria. Evolutionary analysis indicated that gene gain and loss events, and horizontal gene transfer (HGT) play important roles in shaping the genomic and metabolic features of the three new phyla. This study presents the genomic insight into the ecology, metabolism, and evolution of three new phyla, which broadens the phylum-level diversity within the domain Bacteria.

     

Targeting metabolic pathways proteins of Orientia tsutsugamushi using combined hierarchical approach to combat scrub typhus

Orientia tsutsugamushi (Ott) is a causative agent of chigger‐borne zoonosis, scrub typhus which is life threatening and highly pervasive illness in humans. In this report, we have mined and classified the proteins involved in pathways unique to Ott by using high‐throughput computational techniques. The 12 metabolic pathways were found to be unique to the pathogen. Forty‐six proteins were reported to be essential for the pathogen's survival and non‐homologous to the humans. The proteins were categorized into different classes, ie, enzymes, transporters, DNA‐binding, secretory, and outer membrane proteins. Further, in silico analysis of 46 proteins showed that 25 proteins were suitable therapeutic targets with known druggable properties. The structural modeling of B3CSG3 (MurA) protein was carried out and catalytic site essential for its functioning was analyzed. Virtual screening of chemical compounds was performed against modeled structure. The docking study by AutodockVina reported compound from PubChem with CID: 16036947 as best and potential inhibitor by means of docking score and binding affinity. The reliability and stability of the MurA‐16036947 complex were confirmed with molecular dynamics simulation. The report will provide insight to understand the mechanism of pathogenesis of Ott and instigate the development of effective treatment strategies against this disease.     

Pollen wall development: the associated enzymes and metabolic pathways

Pollen grains are surrounded by a sculpted wall, which protects male gametophytes from various environmental stresses and microbial attacks, and also facilitates pollination. Pollen wall development requires lipid and polysaccharide metabolism, and some key genes and proteins that participate in these processes have recently been identified. Here, we summarise the genes and describe their functions during pollen wall development via several metabolic pathways. A working model involving substances and catalytic enzyme reactions that occur during pollen development is also presented. This model provides information on the complete process of pollen wall development with respect to metabolic pathways.     

Automatic detection of conserved gene clusters in multiple genomes by graph comparison and P-quasi grouping

We previously reported two graph algorithms for analysis of genomic information: a graph comparison algorithm to detect locally similar regions called correlated clusters and an algorithm to find a graph feature called P-quasi complete linkage. Based on these algorithms we have developed an automatic procedure to detect conserved gene clusters and align orthologous gene orders in multiple genomes. In the first step, the graph comparison is applied to pairwise genome comparisons, where the genome is considered as a one-dimensionally connected graph with genes as its nodes, and correlated clusters of genes that share sequence similarities are identified. In the next step, the P-quasi complete linkage analysis is applied to grouping of related clusters and conserved gene clusters in multiple genomes are identified. In the last step, orthologous relations of genes are established among each conserved cluster. We analyzed 17 completely sequenced microbial genomes and obtained 2313 clusters when the completeness parameter P was 40%. About one quarter contained at least two genes that appeared in the metabolic and regulatory pathways in the KEGG database. This collection of conserved gene clusters is used to refine and augment ortholog group tables in KEGG and also to define ortholog identifiers as an extension of EC numbers.     

Nuclear and chloroplast transformation inChlamydomonas reinhardtii: strategies for genetic manipulation and gene expression

Transformation of the nuclear, chloroplast, and mitochondrial genomes can now be accomplished inChlamydomonas reinhardtii. Many biosynthetic pathways are carried out in the chloroplast, and efforts to manipulate these pathways will require that gene products be directed to this compartment. Chloroplast proteins are encoded in either the chloroplast or nuclear genome. In the latter case they are synthesized in the cytoplasm and imported post-translationally into the chloroplast. Thus, strategies for expressing foreign genes or overexpressing endogenous genes whose products reside in the chloroplast could involve either genome. This paper reviews the present status of transformation methodology for the nuclear and chloroplast genomes inChlamydomonas. Considerations for expressing gene products in the chloroplast are discussed. Experimental evidence for homologous recombination during transformation of the nuclear genome is presented.     

Expansion of the <Emphasis Type="Italic">Ago</Emphasis> gene family in the teleost clade

AGO proteins are universal effectors of eukaryotic small RNA-directed regulatory pathways. In this study, we used a comparative genomics approach to explore the AGO sub-family in the teleost clade. We identified five Ago homologues in teleost genomes, one more than encoded in other vertebrate clades. The additional teleost homologue was preserved most likely due to the differential retention of regulatory elements following the fish-specific genome duplication event that occurred approximately 350 million years ago. Analysis of all five Ago genomic loci in teleosts revealed that orthologues contain specific, conserved sequence elements in non-coding regions indicating that the teleost Ago paralogues are differentially regulated. This was supported by qRT-PCR analysis that showed differential expression of the zebrafish homologues across development and between adult tissues indicating stage and tissue-specific function of individual AGO proteins. Multiple sequence alignments showed not only that all teleost homologues possess critical residues for AGO function, but also that teleost homologues contain multiple orthologue-specific features, indicative of structural diversification. Notably, these are retained throughout the vertebrate lineage arguing these may be important for orthologue-specific functions.     

Molecular genetics of nucleotide sugar interconversion pathways in plants

Nucleotide sugar interconversion pathways represent a series of enzymatic reactions by which plants synthesize activated monosaccharides for the incorporation into cell wall material. Although biochemical aspects of these metabolic pathways are reasonably well understood, the identification and characterization of genes encoding nucleotide sugar interconversion enzymes is still in its infancy. Arabidopsis mutants defective in the activation and interconversion of specific monosaccharides have recently become available, and several genes in these pathways have been cloned and characterized. The sequence determination of the entire Arabidopsis genome offers a unique opportunity to identify candidate genes encoding nucleotide sugar interconversion enzymes via sequence comparisons to bacterial homologues. An evaluation of the Arabidopsis databases suggests that the majority of these enzymes are encoded by small gene families, and that most of these coding regions are transcribed. Although most of the putative proteins are predicted to be soluble, others contain N-terminal extensions encompassing a transmembrane domain. This suggests that some nucleotide sugar interconversion enzymes are targeted to an endomembrane system, such as the Golgi apparatus, where they may co-localize with glycosyltransferases in cell wall synthesis. The functions of the predicted coding regions can most likely be established via reverse genetic approaches and the expression of proteins in heterologous systems. The genetic characterization of nucleotide sugar interconversion enzymes has the potential to understand the regulation of these complex metabolic pathways and to permit the modification of cell wall material by changing the availability of monosaccharide precursors.     

CoPS: Comprehensive Peptide Signature database

We present the development of a Comprehensive database of 12 076 invariant Peptide Signatures (CoPS) derived from 52 bacterial genomes with a minimum occurrence in at least seven organisms. These peptides were observed in functionally similar proteins and are distributed over nearly 1250 different functional proteins. The database provides function, structure and occurrence in biochemical pathways of the proteins containing these signature peptides. It houses additional information on the signature peptides, such as identical match in other motif/pattern (e.g. PROSITE, BLOCKS, PRINTS and Pfam) databases and the database of interacting proteins, human proteome and mutation effect on these signature peptides. There is a wide applicability of this database in the identification of critical functional residues in proteins. The database also facilitates the identification of folding nucleus/structural determinants in proteins and functional assignment to yet unknown proteins. We demonstrate functional assignment to 2605 hypothetical proteins in bacterial genomes and 112 unknown proteins in human using this database. AVAILABILITY: The database can be freely accessed through the following URL: http://203.195.151.46/copsv2/index.html or http://203.90.127.70/copsv2/index.html     

Inorganic pyrophosphatase in uncultivable hemotrophic mycoplasmas: identification and properties of the enzyme from <Emphasis Type="Italic">Mycoplasma suis</Emphasis>

Background  

Mycoplasma suis belongs to a group of highly specialized hemotrophic bacteria that attach to the surface of host erythrocytes. Hemotrophic mycoplasmas are uncultivable and the genomes are not sequenced so far. Therefore, there is a need for the clarification of essential metabolic pathways which could be crucial barriers for the establishment of an in vitro cultivation system for these veterinary significant bacteria.     

Metabolic reconstruction of the near complete microbiome of the model sponge Ianthella basta

Many marine sponges host highly diverse microbiomes that contribute to various aspects of host health. Although the putative function of individual groups of sponge symbionts has been increasingly described, the extreme diversity has generally precluded in-depth characterization of entire microbiomes, including identification of syntrophic partnerships. The Indo-Pacific sponge Ianthella basta is emerging as a model organism for symbiosis research, hosting only three dominant symbionts: a Thaumarchaeotum, a Gammaproteobacterium, and an Alphaproteobacterium and a range of other low abundance or transitory taxa. Here, we retrieved metagenome assembled genomes (MAGs) representing >90% of I. basta's microbial community, facilitating the metabolic reconstruction of the sponge's near complete microbiome. Through this analysis, we identified metabolic complementarity between microbes, including vitamin sharing, described the importance of low abundance symbionts, and characterized a novel microbe–host attachment mechanism in the Alphaproteobacterium. We further identified putative viral sequences, highlighting the role viruses can play in maintaining symbioses in I. basta through the horizontal transfer of eukaryotic-like proteins, and complemented this data with metaproteomics to identify active metabolic pathways in bacteria, archaea, and viruses. This data provide the framework to adopt I. basta as a model organism for studying host–microbe interactions and provide a basis for in-depth physiological experiments.     

Higher Gene Duplicabilities for Metabolic Proteins Than for Nonmetabolic Proteins in Yeast and <Emphasis Type="Italic">E. coli</Emphasis>

Although the evolutionary significance of gene duplication has long been appreciated, it remains unclear what factors determine gene duplicability. In this study we investigated whether metabolism is an important determinant of gene duplicability because cellular metabolism is crucial for the survival and reproduction of an organism. Using genomic data and metabolic pathway data from the yeast (Saccharomyces cerevisiae) and Escherichia coli, we found that metabolic proteins indeed tend to have higher gene duplicability than nonmetabolic proteins. Moreover, a detailed analysis of metabolic pathways in these two organisms revealed that genes in the central metabolic pathways and the catabolic pathways have, on average, higher gene duplicability than do other genes and that most genes in anabolic pathways are single-copy genes.Reviewing Editor: Dr. Rüdiger Cerff