Computational structural and functional analysis of hypothetical proteins of Staphylococcus aureus

Genome sequencing projects has led to an explosion of large amount of gene products in which many are of hypothetical proteins with unknown function. Analyzing and annotating the functions of hypothetical proteins is important in Staphylococcus aureus which is a pathogenic bacterium that cause multiple types of diseases by infecting various sites in humans and animals. In this study, ten hypothetical proteins of Staphylococcus aureus were retrieved from NCBI and analyzed for their structural and functional characteristics by using various bioinformatics tools and databases. The analysis revealed that some of them possessed functionally important domains and families and protein-protein interacting partners which were ABC transporter ATP-binding protein, Multiple Antibiotic Resistance (MAR) family, export proteins, Helix-Turn-helix domains, arsenate reductase, elongation factor, ribosomal proteins, Cysteine protease precursor, Type-I restriction endonuclease enzyme and plasmid recombination enzyme which might have the same functions in hypothetical proteins. The structural prediction of those proteins and binding sites prediction have been done which would be useful in docking studies for aiding in the drug discovery.     

LC-MS/MS based proteomic analysis and functional inference of hypothetical proteins in Desulfovibrio vulgaris

High efficiency capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to examine the proteins extracted from Desulfovibrio vulgaris cells across six treatment conditions. While our previous study provided a proteomic overview of the cellular metabolism based on proteins with known functions [W. Zhang, M.A. Gritsenko, R.J. Moore, D.E. Culley, L. Nie, K. Petritis, E.F. Strittmatter, D.G. Camp II, R.D. Smith, F.J. Brockman, A proteomic view of the metabolism in Desulfovibrio vulgaris determined by liquid chromatography coupled with tandem mass spectrometry, Proteomics 6 (2006) 4286-4299], this study describes the global detection and functional inference for hypothetical D. vulgaris proteins. Using criteria that a given peptide of a protein is identified from at least two out of three independent LC-MS/MS measurements and that for any protein at least two different peptides are identified among the three measurements, 129 open reading frames (ORFs) originally annotated as hypothetical proteins were found to encode expressed proteins. Functional inference for the conserved hypothetical proteins was performed by a combination of several non-homology based methods: genomic context analysis, phylogenomic profiling, and analysis of a combination of experimental information, including peptide detection in cells grown under specific culture conditions and cellular location of the proteins. Using this approach we were able to assign possible functions to 20 conserved hypothetical proteins. This study demonstrated that a combination of proteomics and bioinformatics methodologies can provide verification of the expression of hypothetical proteins and improve genome annotation.     

Functional prediction of hypothetical proteins in human adenoviruses

Assigning functional information to hypothetical proteins in virus genomes is crucial for gaining insight into their proteomes. Human adenoviruses are medium sized viruses that cause a range of diseases. Their genomes possess proteins with uncharacterized function known as hypothetical proteins. Using a wide range of protein function prediction servers, functional information was obtained about these hypothetical proteins. A comparison of functional information obtained from these servers revealed that some of them produced functional information, while others provided little functional information about these human adenovirus hypothetical proteins. The PFP, ESG, PSIPRED, 3d2GO, and ProtFun servers produced the most functional information regarding these hypothetical proteins.     

Novel identification of expressed genes and functional classification of hypothetical proteins from Neisseria meningitidis serogroup A

To implement the 2-DE database of serogroup A Neisseria meningitidis (MenA) and improve its potential of investigation in bacterial biology, cell extracts were separated by tricine-SDS-PAGE and 131 novel proteins were identified by microLC-ESI-IT-MS/MS. These identifications extended to 404, the number of MenA gene expression products characterized at the proteome level, approximately covering 20% of the total ORFs predicted from genome sequence. This technical approach was particularly useful in ascertaining expression of ribosomal as well as hypothetical proteins. Particular attention was paid to functional characterization of hypothetical proteins by means of software analyses and database searches.     

Evolutionary conservation and disease gene association of the human genes composing pseudogenes

Pseudogenes, the 'genomic fossils' present portrayal of evolutionary history of human genome. The human genes configuring pseudogenes are also now coming forth as important resources in the study of human protein evolution. In this communication, we explored evolutionary conservation of the genes forming pseudogenes over the genes lacking any pseudogene and delving deeper, we probed an evolutionary rate difference between the disease genes in the two groups. We illustrated this differential evolutionary pattern by gene expressivity, number of regulatory miRNA targeting per gene, abundance of protein complex forming genes and lesser percentage of protein intrinsic disorderness. Furthermore, pseudogenes are observed to harbor sequence variations, over their entirety, those become degenerative disease-causing mutations though the disease involvement of their progenitors is still unexplored. Here, we unveiled an immense association of disease genes in the genes casting pseudogenes in human. We interpreted the issue by disease associated miRNA targeting, genes containing polymorphisms in miRNA target sites, abundance of genes having disease causing non-synonymous mutations, disease gene specific network properties, presence of genes having repeat regions, affluence of dosage sensitive genes and the presence of intrinsically unstructured protein regions.     

Towards a two‐dimensional proteomic reference map of Bradyrhizobium japonicum CPAC 15: Spotlighting “hypothetical proteins”

The economic and ecological importance of the symbiosis of soybean with Bradyrhizobium japonicum strains is significant in several countries, particularly Brazil; however, up to now, only one complete and a draft genome for this species are available. In this study, we have obtained a proteomic reference map of B. japonicum strain CPAC 15 (=SEMIA 5079) – used in commercial inoculants for application to soybean crops in Brazil – grown under in vitro conditions. CPAC 15 belongs to the same serogroup as strain USDA 123, and both are known as the soybean bradyrhizobial strains with highest competitive and saprophytic known so far. To increase the precision of the proteomic map, we compared whole‐cell 2‐D protein gel‐electrophoresis profiles of CPAC 15 and of two related strains. One‐hundred and seventy representative spots, selected from the three profiles, were analyzed by MS. In total, 148 spots were successfully identified as cytoplasmic and periplasmic proteins belonging to diverse metabolic pathways, several of them related to the saprophytic and competitive abilities of CPAC 15. We attributed probable functions to 26 hypothetical proteins, including those involved in polyhydroxybutyrate metabolism, β‐lactamase, stress responses and aromatic compound degradation, all with high probability of being related to the saprophytic ability of CPAC 15. In addition, by providing valuable information about expressed proteins in B. japonicum in vitro, our results emphasize the importance of accurate functional annotation of uncharacterized expressed proteins, improving considerably our understanding of the legume–rhizobia symbiosis.     

The crystal structure of hypothetical protein MTH1491 from Methanobacterium thermoautotrophicum

As part of our structural proteomics initiative, we have determined the crystal structure of MTH1491, a previously uncharacterized hypothetical protein from Methanobacterium thermoautotrophicum. MTH1491 is one of numerous structural genomics targets selected in a genome-wide survey of uncharacterized proteins. It belongs to a family of proteins whose biological function is not known. The crystal structure of MTH1491, the first structure for this family of proteins, consists of an overall five-stranded parallel beta-sheet with strand order 51234 and flanking helices. The oligomeric form of this molecule is a trimer as seen from both crystal contacts and gel filtration studies. Analysis revealed that the structure of MTH1491 is similar to that of dehydrogenases, amidohydrolases, and oxidoreductases. Using a combination of sequence and structural analyses, we showed that MTH1491 does not belong to either the dehydrogenase or the amidohydrolase superfamilies of proteins.     

Silk moth chorion pseudogenes: Hallmarks of genomic evolution by sequence duplication and gene conversion

The part of the genetic locus of the domesticated silk moth,Bombyx mori, in which high cysteine (Hc) chorion genes of late developmental specificity reside contains regions encompassing genelike sequences which exhibit properties distinct from those of functional Hc genes. One of these regions has been characterized and shown to contain a chorion pseudogene, ψHcB.15, which shares pronounced similarities with a transcribed chorion pseudogene, ψHcB.12/13, which was characterized previously. Both pseudogenes are homologous to HcB chorion genes but bear multiple single nucleotide substitutions and short segmental mutations (insertions and deletions) which introduce translational frame shifts and termination codons in the coding regions. Structural characteristics unique to the two pseudogenes suggest that ψHcB.15 was generated first from a functional HcB gene and gave rise subsequently to ψHcB 12/13 as a result of a sequence duplication event. The two pseudogenes can be distinguished from each other by the presence of distinct regions of similarity to the consensus sequence of functional HcB genes which appear to have arisen from gene-conversionmediated correctional events. These findings lend support to the hypothesis that chorion pseudogene sequences represent reservoirs of genetic information that participates in the evolution of the chorion locus rather than relics of inactivated genes passively awaiting extinction. Presented at the NATO Advanced Research Workshop onGenome Organization and Evolution, Spetsai, Greece, 16–22 September 1992     

Cloning and characterization of three hypothetical secretion chaperone proteins from Xanthomonas axonopodis pv. citri

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker in plantations around the world and is of particular significance in Brazil where its incidence has risen exponentially over the past decade. Approximately one third of the predicted Xac open reading frames show no homology, or homology with very low score with that of known sequences. It is believed that Xac utilizes secretion systems to transfer virulence proteins into susceptible eukaryotic cells. This process is assisted by secretion chaperones that maintain virulence proteins partly or completely unfolded during translocation. We have cloned three of these hypothetical secretion chaperones: XAC0419 and XAC1346 from type III secretion system (TTSS) and XACb0033 from type IV secretion system (TFSS). All proteins were cloned in a pET23a vector (Novagen), expressed at 37 degrees C using a BL21(DE3)pLysS Escherichia coli strain and purified by ion exchange and gel-filtration chromatographic methods. Pure proteins were characterized using spectroscopic measurements: circular dichroism, and both static and lifetime emission fluorescence in the case of XACb0033. The analyzed proteins are stable at elevated temperatures (up to 65 degrees C) and exhibit alpha-helix content from approximately 30% (XACb003) to approximately 87% (XAC1346). XACb0033 exhibits lifetimes in the fluorescence experiments that indicate different neighborhoods for its tryptophan residues. These chaperones have the characteristics of TTSS and TFSS: all are small, with a high alpha-helix content, and without ATP-binding or ATP-hydrolyzing activity.     

Functional assignment to hypothetical proteins in Orientia tsutsugamushistrain Ikeda

Orientia tsutsugamushi(O. tsutsugamushi) is an intracellular bacterial pathogen which causes zoonosis scrub typhus in humans. Genome of O. tsutsugamushi strain Ikeda contains 214 hypothetical proteins (HPs) which is nearly 20% of the total proteins. Domain and family based functional analysis of HPs results in the annotation of 44 hypothetical proteins. The annotated HPs were classified in to five main classes namely, gene expression and regulation, transport, metabolism, cell signaling and proteolysis. Thus, computational analysis of HPs helps to understand their putative roles in various biological and cellular processes, including pathogenesis for further consideration as potential therapeutic targets.     

Genome wide survey and molecular modeling of hypothetical proteins containing 2Fe-2S and FMN binding domains suggests Rieske Dioxygenase Activity highlighting their potential roles in bioremediation

‘Conserved hypothetical’ proteins pose a challenge not just for functional genomics, but also to biology in general. As long as there are hundreds of conserved proteins with unknown function in model organisms such as Escherichia coli, Bacillus subtilis or Saccharomyces cerevisiae, any discussion towards a ‘complete’ understanding of these biological systems will remain a wishful thinking. Insilico approaches exhibit great promise towards attempts that enable appreciating the plausible roles of these hypothetical proteins. Among the majority of genomic proteins, two-thirds in unicellular organisms and more than 80% in metazoa, are multi-domain proteins, created as a result of gene duplication events. Aromatic ring-hydroxylating dioxygenases, also called Rieske dioxygenases (RDOs), are class of multi-domain proteins that catalyze the initial step in microbial aerobic degradation of many aromatic compounds. Investigations here address the computational characterization of hypothetical proteins containing Ferredoxin and Flavodoxin signatures. Consensus sequence of each class of oxidoreductase was obtained by a phylogenetic analysis, involving clustering methods based on evolutionary relationship. A synthetic sequence was developed by combining the consensus, which was used as the basis to search for their homologs via BLAST. The exercise yielded 129 multidomain hypothetical proteins containing both 2Fe-2S (Ferredoxin) and FNR (Flavodoxin) domains. In the current study, 17 proteins with N-terminus FNR domain and C-terminus 2Fe-2S domain are characterized, through homology modelling and docking exercises which suggest dioxygenase activity indicate their plausible roles in degradation of aromatic moieties.     

Identifying kinetically stable proteins with capillary electrophoresis

Unlike most proteins, which are in equilibrium with partially and globally unfolded conformations, kinetically stable proteins (KSPs) are trapped in their native conformations and are often resistant to harsh environment. Based on a previous correlation between kinetic stability (KS) and a protein's resistance to sodium dodecyl sulfate (SDS), we show here a simple method to identify KSPs by SDS‐capillary electrophoresis (CE). Control non‐KSPs were fully denatured by SDS and formed protein:SDS complexes that exhibited similar mobility in CE. In contrast, KSPs bound fewer SDS molecules, and showed a very different migration time and peak pattern in CE, thereby providing some insight about the structural heterogeneity of SDS:protein complexes and the relative KS of the various proteins.     

The size distribution of insertions and deletions in human and rodent pseudogenes suggests the logarithmic gap penalty for sequence alignment

The size distributions of deletions, insertions, and indels (i.e., insertions or deletions) were studied, using 78 human processed pseudogenes and other published data sets. The following results were obtained: (1) Deletions occur more frequently than do insertions in sequence evolution; none of the pseudogenes studied shows significantly more insertions than deletions. (2) Empirically, the size distributions of deletions, insertions, and indels can be described well by a power law, i.e., f _k = Ck ^–b , where f _k is the frequency of deletion, insertion, or indel with gap length k, b is the power parameter, and C is the normalization factor. (3) The estimates of b for deletions and insertions from the same data set are approximately equal to each other, indicating that the size distributions for deletions and insertions are approximately identical. (4) The variation in the estimates of b among various data sets is small, indicating that the effect of local structure exists but only plays a secondary role in the size distribution of deletions and insertions. (5) The linear gap penalty, which is most commonly used in sequence alignment, is not supported by our analysis; rather, the power law for the size distribution of indels suggests that an appropriate gap penalty is w _k = a + b ln k, where a is the gap creation cost and blnk is the gap extension cost. (6) The higher frequency of deletion over insertion suggests that the gap creation cost of insertion (a _i ) should be larger than that of deletion (a _d ); that is, a _i – a _d = In R, where R is the frequency ratio of deletions to insertions. Correspondence to: W.-H. Li     

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.     

Comprehensive validation of a diagnostic strategy for sequencing genes with one or multiple pseudogenes using pseudoxanthoma elasticum as a model

Pseudogenes are frequently encountered noncoding sequences with a high sequence similarity to their protein-coding paralogue. For this reason, their presence is often considered troublesome in molecular diagnostics. In pseudoxanthoma elasticum(PXE), a disease predominantly caused by mutations in ATPbinding cassette family C member 6(ABCC6), the presence of two pseudogenes complicates the analysis of sequence data. With whole-exome sequencing(WES) becoming the standard of care in molecular diagnostics, we wanted to evaluate whether this technique is as reliable as gene-specific targeted enrichment analysis for the analysis of ABCC6. We established a PCR-based targeted enrichment and next-generation sequencing testing approach and demonstrated that the ABCC6-specific enrichment combined with the applied mapping algorithm overcomes the complication of ABCC6 pseudogene aspecificities, contrary to WES. We propose a time-and cost-efficient diagnostic strategy for comprehensive and accurate molecular genetic testing of PXE, which is highly automatable.     

Unusual evolutionary conservation of 5S rRNA pseudogenes inAspergillus nidulans: Similarity of the DNA sequence associated with the pseudogenes with the mouse immunoglobulin switch region

Summary AllAspergillus nidulans 5S rRNA pseudogenes known so far are the result of integration of an approx. 0.2-kbp-long DNA sequence into the 5S rRNA genes. This sequence, called block C, is present in at least five copies in theA. nidulans genome and seems to be associated either with 5S rRNA genes or pseudogenes. In contrast to the 78% sequence conservation of the C-block in pseudogenes, the truncated 5 halves of the pseudogenes are very highly conserved (96.9–100%). We postulate that the 5S rRNA pseudogenes are still a subject of concerted evolution. The C-block sequence shows similarity to the switch region of the mouse heavy chain immunoglobulin gene. A characteristic motif GGGTGAG is repeated several times in both sequences; the sequence conservation is 63%.     

Search for fucose binding domains in recently sequenced hypothetical proteins using molecular modeling techniques and structural analysis

The crystal structure of a fucose-binding lectin from the bacteria Pseudomonas aeruginosa in complex with α-L-fucose has been recently determined. It is a tetramer; each monomer displays a nine-stranded, antiparallel, β-sandwiched arrangement and contains two calcium ions that mediate the binding of fucose in a recognition mode unique among protein-carbohydrate interactions. In search of this type of unique interactions in other newly discovered protein sequences, we have used molecular modeling techniques to predict and analyze the 3-D structures of some proteins, which exhibited reasonable degree of homology with the amino acid sequence of the bacterial protein. A BLAST search with the sequence of Pseudomonas aeruginosa as query in the non-redundant sequence database identified four proteins from different species, three organisms from bacteria and one from archaea. We have modeled the structures of these proteins as well as those of the complexes with carbohydrates and studied the nature of physicochemical forces involved in the complex formation both in presence and absence of calcium. The calcium-binding loops have been found to be highly conserved both in terms of primary and tertiary structures in these proteins, although a less acidic character is observed in Photorhabdus lectin due to the absence of two aspartic acid residues on the calcium-binding loop which also resulted in lower binding affinity. All these structures exhibited highly negative electrostatic environment in the vicinity of the calcium-binding loops which was essential for neutralizing the positive charges of two closely situated Ca⁺² ions. The comparison of the binding affinities of some monosaccharides other than fucose, e.g. mannose and fructose, showed higher binding energies confirming the fucose specificity of these proteins.     

NMR structure of the conserved hypothetical protein TM0487 from Thermotoga maritima: implications for 216 homologous DUF59 proteins

The NMR structure of the conserved hypothetical protein TM0487 from Thermotoga maritima represents an alpha/beta-topology formed by the regular secondary structures alpha1-beta1-beta2-alpha2-beta3-beta4-alpha3- beta5-3(10)-alpha4, with a small anti-parallel beta-sheet of beta-strands 1 and 2, and a mixed parallel/anti-parallel beta-sheet of beta-strands 3-5. Similar folds have previously been observed in other proteins, with amino acid sequence identity as low as 3% and a variety of different functions. There are also 216 sequence homologs of TM0487, which all have the signature sequence of domains of unknown function 59 (DUF59), for which no three-dimensional structures have as yet been reported. The TM0487 structure thus presents a platform for homology modeling of this large group of DUF59 proteins. Conserved among most of the DUF59s are 13 hydrophobic residues, which are clustered in the core of TM0487. A putative active site of TM0487 consisting of residues D20, E22, L23, T51, T52, and C55 is conserved in 98 of the 216 DUF59 sequences. Asp20 is buried within the proposed active site without any compensating positive charge, which suggests that its pK(a) value may be perturbed. Furthermore, the DUF59 family includes ORFs that are part of a conserved chromosomal group of proteins predicted to be involved in Fe-S cluster metabolism.     

Chaperone proteins identified from synthetic proteasome inhibitor-induced inclusions in PC12 cells by proteomic analysis

Chaperone proteins are significant in Lewy bodies, but the profile of chaperone proteins is incompletely unraveled. Proteomic analysis is used to determine protein candidates for further study. Here, to identify potential chaperone proteins from agent-induced inclusions, we carried out proteomic analysis of artificially synthetic proteasome inhibitor (PSI)-induced inclusions formed in PC12 cells exposed to 10 μM PSI for 48 h. Using biochemical fractionation, 2-D electrophoresis, and identification through peptide mass fingerprints searched against multiple protein databases, we repeatedly identified eight reproducible chaperone proteins from the PSI-induced inclusions. Of these, 58 kDa glucose regulated protein, 75 kDa glucose regulated protein, and calcium-binding protein 1 were newly identified. The other five had been reported to be consistent components of Lewy bodies. These findings suggested that the three potential chaperone proteins might be recruited to PSI-induced inclusions in PC12 cells under proteasome inhibition.