Comparative genomic assessment of novel broad-spectrum targets for antibacterial drugs

Single and multiple resistance to antibacterial drugs currently in use is spreading, since they act against only a very small number of molecular targets; finding novel targets for anti-infectives is therefore of great importance. All protein sequences from three pathogens (Staphylococcus aureus, Mycobacterium tuberculosis and Escherichia coli O157:H7 EDL993) were assessed via comparative genomics methods for their suitability as antibacterial targets according to a number of criteria, including the essentiality of the protein, its level of sequence conservation, and its distribution in pathogens, bacteria and eukaryotes (especially humans). Each protein was scored and ranked based on weighted variants of these criteria in order to prioritize proteins as potential novel broad-spectrum targets for antibacterial drugs. A number of proteins proved to score highly in all three species and were robust to variations in the scoring system used. Sensitivity analysis indicated the quantitative contribution of each metric to the overall score. After further analysis of these targets, tRNA methyltransferase (trmD) and translation initiation factor IF-1 (infA) emerged as potential and novel antimicrobial targets very worthy of further investigation. The scoring strategy used might be of value in other areas of post-genomic drug discovery.     

A novel in silico approach to identify potential therapeutic targets in human bacterial pathogens

In recent years, genome-sequencing projects of pathogens and humans have revolutionized microbial drug target identification. Of the several known genomic strategies, subtractive genomics has been successfully utilized for identifying microbial drug targets. The present work demonstrates a novel genomics approach in which codon adaptation index (CAI), a measure used to predict the translational efficiency of a gene based on synonymous codon usage, is coupled with subtractive genomics approach for mining potential drug targets. The strategy adopted is demonstrated using respiratory pathogens, namely, Streptococcus pneumoniae and Haemophilus influenzae as examples. Our approach identified 8 potent target genes (Streptococcus pneumoniae?C2, H. influenzae?C6), which are functionally significant and also play key role in host-pathogen interactions. This approach facilitates swift identification of potential drug targets, thereby enabling the search for new inhibitors. These results underscore the utility of CAI for enhanced in silico drug target identification.     

Proteomic approach to identify novel mitochondrial proteins in Arabidopsis.

An Arabidopsis mitochondrial proteome project was started for a comprehensive investigation of mitochondrial functions in plants. Mitochondria were prepared from Arabidopsis stems and leaves or from Arabidopsis suspension cell cultures, and the purity of the generated fractions was tested by the resolution of organellar protein complexes applying two-dimensional blue-native/N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine (Tricine) sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Arabidopsis mitochondrial proteome was analyzed by two-dimensional isoelectric focusing/ Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 650 different proteins in a pI range of pH 3 to 10 were separated on single gels. Solubilization conditions, pH gradients for isoelectric focusing, and gel staining procedures were varied, and the number of separable proteins increased to about 800. Fifty-two protein spots were identified by immunoblotting, direct protein sequencing, and mass spectrometry. The characterized proteins cooperate in various processes, such as respiration, citric acid cycle, amino acid and nucleotide metabolism, protection against O(2), mitochondrial assembly, molecular transport, and protein biosynthesis. More than 20% of the identified proteins were not described previously for plant mitochondria, indicating novel mitochondrial functions. The map of the Arabidopsis mitochondrial proteome should be useful for the analysis of knockout mutants concerning nuclear-encoded mitochondrial genes. Considerations of the total complexity of the Arabidopsis mitochondrial proteome are discussed. The data from this investigation will be made available at http://www.gartenbau.uni-hannover.de/genetik/AMPP.     

A multidimensional proteomic approach to identify hypertrophy-associated proteins

Left ventricular hypertrophy (LVH) is a leading cause of congestive heart failure. The exact mechanisms that control cardiac growth and regulate the transition to failure are not fully understood, in part due to the lack of a complete inventory of proteins associated with LVH. We investigated the proteomic basis of LVH using the transverse aortic constriction model of pressure overload in mice coupled with a multidimensional approach to identify known and novel proteins that may be relevant to the development and maintenance of LVH. We identified 123 proteins that were differentially expressed during LVH, including LIM proteins, thioredoxin, myoglobin, fatty acid binding protein 3, the abnormal spindle-like microcephaly protein (ASPM), and cytoskeletal proteins such as actin and myosin. In addition, proteins with unknown functions were identified, providing new directions for future research in this area. We also discuss common pitfalls and strategies to overcome the limitations of current proteomic technologies. Together, the multidimensional approach provides insight into the proteomic changes that occur in the LV during hypertrophy.     

A novel approach to studying the structural and functional properties of proteins with unknown functions

Three proteins from extremophilic bacteria—hypothetical monooxygenase from Deinococcus radiodurans, hypothetical nucleotidyl transferase from Thermotoga maritime, and hypothetical oxidoreductase from Exiguobacterium sibiricum—and the DJ-1 chaperone protein from Homo sapiens have been produced in Escherichia coli. The isolation and purification procedures developed for the recombinant proteins allowed us to achieve yields higher than 96%. Crystallization conditions enabling stable growth of crystals have been determined. X-ray experiments have been performed to test the quality of the crystals and the resolution achieved ranged from 1.2 to 1.8 Å.     

A novel proteomics approach to identify SUMOylated proteins and their modification sites in human cells

The small ubiquitin-related modifier (SUMO) is a small group of proteins that are reversibly attached to protein substrates to modify their functions. The large scale identification of protein SUMOylation and their modification sites in mammalian cells represents a significant challenge because of the relatively small number of in vivo substrates and the dynamic nature of this modification. We report here a novel proteomics approach to selectively enrich and identify SUMO conjugates from human cells. We stably expressed different SUMO paralogs in HEK293 cells, each containing a His(6) tag and a strategically located tryptic cleavage site at the C terminus to facilitate the recovery and identification of SUMOylated peptides by affinity enrichment and mass spectrometry. Tryptic peptides with short SUMO remnants offer significant advantages in large scale SUMOylome experiments including the generation of paralog-specific fragment ions following CID and ETD activation, and the identification of modified peptides using conventional database search engines such as Mascot. We identified 205 unique protein substrates together with 17 precise SUMOylation sites present in 12 SUMO protein conjugates including three new sites (Lys-380, Lys-400, and Lys-497) on the protein promyelocytic leukemia. Label-free quantitative proteomics analyses on purified nuclear extracts from untreated and arsenic trioxide-treated cells revealed that all identified SUMOylated sites of promyelocytic leukemia were differentially SUMOylated upon stimulation.     

A novel approach to identify proteins modified by nitric oxide: the HIS-TAG switch method

S-nitrosylation is emerging as an important signaling mechanism that regulates a broad range of cellular functions. The recognition of Cysteine residues that undergo S-nitrosylation is crucial to elucidate how this modification modulates protein activity. We report here a novel strategy, defined His-tag switch, which allows the purification and identification of S-nitrosylated proteins and the unambiguous localization of the modified cysteine residues by mass spectrometry analysis.     

Structural genomics of highly conserved microbial genes of unknown function in search of new antibacterial targets

With more than 100 antibacterial drugs at our disposal in the 1980s, the problem of bacterial infection was considered solved. Today, however, most hospital infections are insensitive to several classes of antibacterial drugs, and deadly strains of Staphylococcus aureus resistant to vancomycin – the last resort antibiotic – have recently begin to appear. Other life-threatening microbes, such as Enterococcus faecalis and Mycobacterium tuberculosis are already able to resist every available antibiotic. There is thus an urgent, and continuous need for new, preferably large-spectrum, antibacterial molecules, ideally targeting new biochemical pathways. Here we report on the progress of our structural genomics program aiming at the discovery of new antibacterial gene targets among evolutionary conserved genes of uncharacterized function. A series of bioinformatic and comparative genomics analyses were used to identify a set of 221 candidate genes common to Gram-positive and Gram-negative bacteria. These genes were split between two laboratories. They are now submitted to a systematic 3-D structure determination protocol including cloning, protein expression and purification, crystallization, X-ray diffraction, structure interpretation, and function prediction. We describe here our strategies for the 111 genes processed in our laboratory. Bioinformatics is used at most stages of the production process and out of 111 genes processed – and 17 months into the project – 108 have been successfully cloned, 103 have exhibited detectable expression, 84 have led to the production of soluble protein, 46 have been purified, 12 have led to usable crystals, and 7 structures have been determined.     

A proteomic approach to identify developmentally regulated proteins in Leishmania infantum

We used comparative two-dimensional gel electrophoresis (2-DE) and mass spectrometry methodologies to highlight and identify proteins that are differentially expressed in the intracellular stage of the parasite Leishmania donovani infantum, a causative agent of visceral leishmaniasis. During its digenetic life cycle, Leishmania alternates between the alimentary tract of the sandfly vector as an extracellular promastigote and the acidic phagolysosomes of macrophage cells as an intracellular amastigote. Proteins differentially expressed in the intracellular form of the parasite are thought to be important for intracellular survival and pathogenesis. We used narrow pH range strips for isoelectric focusing to resolve soluble proteins of both developmental stages of L. infantum. More than 62 proteins differentially expressed in amastigotes were detected among approximately 2000 protein spots resolved by 2-DE. A quadrupole time-of-flight analysis of few selected protein spots, specifically expressed in the amastigote stage, permitted the identification of two proteins, part of the energetic metabolism pathways, the isocitrate dehydrogenase and the glycolytic enzyme triosephosphate isomerase. The kinetic parameters of these two enzymes were measured in both developmental stages of the parasite and their activity was indeed found to be higher in amastigotes. These findings bring a new insight in our understanding of metabolic and energy requirements of the intracellular form of Leishmania. Comparative analysis of the proteome of both developmental stages of the protozoan parasite Leishmania should permit the identification of protein candidates for the development of vaccines and new drugs.     

A machine learning approach to identify hydrogenosomal proteins in Trichomonas vaginalis

The protozoan parasite Trichomonas vaginalis is the causative agent of trichomoniasis, the most widespread nonviral sexually transmitted disease in humans. It possesses hydrogenosomes-anaerobic mitochondria that generate H(2), CO(2), and acetate from pyruvate while converting ADP to ATP via substrate-level phosphorylation. T. vaginalis hydrogenosomes lack a genome and translation machinery; hence, they import all their proteins from the cytosol. To date, however, only 30 imported proteins have been shown to localize to the organelle. A total of 226 nuclear-encoded proteins inferred from the genome sequence harbor a characteristic short N-terminal presequence, reminiscent of mitochondrial targeting peptides, which is thought to mediate hydrogenosomal targeting. Recent studies suggest, however, that the presequences might be less important than previously thought. We sought to identify new hydrogenosomal proteins within the 59,672 annotated open reading frames (ORFs) of T. vaginalis, independent of the N-terminal targeting signal, using a machine learning approach. Our training set included 57 gene and protein features determined for all 30 known hydrogenosomal proteins and 576 nonhydrogenosomal proteins. Several classifiers were trained on this set to yield an import score for all proteins encoded by T. vaginalis ORFs, predicting the likelihood of hydrogenosomal localization. The machine learning results were tested through immunofluorescence assay and immunodetection in isolated cell fractions of 14 protein predictions using hemagglutinin constructs expressed under the homologous SCSα promoter in transiently transformed T. vaginalis cells. Localization of 6 of the 10 top predicted hydrogenosome-localized proteins was confirmed, and two of these were found to lack an obvious N-terminal targeting signal.     

A proteomics approach to identify the ubiquitinated proteins in mouse heart

There is a growing need for the large-scale identification of the ubiquitinated proteins and ubiquitin attachment sites. As part of this effort, we generated a transgenic mouse expressing a tagged ubiquitin in the heart. We found that the majority of ubiquitinated proteins in mouse heart are insoluble in detergent-free buffer and were chemically cleaved after methionine with CNBr. CNBr cleaved the proteins into smaller polypeptides while preserving the ubiquitin chains. Ubiquitin-conjugated polypeptides were then purified under denaturing conditions, digested with Lys-C and trypsin, and analyzed by liquid chromatography-tandem mass spectrometry. We identified 121 proteins that were ubiquitinated in mouse heart, and we detected 33 ubiquitination sites in 21 of the proteins. Components of cardiac muscle and many mitochondrial proteins were identified as substrates for ubiquitination, strongly suggesting that proteins related to major heart functions such as contraction and energy production are under continuous quality control by the ubiquitin system.     

A combined approach to data mining of textual and structured data to identify cancer-related targets

Background  

We present an effective, rapid, systematic data mining approach for identifying genes or proteins related to a particular interest. A selected combination of programs exploring PubMed abstracts, universal gene/protein databases (UniProt, InterPro, NCBI Entrez), and state-of-the-art pathway knowledge bases (LSGraph and Ingenuity Pathway Analysis) was assembled to distinguish enzymes with hydrolytic activities that are expressed in the extracellular space of cancer cells. Proteins were identified with respect to six types of cancer occurring in the prostate, breast, lung, colon, ovary, and pancreas.     

A computational approach to finding novel targets for existing drugs

Repositioning existing drugs for new therapeutic uses is an efficient approach to drug discovery. We have developed a computational drug repositioning pipeline to perform large-scale molecular docking of small molecule drugs against protein drug targets, in order to map the drug-target interaction space and find novel interactions. Our method emphasizes removing false positive interaction predictions using criteria from known interaction docking, consensus scoring, and specificity. In all, our database contains 252 human protein drug targets that we classify as reliable-for-docking as well as 4621 approved and experimental small molecule drugs from DrugBank. These were cross-docked, then filtered through stringent scoring criteria to select top drug-target interactions. In particular, we used MAPK14 and the kinase inhibitor BIM-8 as examples where our stringent thresholds enriched the predicted drug-target interactions with known interactions up to 20 times compared to standard score thresholds. We validated nilotinib as a potent MAPK14 inhibitor in vitro (IC50 40 nM), suggesting a potential use for this drug in treating inflammatory diseases. The published literature indicated experimental evidence for 31 of the top predicted interactions, highlighting the promising nature of our approach. Novel interactions discovered may lead to the drug being repositioned as a therapeutic treatment for its off-target's associated disease, added insight into the drug's mechanism of action, and added insight into the drug's side effects.     

A computerized database-scan to identify c-MYC targets

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease caused by the expansion of a polyglutamine tract in the protein ataxin-7, a protein of unknown function. In order to analyze the expression pattern of wild type ataxin-7 in detail, the murine SCA7 gene homolog was cloned and the expression pattern in mice analyzed. The SCA7 mouse and human gene exhibit a high degree of identity at both DNA (88.2%) and protein (88.7%) level. The CAG repeat region, known to be polymorphic in man, is conserved in mouse but contained only five repeats in all mouse strains analyzed. The arrestin homology domain and the nuclear localization signal found in human ataxin-7 is also conserved in the murine homolog. Expression of ataxin-7 was detected during mouse embryonic development and in all adult mouse tissues examined by northern and western blots. In brain, immunohistological staining revealed an ataxin-7 expression pattern similar to that in human, with ataxin-7 expression in cerebellum, several brainstem nuclei, cerebral cortex and hippocampus. Our data show high conservation of ataxin-7 both structurally and at the level of expression, suggesting a conserved role for the protein in mice and humans.     

A scaffold hopping approach to identify novel monoamine oxidase B inhibitors

Monoamine oxidase B (MAO-B) inhibitors are used to treat Parkinson's disease. In this study, we searched for novel MAO-B inhibitors using a scaffold hopping approach based on our experience with the thiazolidinedione (TZD) class of compounds as MAO-B inhibitors. Several novel compounds were identified, with potencies in the low nanomolar and low micromolar range. We also found that derivatives of the natural product sulfuretin are potent MAO-A and MAO-B inhibitors.     

A chemical proteomics approach to identify c-di-GMP binding proteins in Pseudomonas aeruginosa

In many bacteria, high levels of the ubiquitous second messenger c-di-GMP have been demonstrated to suppress motility and to promote the establishment of surface-adherent biofilm communities. While molecular mechanisms underlying the synthesis and degradation of c-di-GMP have been comprehensively characterized, little is known about how c-di-GMP mediates its regulatory effects. In this study, we have established a chemical proteomics approach to identify c-di-GMP interacting proteins in the opportunistic pathogen Pseudomonas aeruginosa. A functionalized c-di-GMP analog, 2′-aminohexylcarbamoyl-c-di-GMP (2′-AHC-c-di-GMP), was chemically synthesized and following its immobilization used to perform affinity pull down experiments. Enriched proteins were subsequently identified by high-resolution mass spectrometry. 2′-AHC-c-di-GMP was also employed in surface plasmon resonance studies to evaluate and quantify the interaction of c-di-GMP with its potential target molecules in vitro. The biochemical tools presented here may serve the identification of novel classes of c-di-GMP effectors and thus contribute to a better characterization and understanding of the complex c-di-GMP signaling network.     

TargetScreen: An unbiased approach to identify functionally important microRNA targets

We recently identified miR-19 as the critical activity for leukemogenesis within the oncogenic 17~92 cluster of microRNAs.¹ This finding prompted us to test an unbiased method for pinpointing those miR-19 targets may be key to its oncogenic action. Specifically, we used a large-scale short hairpin RNA screen to identify those miR-19 target genes, whose knockdown could reproduce miR-19's effects on lymphocyte transformation. In this way, we found that miR-19 produces a coordinate clampdown on multiple negative regulators of PI3K-related survival signals. These findings have implications for the therapy of miR-19 expressing tumors. They also validate a new strategy for the unbiased identification of functionally important microRNA target genes. Using the example of miR-19 in leukemia, we will discuss some possibilities and limitations of this new approach.