Crowd sourcing a new paradigm for interactome driven drug target identification in Mycobacterium tuberculosis

A decade since the availability of Mycobacterium tuberculosis (Mtb) genome sequence, no promising drug has seen the light of the day. This not only indicates the challenges in discovering new drugs but also suggests a gap in our current understanding of Mtb biology. We attempt to bridge this gap by carrying out extensive re-annotation and constructing a systems level protein interaction map of Mtb with an objective of finding novel drug target candidates. Towards this, we synergized crowd sourcing and social networking methods through an initiative 'Connect to Decode' (C2D) to generate the first and largest manually curated interactome of Mtb termed 'interactome pathway' (IPW), encompassing a total of 1434 proteins connected through 2575 functional relationships. Interactions leading to gene regulation, signal transduction, metabolism, structural complex formation have been catalogued. In the process, we have functionally annotated 87% of the Mtb genome in context of gene products. We further combine IPW with STRING based network to report central proteins, which may be assessed as potential drug targets for development of drugs with least possible side effects. The fact that five of the 17 predicted drug targets are already experimentally validated either genetically or biochemically lends credence to our unique approach.     

Effect of glutamate and riluzole on manganese-induced apoptotic cell signaling in neuronally differentiated mouse P19 Cells

Excess exposure to Mn causes a neurological disorder known as manganism which is similar to dystonic movements associated with Parkinson's disease. Manganism is largely restricted to occupations in which high atmospheric levels are prevalent which include Mn miners, welders and those employed in the ferroalloy processing or related industrial settings. T1 weighted MRI images reveal that Mn is deposited to the greatest extent in the globus pallidus, an area of the brain that is presumed to be responsible for the major CNS associated symptoms. Neurons within the globus pallidus receive glutamatergic input from the subthalamic nuclei which has been suggested to be involved in the toxic actions of Mn. The neurotoxic actions of Mn and glutamate are similar in that they both affect calcium accumulation in the mitochondria leading to apoptotic cell death. In this paper, we demonstrate that the combination of Mn and glutamate potentiates toxicity of neuronally differentiated P19 cells over that observed with either agent alone. Apoptotic signals ROS, caspase 3 and JNK were increased in an additive fashion when the two neurotoxins were combined. The anti-glutamatergic drug, riluzole, was shown to attenuate these apoptotic signals and prevent P19 cell death. Results of this study confirm, for the first time, that Mn toxicity is potentiated in the presence of glutamate and that riluzole is an effective antioxidant which protects against both Mn and glutamate toxicity.     

Probing the activation sequence of NMDA receptors with lurcher mutations

N-methyl-d-aspartate (NMDA) receptor activation involves a dynamic series of structural rearrangements initiated by glutamate binding to glycine-loaded receptors and culminates with the clearing of the permeation pathway, which allows ionic flux. Along this sequence, three rate-limiting transitions can be quantified with kinetic analyses of single-channel currents, even though the structural determinants of these critical steps are unknown. In inactive receptors, the major permeation barrier resides at the intersection of four M3 transmembrane helices, two from each GluN1 and GluN2 subunits, at the level of the invariant SYTANLAAF sequence, known as the lurcher motif. Because the A7 but not A8 residues in this region display agonist-dependent accessibility to extracellular solutes, they were hypothesized to form the glutamate-sensitive gate. We tested this premise by examining the reaction mechanisms of receptors with substitutions in the lurcher motifs of GluN1 or GluN2A subunits. We found that, consistent with their locations relative to the proposed activation gate, A8Y decreased open-state stability, whereas A7Y dramatically stabilized open states, primarily by preventing gate closure; the equilibrium distribution of A7Y receptors was strongly shifted toward active states and resulted in slower microscopic association and dissociation rate constants for glutamate. In addition, for both A8- and A7-substituted receptors, we noticed patterns of kinetic changes that were specific to GluN1 or GluN2 locations. This may be a first indication that the sequence of discernible kinetic transitions during NMDA receptor activation may reflect subunit-dependent movements of M3 helices. Testing this hypothesis may afford insight into the activation mechanism of NMDA receptors.     

Essentiality Assessment of Cysteinyl and Lysyl-tRNA Synthetases of Mycobacterium smegmatis

Discovery of mupirocin, an antibiotic that targets isoleucyl-tRNA synthetase, established aminoacyl-tRNA synthetase as an attractive target for the discovery of novel antibacterial agents. Despite a high degree of similarity between the bacterial and human aminoacyl-tRNA synthetases, the selectivity observed with mupirocin triggered the possibility of targeting other aminoacyl-tRNA synthetases as potential drug targets. These enzymes catalyse the condensation of a specific amino acid to its cognate tRNA in an energy-dependent reaction. Therefore, each organism is expected to encode at least twenty aminoacyl-tRNA synthetases, one for each amino acid. However, a bioinformatics search for genes encoding aminoacyl-tRNA synthetases from Mycobacterium smegmatis returned multiple genes for glutamyl (GluRS), cysteinyl (CysRS), prolyl (ProRS) and lysyl (LysRS) tRNA synthetases. The pathogenic mycobacteria, namely, Mycobacterium tuberculosis and Mycobacterium leprae, were also found to possess two genes each for CysRS and LysRS. A similar search indicated the presence of additional genes for LysRS in gram negative bacteria as well. Herein, we describe sequence and structural analysis of the additional aminoacyl-tRNA synthetase genes found in M. smegmatis. Characterization of conditional expression strains of Cysteinyl and Lysyl-tRNA synthetases generated in M. smegmatis revealed that the canonical aminoacyl-tRNA synthetase are essential, while the additional ones are not essential for the growth of M. smegmatis.     

Proteomics approach to understand reduced clearance of mycobacteria and high viral titers during HIV–mycobacteria co‐infection

Environmental mycobacteria, highly prevalent in natural and artificial (including chlorinated municipal water) niches, are emerging as new threat to human health, especially to HIV‐infected population. These seemingly harmless non‐pathogenic mycobacteria, which are otherwise cleared, establish as opportunistic infections adding to HIV‐associated complications. Although immune‐evading strategies of pathogenic mycobacteria are known, the mechanisms underlying the early events by which opportunistic mycobacteria establish infection in macrophages and influencing HIV infection are unclear. Proteomics of phagosome‐enriched fractions from Mycobacterium bovis Bacillus Calmette–Guérin (BCG) mono‐infected and HIV–M. bovis BCG co‐infected THP‐1 cells by LC‐MALDI‐MS/MS revealed differential distribution of 260 proteins. Validation of the proteomics data showed that HIV co‐infection helped the survival of non‐pathogenic mycobacteria by obstructing phagosome maturation, promoting lipid biogenesis and increasing intracellular ATP equivalents. In turn, mycobacterial co‐infection up‐regulated purinergic receptors in macrophages that are known to support HIV entry, explaining increased viral titers during co‐infection. The mutualism was reconfirmed using clinically relevant opportunistic mycobacteria, Mycobacterium avium, Mycobacterium kansasii and Mycobacterium phlei that exhibited increased survival during co‐infection, together with increase in HIV titers. Additionally, the catalogued proteins in the study provide new leads that will significantly add to the understanding of the biology of opportunistic mycobacteria and HIV coalition.     

Sterol 24-C-methyltransferase: An enzymatic target for the disruption of ergosterol biosynthesis and homeostasis in Cryptococcus neoformans

Growth of Cryptococcus neoformans was inhibited by nine nitrogen and sulfur-containing sterols with a heteroatom positioned at C3, C7, C24, C25 or C32 in the lanostane frame. Analysis of the sterol composition of control and treated cells by GC-MS and ¹H NMR has proven that the C-methylation reaction catalyzed by the sterol 24-C-methyltransferase (24-SMT) is the crucial first step in a kinetically favored pathway that fails to include obtusifoliol or zymosterol as intermediates. Cultures fed [methyl-²H₃]methionine led to two deuterium atoms into each of the newly biosynthesized sterols forming a route lanosterol, eburicol (24(28)-methylene-24,25-dihydrolanosterol), 32-noreburicol and ergost-7-enol to ergosterol. Examination of the substrate specificity of a soluble 24-SMT from C. neoformans showed lanosterol to be the optimal acceptor molecule. Incubation with the test compounds generated induced amounts of lanosterol, eburicol or 32-noreburicol concurrent with a decrease of ergosterol. Among them 24(R,S),25-epiminolanosterol (inhibitor of 24-SMT) showed the most potent in vitro antifungal activity comparable to those of itraconazole (inhibitor of the 14-demethylase). Taken together, these data indicate that treatment with substrate-based inhibitors of 24-SMT, a catalyst not found in humans, can disrupt ergosterol homeostasis involved with fungal growth and therefore these compounds can provide leads for rational drug design of opportunistic pathogens.     

GTP cyclohydrolase and tetrahydrobiopterin regulate pain sensitivity and persistence

We report that GTP cyclohydrolase (GCH1), the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, is a key modulator of peripheral neuropathic and inflammatory pain. BH4 is an essential cofactor for catecholamine, serotonin and nitric oxide production. After axonal injury, concentrations of BH4 rose in primary sensory neurons, owing to upregulation of GCH1. After peripheral inflammation, BH4 also increased in dorsal root ganglia (DRGs), owing to enhanced GCH1 enzyme activity. Inhibiting this de novo BH4 synthesis in rats attenuated neuropathic and inflammatory pain and prevented nerve injury-evoked excess nitric oxide production in the DRG, whereas administering BH4 intrathecally exacerbated pain. In humans, a haplotype of the GCH1 gene (population frequency 15.4%) was significantly associated with less pain following diskectomy for persistent radicular low back pain. Healthy individuals homozygous for this haplotype exhibited reduced experimental pain sensitivity, and forskolin-stimulated immortalized leukocytes from haplotype carriers upregulated GCH1 less than did controls. BH4 is therefore an intrinsic regulator of pain sensitivity and chronicity, and the GTP cyclohydrolase haplotype is a marker for these traits.     

Solution Structure and Characterisation of the Human Pyruvate Dehydrogenase Complex Core Assembly

Mammalian pyruvate dehydrogenase complex (PDC) is a key multi-enzyme assembly that is responsible for glucose homeostasis maintenance and conversion of pyruvate into acetyl-CoA. It comprises a central pentagonal dodecahedral core consisting of two subunit types (E2 and E3BP) to which peripheral enzymes (E1 and E3) bind tightly but non-covalently. Currently, there are two conflicting models of PDC (E2 + E3BP) core organisation: the ‘addition’ model (60 + 12) and the ‘substitution’ model (48 + 12). Here we present the first ever low-resolution structures of human recombinant full-length PDC core (rE2/E3BP), truncated PDC core (tE2/E3BP) and native bovine heart PDC core (bE2/E3BP) obtained by small-angle X-ray scattering and small-angle neutron scattering. These structures, corroborated by negative-stain and cryo electron microscopy data, clearly reveal open pentagonal core faces, favouring the ‘substitution’ model of core organisation. The native and recombinant core structures are all similar to the truncated bacterial E2 core crystal structure obtained previously. Cryo-electron microscopy reconstructions of rE2/E3BP and rE2/E3BP:E3 directly confirm that the core has open pentagonal faces, agree with scattering-derived models and show density extending outwards from their surfaces, which is much more structurally ordered in the presence of E3. Additionally, analytical ultracentrifugation characterisation of rE2/E3BP, rE2 (full-length recombinant E2-only) and tE2/E3BP supports the substitution model. Superimposition of the small-angle neutron scattering tE2/E3BP and truncated bacterial E2 crystal structures demonstrates conservation of the overall pentagonal dodecahedral morphology, despite evolutionary diversity. In addition, unfolding studies using circular dichroism and tryptophan fluorescence spectroscopy show that the rE2/E3BP is less stable than its rE2 counterpart, indicative of a role for E3BP in core destabilisation. The architectural complexity and lower stability of the E2/E3BP core may be of benefit to mammals, where sophisticated fine-tuning is required for cores with optimal catalytic and regulatory efficiencies.     

Response surface methodology for the optimization of alpha amylase production by Bacillus amyloliquefaciens

The aim of this work was to optimize the cultural and production parameters through the statistical approach for the synthesis of alpha amylase by Bacillus amyloliquefaciens in submerged fermentation (SmF) using a combination of wheat bran and groundnut oil cake (1:1) as the substrate. The process parameters influencing the enzyme production were identified using Plackett-Burman design. Among the various variables screened, the substrate concentration, incubation period and CaCl2 concentration were most significant. The optimum levels of these significant parameters were determined employing the response surface Box-Behnken design, which revealed these as follows: substrate concentration (12.5%), incubation period (42 h) and CaCl2 (0.0275 M).     

Metabolism of carbaryl via 1,2-dihydroxynaphthalene by soil isolates Pseudomonas sp. strains C4, C5, and C6

Pseudomonas sp. strains C4, C5, and C6 utilize carbaryl as the sole source of carbon and energy. Identification of 1-naphthol, salicylate, and gentisate in the spent media; whole-cell O2 uptake on 1-naphthol, 1,2-dihydroxynaphthalene, salicylaldehyde, salicylate, and gentisate; and detection of key enzymes, viz, carbaryl hydrolase, 1-naphthol hydroxylase, 1,2-dihydroxynaphthalene dioxygenase, and gentisate dioxygenase, in the cell extract suggest that carbaryl is metabolized via 1-naphthol, 1,2-dihydroxynaphthalene, and gentisate. Here, we demonstrate 1-naphthol hydroxylase and 1,2-dihydroxynaphthalene dioxygenase activities in the cell extracts of carbaryl-grown cells. 1-Naphthol hydroxylase is present in the membrane-free cytosolic fraction, requires NAD(P)H and flavin adenine dinucleotide, and has optimum activity in the pH range 7.5 to 8.0. Carbaryl-degrading enzymes are inducible, and maximum induction was observed with carbaryl. Based on these results, the proposed metabolic pathway is carbaryl --> 1-naphthol --> 1,2-dihydroxynaphthalene --> salicylaldehyde --> salicylate --> gentisate --> maleylpyruvate.