Nagarajan, Sathya Narayanan; Upadhyay, Sandeep; Chawla, Yogesh; Khan, Shazia; Naz, Saba; Subramanian, Jayashree; Gandotra, Sheetal; Nandicoori, Vinay Kumar
The essential mycobacterial protein kinases PknA and PknB play crucial roles in modulating cell shape and division. However, the precise in vivo functional aspects of PknA have not been investigated. This study aims to dissect the role of PknA in mediating cell survival in vitro as well as in vivo. We observed aberrant cell shape and severe growth defects when PknA was depleted. Using the mouse infection model, we observe that PknA is essential for survival of the pathogen in the host. Complementation studies affirm the importance of the kinase, juxtamembrane, and transmembrane domains of PknA. Surprisingly, the extracytoplasmic domain is dispensable for cell growth and survival in vitro. We find that phosphorylation of the activation loop at Thr(172) of PknA is critical for bacterial growth. PknB has been previously suggested to be the receptor kinase, which activates multiple kinases, including PknA, by trans-phosphorylating their activation loop residues. Using phospho-specific PknA antibodies and conditional pknB mutant, we find that PknA autophosphorylates its activation loop independent of PknB. Fluorescently tagged PknA and PknB show distinctive distribution patterns within the cell, suggesting that although both kinases are known to modulate cell shape and division, their modes of action are likely to be different. This is supported by our findings that expression of kinase-dead PknA versus kinase-dead PknB in mycobacterial cells leads to different cellular phenotypes. Data indicate that although PknA and PknB are expressed as part of the same operon, they appear to be regulating cellular processes through divergent signaling pathways.
Full Text Available The Mycobacterium tuberculosis Ser/Thr kinase PknB has been implicated in the regulation of cell growth and morphology in this organism. The extracytoplasmic domain of this membrane protein comprises four penicillin binding protein and Ser/Thr kinase associated (PASTA domains, which are predicted to bind stem peptides of peptidoglycan. Using a comprehensive library of synthetic muropeptides, we demonstrate that the extracytoplasmic domain of PknB binds muropeptides in a manner dependent on the presence of specific amino acids at the second and third positions of the stem peptide, and on the presence of the sugar moiety N-acetylmuramic acid linked to the peptide. We further show that PknB localizes strongly to the mid-cell and also to the cell poles, and that the extracytoplasmic domain is required for PknB localization. In contrast to strong growth stimulation by conditioned medium, we observe no growth stimulation of M. tuberculosis by a synthetic muropeptide with high affinity for the PknB PASTAs. We do find a moderate effect of a high affinity peptide on resuscitation of dormant cells. While the PASTA domains of PknB may play a role in stimulating growth by binding exogenous peptidoglycan fragments, our data indicate that a major function of these domains is for proper PknB localization, likely through binding of peptidoglycan fragments produced locally at the mid-cell and the cell poles. These data suggest a model in which PknB is targeted to the sites of peptidoglycan turnover to regulate cell growth and cell division.
Full Text Available Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 Å resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation.
Rakette, Sonja; Donat, Stefanie; Ohlsen, Knut; Stehle, Thilo
Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 Å resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation.
Chapman, Timothy M.; Bouloc, Nathalie; Buxton, Roger S.; Chugh, Jasveen; Lougheed, Kathryn E.A.; Osborne, Simon A.; Saxty, Barbara; Smerdon, Stephen J.; Taylor, Debra L.; Whalley, David
A high-throughput screen against PknB, an essential serine–threonine protein kinase present in Mycobacterium tuberculosis (M. tuberculosis), allowed the identification of an aminoquinazoline inhibitor which was used as a starting point for SAR investigations. Although a significant improvement in enzyme affinity was achieved, the aminoquinazolines showed little or no cellular activity against M. tuberculosis. However, switching to an aminopyrimidine core scaffold and the introduction of a basic amine side chain afforded compounds with nanomolar enzyme binding affinity and micromolar minimum inhibitory concentrations against M. tuberculosis. Replacement of the pyrazole head group with pyridine then allowed equipotent compounds with improved selectivity against a human kinase panel to be obtained. PMID:22469702
Um Änderungen in seiner Umwelt wahrnehmen zu können, benötigt S. aureus unterschiedliche Signaltransduktionssysteme. In dieser Arbeit wurde erstmals die Eukaryoten-ähnliche Serin/Threonin-Proteinkinase (STPK) PknB umfassend charakterisiert. Die posttranslationale Proteinmodifikation mittels Phosphorylierung spielt sowohl in Eukaryoten als auch in Prokaryoten eine wichtige Rolle. Man glaubte lange, dass die Phosphorylierung von Serin-, Threonin- und Tyrosinresten ein nur auf Eukaryoten beschrä...
Turapov, Obolbek; Loraine, Jessica; Jenkins, Christopher H; Barthe, Philippe; McFeely, Daniel; Forti, Francesca; Ghisotti, Daniela; Hesek, Dusan; Lee, Mijoon; Bottrill, Andrew R; Vollmer, Waldemar; Mobashery, Shahriar; Cohen-Gonsaud, Martin; Mukamolova, Galina V
PknB is an essential serine/threonine protein kinase required for mycobacterial cell division and cell-wall biosynthesis. Here we demonstrate that overexpression of the external PknB_PASTA domain in mycobacteria results in delayed regrowth, accumulation of elongated bacteria and increased sensitivity to β-lactam antibiotics. These changes are accompanied by altered production of certain enzymes involved in cell-wall biosynthesis as revealed by proteomics studies. The growth inhibition caused by overexpression of the PknB_PASTA domain is completely abolished by enhanced concentration of magnesium ions, but not muropeptides. Finally, we show that the addition of recombinant PASTA domain could prevent regrowth of Mycobacterium tuberculosis, and therefore offers an alternative opportunity to control replication of this pathogen. These results suggest that the PknB_PASTA domain is involved in regulation of peptidoglycan biosynthesis and maintenance of cell-wall architecture.
Miller, Malgorzata; Donat, Stefanie; Rakette, Sonja; Stehle, Thilo; Kouwen, Thijs R. H. M.; Diks, Sander H.; Dreisbach, Annette; Reilman, Ewoud; Gronau, Katrin; Becher, Doerte; Peppelenbosch, Maikel P.; van Dijl, Jan Maarten; Ohlsen, Knut
In eukaryotic cell types, virtually all cellular processes are under control of proline-directed kinases and especially MAP kinases. Serine/threonine kinases in general were originally considered as a eukaryote-specific enzyme family. However, recent studies have revealed that orthologues of eukaryo
Wang, Xiao Ming; Soetaert, Karine; Peirs, Priska; Kalai, Michaël; Fontaine, Véronique; Dehaye, Jean Paul; Lefèvre, Philippe
PknD is one of the eleven eukaryotic-like serine/threonine protein kinases (STPKs) of Mycobacterium tuberculosis (Mtb). In vitro phosphorylation assays with the active recombinant PknD showed that the intracellular protein NAD+-dependent malate dehydrogenase (MDH) is a substrate of this kinase. MDH, an energy-supplying enzyme, catalyzes the interconversion of malate and oxaloacetate and plays crucial roles in several metabolic pathways including the citric acid cycle. The phosphorylation site was identified on threonine residues and the phosphorylation inhibited the MDH activity. In vitro, the recombinant MDH could also be phosphorylated by at least five other STPKs, PknA, PknE, PknH, PknJ, and PknG. Immunoprecipitation analysis revealed that MDH was hyperphosphorylated in the bacteria at the beginning of the stationary and under oxygen-limited conditions by STPKs other than PknD. On the contrary, when PknD-deficient mutant mycobacteria were grown in a phosphate-depleted medium, MDH was not detectably phosphorylated. These results suggest that although the MDH is a substrate of several mycobacterial STPKs, the activity of these kinases can depend on the environment, as we identified PknD as a key element in the MDH phosphorylation assay under phosphate-poor conditions.
Xiao Ming Wang
Full Text Available PknD is one of the eleven eukaryotic-like serine/threonine protein kinases (STPKs of Mycobacterium tuberculosis (Mtb. In vitro phosphorylation assays with the active recombinant PknD showed that the intracellular protein NAD+-dependent malate dehydrogenase (MDH is a substrate of this kinase. MDH, an energy-supplying enzyme, catalyzes the interconversion of malate and oxaloacetate and plays crucial roles in several metabolic pathways including the citric acid cycle. The phosphorylation site was identified on threonine residues and the phosphorylation inhibited the MDH activity. In vitro, the recombinant MDH could also be phosphorylated by at least five other STPKs, PknA, PknE, PknH, PknJ, and PknG. Immunoprecipitation analysis revealed that MDH was hyperphosphorylated in the bacteria at the beginning of the stationary and under oxygen-limited conditions by STPKs other than PknD. On the contrary, when PknD-deficient mutant mycobacteria were grown in a phosphate-depleted medium, MDH was not detectably phosphorylated. These results suggest that although the MDH is a substrate of several mycobacterial STPKs, the activity of these kinases can depend on the environment, as we identified PknD as a key element in the MDH phosphorylation assay under phosphate-poor conditions.
Full Text Available Phosphodiesterase enzymes, involved in cAMP hydrolysis reaction, are present throughout phylogeny and their phosphorylation mediated regulation remains elusive in prokaryotes. In this context, we focused on this enzyme from Mycobacterium tuberculosis. The gene encoded by Rv0805 was PCR amplified and expressed as a histidine-tagged protein (mPDE utilizing Escherichia coli based expression system. In kinase assays, upon incubation with mycobacterial Clade I eukaryotic-type Ser/Thr kinases (PknA, PknB and PknL, Ni-NTA purified mPDE protein exhibited transphosphorylation ability albeit with varying degree. When mPDE was co-expressed one at a time with these kinases in E. coli, it was also recognized by an anti-phosphothreonine antibody, which further indicates its phosphorylating ability. Mass spectrometric analysis identified Thr-309 of mPDE as a phosphosite. In concordance with this observation, anti-phosphothreonine antibody marginally recognized mPDE-T309A mutant protein; however, such alteration did not affect the enzymatic activity. Interestingly, mPDE expressed in Mycobacterium smegmatis yielded a phosphorylated protein that preferentially localized to cell wall. In contrast, mPDE-T309A, the phosphoablative variant of mPDE, did not show such behaviour. On the other hand, phosphomimics of mPDE (T309D or T309E, exhibited similar cell wall anchorage as was observed with the wild-type. Thus, our results provide credence to the fact that eukaryotic-type Ser/Thr kinase mediated phosphorylation of mPDE renders negative charge to the protein, promoting its localization on cell wall. Furthermore, multiple sequence alignment revealed that Thr-309 is conserved among mPDE orthologs of M. tuberculosis complex, which presumably emphasizes evolutionary significance of phosphorylation at this residue.
Full Text Available ATP-dependent Mur ligases (Mur synthetases play essential roles in the biosynthesis of cell wall peptidoglycan (PG as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery.
Ortega, Corrie; Liao, Reiling; Anderson, Lindsey N.; Rustad, Tige; Ollodart, Anja R.; Wright, Aaron T.; Sherman, David R.; Grundner, Christoph
In the majority of cases, Mycobacterium tuberculosis (Mtb) infections are clinically latent, characterized by little or no bacterial replication and drug tolerance. Low oxygen tension is a major host factor inducing bacteriostasis, but the molecular mechanisms driving oxygen-dependent replication are poorly understood. Mtb encodes eleven serine/threonine protein kinases, a family of signaling molecules known to regulate similar replicative adaptations in other bacteria. Here, we tested the role of serine/threonine phosphorylation in the Mtb response to altered oxygen status, using an in vitro model of latency (hypoxia) and reactivation (reaeration). Broad kinase inhibition compromised survival of Mtb in hypoxia. Activity-based protein profiling and genetic mutation identified PknB as the kinase critical for surviving hypoxia. Mtb replication was highly sensitive to changes in PknB levels in aerated culture, and even more so in hypoxia. A mutant overexpressing PknB specifically in hypoxia showed a 10-fold loss in viability in low oxygen conditions. In contrast, chemically reducing PknB activity during hypoxia specifically compromised resumption of growth during reaeration. These data support a model in which PknB activity is reduced to achieve bacteriostasis, and elevated when replication resumes. Together, these data show that phosphosignaling controls replicative transitions associated with latency and reactivation, that PknB is a major regulator of these transitions, and that PknB could provide a highly vulnerable therapeutic target at every step of the Mtb life cycle - active disease, latency, and reactivation.
Kerstin A Wolff
Full Text Available Survival of M. tuberculosis in host macrophages requires the eukaryotic-type protein kinase G, PknG, but the underlying mechanism has remained unknown. Here, we show that PknG is an integral component of a novel redox homeostatic system, RHOCS, which includes the ribosomal protein L13 and RenU, a Nudix hydrolase encoded by a gene adjacent to pknG. Studies in M. smegmatis showed that PknG expression is uniquely induced by NADH, which plays a key role in metabolism and redox homeostasis. In vitro, RenU hydrolyses FAD, ADP-ribose and NADH, but not NAD+. Absence of RHOCS activities in vivo causes NADH and FAD accumulation, and increased susceptibility to oxidative stress. We show that PknG phosphorylates L13 and promotes its cytoplasmic association with RenU, and the phosphorylated L13 accelerates the RenU-catalyzed NADH hydrolysis. Importantly, interruption of RHOCS leads to impaired mycobacterial biofilms and reduced survival of M. tuberculosis in macrophages. Thus, RHOCS represents a checkpoint in the developmental program required for mycobacterial growth in these environments.
Full Text Available NP_542171.1 chr6 Crystal Structure of Protein kinase PknG from Mycobacterium tuberc...ulosis in Complex with Tetrahydrobenzothiophene AX20017 p2pzia_ chr6/NP_542171.1/NP_542171.1_holo_5-542.pdb
Full Text Available NP_076412.3 chr1 Crystal Structure of Protein kinase PknG from Mycobacterium tuberc...ulosis in Complex with Tetrahydrobenzothiophene AX20017 p2pzia_ chr1/NP_076412.3/NP_076412.3_holo_1-581.pdb
Full Text Available NP_055427.2 chr9 Crystal Structure of Protein kinase PknG from Mycobacterium tuberc...ulosis in Complex with Tetrahydrobenzothiophene AX20017 p2pzia_ chr9/NP_055427.2/NP_055427.2_holo_395-1115.p
Prasad, U Venkateswara; Vasu, D; Yeswanth, S; Swarupa, V; Sunitha, M M; Choudhary, A; Sarma, P V G K
Isocitrate dehydrogenase (IDH) gene from Staphylococcus aureus ATCC12600 was cloned, sequenced and characterized (HM067707). PknB site was observed in the active site of IDH; thus, it was predicted as IDH may be regulated by phosphorylation. Therefore, in this study, PknB, alkaline phosphatase III (SAOV 2675) and IDH genes (JN695616, JN645811 and HM067707) of S. aureus ATCC12600 were over expressed from clones PV 1, UVPALP-3 and UVIDH 1. On passing the cytosloic fractions through nickel metal chelate column, pure enzymes were obtained. Phosphorylation of pure IDH by PknB resulted in the complete loss of activity and was restored upon dephosphorylation with SAOV 2675 which indicated that phosphorylation and dephosphorylation regulate IDH activity in S. aureus. Further, when S. aureus ATCC12600 was grown in BHI broth, decreased IDH activity and increased biofilm units were observed; therefore, this regulation of IDH alters redox status in this pathogen favouring biofilm formation.
Mueller, Philipp; Pieters, Jean
Signal transduction in bacteria is generally mediated via two-component systems. These systems depend on the transfer of a phosphate molecule from a donor to an acceptor by histidine kinases, thereby activating the acceptor to allow downstream signaling/activation. Several bacterial genomes, including the genome of M. tuberculosis, were shown to encode eukaryotic-like kinases. To better understand the function of these kinases and the regulatory networks within which they operate, identification of downstream targets is essential. We here present a straightforward approach for the identification of bacterial Ser/Thr-kinase substrates. This approach is based on the KESTREL (Kinase Tracking and Substrate Elucidation) procedure combined with reversed-phase chromatography and two-dimensional gel electrophoresis. Using this method, GarA was identified as one potential substrate for the mycobacterial Ser/Thr-protein kinase G (PknG). These results show that the modified KESTREL approach can be successfully employed for the identification of substrates for bacterial Ser/Thr-kinases.
Grimm, Alejandro; Skoglund, Nils; Eriksson, Gunnar; Bostroem, Dan; Oehman, Marcus
The results from this project show that phosphorous-rich additives could be of interest to reduce fouling and high temperature corrosion without causing increase in slagging and/or bed agglomeration tendency for typical biofuels. General results in this series of experiments show that in order to achieve a good potassium-binding effect the calcium and magnesium content should be low in the phosphorous-rich fuel and additive. If the content of Ca and Mg is high in the final fuel mixture (including both P-fuel/additive and the bulk fuel) the K-binding effect is reduced and more P needs to be added. Of course, this also means that the additive of choice (fuel or chemical) should have a low content of calcium and magnesium. It is therefore probable that the best results will be obtained when using a fuel mix where the final blend has a molar ratio of P/(K+Na+2/3Ca+2/3Mg) approaching 1. For instance, using monoammonium phosphate, this would be equivalent to a cost (autumn 2009) of about 9-14, 10-15 and 30-40 SEK in P-additive cost per MWh of added fuel to achieve this molar ratio for typical logging residue, salix and wheat straw biofuels.
Touchette, Megan H.; Bommineni, Gopal R.; Delle Bovi, Richard J.; Gadbery, John; Nicora, Carrie D.; Shukla, Anil K.; Kyle, Jennifer E.; Metz, Thomas O.; Martin, Dwight W.; Sampson, Nicole S.; Miller, W. T.; Tonge, Peter J.; Seeliger, Jessica C.
Although classified as Gram-positive bacteria, Corynebacterineae possess an asymmetric outer membrane that imparts structural and thereby physiological similarity to more distantly related Gram-negative bacteria. Like lipopolysaccharide in Gram-negative bacteria, lipids in the outer membrane of Corynebacterineae have been associated with the virulence of pathogenic species such as Mycobacterium tuberculosis (Mtb). For example, Mtb strains that lack long, branched-chain alkyl esters known as dimycocerosates (DIMs) are significantly attenuated in model infections. The resultant interest in the biosynthetic pathway of these unusual virulence factors has led to the elucidation of many of the steps leading to the final esterification of the alkyl beta-diol, phthiocerol, with branched-chain fatty acids know as mycocerosates. PapA5 is an acyltransferase implicated in these final reactions. We here show that PapA5 is indeed the terminal enzyme in DIM biosynthesis by demonstrating its dual esterification activity and chain-length preference using synthetic alkyl beta-diol substrate analogues. Applying these analogues to a series of PapA5 mutants, we also revise a model for the substrate binding within PapA5. Finally, we demonstrate that the Mtb Ser/Thr kinase PknB modifies PapA5 on three Thr residues, including two (T196, T198) located on an unresolved loop. These results clarify the DIM biosynthetic pathway and suggest possible mechanisms by which DIM biosynthesis may be regulated by the post-translational modification of PapA5.
Kirubavathy, S. Jone; Velmurugan, R.; Karvembu, R.; Bhuvanesh, N. S. P.; Enoch, Israel V. M. V.; Selvakumar, P. Mosae; Premnath, D.; Chitra, S.
Novel Schiff bases derived from the treatment of mercapto-diamino pyrimidine with two different aldehydes are characterized using elemental analysis, single crystal X-ray diffraction and 1H NMR spectroscopy. The pharmacological action of the synthesized compounds viz., antimicrobial, anticancer and antitubercular activities is studied. The Schiff bases show a very good activity against various test pathogens. DNA and β-CD binding interactions of the compounds are studied using UV-Visible absorption and fluorescence spectral measurements. The binding constants of the compounds towards β-CD are in the order of 103 to 104. Molecular docking is done using MOE program on the 3D structure of the enzymes, viz., human thymidylate synthase complexed with dump and raltitrex, candida albicans N-myristoyltransferasepeptidic inhibitor, catalytic domain of protein kinase pKnb from mycobacterium tuberculosis in complex with mitoxantrone, pare, topoisomerase atpase inhibitor, E. coli and lactobacillus casdihydrofolatereductase. The MIC/IC50 values of the Schiff bases are compared with the glide scores from the molecular docking studies. The number of hydrogen bonding interactions between the Schiff bases and amino acid residues are also reported.
Full Text Available The bacterial divisome is a multiprotein complex. Specific protein-protein interactions specify whether cell division occurs optimally, or whether division is arrested. Little is known about these protein-protein interactions and their regulation in mycobacteria. We have investigated the interrelationship between the products of the Mycobacterium tuberculosis gene cluster Rv0014c-Rv0019c, namely PknA (encoded by Rv0014c and FtsZ-interacting protein A, FipA (encoded by Rv0019c and the products of the division cell wall (dcw cluster, namely FtsZ and FtsQ. M. smegmatis strains depleted in components of the two gene clusters have been complemented with orthologs of the respective genes of M. tuberculosis. Here we identify FipA as an interacting partner of FtsZ and FtsQ and establish that PknA-dependent phosphorylation of FipA on T77 and FtsZ on T343 is required for cell division under oxidative stress. A fipA knockout strain of M. smegmatis is less capable of withstanding oxidative stress than the wild type and showed elongation of cells due to a defect in septum formation. Localization of FtsQ, FtsZ and FipA at mid-cell was also compromised. Growth and survival defects under oxidative stress could be functionally complemented by fipA of M. tuberculosis but not its T77A mutant. Merodiploid strains of M. smegmatis expressing the FtsZ(T343A showed inhibition of FtsZ-FipA interaction and Z ring formation under oxidative stress. Knockdown of FipA led to elongation of M. tuberculosis cells grown in macrophages and reduced intramacrophage growth. These data reveal a novel role of phosphorylation-dependent protein-protein interactions involving FipA, in the sustenance of mycobacterial cell division under oxidative stress.
Puniya, Bhanwar Lal; Kulshreshtha, Deepika; Verma, Srikant Prasad; Kumar, Sanjiv; Ramachandran, Srinivasan
We have carried out weighted gene co-expression network analysis of Mycobacterium tuberculosis to gain insights into gene expression architecture during log phase growth. The differentially expressed genes between at least one pair of 11 different M. tuberculosis strains as source of biological variability were used for co-expression network analysis. This data included genes with highest coefficient of variation in expression. Five distinct modules were identified using topological overlap based clustering. All the modules together showed significant enrichment in biological processes: fatty acid biosynthesis, cell membrane, intracellular membrane bound organelle, DNA replication, Quinone biosynthesis, cell shape and peptidoglycan biosynthesis, ribosome and structural constituents of ribosome and transposition. We then extracted the co-expressed connections which were supported either by transcriptional regulatory network or STRING database or high edge weight of topological overlap. The genes trpC, nadC, pitA, Rv3404c, atpA, pknA, Rv0996, purB, Rv2106 and Rv0796 emerged as top hub genes. After overlaying this network on the iNJ661 metabolic network, the reactions catalyzed by 15 highly connected metabolic genes were knocked down in silico and evaluated by Flux Balance Analysis. The results showed that in 12 out of 15 cases, in 11 more than 50% of reactions catalyzed by genes connected through co-expressed connections also had altered fluxes. The modules 'Turquoise', 'Blue' and 'Red' also showed enrichment in essential genes. We could map 152 of the previously known or proposed drug targets in these modules and identified 15 new potential drug targets based on their high degree of co-expressed connections and strong correlation with module eigengenes.
Shao, Qing-Chun; Zhang, Cui-Juan; Li, Jie
The protein kinase C (PKC) is a family of serine/threonine kinases with a broad range of cellular targets. Members of the PKC family participate at the diverse biological events involved in cellular proliferation, differentiation and survival. The PKC isoform zeta (PKCζ) is an atypical member that has recently been found to play an essential role in promoting human uterine contractility and thus been raised as a new target for treating preterm labour and other tocolytic diseases. In this study, an integrative protocol was described to graft hundreds of inhibitor ligands from their complex crystal structures with cognate kinases into the active pocket of PKCζ and, based on the modeled structures, to evaluate the binding strength of these inhibitors to the non-cognate PKCζ receptor by using a consensus scoring strategy. A total of 32 inhibitors with top score were compiled, and eight out of them were tested for inhibitory potency against PKCζ. Consequently, five compounds, i.e. CDK6 inhibitor fisetin, PIM1 inhibitor myricetin, CDK9 inhibitor flavopiridol and PknB inhibitor mitoxantrone as well as the promiscuous kinase inhibitor staurosporine showed high or moderate inhibitory activity on PKCζ, with IC50 values of 58 ± 9, 1.7 ± 0.4, 108 ± 17, 280 ± 47 and 0.019 ± 0.004 μM, respectively, while other three compounds, including two marketed drugs dasatinib and sunitinib as well as the Rho inhibitor fasudil, have not been detected to possess observable activity. Next, based on the modeled structure data we modified three flavonoid kinase inhibitors, i.e. fisetin, myricetin and flavopiridol, to generate a number of more potential molecular entities, two of which were found to have a moderately improved activity as compared to their parent compounds.
Touchette, Megan H; Bommineni, Gopal R; Delle Bovi, Richard J; Gadbery, John E; Nicora, Carrie D; Shukla, Anil K; Kyle, Jennifer E; Metz, Thomas O; Martin, Dwight W; Sampson, Nicole S; Miller, W Todd; Tonge, Peter J; Seeliger, Jessica C
Although they are classified as Gram-positive bacteria, Corynebacterineae possess an asymmetric outer membrane that imparts structural and thereby physiological similarity to more distantly related Gram-negative bacteria. Like lipopolysaccharide in Gram-negative bacteria, lipids in the outer membrane of Corynebacterineae have been associated with the virulence of pathogenic species such as Mycobacterium tuberculosis (Mtb). For example, Mtb strains that lack long, branched-chain alkyl esters known as dimycocerosates (DIMs) are significantly attenuated in model infections. The resultant interest in the biosynthetic pathway of these unusual virulence factors has led to the elucidation of many of the steps leading to the final esterification of the alkyl β-diol, phthiocerol, with branched-chain fatty acids known as mycocerosates. PapA5 is an acyltransferase implicated in these final reactions. Here, we show that PapA5 is indeed the terminal enzyme in DIM biosynthesis by demonstrating its dual esterification activity and chain-length preference using synthetic alkyl β-diol substrate analogues. By applying these analogues to a series of PapA5 mutants, we also revise a model for the substrate binding within PapA5. Finally, we demonstrate that the Mtb Ser/Thr kinases PknB and PknE modify PapA5 on three overlapping Thr residues and that a fourth Thr is unique to PknE phosphorylation. These results clarify the DIM biosynthetic pathway and indicate post-translational modifications that warrant further elucidation for their roles in the regulation of DIM biosynthesis.
赵海燕; 孙志国; 管永祥; 李刚华; 郑青松; 梁永红; 罗朝晖; 金白云
Objective]Fertilizer,especially chemical fertilizer,contributes significantly to the modern agricultural production. However,in recent years,fertilizer consumption has been increasing exponentially throughout the world and,as a result,causing a series of serious environmental problems. The invention and use of water retaining controlled-release fertilizer(WRCRF)is a promising approach to improving utilization of the water resources and fertilizer nutrients,and pursuing sustainable development of the environment and agriculture. Some fertilizers can also be used to alleviate salt stress of crop plants,such as urea,manure,etc. However,little has been reported on application of WRCRF to plants under salt stress. Recently,a study has been done finding that application of WRCRF(ZL 2012 1 0400570.0)may improve salt tolerance of rice seedlings significantly,which may be attributed to its effects on root growth,content of leaf chlorophyll,photosynthesis and water use efficiency.[Method]To validate the finding a pot experiment was carried out on effects of WRCRF on leaf length,leaf temperature,and absorption and translocation of N, P,K,Na of rice seedlings under salt stress for 20,40 and 80 d,separately. Rice seeds were sown in pots filled with natural soil,2.68 g kg-1 in salinity. Twenty days later,half of the pots were amended with sodium chloride(NaCl)through irrigation to make the soil up to 4.68 g kg-1 in salinity. WRCRF was applied at 0, 1,2 and 4 g kg-1,separately to the pots.[Result]Results show as follows.(1)Maximum leaf length of the rice seedlings increased with application rate of WRCRF regardless of salt stress and duration of treatment. WRCRF application decreased leaf surface temperature of the rice plants under salt stress,and the higher the application rate of WRCRF,the higher the effect. However,the plants gradually died of high salinity. (2)The fertilizer increased the contents of N,P and K in the plants under salt stress,but lowered the content of Na