Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Bacillus anthracis has been employed as an agent of bioterrorism, with high mortality, despite anti-microbial treatment, which strongly indicates the need of new drugs to treat anthrax. Shikimate pathway is a seven step biosynthetic route which generates chorismic acid from phosphoenol pyruvate and erythrose-4-phosphate. Chorismic acid is the major branch point in the synthesis of aromatic amino acids, ubiquinone, and secondary metabolites. The shikimate pathway is essential for many pathological organisms, whereas it is absent in mammals. Therefore, these enzymes are potential targets for the development of nontoxic antimicrobial agents and herbicides and have been submitted to intensive structural studies. The forth enzyme of this pathway is responsible for the conversion of dehydroshikimate to shikimate in the presence of NADP. In order to pave the way for structural and functional efforts toward development of new antimicrobials we describe the molecular modeling of shikimate dehydrogenase from Bacillus anthracis complexed with the cofactor NADP. This study was able to identify the main residues of the NADP binding site responsible for ligand affinities. This structural study can be used in the design of more specific drugs against infectious diseases.
J Mol Model 2009 Feb
PMID:Structural studies of shikimate dehydrogenase from Bacillus anthracis complexed with cofactor NADP. 1904 50

Bacillus anthracis causes anthrax disease and exerts its deleterious effects by the release of three exotoxins: lethal factor, protective antigen, and edema factor (EF), a highly active calmodulin-dependent adenylyl cyclase (AC). However, conventional antibiotic treatment is ineffective against either toxemia or antibiotic-resistant strains. Thus, more effective drugs for anthrax treatment are needed. Previous studies from our laboratory showed that mammalian membranous AC (mAC) exhibits broad specificity for purine and pyrimidine nucleotides ( Mol Pharmacol 70: 878-886, 2006 ). Here, we investigated structural requirements for EF inhibition by natural purine and pyrimidine nucleotides and nucleotides modified with N-methylanthraniloyl (MANT)- or anthraniloyl groups at the 2'(3')-O-ribosyl position. MANT-CTP was the most potent EF inhibitor (K(i), 100 nM) among 16 compounds studied. MANT-nucleotides inhibited EF competitively. Activation of EF by calmodulin resulted in effective fluorescence resonance energy transfer (FRET) from tryptophan and tyrosine residues located in the vicinity of the catalytic site to MANT-ATP, but FRET to MANT-CTP was only small. Mutagenesis studies revealed that Phe586 is crucial for FRET to MANT-ATP and MANT-CTP and that the mutations N583Q, K353A, and K353R differentially alter the inhibitory potencies of MANT-ATP and MANT-CTP. Docking approaches relying on crystal structures of EF indicate similar binding modes of the MANT nucleotides with subtle differences in the region of the nucleobases. In conclusion, like mAC, EF accommodates both purine and pyrimidine nucleotides. The unique preference of EF for the base cytosine offers an excellent starting point for the development of potent and selective EF inhibitors.
Mol Pharmacol 2009 Mar
PMID:Molecular analysis of the interaction of anthrax adenylyl cyclase toxin, edema factor, with 2'(3')-O-(N-(methyl)anthraniloyl)-substituted purine and pyrimidine nucleotides. 1905 99

Antibodies provide a sensitive indicator of proteins displayed by bacteria during sepsis. Because signals produced by infection are naturally amplified during the antibody response, host immunity can be used to identify biomarkers for proteins that are present at levels currently below detectable limits. We developed a microarray comprising approximately 70% of the 4066 proteins contained within the Yersinia pestis proteome to identify antibody biomarkers distinguishing plague from infections caused by other bacterial pathogens that may initially present similar clinical symptoms. We first examined rabbit antibodies produced against proteomes extracted from Y. pestis, Burkholderia mallei, Burkholderia cepecia, Burkholderia pseudomallei, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella flexneri, and Escherichia coli, all pathogenic Gram-negative bacteria. These antibodies enabled detection of shared cross-reactive proteins, fingerprint proteins common for two or more bacteria, and signature proteins specific to each pathogen. Recognition by rabbit and non-human primate antibodies involved less than 100 of the thousands of proteins present within the Y. pestis proteome. Further antigen binding patterns were revealed that could distinguish plague from anthrax, caused by the Gram-positive bacterium Bacillus anthracis, using sera from acutely infected or convalescent primates. Thus, our results demonstrate potential biomarkers that are either specific to one strain or common to several species of pathogenic bacteria.
Mol Cell Proteomics 2009 May
PMID:Extensive antibody cross-reactivity among infectious gram-negative bacteria revealed by proteome microarray analysis. 1911 81

Gentamicin is a potent antibiotic that is used in combination therapy for inhalation anthrax disease. The drug is also often used in therapy for methicillin-resistant Staphylococcusaureus. Gentamicin works by flipping a conformational switch on the ribosome, disrupting the reading head (i.e., 16S ribosomal decoding bases 1492-1493) used for decoding messenger RNA. We use explicit solvent all-atom molecular simulation to study the thermodynamics of the ribosomal decoding site and its interaction with gentamicin. The replica exchange molecular dynamics simulations used an aggregate sampling of 15 mus when summed over all replicas, allowing us to explicitly calculate the free-energy landscape, including a rigorous treatment of enthalpic and entropic effects. Here, we show that the decoding bases flip on a timescale faster than that of gentamicin binding, supporting a stochastic gating mechanism for antibiotic binding, rather than an induced-fit model where the bases only flip in the presence of a ligand. The study also allows us to explore the nonspecific binding landscape near the binding site and reveals that, rather than a two-state bound/unbound scenario, drug dissociation entails shuttling between many metastable local minima in the free-energy landscape. Special care is dedicated to validation of the obtained results, both by direct comparison to experiment and by estimation of simulation convergence.
J Mol Biol 2009 Feb 27
PMID:Stochastic gating and drug-ribosome interactions. 1914 58

Regulated expression of the genes for anthrax toxin proteins is essential for the virulence of the pathogenic bacterium Bacillus anthracis. Induction of toxin gene expression depends on several factors, including temperature, bicarbonate levels, and metabolic state of the cell. To identify factors that regulate toxin expression, transposon mutagenesis was performed under non-inducing conditions and mutants were isolated that untimely expressed high levels of toxin. A number of these mutations clustered in the haem biosynthetic and cytochrome c maturation pathways. Genetic analysis revealed that two haem-dependent, small c-type cytochromes, CccA and CccB, located on the extracellular surface of the cytoplasmic membrane, regulate toxin gene expression by affecting the expression of the master virulence regulator AtxA. Deregulated AtxA expression in early exponential phase resulted in increased expression of toxin genes in response to loss of the CccA-CccB signalling pathway. This is the first function identified for these two small c-type cytochromes of Bacillus species. Extension of the transposon screen identified a previously uncharacterized protein, BAS3568, highly conserved across many bacterial and archeal species, as involved in cytochrome c activity and virulence regulation. These findings are significant not only to virulence regulation in B. anthracis, but also to analysis of virulence regulation in many pathogenic bacteria and to the study of cytochrome c activity in Gram-positive bacteria.
Mol Microbiol 2009 Apr
PMID:Two small c-type cytochromes affect virulence gene expression in Bacillus anthracis. 1922 57

Bacillus anthracis is the etiological agent of anthrax. Protective antigen (PA) has been established as the key protective immunogen and is the major component of anthrax vaccine. Prior studies have indicated that C-terminus host cell receptor binding region contains dominant protective epitopes of PA. In the present study, we focused our attention on determining B-cell epitopes from this region, which could be employed as a vaccine. Using B-cell epitope prediction systems, three regions were identified; ID-I: 604-622, ID-II: 626-676 and ID-III: 707-723 aa residues. These epitopes elicited potent B-cell response in BALB/c mice. ID-II in particular was found to be highly immunogenic in terms of IgG antibody titre, with a predominantly IgG1/IgG2a subclass distribution indicating Th2 bias and high affinity/avidity index. Effective cellular immunity was additionally generated which also signified its Th2 bias. Further, ID-II induced high level of lethal toxin neutralizing antibodies and robust protective immunity (66%) against in vivo lethal toxin challenge. Thus, ID-II can be classified as an immunodominant B-cell epitope and may prove significant in the development of an effective immunoprophylactic strategy against anthrax.
Mol Immunol 2009 Jun
PMID:Identification and characterization of immunodominant B-cell epitope of the C-terminus of protective antigen of Bacillus anthracis. 1935 2

The virulence of Bacillus anthracis is critically dependent on the cytotoxic components of the anthrax toxin, lethal factor (LF) and edema factor (EF). LF and EF gain entry into host cells through interactions with the protective antigen (PA), which binds to host cellular receptors such as CMG2. Antibodies that neutralize PA have been shown to confer protection in animal models and are undergoing intense clinical development. A murine monoclonal antibody, 14B7, has been reported to interact with domain 4 of PA (PAD4) and block its binding to CMG2. More recently, the 14B7 antibody was used as the platform for the selection of very high affinity, single-chain antibodies that have tremendous potential as a combination anthrax prophylactic and treatment. Here, we report the high-resolution X-ray structures of three high-affinity, single-chain antibodies in the 14B7 family; 14B7 and two high-affinity variants 1H and M18. In addition, we present the first neutralizing antibody-PA structure, M18 in complex with PAD4 at 3.8 A resolution. These structures provide insights into the mechanism of neutralization, and the effect of various mutations on antibody affinity, and enable a comparison between the binding of the M18 antibody and CMG2 with PAD4.
J Mol Biol 2009 Apr 03
PMID:Crystal structure of the engineered neutralizing antibody M18 complexed to domain 4 of the anthrax protective antigen. 1936 25

Solid tumor growth is dependent on angiogenesis, the formation of neovasculature from existing vessels. Endothelial activation of the extracellular signal-regulated kinase 1/2, c-jun NH(2)-terminal kinase, and p38 mitogen-activated protein kinase pathways is central to this process, and thus presents an attractive target for the development of angiogenesis inhibitors. Anthrax lethal toxin (LeTx) has potent catalytic mitogen-activated protein kinase inhibition activity. Preclinical studies showed that LeTx induced potent tumor growth inhibition via the inhibition of xenograft vascularization. However, LeTx receptors and the essential furin-like activating proteases are expressed in many normal tissues, potentially limiting the specificity of LeTx as an antitumor agent. To circumvent nonspecific LeTx activation and simultaneously enhance tumor vascular targeting, a substrate preferably cleaved by the gelatinases class of matrix metalloproteinases (MMP) was substituted for the furin LeTx activation site. In vivo efficacy studies showed that this MMP-activated LeTx inhibited tumor xenografts growth via the reduced migration of endothelial cells into the tumor parenchyma. Here we have expanded on these initial findings by showing that this MMP-activated LeTx reduces endothelial proangiogenic MMP expression, thus causing a diminished proteolytic capacity for extracellular matrix remodeling and endothelial differentiation into capillary networks. Additionally, our data suggest that inhibition of the c-jun NH(2)-terminal kinase and p38, but not extracellular signal-regulated kinase-1/2, pathways is significant in the antiangiogenic activity of the MMP-activated LeTx. Collectively, these results support the clinical development of the MMP-activated LeTx for the treatment of solid tumors.
Mol Cancer Res 2009 Apr
PMID:Matrix metalloproteinase-activated anthrax lethal toxin inhibits endothelial invasion and neovasculature formation during in vitro morphogenesis. 1937 76

The scarcity of methods to visualize the activity of individual cell surface proteases in situ has hampered basic research and drug development efforts. In this chapter, we describe a simple, sensitive, and noninvasive assay that uses nontoxic reengineered bacterial cytotoxins with altered protease cleavage specificity to visualize specific cell surface proteolytic activity in single living cells. The assay takes advantage of the absolute requirement for site-specific endoproteolytic cleavage of cell surface-bound anthrax toxin protective antigen for its capacity to translocate an anthrax toxin lethal factor-beta-lactamase fusion protein to the cytoplasm. A fluorogenic beta-lactamase substrate is then used to visualize the cytoplasmically translocated anthrax toxin lethal factor-beta-lactamase fusion protein. By using anthrax toxin protective antigen variants that are reengineered to be cleaved by furin, urokinase plasminogen activator, or metalloproteinases, the cell surface activities of each of these proteases can be specifically and quantitatively determined with single cell resolution. The imaging assay is excellently suited for fluorescence microscope, fluorescence plate reader, and flow cytometry formats, and it can be used for a variety of purposes.
Methods Mol Biol 2009
PMID:Imaging specific cell surface protease activity in living cells using reengineered bacterial cytotoxins. 1937 67

Anthrax toxin is a three-part toxin secreted by Bacillus anthracis, consisting of protective antigen (PrAg), edema factor (EF), and lethal factor (LF). To intoxicate host mammalian cells, PrAg, the cell-binding moiety of the toxin, binds to cells and is then proteolytically activated by furin on the cell surface, resulting in the active heptameric form of PrAg. This heptamer serves as a protein-conducting channel that translocates EF and LF, the two enzymatic moieties of the toxin, into the cytosol of the cells where they exert cytotoxic effects. The anthrax toxin delivery system has been well characterized. The amino-terminal PrAg-binding domain of LF (residues 1-254, LFn) is sufficient to allow translocation of fused "passenger" polypeptides, such as the ADP-ribosylation domain of Pseudomonas exotoxin A, to the cytosol of the cells in a PrAg-dependent process. The protease specificity of the anthrax toxin delivery system can also be reengineered by replacing the furin cleavage target sequence of PrAg with other protease substrate sequences. PrAg-U2 is such a PrAg variant, one that is selectively activated by urokinase plasminogen activator (uPA). The uPA-dependent proteolytic activation of PrAg-U2 on the cell surface is readily detected by western blotting analysis of cell lysates in vitro, or cell or animal death in vivo. Here, we describe the use of PrAg-U2 as a molecular reporter tool to test the controversial question of what components are required for uPAR-mediated cell surface pro-uPA activation. The results demonstrate that both uPAR and plasminogen play critical roles in pro-uPA activation both in vitro and in vivo.
Methods Mol Biol 2009
PMID:Dissecting the urokinase activation pathway using urokinase-activated anthrax toxin. 1937 74


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