58 research outputs found
Hydrazines as versatile chemical biology probes and drug-discovery tools for cofactor-dependent enzymes [preprint]
Known chemoproteomic probes generally use warheads that tag a single type of amino acid or modified form thereof to identify cases in which its hyper-reactivity underpins function. Much important biochemistry derives from electron-poor enzyme cofactors, transient intermediates and chemically-labile regulatory modifications, but probes for such species are underdeveloped. Here, we have innovated a versatile class of chemoproteomic probes for this less charted hemisphere of the proteome by using hydrazine as the common chemical warhead. Its electron-rich nature allows it to react by both polar and radicaloid mechanisms and to target multiple, pharmacologically important functional classes of enzymes bearing diverse organic and inorganic cofactors. Probe attachment can be blocked by active-site-directed inhibitors, and elaboration of the warhead supports connection of a target to a lead compound. The capacity of substituted hydrazines to profile, discover and inhibit diverse cofactor-dependent enzymes enables cell and tissue imaging and makes this platform useful for enzyme and drug discovery
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Thematic Review Series: Proteomics. An integrated omics analysis of eicosanoid biology.
Eicosanoids have been implicated in a vast number of devastating inflammatory conditions, including arthritis, atherosclerosis, pain, and cancer. Currently, over a hundred different eicosanoids have been identified, with many having potent bioactive signaling capacity. These lipid metabolites are synthesized de novo by at least 50 unique enzymes, many of which have been cloned and characterized. Due to the extensive characterization of eicosanoid biosynthetic pathways, this field provides a unique framework for integrating genomics, proteomics, and metabolomics toward the investigation of disease pathology. To facilitate a concerted systems biology approach, this review outlines the proteins implicated in eicosanoid biosynthesis and signaling in human, mouse, and rat. Applications of the extensive genomic and lipidomic research to date illustrate the questions in eicosanoid signaling that could be uniquely addressed by a thorough analysis of the entire eicosanoid proteome
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Lipopolysaccharide-induced cyclooxygenase-2 expression in human U937 macrophages is phosphatidic acid phosphohydrolase-1-dependent.
Cyclooxygenase (COX) has two isoforms, COX-1 and -2, which catalyze the key step in the conversion of cellular arachidonic acid into prostaglandins. In recent years, interest in COX-2 has significantly increased since it has been a target for the development of specific non-steroidal anti-inflammatory drugs. We report that COX-2 expression is up-regulated in phorbol ester (phorbol myristate acetate, PMA)-differentiated human U937 macrophage-like cells stimulated with lipopolysaccharide (LPS), whereas COX-1 is not up-regulated. We show that the LPS-induced up-regulation of COX-2 depends on the activity of the Mg(+2)-dependent phosphatidic acid phosphohydrolase 1 (PAP-1). Inhibition of PAP-1 by bromoenol lactone, propranolol, or ethanol resulted in a decrease in LPS-induced COX-2 mRNA transcript production, COX-2 protein expression, and prostaglandin E(2) release from U937 macrophages. To ensure that these results did not arise because of PMA treatment of the U937 cells, similar experiments were conducted with the P388D(1) cell line, which does not require PMA differentiation. LPS increased the levels of endogenous cellular diacylglycerol (DAG) within 2 min of stimulation. This increase was observed to be sensitive to the PAP-1 inhibitors. Furthermore, phosphatidic acid phosphohydrolase activity assays showed that the bromoenol lactone-sensitive PAP-1 activity was translocated from the cytosolic fraction to the membrane fraction within 2 min of LPS exposure. Finally, DAG add-back experiments demonstrate that LPS-induced COX-2 expression is enhanced by the addition of exogenous DAG
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Lipopolysaccharide-induced cyclooxygenase-2 expression in human U937 macrophages is phosphatidic acid phosphohydrolase-1-dependent.
Cyclooxygenase (COX) has two isoforms, COX-1 and -2, which catalyze the key step in the conversion of cellular arachidonic acid into prostaglandins. In recent years, interest in COX-2 has significantly increased since it has been a target for the development of specific non-steroidal anti-inflammatory drugs. We report that COX-2 expression is up-regulated in phorbol ester (phorbol myristate acetate, PMA)-differentiated human U937 macrophage-like cells stimulated with lipopolysaccharide (LPS), whereas COX-1 is not up-regulated. We show that the LPS-induced up-regulation of COX-2 depends on the activity of the Mg(+2)-dependent phosphatidic acid phosphohydrolase 1 (PAP-1). Inhibition of PAP-1 by bromoenol lactone, propranolol, or ethanol resulted in a decrease in LPS-induced COX-2 mRNA transcript production, COX-2 protein expression, and prostaglandin E(2) release from U937 macrophages. To ensure that these results did not arise because of PMA treatment of the U937 cells, similar experiments were conducted with the P388D(1) cell line, which does not require PMA differentiation. LPS increased the levels of endogenous cellular diacylglycerol (DAG) within 2 min of stimulation. This increase was observed to be sensitive to the PAP-1 inhibitors. Furthermore, phosphatidic acid phosphohydrolase activity assays showed that the bromoenol lactone-sensitive PAP-1 activity was translocated from the cytosolic fraction to the membrane fraction within 2 min of LPS exposure. Finally, DAG add-back experiments demonstrate that LPS-induced COX-2 expression is enhanced by the addition of exogenous DAG
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