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1.
Redox Biol ; 11: 663-672, 2017 04.
Article in English | MEDLINE | ID: mdl-28160743

ABSTRACT

Activated platelets generate an eicosanoid proposed to be 8-hydroxy-9,10-dioxolane A3 (DXA3). Herein, we demonstrate that significant amounts of DXA3 are rapidly attached to phosphatidylethanolamine (PE) forming four esterified eicosanoids, 16:0p, 18:0p, 18:1p and 18:0a/DXA3-PEs that can activate neutrophil integrin expression. These lipids comprise the majority of DXA3 generated by platelets, are formed in ng amounts (24.3±6.1ng/2×108) and remain membrane bound. Pharmacological studies revealed DXA3-PE formation involves cyclooxygenase-1 (COX), protease-activated receptors (PAR) 1 and 4, cytosolic phospholipase A2 (cPLA2), phospholipase C and intracellular calcium. They are generated primarily via esterification of newly formed DXA3, but can also be formed in vitro via co-oxidation of PE during COX-1 co-oxidation of arachidonate. All four DXA3-PEs were detected in human clots. Purified platelet DXA3-PE activated neutrophil Mac-1 expression, independently of its hydrolysis to the free eicosanoid. This study demonstrates the structures and cellular synthetic pathway for a family of leukocyte-activating platelet phospholipids generated on acute activation, adding to the growing evidence that enzymatic PE oxidation is a physiological event in innate immune cells.


Subject(s)
Blood Platelets/metabolism , Dioxolanes/blood , Integrins/blood , Lipids/blood , Phosphatidylethanolamines/blood , Calcium/blood , Cyclooxygenase 1/blood , Eicosanoids/blood , Gene Expression Regulation , Humans , Integrins/biosynthesis , Macrophage-1 Antigen/genetics , Neutrophils/metabolism , Oxidation-Reduction , Phospholipases A2, Cytosolic/blood , Platelet Activation/genetics , Receptor, PAR-1/blood , Receptors, Thrombin/blood , Thrombin/metabolism , Type C Phospholipases/blood
2.
Anal Chem ; 88(6): 3107-14, 2016 Mar 15.
Article in English | MEDLINE | ID: mdl-26891127

ABSTRACT

Although tumor hypoxia is associated with tumor aggressiveness and resistance to cancer treatment, many details of hypoxia-induced changes in tumors remain to be elucidated. Mass spectrometry imaging (MSI) is a technique that is well suited to study the biomolecular composition of specific tissue regions, such as hypoxic tumor regions. Here, we investigate the use of pimonidazole as an exogenous hypoxia marker for matrix-assisted laser desorption/ionization (MALDI) MSI. In hypoxic cells, pimonidazole is reduced and forms reactive products that bind to thiol groups in proteins, peptides, and amino acids. We show that a reductively activated pimonidazole metabolite can be imaged by MALDI-MSI in a breast tumor xenograft model. Immunohistochemical detection of pimonidazole adducts on adjacent tissue sections confirmed that this metabolite is localized to hypoxic tissue regions. We used this metabolite to image hypoxic tissue regions and their associated lipid and small molecule distributions with MALDI-MSI. We identified a heterogeneous distribution of 1-methylnicotinamide and acetylcarnitine, which mostly colocalized with hypoxic tumor regions. As pimonidazole is a widely used immunohistochemical marker of tissue hypoxia, it is likely that the presented direct MALDI-MSI approach is also applicable to other tissues from pimonidazole-injected animals or humans.


Subject(s)
Biomarkers, Tumor/metabolism , Breast Neoplasms/metabolism , Nitroimidazoles/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Female , Humans
3.
ACS Synth Biol ; 4(7): 796-807, 2015 Jul 17.
Article in English | MEDLINE | ID: mdl-25713978

ABSTRACT

A key problem in the engineering of pathways for the production of pharmaceutical compounds is the limited diversity of biosynthetic enzymes, which restricts the attainability of suitable traits such as less harmful byproducts, enhanced expression features, or different cofactor requirements. A promising synthetic biology approach is to redesign the biosynthetic pathway by replacing the native enzymes by heterologous proteins from unrelated pathways. In this study, we applied this method to effectively re-engineer the biosynthesis of hydroxyphenylglycine (HPG), a building block for the calcium-dependent antibiotic of Streptomyces coelicolor, a nonribosomal peptide. A key step in HPG biosynthesis is the conversion of 4-hydroxymandelate to 4-hydroxyphenylglyoxylate, catalyzed by hydroxymandelate oxidase (HmO), with concomitant generation of H2O2. The same reaction can also be catalyzed by O2-independent mandelate dehydrogenase (MdlB), which is a catabolic enzyme involved in bacterial mandelate utilization. In this work, we engineered alternative HPG biosynthetic pathways by replacing the native HmO in S. coelicolor by both heterologous oxidases and MdlB dehydrogenases from various sources and confirmed the restoration of calcium-dependent antibiotic biosynthesis by biological and UHPLC-MS analysis. The alternative enzymes were isolated and kinetically characterized, confirming their divergent substrate specificities and catalytic mechanisms. These results demonstrate that heterologous enzymes with different physiological contexts can be used in a Streptomyces host to provide an expanded library of enzymatic reactions for a synthetic biology approach. This study thus broadens the options for the engineering of antibiotic production by using enzymes with different catalytic and structural features.


Subject(s)
Alcohol Oxidoreductases/metabolism , Anti-Bacterial Agents/biosynthesis , Glycine/analogs & derivatives , Oxidoreductases/metabolism , Alcohol Oxidoreductases/classification , Anti-Bacterial Agents/analysis , Anti-Bacterial Agents/chemistry , Chromatography, High Pressure Liquid , Glycine/biosynthesis , Glycine/chemistry , Glyoxylates/chemistry , Glyoxylates/metabolism , Hydrogen Peroxide/metabolism , Mass Spectrometry , Oxidoreductases/classification , Phylogeny , Plasmids/metabolism , Streptomyces coelicolor/chemistry , Streptomyces coelicolor/metabolism
4.
J Lipid Res ; 54(11): 3085-97, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23883581

ABSTRACT

Oxidized phospholipids (oxPLs) generated nonenzymatically display pleiotropic biological actions in inflammation. Their generation by cellular cyclooxygenases (COXs) is currently unknown. To determine whether platelets generate prostaglandin (PG)-containing oxPLs, then characterize their structures and mechanisms of formation, we applied precursor scanning-tandem mass spectrometry to lipid extracts of agonist-activated human platelets. Thrombin, collagen, or ionophore activation stimulated generation of families of PGs comprising PGE2 and D2 attached to four phosphatidylethanolamine (PE) phospholipids (16:0p/, 18:1p/, 18:0p/, and 18:0a/). They formed within 2 to 5 min of activation in a calcium, phospholipase C, p38 MAP kinases, MEK1, cPLA2, and src tyrosine kinase-dependent manner (28.1 ± 2.3 pg/2 × 108 platelets). Unlike free PGs, they remained cell associated, suggesting an autocrine mode of action. Their generation was inhibited by in vivo aspirin supplementation (75 mg/day) or in vitro COX-1 blockade. Inhibitors of fatty acyl reesterification blocked generation significantly, while purified COX-1 was unable to directly oxidize PE in vitro. This indicates that they form in platelets via rapid esterification of COX-1 derived PGE2/D2 into PE. In summary, COX-1 in human platelets acutely mediates membrane phospholipid oxidation via formation of PG-esterified PLs in response to pathophysiological agonists.


Subject(s)
Aspirin/pharmacology , Blood Platelets/drug effects , Blood Platelets/metabolism , Cyclooxygenase 1/metabolism , Cyclooxygenase Inhibitors/pharmacology , Phospholipids/metabolism , Prostaglandins/metabolism , Blood Platelets/physiology , Calcium/metabolism , Dinoprostone/metabolism , Dose-Response Relationship, Drug , Esterification/drug effects , Feedback, Physiological/drug effects , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , MAP Kinase Kinase 1/metabolism , Phosphatidylethanolamines/metabolism , Platelet Activation/drug effects , Prostaglandin D2/metabolism , Protein Kinase C/metabolism , Receptor, PAR-1/metabolism , Thrombin/metabolism , src-Family Kinases/metabolism
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