ABSTRACT
In neutrophils, nicotinamide adenine dinucleotide phosphate (NADPH) generated via the pentose phosphate pathway fuels NADPH oxidase NOX2 to produce reactive oxygen species for killing invading pathogens. However, excessive NOX2 activity can exacerbate inflammation, as in acute respiratory distress syndrome (ARDS). Here, we use two unbiased chemical proteomic strategies to show that small-molecule LDC7559, or a more potent designed analog NA-11, inhibits the NOX2-dependent oxidative burst in neutrophils by activating the glycolytic enzyme phosphofructokinase-1 liver type (PFKL) and dampening flux through the pentose phosphate pathway. Accordingly, neutrophils treated with NA-11 had reduced NOX2-dependent outputs, including neutrophil cell death (NETosis) and tissue damage. A high-resolution structure of PFKL confirmed binding of NA-11 to the AMP/ADP allosteric activation site and explained why NA-11 failed to agonize phosphofructokinase-1 platelet type (PFKP) or muscle type (PFKM). Thus, NA-11 represents a tool for selective activation of PFKL, the main phosphofructokinase-1 isoform expressed in immune cells.
Subject(s)
Phagocytosis , Phosphofructokinase-1, Liver Type/metabolism , Respiratory Burst , Adenosine Diphosphate/metabolism , Adenosine Monophosphate/metabolism , Allosteric Regulation/drug effects , Enzyme Activation/drug effects , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Glycolysis/drug effects , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Kinetics , Microbial Viability/drug effects , Models, Molecular , NADPH Oxidases/metabolism , Neutrophils/drug effects , Neutrophils/metabolism , Phagocytosis/drug effects , Phosphate-Binding Proteins/metabolism , Phosphofructokinase-1, Liver Type/antagonists & inhibitors , Phosphofructokinase-1, Liver Type/ultrastructure , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Recombinant Proteins/isolation & purification , Respiratory Burst/drug effects , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
Cancer cells must satisfy the metabolic demands of rapid cell growth within a continually changing microenvironment. We demonstrated that the dynamic posttranslational modification of proteins by O-linked ß-N-acetylglucosamine (O-GlcNAcylation) is a key metabolic regulator of glucose metabolism. O-GlcNAcylation was induced at serine 529 of phosphofructokinase 1 (PFK1) in response to hypoxia. Glycosylation inhibited PFK1 activity and redirected glucose flux through the pentose phosphate pathway, thereby conferring a selective growth advantage on cancer cells. Blocking glycosylation of PFK1 at serine 529 reduced cancer cell proliferation in vitro and impaired tumor formation in vivo. These studies reveal a previously uncharacterized mechanism for the regulation of metabolic pathways in cancer and a possible target for therapeutic intervention.
