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
Despite abundant knowledge of the regulation and biochemistry of glycolytic enzymes, we have limited understanding on how they are spatially organized in the cell. Emerging evidence indicates that nonglycolytic metabolic enzymes regulating diverse pathways can assemble into polymers. We now show tetramer- and substrate-dependent filament assembly by phosphofructokinase-1 (PFK1), which is considered the "gatekeeper" of glycolysis because it catalyzes the step committing glucose to breakdown. Recombinant liver PFK1 (PFKL) isoform, but not platelet PFK1 (PFKP) or muscle PFK1 (PFKM) isoforms, assembles into filaments. Negative-stain electron micrographs reveal that filaments are apolar and made of stacked tetramers oriented with exposed catalytic sites positioned along the edge of the polymer. Electron micrographs and biochemical data with a PFKL/PFKP chimera indicate that the PFKL regulatory domain mediates filament assembly. Quantified live-cell imaging shows dynamic properties of localized PFKL puncta that are enriched at the plasma membrane. These findings reveal a new behavior of a key glycolytic enzyme with insights on spatial organization and isoform-specific glucose metabolism in cells.
