Fatty acid binding protein 5 forms higher order assemblies with FLAP or COX-2 in LPS-stimulated macrophages

Abstract

AbstractImmune cells must integrate multiple extracellular signals to produce an appropriate inflammatory response, including production of a dynamic mix of eicosanoids and related bioactive lipids. Synthesis of these lipids is initiated on the membrane surface of the nuclear envelope and endoplasmic reticulum. One critical question is how the precursor arachidonic acid (AA) is distributed between the initial biosynthetic enzymes of the arachidonate 5-lipoxygenase (5-LO) and prostaglandin-endoperoxidase synthase-1/2 (COX-1/2) related pathways. To understand these balancing mechanisms, we hypothesized that fatty acid binding proteins mediate this process. We employed a multi-modal imaging approach by combining direct stochastic optical reconstruction microscopy (dSTORM) with computational analyses and fluorescence lifetime imaging microscopy (FLIM) to delineate the relationships of fatty acid binding proteins 3, 4, and 5 (FABP3–5) with 5-LO activating protein (FLAP), COX-1, and COX-2 in the presence of a stimulus (lipopolysaccharide, LPS) that triggers the synthesis of prostaglandin E2 (PGE2). LPS triggers a redistribution of FABP5 to higher order assemblies of COX-2 or FLAP. This was evidenced by a decrease in lifetime determined by FLIM. Colocalization between FABP3 and FLAP decreased, but no other changes in distribution were observed for FABP3 and FABP4. In contrast, assemblies of FABP5 with COX-1 were smaller and showed an increase in lifetime. The data indicate that FABP5 is a member of higher order assemblies of eicosanoid biosynthetic enzymes and that FABP5 may play a key role in regulating the organization of these structures. FABP5 is positioned to distribute AA to both the 5-LO and COX-2 pathways.