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The interaction of ceramide 1-phosphate with group IVA cytosolic phospholipase A₂ coordinates acute wound healing and repair.

ABSTRACT: The sphingolipid ceramide 1-phosphate (C1P) directly binds to and activates group IVA cytosolic phospholipase A₂ (cPLA₂α) to stimulate the production of eicosanoids. Because eicosanoids are important in wound healing, we examined the repair of skin wounds in knockout (KO) mice lacking cPLA₂α and in knock-in (KI) mice in which endogenous cPLA₂α was replaced with a mutant form having an ablated C1P interaction site. Wound closure rate was not affected in the KO or KI mice, but wound maturation was enhanced in the KI mice compared to that in wild-type controls. Wounds in KI mice displayed increased infiltration of dermal fibroblasts into the wound environment, increased wound tensile strength, and a higher ratio of type I:type III collagen. In vitro, primary dermal fibroblasts (pDFs) from KI mice showed substantially increased collagen deposition and migration velocity compared to pDFs from wild-type and KO mice. KI mice also showed an altered eicosanoid profile of reduced proinflammatory prostaglandins (PGE₂ and TXB₂) and an increased abundance of certain hydroxyeicosatetraenoic acid (HETE) species. Specifically, an increase in 5-HETE enhanced dermal fibroblast migration and collagen deposition. This gain-of-function role for the mutant cPLA₂α was also linked to the relocalization of cPLA₂α and 5-HETE biosynthetic enzymes to the cytoplasm and cytoplasmic vesicles. These findings demonstrate the regulation of key wound-healing mechanisms in vivo by a defined protein-lipid interaction and provide insights into the roles that cPLA₂α and eicosanoids play in orchestrating wound repair.

SUBMITTER: MacKnight HP

PROVIDER: S-EPMC8103357 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The Group IVA (GIVA) phospholipase A(2) associates with natural membranes in response to an increase in intracellular Ca(2+) along with increases in certain lipid mediators. This enzyme associates with the membrane surface as well as binding a single phospholipid molecule in the active site for catalysis. Employing deuterium exchange mass spectrometry, we have identified the regions of the protein binding the lipid surface and conformational changes upon a single phospholipid binding in the absence of a lipid surface. Experiments were carried out using natural palmitoyl arachidonyl phosphatidylcholine vesicles with the intact GIVA enzyme as well as the isolated C2 and catalytic domains. Lipid binding produced changes in deuterium exchange in eight different regions of the protein. The regions with decreased exchange included Ca(2+) binding loop one, which has been proposed to penetrate the membrane surface, and a charged patch of residues, which may be important in interacting with the polar head groups of phospholipids. The regions with an increase in exchange are all located either in the hydrophobic core underneath the lid region or near the lid and hinge regions from 403 to 457. Using the GIVA phospholipase A(2) irreversible inhibitor methyl-arachidonyl fluorophosphonate, we were able to isolate structural changes caused only by pseudo-substrate binding. This produced results that were very similar to natural lipid binding in the presence of a lipid interface with the exception of the C2 domain and region 466-470. This implies that most of the changes seen in the catalytic domain are due to a substrate-mediated, not interface-mediated, lid opening, which exposes the active site to water. Finally experiments carried out with inhibitor plus phospholipid vesicles showed decreases at the C2 domain as well as charged residues on the putative membrane binding surface of the catalytic domain revealing the binding sites of the enzyme to the lipid surface.

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The interaction of ceramide 1-phosphate with group IVA cytosolic phospholipase A₂ coordinates acute wound healing and repair.

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The interaction of ceramide 1-phosphate with group IVA cytosolic phospholipase A2 coordinates acute wound healing and repair.

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OmicsDI is part of the ELIXIR infrastructure

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The interaction of ceramide 1-phosphate with group IVA cytosolic phospholipase A₂ coordinates acute wound healing and repair.