Cossins, B. | Search bioRxiv

bioRxiv2025

12/15-Lipoxygenase orchestrates murine wound healing via PPARg-activating oxylipins acting holistically to dampen inflammation.

12/15-lipoxygenase (12/15-LOX, Alox15) generates bioactive oxygenated lipids during inflammation, however its homeostatic role(s) in normal healing are unclear. Here, the role of 12/15-LOX in resolving skin wounds was elucidated, focusing on how its lipids act together in physiologically relevant amounts. In mice, wounding caused acute appearance of 12/15-LOX-expressing macrophages and stem cells, coupled to early generation of [~]12 monohydroxy-oxylipins and enzymatically oxygenated phospholipids (eoxPL). Alox15 deletion increased -smooth muscle actin, collagen deposition, stem cell/fibroblast proliferation, IL6/pSTAT3, pSMAD3, and IFN-{gamma} levels. Conversely, CD206 expression, F480+ cells, MMP9 and MMP2 activities were reduced. Alox15-/- skin was deficient in PPAR{gamma}/adiponectin activity. Furthermore, while pro-inflammatory genes were upregulated as normal during wounding, many including Il6, Il1b, ccl4, Cd14, Cd274, Clec4d, Clec4e, Csf3, and Cxcl2 failed to revert to baseline during healing, indicating disruption of an anti-inflammatory brake. Reconstituting Alox15-/- wounds with a physiological mixture of Alox15-derived primary oxylipins generated by healing wounds restored MMP and dampened collagen deposition. The oxylipin mixture activated PPAR{gamma} in vitro, while in vivo, the PPAR{gamma} co-activator, Helz2, was significantly upregulated. Additional inflammatory and proliferative gene networks impacted by Alox15-/-included Elf4, Cebpb and Tcf3, with many of their associated genes significantly dysregulated. In summary, the impact of 12/15-LOX is ascribed to the deficiency of abundantly generated monohydroxy oxylipins acting together via PPAR{gamma}/adiponectin. The identification of multiple gene alterations reveals several new targets for treatment of non-healing wounds. Our studies demonstrate that abundant 12/15-LOX oxylipins act together, dampening inflammation in vivo, revealing a need to consider lipid signaling holistically. Significance statementDefective wound healing is a significant global clinical problem. Macrophage 12/15-lipoxygenase (12/15-LOX, Alox15) generates abundant lipid mediators termed oxylipins during inflammation. However, its physiological role during resolving wound healing is unclear, with studies so far assessing the bioactivity of individual lipids pharmacologically, rather than holistically in physiological amounts. Here, we report that Alox15 deficiency in mice caused a fibrotic response with failure to dampen inflammation, due to a dysregulated PPAR{gamma}/adiponectin axis. Treatment of Alox15-/- wounds with physiological mixtures of PPAR{gamma}-activating 12/15-LOX primary monohydroxy products restored the phenotype. Several transcriptional networks (Elf4, Cebpb and Tcf3) controlled by Alox15 were uncovered, identifying new targets for promoting physiological wound healing.

pharmacology and toxicology↗

bioRxiv2020

Membrane Interactions of α-Synuclein Revealed by Multiscale Molecular Dynamics Simulations, Markov State Models, and NMR

-Synuclein is a presynaptic protein that binds to cell membranes and is linked to Parkinsons disease (PD). Whilst the normal function of remains -synuclein remains uncertain, it is thought that oligomerization of the protein on the cell membrane contributes to cell damage. Knowledge of how -synuclein binds to lipid bilayers is therefore of great interest as a likely first step in the molecular pathophysiology of PD, and may provide insight of the phenotype of PD-promoting mutations. We use coarse-grained and atomistic simulations in conjunction with NMR and cross-linking mass spectrometry studies of -synuclein bound to anionic lipid bilayers to reveal a break in the helical structure of the NAC region, which may give rise to subsequent oligomer formation. Coarse-grained simulations of -synuclein show that the interhelical region leads recognition and binding to both POPG and mixed composition bilayers and identifies important protein-lipid contacts, including those in the region between the two helices in the folded structure. We extend these simulations with all-atom simulations of the initial binding event to reveal details of the time-progression of lipid binding. We present secondary structure analysis that reveals points of possible {beta}-strand formation in the structure, and investigate intramolecular contacts with simulations and mass-spectrometry crosslinking. Additionally we show how Markov state models can be used to investigate possible conformational changes of membrane bound -synuclein in the NAC region, and we extract representative structures. These structural insights will aid the design and development of novel therapeutic approaches.

biophysics↗