Fresnedo, O. | bioRxiv Science

bioRxiv2023

IRF5 regulates microglial myelin clearance and cholesterol metabolism after demyelination

Interferon regulatory factor 5 (IRF5), a transcription factor highly involved in innate immunity that drives microglia/macrophage towards a pro-inflammatory state, has been associated to multiple sclerosis susceptibility but its role in MS pathogenesis is unknown. Here we analysed the role of IRF5 in multiple sclerosis animal models. Irf5-/- mice showed exacerbated damage in the chronic phase of experimental autoimmune encephalomyelitis (EAE) mice, despite an initial delay in its onset, as well as after lysolecithin injection into the spinal cord. Transcriptomic and lipidomic analysis evidence a role of this transcription factor in myelin metabolism and cholesterol homeostasis. Indeed, Irf5-/- mice showed an aberrant accumulation of myelin debris and lipidic structures, such as CE-containing lipid droplets and cholesterol crystals, suggesting that myelin-derived lipids are not properly processed. Cholesterol crystal accumulation leads to an aberrant inflammatory response, which block oligodendrocyte migration into the core of demyelinated lesion and remyelination. Pharmacologically facilitating cholesterol transport reduces lipid droplet accumulation and ameliorates EAE exacerbated damage in Irf5-/- mice. These results reveal for the first time the role of Irf5, a transcription factor necessary to orchestrate the immune responses, in phagocytes lipid metabolism which could be pivotal in regenerative responses such as remyelination.

neuroscience↗

bioRxiv2022

Nonionic surfactants can modify the thermal stability of globular and membrane proteins interfering with the thermal proteome profiling principles to identify protein targets

The membrane proteins are essential targets to understand cellular function. The unbiased identification of membrane protein targets is still the bottleneck for a system-level understanding of cellular response to stimuli or perturbations. It has been suggested to enrich the soluble proteome with membrane proteins by introducing nonionic surfactants in the solubilization solution. This strategy was aiming to simultaneous identify the globular and membrane protein targets by thermal proteome profiling principles. However, the thermal shift assay would surpass the cloud point temperature from the nonionic surfactants frequently utilized for membrane protein solubilization. It is expected that around the cloud point temperature, the surfactant micelles would suffer structural modifications altering protein solubility. Here, we show that the presence of nonionic surfactants can alter protein thermal stability from a mixed, globular and membrane, proteome. In the presence of surfactant micelles, the changes in proteins solubility analyzed after the thermal shift assay were affected by the thermal dependent modification of the micellar size, and its interaction with proteins. We demonstrate that the introduction of nonionic surfactants for the solubilization of membrane proteins is not compatible with the principles of target identification by thermal proteome profiling methodologies. Our results lead to explore thermal-independent strategies for membrane protein solubilization to assure confident membrane protein target identification. The proteome-wide thermal shift methods have already shown their capability to elucidate mechanisms of action from pharma, biomedicine, analytical chemistry, or toxicology and finding strategies, free from surfactants, to identify membrane protein targets would be the next challenge.

biophysics↗