Lipopolysaccharide Upregulates Palmitoylated Enzymes of the Phosphatidylinositol Cycle: An Insight from Proteomic Studies

Abstract

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria that induces strong proinflammatory reactions of mammals. These processes are triggered upon sequential binding of LPS to CD14, a GPI-linked plasma membrane raft protein, and to the TLR4/MD2 receptor complex. We have found earlier that upon LPS binding, CD14 triggers generation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P], a lipid controlling subsequent proinflammatory cytokine production. Here we show that stimulation of RAW264 macrophage-like cells with LPS induces global changes of the level of fatty-acylated, most likely palmitoylated, proteins. Among the acylated proteins that were up-regulated in those conditions were several enzymes of the phosphatidylinositol cycle. Global profiling of acylated proteins was performed by metabolic labeling of RAW264 cells with 17ODYA, an analogue of palmitic acid functionalized with an alkyne group, followed by detection and enrichment of labeled proteins using biotin-azide/streptavidin and their identification with mass spectrometry. This proteomic approach revealed that 154 fatty-acylated proteins were up-regulated, 186 downregulated, and 306 not affected in cells stimulated with 100 ng/ml LPS for 60 min. The acylated proteins affected by LPS were involved in diverse biological functions, as found by Ingenuity Pathway Analysis. Detailed studies of 17ODYA-labeled and immunoprecipitated proteins revealed that LPS induces -palmitoylation, hence activation, of type II phosphatidylinositol 4-kinase (PI4KII) β, which phosphorylates phosphatidylinositol to phosphatidylinositol 4-monophosphate, a PI(4,5)P precursor. Silencing of PI4KIIβ and PI4KIIα inhibited LPS-induced expression and production of proinflammatory cytokines, especially in the TRIF-dependent signaling pathway of TLR4. Reciprocally, this LPS-induced signaling pathway was significantly enhanced after overexpression of PI4KIIβ or PI4KIIα; this was dependent on palmitoylation of the kinases. However, the -palmitoylation of PI4KIIα, hence its activity, was constitutive in RAW264 cells. Taken together the data indicate that LPS triggers-palmitoylation and activation of PI4KIIβ, which generates PI(4)P involved in signaling pathways controlling production of proinflammatory cytokines.

Flotillins: At the Intersection of Protein S-Palmitoylation and Lipid-Mediated Signaling

“…Thus, while the involvement of flotillin-1 in the functioning of the endoplasmic reticulum is firmly established, its mechanism(s) requires clarification. It is worth mentioning that activation of TLR4 with bacterial lipopolysaccharide (LPS), a strong pro-inflammatory agent, increases the level of palmitoylated flotillin-1 but not flotillin-2 in Raw264 macrophage-like cells, suggesting that S-palmitoylation of flotillin-1 could be important for the signaling activity of TLR4 [129].…”

Udział lipidów w regulacji prozapalnych szlaków sygnałowych indukowanych przez lipopolisacharyd

“…Among those proteins, 154 were up-regulated and 186 downregulated in cells stimulated with 100 ng/ml LPS for 60 min. This is a time window where the signaling events related to both the MyD88-and TRIF-dependent pathways of TLR4 occur [49]. The obtained results indicated a global character of changes in the palmitoylation of proteins associated with the stimulation of cells by LPS.…”

“…Overproduction of palmitoylatable forms of both kinases up-regulated the endosomal TRIF-dependent signaling of TLR4, and conversely, depletion of the kinases inhibited especially strongly this signaling pathway. These data indicate that LPS induces palmitoylation and activation of PI4KIIβ kinase, which together with PI4KIIα produces PI(4)P participating in signaling pathways that control the synthesis of pro-inflammatory cytokines [19,49].…”

“…A pool of PI(4,5)P 2 -generating kinases, the PI(4,5)P 2 newly formed in LPS-stimulated cells and CD14, all accumulated in the cellular fraction insoluble in nonionic detergents, enriched in the components of membrane rafts [18,49]. A few other studies, including those on Fcγ receptor IIa sig-naling, indicated that PI(4,5)P 2 can be enriched in rafts of the plasma membrane [17,50] and that clustering of GPI-anchored CD59 protein can induce PI(4,5)P 2 hydrolysis and the release of Ca 2+ from intracellular stores [51].…”

“…The obtained results indicated a global character of changes in the palmitoylation of proteins associated with the stimulation of cells by LPS. The palmitoylated proteins affected were involved in several processes, including transcription and translation, amino acid and lipid metabolism, cell reorganization and intercellular interactions [49]. Among them was also Lyn tyrosine kinase of the Src family.…”