Uncontrolled activation of tumor necrosis factor receptor-associated factor (TRAF) proteins may result in profound tissue injury by linking surface signals to cytokine release. Here we show that a ubiquitin E3 ligase component, Fbxo3, potently stimulates cytokine secretion from human inflammatory cells by destabilizing a sentinel TRAF inhibitor, Fbxl2. Fbxo3 and TRAF protein in circulation positively correlated with cytokine responses in septic subjects and we furthermore identified a hypofunctional Fbxo3 human polymorphism. A small molecule inhibitor targeting Fbxo3 was sufficient to lessen severity of cytokine-driven inflammation in several murine disease models. These studies identify a pathway of innate immunity that may characterize subjects with altered immune responses during critical illness or provide a basis for therapeutic intervention targeting TRAF protein abundance.
The ST2L receptor for interleukin 33 (IL-33) mediates pulmonary inflammation and immune system–related disorders, such as asthma and rheumatoid arthritis. At present, very little is known about the molecular regulation of ST2L expression. Here we found that FBXL19, an ‘orphan’ member of the Skp1–Cullin–F-box family of E3 ubiquitin ligases, selectively bound to ST2L to mediate its polyubiquitination and elimination in the proteasome. Degradation of ST2L involved phosphorylation of ST2L at Ser442 catalyzed by the kinase GSK3β. Overexpression of FBXL19 abrogated the proapoptotic and inflammatory effects of IL-33 and lessened the severity of lung injury in mouse models of pneumonia. Our results suggest that modulation of the IL-33–ST2L axis by ubiquitin ligases might serve as a unique strategy for lessening pulmonary inflammation.
Background: LPS increases NALP3 levels, but the mechanisms remain unknown. Results: LPS prolongs the lifespan of NALP3 protein by reducing E3 ligase (SCF FBXL2 )-mediated ubiquitination. Conclusion: Proinflammatory cytokine release is reduced by a small molecule that restores cellular SCF FBXL2 levels. Significance: We identified a novel pathway of inflammasome priming that may serve as a springboard for future translational studies.
Rac1, a member of the Rho family of GTPases, regulates diverse cellular functions, including cytoskeleton reorganization and cell migration. F-box proteins are major subunits within the Skp1-Cul1-F-box (SCF) E3 ubiquitin ligases that recognize specific substrates for ubiquitination. The role of F-box proteins in regulating Rac1 stability has not been studied. Mouse lung epithelial (MLE12) cells were used to investigate Rac1 stability and cell migration. Screening of an F-box protein library and in vitro ubiquitination assays identified FBXL19, a relatively new member of the F-box protein family that targets Rac1 for its polyubiquitination and proteasomal degradation. Overexpression of FBXL19 decreased both Rac1 active and inactive forms and significantly reduced cellular migration. Protein kinase AKT-mediated phosphorylation of Rac1 at serine(71) was essential for FBXL19-mediated Rac1 ubiquitination and depletion. Lysine(166) within Rac1 was identified as a polyubiquitination acceptor site. Rac1(S71A) and Rac1(K166R) mutant proteins were resistant to FBXL19-mediated ubiquitination and degradation. Further, ectopically expressed FBXL19 reduced cell migration in Rac1-overexpressing cells (P<0.01, Rac1 cells vs. FBXL19+Rac1 cells), but not in Rac1 lysine(166) mutant-overexpressing cells. FBXL19 diminished formation of the migratory leading edge. Thus, SCF(FBXL19) targets Rac1 for its disposal, a process regulated by AKT. These findings provide the first evidence of an F-box protein targeting a small G protein for ubiquitination and degradation to modulate cell migration.
The enzyme acyl-CoA:lysophosphatidylcholine acyltransferase (Lpcat1) is a critical cytosolic enzyme needed for lung surfactant synthesis that catalyzes an acyltransferase reaction by adding a palmitate to the sn-2 position of lysophospholipids. Here we report that histone H4 protein is subject to palmitoylation catalyzed by Lpcat1 in a calcium-regulated manner. The enzyme acyl-CoA:lysophosphatidylcholine acyltransferase (Lpcat1) was recently cloned from lung epithelia and is indispensable for generation of pulmonary surfactant. Lpcat1 catalyzes an O-acyltransferase reaction by covalently adding saturated acyl-CoAs (palmitoyl groups, 16:0) to its acceptor lysophospholipids. Specifically, in the lung, it catalyzes an oxyester linkage between palmitate and lysophosphatidylcholine to generate dipalmitoylphosphatidylcholine, the major surface tension-lowering component of pulmonary surfactant in the remodeling pathway (1, 2). However, Lpcat1 appears to be somewhat promiscuous and acts on substrates that include lysophosphatidylcholine, lysoplasmanylcholine, and lysophosphatidylglycercol (1-3). Notably, in addition to lipid substrates, O-acyltransferases can also lipidate some protein substrates (4 -7). These enzymes share a highly conserved histidine residue within a catalytic core that is essential for their functionality.Protein palmitoylation, a prototypical form of lipidation, is an important post-translational modification that occurs ubiquitously in eukaryotes. Protein palmitoylation is generally categorized as S-palmitoylation, N-palmitoylation, and O-palmitoylation based on the chemical linkage between donor and acceptor substrates. Among these, S-palmitoylation is best characterized and involves generation of a reversible thioester bond between a cysteine residue and a palmitate group (8). As many as 250 proteins were found to be modified by S-palmitoylation in mammalian neurons, and these reactions are largely catalyzed by aspartate-histidine-histidine-cysteine (DHHC) palmitoyl acyltransferase family members (8, 9). The biochemistry of N-palmitoylation and O-palmitoylation, however, is less studied. One known substrate for N-palmitoylation is the Sonic Hedgehog protein because its NH 2 -terminal cysteine is palmitoylated via an amide linkage by Hedgehog acyltransferase (Hhat) (5). O-Palmitoylation is exemplified by Wnt/Wg proteins that harbor an oxyester linkage between monounsaturated palmitate and a serine residue. Another recently described O-palmitoylation target, the peptide hormone preghrelin, is modified by octanoylation at a serine residue. The enzymes that catalyze the oxyester linkages within Wnt/Wg proteins and preghrelin are porcupine (6) and ghrelin O-acyltransferase (7), respectively. The physiological consequences of palmitoylation are diverse; by increasing substrate hydrophobicity, these lipidation reactions appear to modulate interactions of substrates with other biomolecules, often affecting signal transduction, protein stability, intracellular trafficking, and localization (10 -12).Histo...
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