Flotillins affect LPS-induced TLR4 signaling by modulating the trafficking and abundance of CD14.

Lipopolysaccharide (LPS) induces a strong pro-inflammatory reaction of macrophages upon activation of Toll-like receptor 4 (TLR4) with the assistance of CD14 protein. Considering a key role of plasma membrane rafts in CD14 and TLR4 activity and the significant impact exerted on that activity by endocytosis and intracellular trafficking of the both LPS acceptors, it seemed likely that the pro-inflammatory reaction could be modulated by flotillins. Flotillin-1 and -2 are scaffolding proteins associated with the plasma membrane and also with endo-membranes, affecting both the plasma membrane dynamics and intracellular protein trafficking. To verify the above hypothesis, a set of shRNA was used to down-regulate flotillin-2 in Raw264 cells, which were found to also become deficient in flotillin-1. The flotillin deficiency inhibited strongly the TRIF-dependent endosomal signaling of LPS-activated TLR4, and to a lower extent also the MyD88-dependent one, without affecting the cellular level of TLR4. The flotillin depletion also inhibited the pro-inflammatory activity of TLR2/TLR1 and TLR2/TLR6 but not TLR3. In agreement with those effects, the depletion of flotillins down-regulated the CD14 mRNA level and the cellular content of CD14 protein, and also inhibited constitutive CD14 endocytosis thereby facilitating its shedding. Ultimately, the cell-surface level of CD14 was markedly diminished. Concomitantly, CD14 recycling was enhanced via EEA1-positive early endosomes and golgin-97-positive trans-Golgi network, likely to compensate for the depletion of the cell-surface CD14. We propose that the paucity of surface CD14 is the reason for the down-regulated signaling of TLR4 and the other TLRs depending on CD14 for ligand binding.

Diacylglycerol kinase-ε is S-palmitoylated on cysteine in the cytoplasmic end of its N-terminal transmembrane fragment.

Diacylglycerol kinase-ε (DGKε) catalyzes phosphorylation of diacylglycerol to phosphatidic acid with a unique specificity toward 1-stearoyl-2-arachidonoyl-sn-glycerol, which is a backbone of phosphatidylinositol (PI). Owing to this specificity, DGKε is involved in the PI cycle maintaining the cellular level of phosphorylated PI derivatives of signaling activity and was also found crucial for lipid metabolism. DGKε dysfunction is linked with the development of atypical hemolytic uremic syndrome (aHUS) and possibly other human diseases. Despite the DGKε significance, data on its regulation by cotranslational and/or post-translational modifications are scarce. Here, we report that DGKε is S-palmitoylated at Cys38/40 (mouse/human DGKε) located in the cytoplasmic end of its N-terminal putative transmembrane fragment. The S-palmitoylation of DGKε was revealed by metabolic labeling of cells with a palmitic acid analogue followed by click chemistry and with acyl-biotin and acyl-polyethylene glycol exchange assays. The S-acyltransferases zDHHC7 (zinc finger DHHC domain containing) and zDHHC17 and the zDHHC6/16 tandem were found to catalyze DGKε S-palmitoylation, which also increased the DGKε abundance. Mouse DGKε-Myc ectopically expressed in human embryonic kidney 293 cells localized to the endoplasmic reticulum where zDHHC6/16 reside and in small amounts also to the Golgi apparatus where zDHHC7 and zDHHC17 are present. The Cys38Ala substitution upregulated, whereas hyperpalmitoylation of wild-type DGKε reduced the kinase activity, indicating an inhibitory effect of the Cys38 S-palmitoylation. In addition, the substitution of neighboring Pro31 with Ala also diminished the activity of DGKε. Taken together, our data indicate that S-palmitoylation can fine-tune DGKε activity in distinct cellular compartments, possibly by affecting the distance between the kinase and its substrate in a membrane.

CD14 recycling modulates LPS-induced inflammatory responses of murine macrophages.

TLR4 is activated by the bacterial endotoxin lipopolysaccharide (LPS) and triggers two proinflammatory signaling cascades: a MyD88-dependent one in the plasma membrane, and the following TRIF-dependent one in endosomes. An inadequate inflammatory reaction can be detrimental for the organism by leading to sepsis. Therefore, novel approaches to therapeutic modulation of TLR4 signaling are being sought after. The TLR4 activity is tightly connected with the presence of CD14, a GPI-anchored protein that transfers LPS monomers to the receptor and controls its endocytosis. In this study we focused on CD14 trafficking as a still poorly understood factor affecting TLR4 activity. Two independent assays were used to show that after endocytosis CD14 can recycle back to the plasma membrane in both unstimulated and stimulated cells. This route of CD14 trafficking can be controlled by sorting nexins (SNX) 1, 2 and 6, and is important for maintaining the surface level and the total level of CD14, but can also affect the amount of TLR4. Silencing of these SNXs attenuated especially the CD14-dependent endosomal signaling of TLR4, making them a new target for therapeutic regulation of the inflammatory response of macrophages to LPS.

Palm Oil-Rich Diet Affects Murine Liver Proteome and S-Palmitoylome.

Palmitic acid (C16:0) is the most abundant saturated fatty acid in animals serving as a substrate in synthesis and ß-oxidation of other lipids, and in the modification of proteins called palmitoylation. The influence of dietary palmitic acid on protein S-palmitoylation remains largely unknown. In this study we performed high-throughput proteomic analyses of a membrane-enriched fraction of murine liver to examine the influence of a palm oil-rich diet (HPD) on S-palmitoylation of proteins. HPD feeding for 4 weeks led to an accumulation of C16:0 and C18:1 fatty acids in livers which disappeared after 12-week feeding, in contrast to an accumulation of C16:0 in peritoneal macrophages. Parallel proteomic studies revealed that HPD feeding induced a sequence of changes of the level and/or S-palmitoylation of diverse liver proteins involved in fatty acid, cholesterol and amino acid metabolism, hemostasis, and neutrophil degranulation. The HPD diet did not lead to liver damage, however, it caused progressing obesity, hypercholesterolemia and hyperglycemia. We conclude that the relatively mild negative impact of such diet on liver functioning can be attributed to a lower bioavailability of palm oil-derived C16:0 vs. that of C18:1 and the efficiency of mechanisms preventing liver injury, possibly including dynamic protein S-palmitoylation.

TLR4 and CD14 trafficking and its influence on LPS-induced pro-inflammatory signaling.

Toll-like receptor (TLR) 4 belongs to the TLR family of receptors inducing pro-inflammatory responses to invading pathogens. TLR4 is activated by lipopolysaccharide (LPS, endotoxin) of Gram-negative bacteria and sequentially triggers two signaling cascades: the first one involving TIRAP and MyD88 adaptor proteins is induced in the plasma membrane, whereas the second engaging adaptor proteins TRAM and TRIF begins in early endosomes after endocytosis of the receptor. The LPS-induced internalization of TLR4 and hence also the activation of the TRIF-dependent pathway is governed by a GPI-anchored protein, CD14. The endocytosis of TLR4 terminates the MyD88-dependent signaling, while the following endosome maturation and lysosomal degradation of TLR4 determine the duration and magnitude of the TRIF-dependent one. Alternatively, TLR4 may return to the plasma membrane, which process is still poorly understood. Therefore, the course of the LPS-induced pro-inflammatory responses depends strictly on the rates of TLR4 endocytosis and trafficking through the endo-lysosomal compartment. Notably, prolonged activation of TLR4 is linked with several hereditary human diseases, neurodegeneration and also with autoimmune diseases and cancer. Recent studies have provided ample data on the role of diverse proteins regulating the functions of early, late, and recycling endosomes in the TLR4-induced inflammation caused by LPS or phagocytosis of E. coli. In this review, we focus on the mechanisms of the internalization and intracellular trafficking of TLR4 and CD14, and also of LPS, in immune cells and discuss how dysregulation of the endo-lysosomal compartment contributes to the development of diverse human diseases.

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

Flotillin-1 and flotillin-2 are ubiquitously expressed, membrane-associated proteins involved in multifarious cellular events from cell signaling, endocytosis, and protein trafficking to gene expression. They also contribute to oncogenic signaling. Flotillins bind the cytosolic leaflet of the plasma membrane and endomembranes and, upon hetero-oligomerization, serve as scaffolds facilitating the assembly of multiprotein complexes at the membrane-cytosol interface. Additional functions unique to flotillin-1 have been discovered recently. The membrane-binding of flotillins is regulated by S-palmitoylation and N-myristoylation, hydrophobic interactions involving specific regions of the polypeptide chain and, to some extent, also by their oligomerization. All these factors endow flotillins with an ability to associate with the sphingolipid/cholesterol-rich plasma membrane domains called rafts. In this review, we focus on the critical input of lipids to the regulation of the flotillin association with rafts and thereby to their functioning. In particular, we discuss how the recent developments in the field of protein S-palmitoylation have contributed to the understanding of flotillin1/2-mediated processes, including endocytosis, and of those dependent exclusively on flotillin-1. We also emphasize that flotillins affect directly or indirectly the cellular levels of lipids involved in diverse signaling cascades, including sphingosine-1-phosphate and PI(4,5)P2. The mutual relations between flotillins and distinct lipids are key to the regulation of their involvement in numerous cellular processes.

Sphingomyelin synthase activity affects TRIF-dependent signaling of Toll-like receptor 4 in cells stimulated with lipopolysaccharide.

Bacterial lipopolysaccharide (LPS) is recognized by CD14 protein and the Toll-like receptor (TLR)4/MD2 complex localized in the plasma membrane of immune cells. TLR4 triggers two signaling pathways engaging the MyD88 and TRIF adaptor proteins which lead to production of various pro-inflammatory cytokines. These processes are likely to be modulated by sphingomyelin, as the CD14 - TLR4 interaction takes place in plasma membrane rafts enriched in this lipid. To verify this assumption, we analyzed the influence of tricyclodecane-9-yl xanthogenate (D609), which was proven here to be an SMS inhibitor, and silencing of sphingomyelin synthase (SMS) 1 and/or SMS2 on LPS-induced signaling in macrophages. LPS up-regulated the expression and activity of SMS while exposure to D609 or silencing of SMS1 and SMS2 counteracted this action and led (except for SMS2 silencing) to a depletion of sphingomyelin in cells. Concomitantly, the MyD88- and TRIF-dependent signaling pathways of TLR4 were inhibited with the latter being especially sensitive to the reduction of the SMS1 and/or SMS2 activity. The D609 treatment and SMS1 and/or SMS2 depletion all reduced the level of CD14 protein in cells, which likely was an important determinant of the reduction of the LPS-induced pro-inflammatory responses.

Lysophosphatidic acid up-regulates IL-10 production to inhibit TNF-α synthesis in Mϕs stimulated with LPS.

Bacterial LPS strongly induces pro-inflammatory responses of Mϕs after binding to CD14 protein and the TLR4/MD-2 receptor complex. The LPS-triggered signaling can be modulated by extracellular lysophosphatidic acid (LPA), which is of substantial importance for Mϕ functioning under specific pathophysiological conditions, such as atherosclerosis. The molecular mechanisms of the crosstalk between the LPS- and LPA-induced signaling, and the LPA receptors involved, are poorly known. In this report, we show that LPA strongly inhibits the LPS-induced TNF-α production at the mRNA and protein levels in primary Mϕs and Mϕ-like J774 cells. The decreased TNF-α production in LPA/LPS-stimulated cells is to high extent independent of NF-κB but is preceded by enhanced expression and secretion of the anti-inflammatory cytokine IL-10. The IL-10 elevation and TNF-α reduction are both abrogated upon depletion of the LPA5 and LPA6 receptors in J774 cells and can be linked with LPA-mediated activation of p38. We propose that the binding of LPA to LPA5 and LPA6 fine-tunes the LPS-induced inflammatory response by activating p38, and up-regulating IL-10 and down-regulating TNF-α production.

Association of Lyn kinase with membrane rafts determines its negative influence on LPS-induced signaling.

Lipopolysaccharide (LPS) is the component of Gram-negative bacteria that activates Toll-like receptor 4 (TLR4) to trigger proinflammatory responses. We examined the involvement of Lyn tyrosine kinase in TLR4 signaling of macrophages, distinguishing its catalytic activity and intermolecular interactions. For this, a series of Lyn-GFP constructs bearing point mutations in particular domains of Lyn were overexpressed in RAW264 macrophage-like cells or murine peritoneal macrophages, and their influence on LPS-induced responses was analyzed. Overproduction of wild-type or constitutively active Lyn inhibited production of TNF-α and CCL5/RANTES cytokines and down-regulated the activity of NFκB and IRF3 transcription factors in RAW264 cells. The negative influence of Lyn was nullified by point mutations of Lyn catalytic domain or Src homology 2 (SH2) or SH3 domains or of the cysteine residue that undergoes LPS-induced palmitoylation. Depending on the cell type, overproduction of those mutant forms of Lyn could even up-regulate LPS-induced responses, and this effect was reproduced by silencing of endogenous Lyn expression. Simultaneously, the Lyn mutations blocked its LPS-induced accumulation in the raft fraction of RAW264 cells. These data indicate that palmitoylation, SH2- and SH3-mediated intermolecular interactions, and the catalytic activity of Lyn are required for its accumulation in rafts, thereby determining the negative regulation of TLR4 signaling.

Protein Palmitoylation and Its Role in Bacterial and Viral Infections.

S-palmitoylation is a reversible, enzymatic posttranslational modification of proteins in which palmitoyl chain is attached to a cysteine residue via a thioester linkage. S-palmitoylation determines the functioning of proteins by affecting their association with membranes, compartmentalization in membrane domains, trafficking, and stability. In this review, we focus on S-palmitoylation of proteins, which are crucial for the interactions of pathogenic bacteria and viruses with the host. We discuss the role of palmitoylated proteins in the invasion of host cells by bacteria and viruses, and those involved in the host responses to the infection. We highlight recent data on protein S-palmitoylation in pathogens and their hosts obtained owing to the development of methods based on click chemistry and acyl-biotin exchange allowing proteomic analysis of protein lipidation. The role of the palmitoyl moiety present in bacterial lipopolysaccharide and lipoproteins, contributing to infectivity and affecting recognition of bacteria by innate immune receptors, is also discussed.

Bis(monoacylglycero)phosphate inhibits TLR4-dependent RANTES production in macrophages.

Toll-like receptor 4 (TLR4) is the receptor for bacterial lipopolysaccharide (LPS) triggering production of pro-inflammatory cytokines which help eradicate the bacteria but could also be harmful when overproduced. The signaling activity of TLR4 is modulated by cholesterol level in cellular membranes, which in turn is affected by bis(monoacylglycero)phosphate (BMP), a phospholipid enriched in late endosomes. We found that exogenously added BMP isomers become incorporated into the plasma membrane and intracellular vesicles of macrophages and strongly reduced LPS-stimulated production of a chemokine RANTES, which was correlated with inhibition of interferon regulatory factor 3 (IRF3) controlling Rantes expression. To investigate the mechanism underlying the influence of BMP on TLR4 signaling we applied Laurdan and studied the impact of BMP incorporation on lipid packing, a measure for membrane order. Enrichment of model and cellular membranes with BMP significantly reduced their order and the reduction was maintained during stimulation of cells with LPS. This effect of BMP was abolished by enrichment of macrophages with cholesterol. In parallel, the inhibitory effect of BMP exerted on the TLR4-dependent phosphorylation of IRF3 was also reversed. Taken together our results indicate that BMP reduces the order of macrophage membranes which contributes to the inhibition of TLR4-dependent RANTES production.

Structural evidence of the species-dependent albumin binding of the modified cyclic phosphatidic acid with cytotoxic properties.

Cyclic phosphatidic acids (cPAs) are naturally occurring, very active signalling molecules, which are involved in several pathological states, such as cancer, diabetes or obesity. As molecules of highly lipidic character found in the circulatory system, cPAs are bound and transported by the main extracellular lipid binding protein-serum albumin. Here, we present the detailed interactions between human serum albumin (HSA) and equine serum albumin (ESA) with a derivative of cPA, 1-O-myristoyl-sn-glycerol-2,3-cyclic phosphorodithioate (Myr-2S-cPA). Initial selection of the ligand used for the structural study was made by the analysis of the therapeutically promising properties of the sulfur containing analogues of cPA in respect to the unmodified lysophospholipids (LPLs). Substitution of one or two non-bridging oxygen atoms in the phosphate group with one or two sulfur atoms increases the cytotoxic effect of cPAs up to 60% on the human prostate cancer (PC) cells. Myr-2S-cPA reduces cancer cell viability in a dose-dependent manner, with IC50 value of 29.0 µM after 24 h incubation, which is almost 30% lower than IC50 of single substituted phosphorothioate cPA. Although, the structural homology between HSA and ESA is big, their crystal complexes with Myr-2S-cPA demonstrate significantly different mode of binding of this LPL analogue. HSA binds three molecules of Myr-2S-cPA, whereas ESA only one. Moreover, none of the identified Myr-2S-cPA binding sites overlap in both albumins.

Sulfur- and acyl chain-dependent influence of 2-methoxy-lysophosphatidylcholine analogues on ß pancreatic cells.

Nutrient-induced increase in intracellular Ca(2+) concentration ([Ca(2+)]i) is one of the key mechanisms responsible for insulin release from pancreatic islet ß cells. Lysophosphatidylcholine (LPC) was demonstrated to induce insulin secretion from ß cells, activate glucose uptake and effectively lower blood glucose levels in mouse models of type 1 and 2 diabetes mellitus. The article hereby presents the results of a characterization of 2-OMe-LPC sulfur analogues with defined acyl residues in terms of their effect on intracellular Ca(2+) concentration and cellular membrane integrity in the murine ßTC-3 cell model. Active LPC series that could induce calcium flux in ßTC-3 cell model include unmodified LPC 12:0, 14:0, 16:0, and 18:0 as well as phosphorothioate analogues of LPC 12:0, 14:0 and 16:0. However, in the case of species bearing mirystoyl and palmitoyl residues [Ca(2+)]i was associated with membrane permeabilization as demonstrated by propidium iodide incorporation and lactate dehydrogenase release. LPC 12:0 (both unmodified and a sulfurcontaining counterpart) and unmodified LPC 18:0 did not demonstrate membrane disruption but acted as calcium inducers. Interestingly, no stimulation of calcium flux or membrane disruption was observed in the case of LPC analogues with two sulfur atoms introduced into a phosphate group. Experiments with nitrendipine and NiCl2 blocking voltage-dependent calcium channels and the general calcium influx, respectively, revealed remarkably that the compounds studied were involved in different signaling mechanisms while administered to the cell culture, which is clearly related to their chemical structure, both acyl chain and modification dependently.

Modification of pro-inflammatory signaling by dietary components: The plasma membrane as a target.

You are what you eat - this well-known phrase properly describes the phenomenon of the effects of diet on acute and chronic inflammation. Several lipids and lipophilic compounds that are delivered with food or are produced in situ in pathological conditions exert immunomodulatory activity due to their interactions with the plasma membrane. This group of compounds includes cholesterol and its oxidized derivatives, fatty acids, α-tocopherol, and polyphenols. Despite their structural heterogeneity, all these compounds ultimately induce changes in plasma membrane architecture and fluidity. By doing this, they modulate the dynamics of plasma membrane receptors, such as TLR4. This receptor is activated by lipopolysaccharide, triggering acute inflammation during bacterial infection, which often leads to sepsis and is linked with diverse chronic inflammatory diseases. In this review, we discuss how the impact on plasma membrane properties contributes to the immunomodulatory activity of dietary compounds, pointing to the therapeutic potential of some of them. Also watch the Video Abstract.

Melatonin and cortisol profiles in patients with pituitary tumors.

The optic tract section at the optic chiasm is expected to disturb the suprachiasmatic nucleus (SCN) rhythm, circadian rhythm and melatonin secretion rhythms in humans, although detailed studies have never been conducted. The aim of this paper was to describe melatonin and cortisol profiles in patients with a pituitary tumor exerting optic chiasm compression. Six patients with pituitary tumors of different size, four of whom had significant optic chiasm compression, were examined. In each brain, MRI, an ophthalmological examination including the vision field and laboratory tests were performed. Melatonin and cortisol concentrations were measured at 22:00 h, 02:00 h, 06:00 h, and 10:00 h in patients lying in a dark, isolated room. One of the four cases with significant optic chiasm compression presented a flattened melatonin rhythm. The melatonin rhythm was also disturbed in one patient without optic chiasm compression. Larger tumors may play a role in the destruction of neurons connecting the retina with the suprachiasmatic nucleus (SCN) and breaking of basic way for inhibiting effect to the SCN from the retina.

The prevalence of tobacco smoking and attitudes of Polish pulmonologists towards smoking.

INTRODUCTION: Pulmonologists can play an important role in smoking prevention and control. The aim of this study was to assess smoking prevalence among Polish pulmonologists and physicians' behaviour towards smoking patients. MATERIAL AND METHODS: The study was performed in 2006 during national congress of Polish Respiratory Society. The survey was performed using a questionnaire including questions regarding age, sex, professional activities, specialty, academic degree and respondents' nicotine status, attitude and activity towards smoking patients. RESULTS: Questionnaires were filled in by 498 physicians: 160 (32.1%) men and 338 (67.9%) women, out of 852 participating physicians (58.5%) who received the questionnaire. There were 11.3% smokers (13.8% men and 10% women), 25.1% of surveyed physicians were ex-smokers, and 63.6% never smokers. 91.4% of respondents always ask patients if they smoke. 87.4% physicians always advise smoking cessation to their patients, but smoking cessation support is offered only by 48% of respondents. Almost half respondents use drugs in nicotine dependence treatment. Majority of respondents (81.1%) think that physician's attitude influences patient's behaviour. More than a half (55.7%) think that smoking by physicians is not in accordance with physician's ethics. 91.4% support smoking ban in public places. CONCLUSIONS: This study found a lower prevalence of smoking among Polish pulmonologists compared with the general population, and with the previous surveys and demonstrated the impact of personal smoking on physicians' attitudes towards smoking.