c-Abl Inhibition Activates TFEB and Promotes Cellular Clearance in a Lysosomal Disorder.

The transcription factor EB (TFEB) has emerged as a master regulator of lysosomal biogenesis, exocytosis, and autophagy, promoting the clearance of substrates stored in cells. c-Abl is a tyrosine kinase that participates in cellular signaling in physiological and pathophysiological conditions. In this study, we explored the connection between c-Abl and TFEB.

R468A mutation in perfringolysin O destabilizes toxin structure and induces membrane fusion.

Perfringolysin O (PFO) belongs to the family of cholesterol-dependent cytolysins. Upon binding to a cholesterol-containing membrane, PFO undergoes a series of structural changes that result in the formation of a β-barrel pore and cell lysis. Recognition and binding to cholesterol are mediated by the D4 domain, one of four domains of PFO.

oxLDL and eLDL Induced Membrane Microdomains in Human Macrophages.

Background: Extravasation of macrophages and formation of lipid-laden foam cells are key events in the development and progression of atherosclerosis. The degradation of atherogenic lipoproteins subsequently leads to alterations in cellular lipid metabolism that influence inflammatory signaling. Especially sphingolipids and ceramides are known to be involved in these processes.

Annexin A1 Tethers Membrane Contact Sites that Mediate ER to Endosome Cholesterol Transport.

Membrane contact sites between the ER and multivesicular endosomes/bodies (MVBs) play important roles in endosome positioning and fission and in neurite outgrowth. ER-MVB contacts additionally function in epidermal growth factor receptor (EGFR) tyrosine kinase downregulation by providing sites where the ER-localized phosphatase, PTP1B, interacts with endocytosed EGFR before the receptor is sorted onto intraluminal vesicles (ILVs). Here we show that these contacts are tethered by annexin A1 and its Ca(2+)-dependent ligand, S100A11, and form a subpopulation of differentially regulated contact sites between the ER and endocytic organelles.

Ceramide generation during curcumin-induced apoptosis is controlled by crosstalk among Bcl-2, Bcl-xL, caspases and glutathione.

Curcumin exhibits anti-cancer properties manifested by activation of pro-apoptotic signaling. We have demonstrated earlier that apoptosis of HL-60 human leukemia cells induced by curcumin is controlled by ceramide generated by neutral sphingomyelinase (nSMase) which contributes to sphingomyelin synthase (SMS) inhibition favoring accumulation of ceramide in cells. Here we report that the activity of nSMase, ceramide accumulation and death of HL-60 cells are inhibited by overexpression of Bcl-xL or Bcl-2 proteins, while down-regulation of nSMase interferes with degradation of Bcl-2 but not Bcl-xL.

Curcumin induces apoptosis of multidrug-resistant human leukemia HL60 cells by complex pathways leading to ceramide accumulation.

Most anti-cancer agents induce apoptosis, however, a development of multidrug resistance in cancer cells and defects in apoptosis contribute often to treatment failure. Here, the mechanism of curcumin-induced apoptosis was investigated in human leukemia HL60 cells and their HL60/VCR multidrug-resistant counterparts. In both cell lines curcumin induced a bi-phasic ceramide generation with a slow phase until 6 h followed by a more rapid one.

Crucial role of perfringolysin O D1 domain in orchestrating structural transitions leading to membrane-perforating pores: a hydrogen-deuterium exchange study.

Perfringolysin O (PFO) is a toxic protein that binds to cholesterol-containing membranes, oligomerizes, and forms a β-barrel transmembrane pore, leading to cell lysis. Previous studies have uncovered the sequence of events in this multistage structural transition to a considerable detail, but the underlying molecular mechanisms are not yet fully understood. By measuring hydrogen-deuterium exchange patterns of peptide bond amide protons monitored by mass spectrometry (MS), we have mapped structural changes in PFO and its variant bearing a point mutation during incorporation to the lipid environment.

Visualization of cholesterol deposits in lysosomes of Niemann-Pick type C fibroblasts using recombinant perfringolysin O.

Background: Niemann-Pick disease type C (NPC) is caused by defects in cholesterol efflux from lysosomes due to mutations of genes coding for NPC1 and NPC2 proteins. As a result, massive accumulation of unesterified cholesterol in late endosomes/lysosomes is observed. At the level of the organism these cholesterol metabolism disorders are manifested by progressive neurodegeneration and hepatosplenomegaly.

Tracking cholesterol/sphingomyelin-rich membrane domains with the ostreolysin A-mCherry protein.

Ostreolysin A (OlyA) is an ∼15-kDa protein that has been shown to bind selectively to membranes rich in cholesterol and sphingomyelin. In this study, we investigated whether OlyA fluorescently tagged at the C-terminal with mCherry (OlyA-mCherry) labels cholesterol/sphingomyelin domains in artificial membrane systems and in membranes of Madin-Darby canine kidney (MDCK) epithelial cells. OlyA-mCherry showed similar lipid binding characteristics to non-tagged OlyA.

CD14 mediates binding of high doses of LPS but is dispensable for TNF-α production.

Activation of macrophages with lipopolysaccharide (LPS) involves a sequential engagement of serum LPS-binding protein (LBP), plasma membrane CD14, and TLR4/MD-2 signaling complex. We analyzed participation of CD14 in TNF-α production stimulated with 1-1000 ng/mL of smooth or rough LPS (sLPS or rLPS) and in sLPS binding to RAW264 and J744 cells. CD14 was indispensable for TNF-α generation induced by a low concentration, 1 ng/mL, of sLPS and rLPS.

Species differences take shape at nanoparticles: protein corona made of the native repertoire assists cellular interaction.

Cells recognize the biomolecular corona around a nanoparticle, but the biological identity of the complex may be considerably different among various species. This study explores the importance of protein corona composition for nanoparticle recognition by coelomocytes of the earthworm Eisenia fetida using E. fetida coelomic proteins (EfCP) as a native repertoire and fetal bovine serum (FBS) as a non-native reference.

An interplay between scavenger receptor A and CD14 during activation of J774 cells by high concentrations of LPS.

Lipopolysaccharide (LPS) activates macrophages by binding to the TLR4/MD-2 complex and triggers two pro-inflammatory signaling pathways: one relies on MyD88 at the plasma membrane, and the other one depends on TRIF in endosomes. When present in high doses, LPS is internalized and undergoes detoxification. We found that the uptake of a high concentration of LPS (1000ng/ml) in macrophage-like J774 cells was upregulated upon inhibition of clathrin- and dynamin-mediated endocytosis which, on the other hand, strongly reduced the production of pro-inflammatory mediators TNF-α and RANTES.

Raft coalescence and FcγRIIA activation upon sphingomyelin clustering induced by lysenin.

Activation of immunoreceptor FcγRIIA by cross-linking with antibodies is accompanied by coalescence of sphingolipid/cholesterol-rich membrane rafts leading to the formation of signaling platforms of the receptor. In this report we examined whether clustering of the raft lipid sphingomyelin can reciprocally induce partition of FcγRIIA to rafts. To induce sphingomyelin clustering, cells were exposed to non-lytic concentrations of GST-lysenin which specifically recognizes sphingomyelin.

Cell surface ceramide controls translocation of transferrin receptor to clathrin-coated pits.

Transferrin receptor mediates internalization of transferrin with bound ferric ions through the clathrin-dependent pathway. We found that binding of transferrin to the receptor induced rapid generation of cell surface ceramide which correlated with activation of acid, but not neutral, sphingomyelinase. At the onset of transferrin internalization both ceramide level and acid sphingomyelinase activity returned to their basic levels.

LPS induces phosphorylation of actin-regulatory proteins leading to actin reassembly and macrophage motility.

Upon bacterial infection lipopolysaccharide (LPS) induces migration of monocytes/macrophages to the invaded region and production of pro-inflammatory mediators. We examined mechanisms of LPS-stimulated motility and found that LPS at 100 ng/ml induced rapid elongation and ruffling of macrophage-like J774 cells. A wound-healing assay revealed that LPS also activated directed cell movement that was followed by TNF-α production.

Mycobacterium tuberculosis lipoarabinomannan enhances LPS-induced TNF-α production and inhibits NO secretion by engaging scavenger receptors.

Lipoarabinomannan capped with terminal oligomannosides (ManLAM) is a component of mycobacteria cell wall enabling Mycobacterium tuberculosis to infect macrophages. We found that short treatment (3.5h) of macrophage-like J774 cells and thioglycollate-elicited peritoneal murine macrophages with ManLAM and its deacylated form enhanced LPS-stimulated release of tumor necrosis factor-α (TNF-α).

Determination of cell surface expression of Toll-like receptor 4 by cellular enzyme-linked immunosorbent assay and radiolabeling.

Lipopolysaccharide (LPS) is recognized by Toll-like receptor 4 (TLR4) of macrophages triggering production of pro-inflammatory mediators. One of the factors determining the magnitude of responses to LPS, which may even lead to life-threatening septic shock, is the cell surface abundance of TLR4. However, quantitation of the surface TLR4 is difficult due to the low level of receptor expression.

Ceramide and ceramide 1-phosphate are negative regulators of TNF-α production induced by lipopolysaccharide.

LPS is a constituent of cell walls of Gram-negative bacteria that, acting through the CD14/TLR4 receptor complex, causes strong proinflammatory activation of macrophages. In murine peritoneal macrophages and J774 cells, LPS at 1-2 ng/ml induced maximal TNF-α and MIP-2 release, and higher LPS concentrations were less effective, which suggested a negative control of LPS action. While studying the mechanism of this negative regulation, we found that in J774 cells, LPS activated both acid sphingomyelinase and neutral sphingomyelinase and moderately elevated ceramide, ceramide 1-phosphate, and sphingosine levels.

Sphingomyelin-rich domains are sites of lysenin oligomerization: implications for raft studies.

Lysenin is a self-assembling, pore-forming toxin which specifically recognizes sphingomyelin. Mutation of tryptophan 20 abolishes lysenin oligomerization and cytolytic activity. We studied the interaction of lysenin WT and W20A with sphingomyelin in membranes of various lipid compositions which, according to atomic force microscopy studies, generated either homo- or heterogeneous sphingomyelin distribution.

Contribution of PIP-5 kinase Ialpha to raft-based FcgammaRIIA signaling.

Receptor FcgammaIIA (FcgammaRIIA) associates with plasma membrane rafts upon activation to trigger signaling cascades leading to actin polymerization. We examined whether compartmentalization of PI(4,5)P(2) and PI(4,5)P(2)-synthesizing PIP5-kinase Ialpha to rafts contributes to FcgammaRIIA signaling. A fraction of PIP5-kinase Ialpha was detected in raft-originating detergent-resistant membranes (DRM) isolated from U937 monocytes and other cells.

How Mycobacterium tuberculosis subverts host immune responses.

Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis which has infected one third of the mankind and causes 2-3 million deaths worldwide each year. The persistence of the infection ensues from the ability of M. tuberculosis to subvert host immune responses in favor of survival and growth of mycobacteria in macrophages.

Disturbance of smooth muscle regulatory function by Eisenia foetida toxin lysenin: insight into the mechanism of smooth muscle contraction.

Lysenin, a toxin present in the coelomic fluid of the earthworm Eisenia foetida, is known to cause a long-lasting contraction of rat aorta smooth muscle strips. We addressed the mechanisms underlying its action on smooth muscle cells and present the first report demonstrating a completely new property of lysenin unrelated to its basic sphingomyelin-binding ability. Here we report lysenin enhancement effect on smooth muscle actomyosin ATPase activity and the ability of networking the actin filaments.

Secondary structure and orientation of the pore-forming toxin lysenin in a sphingomyelin-containing membrane.

Lysenin is a sphingomyelin-recognizing toxin which forms stable oligomers upon membrane binding and causes cell lysis. To get insight into the mechanism of the transition of lysenin from a soluble to a membrane-bound form, surface activity of the protein and its binding to lipid membranes were studied using tensiometric measurements, Fourier-transform infrared spectroscopy (FTIR) and FTIR-linear dichroism. The results showed cooperative adsorption of recombinant lysenin-His at the argon-water interface from the water subphase which suggested self-association of lysenin-His in solution.

Expression of PI(4,5)P2-binding proteins lowers the PI(4,5)P2level and inhibits FcgammaRIIA-mediated cell spreading and phagocytosis.

We found that FcgammaRII-mediated cell spreading and phagocytosis were correlated with an increase of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] level in cells. During the spreading, a long-lasting elevation of PI(4,5)P(2) and concomitant actin polymerization occurred. Filopodia and lamellae of spreading cells were enriched in phosphatidylinositol 4-phosphate 5-kinase Ialpha (PIP5-kinase Ialpha) that colocalized with PI(4,5)P(2 )and actin filaments.

Fc gamma RII activation induces cell surface ceramide production which participates in the assembly of the receptor signaling complex.

We studied an involvement of various cellular ceramide pools in signaling of immunoreceptor Fc gamma II (Fc gamma RII). The cell surface ceramide level was assessed by a technique based on binding of ceramide probes to intact cells. Total cellular ceramide was estimated by radioactive measurements.