Caveolin-1 Endows Order in Cholesterol-Rich Detergent Resistant Membranes.

Cholesterol-enriched functional portions of plasma membranes, such as caveolae and rafts, were isolated from lungs of wild-type (WT) and caveolin-1 knockout (Cav-1 KO) mice within detergent resistant membranes (DRMs). To gain insight into their molecular composition we performed proteomic and lipid analysis on WT and Cav-1 KO-DRMs that showed predicted variations of proteomic profiles and negligible differences in lipid composition, while Langmuir monolayer technique and small and wide-angle X-ray scattering (SAXS-WAXS) were here originally introduced to study DRMs biophysical association state. Langmuir analysis of Cav-1 containing DRMs displayed an isotherm with a clear-cut feature, suggesting the coexistence of the liquid-ordered () phase typical of the raft structure, namely "cholesterol-rich phase," with a phase fully missing in Cav-1 KO that we named "caveolin-induced phase.

Dexamethasone Predisposes Human Erythroblasts Toward Impaired Lipid Metabolism and Renders Their Expansion Highly Dependent on Plasma Lipoproteins.

Cultures of stem cells from discarded sources supplemented with dexamethasone, a synthetic glucocorticoid receptor agonist, generate cultured red blood cells (cRBCs) in numbers sufficient for transfusion. According to the literature, however, erythroblasts generated with dexamethasone exhibit low enucleation rates giving rise to cRBCs that survive poorly . The knowledge that the glucocorticoid receptor regulates lipid metabolism and that lipid composition dictates the fragility of the plasma membrane suggests that insufficient lipid bioavailability restrains generation of cRBCs.

Disruption of IFN-I Signaling Promotes HER2/Neu Tumor Progression and Breast Cancer Stem Cells.

Type I interferon (IFN-I) is a class of antiviral immunomodulatory cytokines involved in many stages of tumor initiation and progression. IFN-I acts directly on tumor cells to inhibit cell growth and indirectly by activating immune cells to mount antitumor responses. To understand the role of endogenous IFN-I in spontaneous, oncogene-driven carcinogenesis, we characterized tumors arising in HER2/neu transgenic (neuT) mice carrying a nonfunctional mutation in the IFNI receptor (IFNAR1).

The thrombopoietin/MPL axis is activated in the Gata1 mouse model of myelofibrosis and is associated with a defective RPS14 signature.

Myelofibrosis (MF) is characterized by hyperactivation of thrombopoietin (TPO) signaling, which induces a RPS14 deficiency that de-regulates GATA1 in megakaryocytes by hampering its mRNA translation. As mice carrying the hypomorphic Gata1 mutation, which reduces the levels of Gata1 mRNA in megakaryocytes, develop MF, we investigated whether the TPO axis is hyperactive in this model. Gata1 mice contained two times more Tpo mRNA in liver and TPO in plasma than wild-type littermates.

2p15-p16.1 microdeletions encompassing and proximal to BCL11A are associated with elevated HbF in addition to neurologic impairment.

Elevated fetal hemoglobin (HbF) ameliorates the clinical severity of hemoglobinopathies such as β-thalassemia and sickle cell anemia. Currently, the only curative approach for individuals under chronic transfusion/chelation support therapy is allogeneic stem cell transplantation. However, recent analyses of heritable variations in HbF levels have provided a new therapeutic target for HbF reactivation: the transcriptional repressor BCL11A.

Activation of non-canonical TGF-β1 signaling indicates an autoimmune mechanism for bone marrow fibrosis in primary myelofibrosis.

Primary myelofibrosis (PMF) is characterized by megakaryocyte hyperplasia, dysplasia and death with progressive reticulin/collagen fibrosis in marrow and hematopoiesis in extramedullary sites. The mechanism of fibrosis was investigated by comparing TGF-β1 signaling of marrow and spleen of patients with PMF and of non-diseased individuals. Expression of 39 (23 up-regulated and 16 down-regulated) and 38 (8 up-regulated and 30 down-regulated) TGF-β1 signaling genes was altered in the marrow and spleen of PMF patients, respectively.

Transcriptomic and phospho-proteomic analyzes of erythroblasts expanded in vitro from normal donors and from patients with polycythemia vera.

Erythropoiesis is a tightly regulated process which becomes decoupled from its normal differentiation program in patients with polycythemia vera (PV). Somatic mutations in JAK2 are commonly associated with this myeloid proliferative disorder. To gain insight into the molecular events that are required for abnormally developing erythroid cells to escape dependence on normal growth signals, we performed in vitro expansion of mature erythroblasts (ERY) from seven normal healthy donors and from seven polycythemic patients in the presence of IL3, EPO, SCF for 10, 11, or 13 days.

Characterization of the TGF-β1 signaling abnormalities in the Gata1low mouse model of myelofibrosis.

Primary myelofibrosis (PMF) is characterized by fibrosis, ineffective hematopoiesis in marrow, and hematopoiesis in extramedullary sites and is associated with abnormal megakaryocyte (MK) development and increased transforming growth factor (TGF)-β1 release. To clarify the role of TGF-β1 in the pathogenesis of this disease, the TGF-β1 signaling pathway of marrow and spleen of the Gata1(low) mouse model of myelofibrosis (MF) was profiled and the consequences of inhibition of TGF-β1 signaling on disease manifestations determined. The expression of 20 genes in marrow and 36 genes in spleen of Gata1(low) mice was altered.

Evaluation of the spontaneous reversibility of carbon tetrachloride-induced liver cirrhosis in rabbits.

There is a general consensus that liver fibrosis in humans is potentially reversible, while scepticism prevails on the concept that cirrhosis can be truly reversed. The availability of suitable experimental models is fundamental for disease research. The experimental murine model of liver cirrhosis induced by carbon tetrachloride (CCl(4)) reproduces both the histological picture of the postnecrotic cirrhosis and its biochemical and clinical parameters.

Hepatic VLDL assembly is disturbed in a rat model of nonalcoholic fatty liver disease: is there a role for dietary coenzyme Q?

The overproduction of very-low-density lipoprotein (VLDL) is a characteristic feature of nonalcoholic fatty liver disease (NAFLD). The aim of this study was to use a high-fat diet-induced model of NAFLD in rats to investigate 1) the influence of the disease on hepatic VLDL processing in the endoplasmic reticulum and 2) the potential modulatory effects of dietary coenzyme Q (CoQ). Rats were fed a standard low-fat diet (control) or a diet containing 35% fat (57% metabolizable energy).

Saposin B binds and transfers phospholipids.

Saposin B (Sap B) is a member of a family of four small glycoproteins, Sap A, B, C, and D. Like the other three saposins, Sap B plays a physiological role in the lysosomal degradation of sphingolipids (SLs). Although the interaction of Sap B with SLs has been investigated extensively, that with the main membrane lipid components, namely phospholipids and cholesterol (Chol), is scarcely known.

The N370S (Asn370-->Ser) mutation affects the capacity of glucosylceramidase to interact with anionic phospholipid-containing membranes and saposin C.

The properties of the endolysosomal enzyme GCase (glucosylceramidase), carrying the most prevalent mutation observed in Gaucher patients, namely substitution of an asparagine residue with a serine at amino acid position 370 [N370S (Asn370-->Ser) GCase], were investigated in the present study. We previously demonstrated that Sap (saposin) C, the physiological GCase activator, promotes the association of GCase with anionic phospholipid-containing membranes, reconstituting in this way the enzyme activity. In the present study, we show that, in the presence of Sap C and membranes containing high levels of anionic phospholipids, both normal and N370S GCases are able to associate with the lipid surface and to express their activity.

Glucosylceramidase mass and subcellular localization are modulated by cholesterol in Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) is characterized by the accumulation of cholesterol and sphingolipids in the late endosomal/lysosomal compartment. The mechanism by which the concentration of sphingolipids such as glucosylceramide is increased in this disease is poorly understood. We have found that, in NPC fibroblasts, the cholesterol storage affects the stability of glucosylceramidase (GCase), decreasing its mass and activity; a reduction of cholesterol raises the level of GCase to nearly normal values.

Interaction of saposin D with membranes: effect of anionic phospholipids and sphingolipids.

Saposin (Sap) D is an endolysosomal protein that, together with three other similar proteins, Sap A, Sap B and Sap C, is involved in the degradation of sphingolipids and, possibly, in the solubilization and transport of gangliosides. We found that Sap D is able to destabilize and disrupt membranes containing each of the three anionic phospholipids most abundant in the acidic endolysosomal compartment, namely lysobisphosphatidic acid (LBPA), phosphatidylinositol (PI) and phosphatidylserine (PS). The breakdown of the membranes, which occurs when the Sap D concentration on the lipid surface reaches a critical value, is a slow process that gives rise to small particles.

Saposin D solubilizes anionic phospholipid-containing membranes.

Saposin (Sap) D is a late endosomal/lysosomal small protein, generated together with three other similar proteins, Sap A, B, and C, from the common precursor, prosaposin. Although the functions of saposins such as Sap B and C are well known (Sap B promotes the hydrolysis of sulfatides and Sap C that of glucosylceramide), neither the physiological function nor the mechanism of action of Sap D are yet fully understood. We previously found that a dramatic increase of Sap D superficial hydrophobicity, occurring at the low pH values characteristic of the late endosomal/lysosomal environment, triggers the interaction of the saposin with anionic phospholipid-containing vesicles.

Further studies on the reconstitution of glucosylceramidase activity by Sap C and anionic phospholipids.

The reconstitution of the activity of the lysosomal enzyme glucosylceramidase requires anionic phospholipids and, at least, a protein factor, saposin C (Sap C). We have previously proposed a mechanism for the glucosylceramidase activation [Vaccaro et al. (1993) FEBS Lett.

Structural and membrane-binding properties of saposin D.

Saposin D is generated together with three similar proteins, saposins A, B and C, from a common precursor, called prosaposin, in acidic organelles such as late endosomes and lysosomes. Although saposin D has been reported to stimulate the enzymatic hydrolysis of sphingomyelin and ceramide, its physiological role has not yet been clearly established. In the present study we examined structural and membrane-binding properties of saposin D.

Saposins and their interaction with lipids.

The lysosomal degradation of several sphingolipids requires the presence of four small glycoproteins called saposins, generated by proteolytic processing of a common precursor, prosaposin. Saposins share several structural properties, including six similarly located cysteines forming three disulfide bridges with the same cysteine pairings. Recently it has been noted that also other proteins have the same polypeptide motif characterized by the similar location of six cysteines.

Effect of saposins A and C on the enzymatic hydrolysis of liposomal glucosylceramide.

The degradation of glucosylceramide in lysosomes is accomplished by glucosylceramidase with the assistance of, at least, another protein, saposin C (Sap C), which is generated from a large precursor together with three other similar proteins, saposins A, B, and D. In the present study, we have examined the effects of saposins on the enzymatic hydrolysis of glucosylceramide inserted in large and small phospholipid liposomes. The glucosylceramide contained in large unilamellar vesicles (LUV) was degraded by glucosylceramidase at a rate 7-8-fold lower than glucosylceramide inserted in small unilamellar vesicles (SUV).

pH-dependent conformational properties of saposins and their interactions with phospholipid membranes.

Saposins A, B, C, and D are small lysosomal glycoproteins released by proteolysis from a single precursor polypeptide, prosaposin. We have presently investigated the conformational states of saposins and their interaction with membranes at acidic pH values similar to those present in lysosomes. With the use of phase partitioning in Triton X-114, experimental evidence was provided that, upon acidification, saposins (Sap) A, C, and D acquire hydrophobic properties, while the hydrophilicity of Sap B is apparently unchanged.

Structural analysis of saposin C and B. Complete localization of disulfide bridges.

Saposins A, B, C, and D are a group of homologous glycoproteins derived from a single precursor, prosaposin, and apparently involved in the stimulation of the enzymatic degradation of sphingolipids in lysosomes. All saposins have six cysteine residues at similar positions. In the present study we have investigated the disulfide structure of saposins B and C using advanced mass spectrometric procedures.

Saposin C induces pH-dependent destabilization and fusion of phosphatidylserine-containing vesicles.

We have previously shown that saposin C (Sap C), a glucosylceramidase activator protein, interacts with phosphatidylserine (PS) large unilamellar vesicles (LUV), promoting the glucosylceramidase binding to the bilayer [(1993) FEBS Lett. 336, 159-162]. In the present paper the consequences of the Sap C interaction on the lipid organization of the vesicles are reported.

Function of saposin C in the reconstitution of glucosylceramidase by phosphatidylserine liposomes.

The function of saposin C (Sap C), a glucosylceramidase activator protein, in the enzyme stimulation by phosphatidylserine (PS) liposomes has been investigated. Using gel filtration experiments evidence was obtained for Sap C binding to PS large unilamellar vesicles (LUV) but not to glucosylceramidase. PS LUV, which by themselves are unable to tightly bind and stimulate the enzyme, acquire the capacity to also bind the enzyme after interaction with Sap C, making it express its full activity.

Studies on glucosylceramidase binding to phosphatidylserine liposomes: the role of bilayer curvature.

The influence of phosphatidylserine (PS) liposome size on their capacity to activate and bind purified glucosylceramidase was investigated. Gel filtration and flotation experiments showed that large unilamellar vesicles (LUV) of either pure PS or PS in admixture with phosphatidylcholine (PC) are unable to tightly bind purified glucosylceramidase, and thus, to fully stimulate its activity. By contrast, small unilamellar vesicles (SUV) of PS adsorb glucosylceramidase can either be favoured or inhibited by factors affecting the bilayer curvature of PS liposomes.

Reconstitution of glucosylceramidase on binding to acidic phospholipid-containing vesicles.

Studies were conducted to investigate the mechanism by which acidic phospholipid-containing vesicles stimulate purified placental glucosylceramidase activity towards the water-soluble substrate 4-methylumbelliferyl-beta-D-glucopyranoside (MUGlc). Vesicles composed of pure phosphatidic acid (PA) or pure phosphatidylserine (PS) stimulated the activity of the enzyme about 20-fold. The inclusion of cholesterol and phosphatidylcholine (PC), beside PA, into the vesicles slightly improved their stimulatory effect.