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. GCase is activated and stabilized by saposin C (Sap C) and anionic phospholipids. Here we show by immunofluorescence microscopy that in normal fibroblasts, GCase, Sap C, and lysobisphosphatidic acid (LBPA), the most abundant anionic phospholipid in the endolysosomal system, reside in the same intracellular vesicular structures. In contrast, the colocalization of GCase, Sap C, and LBPA is markedly impaired in NPC fibroblasts but can be re-established by cholesterol depletion. These data show for the first time that the level of cholesterol modulates the interaction of GCase with its protein and lipid activators, namely Sap C and LBPA, regulating the GCase activity and stability

Improved pregnancy outcome in patients with Rheumatoid Arthritis who followed an ideal clinical pathway

Among women with rheumatoid arthritis (RA) we aimed to assess the effect of optimal management of pregnancy, on a composite outcome of miscarriage and complicated birth

Cellular Localization and Functional Role of Phosphatidylcholine-Specific Phospholipase C in NK Cells

AbstractAlthough several classes of phospholipases have been implicated in NK cell-mediated cytotoxicity, no evidence has been reported to date on involvement of phosphatidylcholine-specific phospholipase C (PC-PLC) in NK activation by lymphokines and/or in lytic granule exocytosis. This study demonstrated the expression of two PC-PLC isoforms (Mr 40 and 66 kDa) and their IL-2-dependent distribution between cytoplasm and ectoplasmic membrane surface in human NK cells. Following cell activation by IL-2, cytoplasmic PC-PLC translocated from the microtubule-organizing center toward cell periphery, essentially by kinesin-supported transport along microtubules, while PC-PLC exposed on the outer cell surface increased 2-fold. Preincubation of NK cells with a PC-PLC inhibitor, tricyclodecan-9-yl-xanthogenate, strongly reduced NK-mediated cytotoxicity. In IL-2-activated cells, this loss of cytotoxicity was associated with a decrease of PC-PLC exposed on the cell surface, and accumulation of cytoplasmic PC-PLC in the Golgi region. Massive colocalization of PC-PLC-rich particles with perforin-containing granules was found in the cytoplasm of NK-activated (but not NK-resting) cells; both organelles clustered at the intercellular contact region of effector-target cell conjugates. These newly detected mechanisms of PC-PLC translocation and function support an essential role of this enzyme in regulated granule exocytosis and NK-mediated cytotoxicity

Role of Cholesterol in Human Immunodeficiency Virus Type 1 Envelope Protein-Mediated Fusion with Host Cells

In this study we examined the effects of target membrane cholesterol depletion and cytoskeletal changes on human immunodeficiency virus type 1 (HIV-1) Env-mediated membrane fusion by dye redistribution assays. We found that treatment of peripheral blood lymphocytes (PBL) with methyl-β-cyclodextrin (MβCD) or cytochalasin reduced their susceptibility to membrane fusion with cells expressing HIV-1 Env that utilize CXCR4 or CCR5. However, treatment of human osteosarcoma (HOS) cells expressing high levels of CD4 and coreceptors with these agents did not affect their susceptibility to HIV-1 Env-mediated membrane fusion. Removal of cholesterol inhibited stromal cell-derived factor-1α- and macrophage inflammatory protein 1β-induced chemotaxis of both PBL and HOS cells expressing CD4 and coreceptors. The fusion activity as well as the chemotactic activity of PBL was recovered by adding back cholesterol to these cells. Confocal laser scanning microscopy analysis indicated that treatment of lymphocytes with MβCD reduced the colocalization of CD4 or of CXCR4 with actin presumably in microvilli. These findings indicate that, although cholesterol is not required for HIV-1 Env-mediated membrane fusion per se, its depletion from cells with relatively low coreceptor densities reduces the capacity of HIV-1 Env to engage coreceptor clusters required to trigger fusion. Furthermore, our results suggest that coreceptor clustering may occur in microvilli that are supported by actin polymerization