Effects of TSPO and STAR inhibitors on cell death in a cardiomyocyte model of hypoxia-reoxygenation

International audienceHypoxia-reoxygenation TSPO-STAR Introduction We previously demonstrated that reperfusion of an ischemic myocardium induces an increase in mitochondrial cholesterol (CL) content accompanied by a generation of oxysterols. The translocator protein (TSPO) and the steroid acute regulatory protein (STAR) are involved in CL transport at the mitochondrial membrane in steroidogenic tissues but in the heart their role remain uncertain. The TSPO ligand 4'-chlorodiazepam (4'CDZ) has been demonstrated to inhibit STAR and sterols mitochondrial accumulations and to reduce infarct size. These data suggest that targeting mitochondrial sterol accumulation could participate to the protective effects of TSPO ligands. Objective To analyze the mechanisms and the role of the mitochondrial CL transport in cell death with a cardiomyocyte model of hypoxiareoxygenation (HR). Method AC16 human cardiomyocytes were submitted to different durations of hypoxia (1% O2) followed by reoxygenation (21% O2) to achieve 50% mortality. Cells were then treated at reoxygenation with 4'CDZ and novel TSPO and STAR inhibitors, known to delay steroidogenesis in vitro by targeting CL specific binding sites of these proteins (CRAC and START, respectively). Cell mortality was assessed with MTT and crystal violet assay and CL was identified by means of fluorescent probes. Results HR induced 41±3% mortality and modified membrane CL pattern in the cells. In this model, 4'-CDZ (10 µM) did not display cardioprotective effect (44±3% mortality). Similar results were observed with the TSPO inhibitors (CRAC benzamide 100 µM, CRAC triol 100 µM) and the STAR inhibitor (START triol 100 µM) (46±1% 43±3% and 54±3% mortality, respectively). Conclusion This preliminary study suggests that TSPO ligands do not exert cardioprotection through a direct action on cardiomyocytes. This conclusion needs to be confirmed with the use of adult primary cardiomyocytes

Involvement of a cluster of basic amino acids in phosphoryla-tion-dependent functional repression of the ceramide transport protein CERT

The uncropped images of western blots, the unprocessed images of immunofluorescence, and raw data obtained in this study are deposited in this Figshare site

HIV-1 Gag targeting to the plasma membrane reorganizes sphingomyelin-rich and cholesterol-rich lipid domains

Abstract Although the human immunodeficiency virus type 1 lipid envelope has been reported to be enriched with host cell sphingomyelin and cholesterol, the molecular mechanism of the enrichment is not well understood. Viral Gag protein plays a central role in virus budding. Here, we report the interaction between Gag and host cell lipids using different quantitative and super-resolution microscopy techniques in combination with specific probes that bind endogenous sphingomyelin and cholesterol. Our results indicate that Gag in the inner leaflet of the plasma membrane colocalizes with the outer leaflet sphingomyelin-rich domains and cholesterol-rich domains, enlarges sphingomyelin-rich domains, and strongly restricts the mobility of sphingomyelin-rich domains. Moreover, Gag multimerization induces sphingomyelin-rich and cholesterol-rich lipid domains to be in close proximity in a curvature-dependent manner. Our study suggests that Gag binds, coalesces, and reorganizes pre-existing lipid domains during assembly

Formation of tubules and helical ribbons by ceramide phosphoethanolamine-containing membranes

Abstract Ceramide phosphoethanolamine (CPE), a major sphingolipid in invertebrates, is crucial for axonal ensheathment in Drosophila. Darkfield microscopy revealed that an equimolar mixture of bovine buttermilk CPE (milk CPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (diC18:1 PC) tends to form tubules and helical ribbons, while pure milk CPE mainly exhibits amorphous aggregates and, at low frequency, straight needles. Negative staining electron microscopy indicated that helices and tubules were composed of multilayered 5–10 nm thick slab-like structures. Using different molecular species of PC and CPE, we demonstrated that the acyl chain length of CPE but not of PC is crucial for the formation of tubules and helices in equimolar mixtures. Incubation of the lipid suspensions at the respective phase transition temperature of CPE facilitated the formation of both tubules and helices, suggesting a dynamic lipid rearrangement during formation. Substituting diC18:1 PC with diC18:1 PE or diC18:1 PS failed to form tubules and helices. As hydrated galactosylceramide (GalCer), a major lipid in mammalian myelin, has been reported to spontaneously form tubules and helices, it is believed that the ensheathment of axons in mammals and Drosophila is based on similar physical processes with different lipids

Source Data.xlsx

Source Data file for the study entitled 'HIV-1 Gag targeting to the plasma membrane reorganizes sphingomyelin-rich and cholesterol-rich lipid domains'</p

MOSPD2 is an endoplasmic reticulum–lipid droplet tether functioning in LD homeostasis

International audienceMembrane contact sites between organelles are organized by protein bridges. Among the components of these contacts, the VAP family comprises ER–anchored proteins, such as MOSPD2, that function as major ER–organelle tethers. MOSPD2 distinguishes itself from the other members of the VAP family by the presence of a CRAL-TRIO domain. In this study, we show that MOSPD2 forms ER–lipid droplet (LD) contacts, thanks to its CRAL-TRIO domain. MOSPD2 ensures the attachment of the ER to LDs through a direct protein–membrane interaction. The attachment mechanism involves an amphipathic helix that has an affinity for lipid packing defects present at the surface of LDs. Remarkably, the absence of MOSPD2 markedly disturbs the assembly of lipid droplets. These data show that MOSPD2, in addition to being a general ER receptor for inter-organelle contacts, possesses an additional tethering activity and is specifically implicated in the biology of LDs via its CRAL-TRIO domain

Casein Kinase Iγ2 Down-Regulates Trafficking of Ceramide in the Synthesis of Sphingomyelin

Intracellullar trafficking of lipids is fundamental to membrane biogenesis. For the synthesis of sphingomyelin, ceramide is transported from the endoplasmic reticulum to the Golgi apparatus by the ceramide transfer protein CERT. CERT is phosphorylated by protein kinase D at S132 and subsequently multiple times in a serine-repeat motif, resulting in its inactivation. However, the kinase involved in the multiple phosphorylation remains unclear. Here, we identify the γ2 isoform of casein kinase I (CKIγ2) as a kinase whose overexpression confers sphingomyelin-directed toxin-resistance to Chinese hamster ovary cells. In a transformant stably expressing CKIγ2, CERT was hyperphosphorylated, and the intracellular trafficking of ceramide was retarded, thereby reducing de novo sphingomyelin synthesis. The reduction in the synthesis of sphingomyelin caused by CKIγ2 was reversed by the expression of CERT mutants that are not hyperphosphorylated. Furthermore, CKIγ2 directly phosphorylated CERT in vitro. Among three γ isoforms, only knockdown of γ2 isoform caused drastic changes in the ratio of hypo- to hyperphosphorylated form of CERT in HeLa cells. These results indicate that CKIγ2 hyperphosphorylates the serine-repeat motif of CERT, thereby inactivating CERT and down-regulating the synthesis of sphingomyelin