Mechanisms of nonvesicular lipid transport.
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ABSTRACT:
SUBMITTER: Reinisch KM
PROVIDER: S-EPMC7901144 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The Aster proteins (encoded by the Gramd1a-c genes) contain a ligand-binding fold structurally similar to a START domain and mediate nonvesicular plasma membrane (PM) to endoplasmic reticulum (ER) cholesterol transport. In an effort to develop small molecule modulators of Asters, we identified 20α-hydroxycholesterol (HC) and U18666A as lead compounds. Unfortunately, both 20α-HC and U18666A target other sterol homeostatic proteins, limiting their utility. 20α-HC inhibits sterol regulatory element-binding protein 2 (SREBP2) processing, and U18666A is an inhibitor of the vesicular trafficking protein Niemann-Pick C1 (NPC1). To develop potent and selective Aster inhibitors, we synthesized a series of compounds by modifying 20α-HC and U18666A. Among these, AI (Aster inhibitor)-1l, which has a longer side chain than 20α-HC, selectively bound to Aster-C. The crystal structure of Aster-C in complex with AI-1l suggests that sequence and flexibility differences in the loop that gates the binding cavity may account for the ligand specificity for Aster C. We further identified the U18666A analog AI-3d as a potent inhibitor of all three Aster proteins. AI-3d blocks the ability of Asters to bind and transfer cholesterol in vitro and in cells. Importantly, AI-3d also inhibits the movement of low-density lipoprotein (LDL) cholesterol to the ER, although AI-3d does not block NPC1. This finding positions the nonvesicular Aster pathway downstream of NPC1-dependent vesicular transport in the movement of LDL cholesterol to the ER. Selective Aster inhibitors represent useful chemical tools to distinguish vesicular and nonvesicular sterol transport mechanisms in mammalian cells.
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Project description:In cell models, changes in the “accessible” pool of plasma membrane (PM) cholesterol are linked with the regulation of ER sterol synthesis and metabolism by the Aster family of nonvesicular transporters. However, the relevance of such nonvesicular transport mechanisms for lipid homeostasis in vivo has not been defined. Here we reveal two physiological contexts that generate accessible PM cholesterol and engage the Aster pathway in liver: fasting and reverse cholesterol transport (RCT). During fasting, adipose tissue–derived fatty acids activate hepatocyte sphingomyelinase to liberate sequestered PM cholesterol. Aster-dependent cholesterol transport during fasting facilitates cholesteryl ester (CE) formation, cholesterol movement into bile, and VLDL production. During RCT, HDL delivers excess cholesterol to the hepatocyte PM through SR-BI. Loss of hepatic Asters impairs cholesterol movement into feces, raises plasma cholesterol levels, and causes cholesterol accumulation in peripheral tissues. These results reveal fundamental mechanisms by which Aster cholesterol flux contributes to hepatic and systemic lipid homeostasis.
2022-10-24 | GSE206278 | GEO
Project description:Membrane contact sites (MCSs) in eukaryotic cells are hotspots for lipid exchange, which is essential for many biological functions, including regulation of membrane properties and protein trafficking. Lipid transfer proteins anchored at membrane contact sites (LAMs) contain sterol-specific lipid transfer domains [StARkin domain (SD)] and multiple targeting modules to specific membrane organelles. Elucidating the structural mechanisms of targeting and ligand recognition by LAMs is important for understanding the interorganelle communication and exchange at MCSs. Here, we determined the crystal structures of the yeast Lam6 pleckstrin homology (PH)-like domain and the SDs of Lam2 and Lam4 in the apo form and in complex with ergosterol. The Lam6 PH-like domain displays a unique PH domain fold with a conserved N-terminal ?-helix. The Lam6 PH-like domain lacks the basic surface for phosphoinositide binding, but contains hydrophobic patches on its surface, which are critical for targeting to endoplasmic reticulum (ER)-mitochondrial contacts. Structures of the LAM SDs display a helix-grip fold with a hydrophobic cavity and a flexible ?1-loop as a lid. Ergosterol is bound to the pocket in a head-down orientation, with its hydrophobic acyl group located in the tunnel entrance. The ?1-loop in an open conformation is essential for ergosterol binding by direct hydrophobic interaction. Structural comparison suggested that the sterol binding mode of the Lam2 SD2 is likely conserved among the sterol transfer proteins of the StARkin superfamily. Structural models of full-length Lam2 correlated with the sterol transport function at the membrane contact sites.
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Project description:The mechanisms underlying sterol transport in mammalian cells are poorly understood. In particular, how cholesterol internalized from HDL is made available to the cell for storage or modification is unknown. Here, we describe three ER-resident proteins (Aster-A, -B, -C) that bind cholesterol and facilitate its removal from the plasma membrane. The crystal structure of the central domain of Aster-A broadly resembles the sterol-binding fold of mammalian StARD proteins, but sequence differences in the Aster pocket result in a distinct mode of ligand binding. The Aster N-terminal GRAM domain binds phosphatidylserine and mediates Aster recruitment to plasma membrane-ER contact sites in response to cholesterol accumulation in the plasma membrane. Mice lacking Aster-B are deficient in adrenal cholesterol ester storage and steroidogenesis because of an inability to transport cholesterol from SR-BI to the ER. These findings identify a nonvesicular pathway for plasma membrane to ER sterol trafficking in mammals.
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Project description:Intestinal cholesterol absorption is an important determinant of systemic cholesterol homeostasis. Niemann-Pick C1 Like 1 (NPC1L1), the target of the drug ezetimibe, is a critical player in dietary cholesterol uptake. But how cholesterol moves within the cell downstream of NPC1L1 is unknown. Here we show that the nonvesicular sterol transporters Aster-B and -C cooperate with NPC1L1 to deliver dietary cholesterol from the gut lumen to the enterocyte ER for chylomicron packaging. Aster proteins are recruited to the enterocyte plasma membrane (PM) in response to NPC1L1-dependent cholesterol accumulation. Mice lacking Asters in intestine have impaired cholesterol absorption, and reduced plasma cholesterol. NanoSIMS imaging and tracer studies reveal delayed lipid trafficking into chylomicrons in Aster-deficient enterocytes. Interestingly, in addition to potently blocking NPC1L1, ezetimibe is also a low-affinity inhibitor of Aster-B and -C but not -A, and the structure of the Aster-C-ezetimibe complex reveals the basis for this selectivity. Our findings support a model in which NPC1L1 enriches dietary cholesterol at the apical PM, and ASTERs subsequently traffic this cholesterol to the ER. The findings identify the enterocyte Aster pathway as potential target for treatment of hypercholesterolemia. Alessandra Ferrari, PhD
2023-12-21 | GSE206780 | GEO
Project description:In mammalian cells, ceramide is synthesized in the endoplasmic reticulum and transferred to the Golgi apparatus for conversion to sphingomyelin. Ceramide transport occurs in a nonvesicular manner and is mediated by CERT, a cytosolic 68-kDa protein with a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. The CERT START domain efficiently transfers natural D-erythro-C16-ceramide, but not lipids with longer (C20) amide-acyl chains. The molecular mechanisms of ceramide specificity, both stereo-specific recognition and length limit, are not well understood. Here we report the crystal structures of the CERT START domain in its apo-form and in complex with ceramides having different acyl chain lengths. In these complex structures, one ceramide molecule is buried in a long amphiphilic cavity. At the far end of the cavity, the amide and hydroxyl groups of ceramide form a hydrogen bond network with specific amino acid residues that play key roles in stereo-specific ceramide recognition. At the head of the ceramide molecule, there is no extra space to accommodate additional bulky groups. The two aliphatic chains of ceramide are surrounded by the hydrophobic wall of the cavity, whose size and shape dictate the length limit for cognate ceramides. Furthermore, local high-crystallographic B-factors suggest that the alpha-3 and the Omega1 loop might work as a gate to incorporate the ceramide into the cavity. Thus, the structures demonstrate the structural basis for the mechanism by which CERT can distinguish ceramide from other lipid types yet still recognize multiple species of ceramides.
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Project description:BACKGROUND:In recent years, nanoparticles (NPs) including nanostructured lipid carries (NLC) and solid lipid nanoparticles (SLN) captured an increasing amount of attention in the field of transdermal drug delivery system. However, the mechanisms of penetration enhancement and transdermal transport properties of NPs are not fully understood. Therefore, this work applied different platforms to evaluate the interactions between skin and NPs loading triptolide (TPL, TPL-NLC and TPL-SLN). Besides, NPs labeled with fluorescence probe were tracked after administration to investigate the dynamic penetration process in skin and skin cells. In addition, ELISA assay was applied to verify the in vitro anti-inflammatory effect of TPL-NPs. RESULTS:Compared with the control group, TPL-NPs could disorder skin structure, increase keratin enthalpy and reduce the SC infrared absorption peak area. Besides, the work found that NPs labeled with fluorescence probe accumulated in hair follicles and distributed throughout the skin after 1 h of administration and were taken into HaCaT cells cytoplasm by transcytosis. Additionally, TPL-NLC could effectively inhibit the expression of IL-4, IL-6, IL-8, IFN-?, and MCP-1 in HaCaT cells, while TPL-SLN and TPL solution can only inhibit the expression of IL-6. CONCLUSIONS:TPL-NLC and TPL-SLN could penetrate into skin in a time-dependent manner and the penetration is done by changing the structure, thermodynamic properties and components of the SC. Furthermore, the significant anti-inflammatory effect of TPL-NPs indicated that nanoparticles containing NLC and SLN could serve as safe prospective agents for transdermal drug delivery system.
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