In humans, mutations in ATGL lead to TG accumulation in LDs of most tissues and cells, including peripheral blood leukocytes. This pathologic condition is called Jordans’ anomaly, in which functional consequences have not been investigated. In the present study, we tested the hypothesis that ATGL plays a role in leukocyte LD metabolism and immune cell function. Similar to humans with loss-of-function mutations in ATGL, we found that global and myeloid-specific Atgl−/− mice exhibit Jordans’ anomaly with increased abundance of intracellular TG-rich LDs in neutrophil granulocytes. In a model of inflammatory peritonitis, lipid accumulation was also observed in monocytes and macrophages but not in eosinophils or lymphocytes. Neutrophils from Atgl−/− mice showed enhanced immune responses in vitro, which were more prominent in cells from global compared with myeloid-specific Atgl−/− mice. Mechanistically, ATGL−/− as well as pharmacological inhibition of ATGL led to an impaired release of lipid mediators from neutrophils. These findings demonstrate that the release of lipid mediators is dependent on the liberation of precursor molecules from the TG-rich pool of LDs by ATGL. Our data provide mechanistic insights into Jordans’ anomaly in neutrophils and suggest that ATGL is a potent regulator of immune cell function and inflammatory diseases.
Lysosome-associated protein transmembrane-4b (LAPTM4B) associates with poor prognosis in several cancers, but its physiological function is not well understood. Here we use novel ceramide probes to provide evidence that LAPTM4B interacts with ceramide and facilitates its removal from late endosomal organelles (LEs). This lowers LE ceramide in parallel with and independent of acid ceramidase-dependent catabolism. In LAPTM4B-silenced cells, LE sphingolipid accumulation is accompanied by lysosomal membrane destabilization. However, these cells resist ceramide-driven caspase-3 activation and apoptosis induced by chemotherapeutic agents or gene silencing. Conversely, LAPTM4B overexpression reduces LE ceramide and stabilizes lysosomes but sensitizes to drug-induced caspase-3 activation. Together, these data uncover a cellular ceramide export route from LEs and identify LAPTM4B as its regulator. By compartmentalizing ceramide, LAPTM4B controls key sphingolipid-mediated cell death mechanisms and emerges as a candidate for sphingolipid-targeting cancer therapies.
Membrane proteins are functionally regulated by the composition of the surrounding lipid bilayer. The late endosomal compartment is a central site for the generation of ceramide, a bioactive sphingolipid, which regulates responses to cell stress. The molecular interactions between ceramide and late endosomal transmembrane proteins are unknown. Here, we uncover in atomistic detail the ceramide interaction of Lysosome Associated Protein Transmembrane 4B (LAPTM4B), implicated in ceramide-dependent cell death and autophagy, and its functional relevance in lysosomal nutrient signaling. The ceramide-mediated regulation of LAPTM4B depends on a sphingolipid interaction motif and an adjacent aspartate residue in the protein’s third transmembrane (TM3) helix. The interaction motif provides the preferred contact points for ceramide while the neighboring membrane-embedded acidic residue confers flexibility that is subject to ceramide-induced conformational changes, reducing TM3 bending. This facilitates the interaction between LAPTM4B and the amino acid transporter heavy chain 4F2hc, thereby controlling mTORC signaling. These findings provide mechanistic insights into how transmembrane proteins sense and respond to ceramide.
Objective-The purpose of this study was to identify rare APOA5 variants in 130 severe hypertriglyceridemic patients by sequencing, and to test their functionality, since no patient recall was possible. Methods and Results-We studied the impact in vitro on LPL activity and receptor binding of 3 novel heterozygous variants, apoAV-E255G, -G271C, and -H321L, together with the previously reported -G185C, -Q139X, -Q148X, and a novel construct -⌬139 to 147. Using VLDL as a TG-source, compared to wild type, apoAV-G255, -L321 and -C185 showed reduced LPL activation (Ϫ25% [Pϭ0.005], Ϫ36% [PϽ0.0001], and Ϫ23% [Pϭ0.02]), respectively). ApoAV-C271, -X139, -X148, and ⌬139 to 147 had little affect on LPL activity, but apoAV-X139, -X148, and -C271 showed no binding to LDL-family receptors, LR8 or LRP1. Although the G271C proband carried no LPL and APOC2 mutations, the H321L carrier was heterozygous for LPL P207L. The E255G carrier was homozygous for LPL W86G, yet only experienced severe hypertriglyceridemia when pregnant. Key Words: apolipoprotein AV Ⅲ LDL-R family Ⅲ LR8 Ⅲ LRP1 Ⅲ HSPG-bound LPL P remature truncations of APOA5, Q139X, Q148X, and the IVS3ϩ3gϾc are associated with severe hypertriglyceridemia (HyperTG), 1-3 behaving as phenocopies of lipoprotein lipase (LPL) deficiency. These rare APOA5 variants do not always lead to a deficiency in circulating plasma apoAV, and carriers present with a range of apoAV levels (reviewed in 4 ). Kao et al 5 identified a common polymorphism G185C in a Taiwanese study which occurs at a minor allele frequency of 0.04 in controls but at a 6.3-fold higher frequency of 0.27 in hypertriglyceridemic patients (PϽ0.001). Conclusion-TheApoAV is present on chylomicrons, VLDL, and HDL, but not on IDL or LDL, suggesting that VLDL-containing apoAV is cleared before the lipolytic cascade. 6 One function of apoAV is to activate LPL, and Apoa5 knockout mice have 4-fold higher TG levels than wild type. [7][8][9] Whereas LPL transgenic mice can rescue Apoa5 knockout mice from HyperTG, this is not entirely reciprocal 8 suggesting that the effect of apoAV on plasma TG is dependent on heparinsulfate proteoglycan (HSPGs)-bound LPL. 7,8 In addition, apoAV-dependent TG catabolism acts by enhancing receptormediated endocytosis via members of the LDL-receptor (LDLR) family. 10,11 We have identified 3 novel APOA5 missense variants (E255G, G271C, H321L) in patients with TG levels Ͼ10 mmol/L. Although LPL and APOC2 variants had been excluded for the G271C carrier, the coding exons of LPL and APOC2 were sequenced in the 2 other probands. Because family studies were not possible, the APOA5 variants were expressed in vitro together with G185C, Q139X, Q148X. 1,2,5 In addition we designed a deletion construct ⌬139 to 147, to MethodsSee supplemental methods (available online at http://atvb.ahajournals. org) for full details. PatientsIn total 130 patients with TG levels Ͼ10 mmol/L were recruited into the study; seven patients from the UK, 28 patients from the Netherlands with LPL and APOC2 mutations excluded, and 95 p...
This article is available online at http://www.jlr.org can be found in almost any type of cell ( 1 ). Currently, the role of lipid droplets in the pathophysiology of obesitydependent metabolic diseases involving insulin resistance is studied intensively ( 2 ). Lipid droplets are also present in various types of infl ammatory cells ( 3-6 ), where they are usually called lipid bodies (LB) and participate in cell signaling and in the generation of biologically active lipid mediators evoked by infl ammatory and infectious conditions ( 7-11 ).Lipid bodies consist of a neutral lipid core that is surrounded by a monolayer of amphipathic lipids (phospholipids and unesterifi ed cholesterol) and by proteins involved in the formation and traffi cking of the LBs and in the turnover of their lipids. Depending on the type and metabolic state of a cell, the protein and lipid compositions of the LBs may vary considerably, refl ecting active metabolism of their lipid components ( 12 ). The main proteins known to regulate the metabolism of the LB lipids are the members of the PAT protein family, mostly studied in adipocytes. This family includes fi ve perilipins: perilipin 1 (PLIN1; formerly perilipin), perilipin 2 (PLIN2; formerly adipose differentiation-related protein), perilipin 3 (PLIN3; formerly tail-interacting protein of 47 kDa), perilipin 4 (PLIN4; formerly S3-12), and perilipin 5 (PLIN5; formerly lipid storage droplet protein 5) ( 13 ). PLIN1 regulates the lipolytic activity of adipose triglyceride lipase (ATGL) during triacylglycerol (TG) mobilization via interacting with its coactivating factor, the comparative gene identifi cation 58 (CGI-58) ( 14 ), whereas overexpression Abstract Lipid droplets, also called lipid bodies (LB) in infl ammatory cells, are important cytoplasmic organelles. However, little is known about the molecular characteristics and functions of LBs in human mast cells (MC). Here, we have analyzed the genesis and components of LBs during differentiation of human peripheral blood-derived CD34+ progenitors into connective tissue-type MCs. In our serumfree culture system, the maturing MCs, derived from 18 different donors, invariably developed triacylglycerol (TG)-rich LBs. Not known heretofore, the MCs transcribe the genes for perilipins (PLIN)1-4, but not PLIN5, and PLIN2 and PLIN3 display different degrees of LB association. Upon MC activation and ensuing degranulation, the LBs were not cosecreted with the cytoplasmic secretory granules. Exogenous arachidonic acid (AA) enhanced LB genesis in Triacsin C-sensitive fashion, and it was found to be preferentially incorporated into the TGs of LBs. The large TG-associated pool of AA in LBs likely is a major precursor for eicosanoid production by MCs. In summary, we demonstrate that cultured human MCs derived from CD34 + progenitors in peripheral blood provide a new tool to study regulatory mechanisms involving LB functions, with particular emphasis on AA metabolism, eicosanoid biosynthesis, and subsequent release of proinfl ammatory lipid mediators from t...
Apolipoprotein A-V (apoA-V) affects plasma triglyceride (TG) levels; however, the properties of apoA-V that mediate its action(s) are still incompletely understood. It is unclear how apoA-V, whose plasma concentration is extremely low, can affect the pronounced TG differences observed in individuals with various apoA-V dysfunctions. To gain novel insights into apoA-V biology, we expanded our previous studies in the chicken to this apolipoprotein. First, we characterized the first avian apoA-V, revealing its expression not only in liver and small intestine but also in brain, kidney, and ovarian follicles and showing its presence in the circulation. Second, we demonstrate directly that galline apoA-V binds to the major LDL receptor family member (LR) of the laying hen and that this interaction does not depend on the association of the apolipoprotein with lipid or lipoproteins. We propose that a direct interaction with LRs may represent a novel, additional mechanism for the modulation of TG levels by apoA-V. Apolipoprotein A-V (apoA-V) was discovered by comparative genomic analysis of human and murine DNA and by a cDNA subtraction approach in the rat (1). In murine models and human subjects, strong correlations of apoA-V expression levels and DNA sequence polymorphisms, respectively, with plasma triglyceride (TG) levels have been observed (2; reviewed in Ref.3). In the rat, hepatic apoA-V expression is induced significantly after partial hepatectomy (1). The effects of genetically manipulating murine apoA-V underscore the importance of this exchangeable apolipoprotein (4) in the maintenance of normal TG levels: overexpressing mice are hypotriglyceridemic, and knockout mice are hypertriglyceridemic (2). However, in contrast to results in animal studies, human plasma apoA-V positively correlates with plasma TG levels (5), and APOAV may thus define a TG-modifier gene (6).Despite many recent studies, the apoA-V activity responsible for the effects on TG levels and metabolic events remains unclear. As discussed previously (7), possible functions include proteoglycan-dependent direct modulation of LPL activity, interference with the secretion of nascent TG-rich lipoproteins, and/or indirect effects on lipolysis via apoA-V binding to heparan sulfate proteoglycans. Another possibility for the action of apoA-V has been indicated by the finding (8) that VLDL particles from apoa5 2/2 mice are poorer competitors for binding to the LDL receptor than those from normal mice, implying a possible role for apoA-V in mediating or modulating lipoprotein receptor binding. This mode of action is supported by a report (9) that appeared during the revision of this article. By surface plasmon resonance measurements, the authors demonstrated the interaction of human recombinant apoA-V with two membrane receptors, the LDL receptor-related protein (10) and the mosaic receptor SorLA/LR11 (11).Studies in nonmammalian species can reveal novel aspects of apoA-V biology. We and others are exploiting the laying hen to answer questions conc...
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