BBA - Molecular and Cell Biology of Lipids (v.1831, #11)
Editorial Board (i).
Thermodynamic and solution state NMR characterization of the binding of secondary and conjugated bile acids to STARD5 by Danny Létourneau; Aurélien Lorin; Andrée Lefebvre; Jérôme Cabana; Pierre Lavigne; Jean-Guy LeHoux (1589-1599).
STARD5 is a member of the STARD4 sub-family of START domain containing proteins specialized in the non-vesicular transport of lipids and sterols. We recently reported that STARD5 binds primary bile acids. Herein, we report on the biophysical and structural characterization of the binding of secondary and conjugated bile acids by STARD5 at physiological concentrations. We found that the absence of the 7α-OH group and its epimerization increase the affinity of secondary bile acids for STARD5. According to NMR titration and molecular modeling, the affinity depends mainly on the number and positions of the steroid ring hydroxyl groups and to a lesser extent on the presence or type of bile acid side-chain conjugation. Primary and secondary bile acids have different binding modes and display different positioning within the STARD5 binding pocket. The relative STARD5 affinity for the different bile acids studied is: DCA > LCA > CDCA > GDCA > TDCA > CA > UDCA. TCA and GCA do not bind significantly to STARD5. The impact of the ligand chemical structure on the thermodynamics of binding is discussed. The discovery of these new ligands suggests that STARD5 is involved in the cellular response elicited by bile acids and offers many entry points to decipher its physiological role.
Keywords: Cholesterol metabolism; Bile acid; Steroidogenic acute regulatory protein (StAR); Isothermal titration calorimetry; Circular dichroism; NMR spectroscopy;
Cardiac oxidative stress in a mouse model of neutral lipid storage disease by Astrid Schrammel; Marion Mussbacher; Sarah Winkler; Guenter Haemmerle; Heike Stessel; Gerald Wölkart; Rudolf Zechner; Bernd Mayer (1600-1608).
Cardiac oxidative stress has been implicated in the pathogenesis of hypertrophy, cardiomyopathy and heart failure. Systemic deletion of the gene encoding adipose triglyceride lipase (ATGL), the enzyme that catalyzes the rate-limiting step of triglyceride lipolysis, results in a phenotype characterized by severe steatotic cardiac dysfunction. The objective of the present study was to investigate a potential role of oxidative stress in cardiac ATGL deficiency. Hearts of mice with global ATGL knockout were compared to those of mice with cardiomyocyte-restricted overexpression of ATGL and to those of wildtype littermates. Our results demonstrate that oxidative stress, measured as lucigenin chemiluminescence, was increased ~ 6-fold in ATGL-deficient hearts. In parallel, cytosolic NADPH oxidase subunits p67phox and p47phox were upregulated 4–5-fold at the protein level. Moreover, a prominent upregulation of different inflammatory markers (tumor necrosis factor α, monocyte chemotactant protein-1, interleukin 6, and galectin-3) was observed in those hearts. Both the oxidative and inflammatory responses were abolished upon cardiomyocyte-restricted overexpression of ATGL. Investigating the effect of oxidative and inflammatory stress on nitric oxide/cGMP signal transduction we observed a ~ 2.5-fold upregulation of soluble guanylate cyclase activity and a ~ 2-fold increase in cardiac tetrahydrobiopterin levels. Systemic treatment of ATGL-deficient mice with the superoxide dismutase mimetic Mn(III)tetrakis (4-benzoic acid) porphyrin did not ameliorate but rather aggravated cardiac oxidative stress. Our data suggest that oxidative and inflammatory stress seems involved in lipotoxic heart disease. Upregulation of soluble guanylate cyclase and cardiac tetrahydrobiopterin might be regarded as counterregulatory mechanisms in cardiac ATGL deficiency.Display Omitted
Keywords: Adipose triglyceride lipase; Cardiac hypertrophy; Oxidative stress; Inflammation; NADPH oxidase;
Dyslipidemia, but not hyperglycemia and insulin resistance, is associated with marked alterations in the HDL lipidome in type 2 diabetic subjects in the DIWA cohort: Impact on small HDL particles by Marcus Ståhlman; Björn Fagerberg; Martin Adiels; Kim Ekroos; John M. Chapman; Anatol Kontush; Jan Borén (1609-1617).
In this study we have used mass spectrometry in order to characterize the HDL lipidome in three groups of women from the DIWA cohort; one control group, plus two groups with type 2 diabetes with insulin resistance; one dyslipidemic and one normolipidemic. The aim was to investigate whether dyslipidemia is required in addition to insulin resistance for the occurrence of an altered HDL lipidome, which in turn might impact HDL functionality. The dyslipidemic type 2 diabetic subjects were distinguished by obesity, hypertriglyceridemia with elevated apoC3, low HDL-cholesterol and chronic low grade inflammation. In a stepwise multivariate linear regression analysis, including biomarkers of dyslipidemia and insulin resistance as independent variables, only dyslipidemia showed a significant correlation with HDL lipid classes. Small HDL-particles predominated in dyslipidemic subjects in contrast to the normolipidemic diabetic and control groups, and were enriched in lysophosphatidylcholine (+ 13%), a product of proinflammatory phospholipases, and equally in two core lipids, palmitate-rich triacylglycerols and diacylglycerols (+ 77 %), thereby reflecting elevated CETP activity. Dyslipidemic small HDL particles were further distinguished not only as the primary carrier of ceramides, which promote inflammation and insulin resistance, but also by a subnormal plasmalogen/apoAI ratio, consistent with elevated oxidative stress typical of type 2 diabetes. From these data we conclude that in type 2 diabetes, dyslipidemia predominates relative to hyperglycemia for the occurrence of an altered HDL lipidome. Furthermore, dyslipidemia alters the cargo of bioactive lipids, with implications for HDL function.
Keywords: HDL; Lipids; Lipidomics; Mass spectrometry;
PPARβ mRNA expression, reduced by n − 3 PUFA diet in mammary tumor, controls breast cancer cell growth by Ramez Wannous; Emeline Bon; Karine Mahéo; Caroline Goupille; Julie Chamouton; Philippe Bougnoux; Sébastien Roger; Pierre Besson; Stephan Chevalier (1618-1625).
The effect of numerous anticancer drugs on breast cancer cell lines and rodent mammary tumors can be enhanced by a treatment with long-chain n − 3 polyunsaturated fatty acids (n − 3 PUFA) such as docosahexaenoic acid (DHA, 22:6n − 3) which is a natural ligand of peroxisome proliferator-activated receptors (PPAR). In order to identify the PPAR regulating breast cancer cell growth, we tested the impact of siRNA, selected to suppress PPARα, PPARβ or PPARγ mRNA in MDA-MB-231 and MCF-7 breast cancer cell lines. The siPPARβ was the most effective to inhibit breast cancer cell growth in both cell lines. Using PPARα, PPARβ and PPARγ pharmacological antagonists, we showed that PPARβ regulated DHA-induced inhibition of growth in MDA-MB-231 and MCF-7 cells. In addition, the expressions of all 3 PPAR mRNA were co-regulated in both cell lines, upon treatments with siRNA or PPAR antagonists. PPAR mRNA expression was also examined in the NitrosoMethylUrea (NMU)-induced rat mammary tumor model. The expressions of PPARα and PPARβ mRNAs were correlated in the control group but not in the n − 3 PUFA group in which the expression of PPARβ mRNA was reduced. Although PPARα expression was also increased in the n − 3 PUFA-enriched diet group under docetaxel treatment, it is only the expression of PPARβ mRNA that correlated with the regression of mammary tumors: those that most regressed displayed the lowest PPARβ mRNA expression. Altogether, these data identify PPARβ as an important player capable of modulating other PPAR mRNA expressions, under DHA diet, for inhibiting breast cancer cell growth and mammary tumor growth.
Keywords: PPAR; MDA-MB-231; MCF-7; Breast tumor; DHA; n − 3 PUFA;
HDL endocytosis and resecretion by Clemens Röhrl; Herbert Stangl (1626-1633).
HDL removes excess cholesterol from peripheral tissues and delivers it to the liver and steroidogenic tissues via selective lipid uptake without catabolism of the HDL particle itself. In addition, endocytosis of HDL holo-particles has been debated for nearly 40 years. However, neither the connection between HDL endocytosis and selective lipid uptake, nor the physiological relevance of HDL uptake has been delineated clearly. This review will focus on HDL endocytosis and resecretion and its relation to cholesterol transfer. We will discuss the role of HDL endocytosis in maintaining cholesterol homeostasis in tissues and cell types involved in atherosclerosis, focusing on liver, macrophages and endothelium. We will critically summarize the current knowledge on the receptors mediating HDL endocytosis including SR-BI, F1-ATPase and CD36 and on intracellular HDL transport routes. Dependent on the tissue, HDL is either resecreted (retro-endocytosis) or degraded after endocytosis. Finally, findings on HDL transcytosis across the endothelial barrier will be summarized. We suggest that HDL endocytosis and resecretion is a rather redundant pathway under physiologic conditions. In case of disturbed lipid metabolism, however, HDL retro-endocytosis represents an alternative pathway that enables tissues to maintain cellular cholesterol homeostasis.
Keywords: Lipoprotein; Holo-particle uptake; Cholesterol; Transcytosis; Resecretion; Degradation;
PPARγ agonists upregulate sphingosine 1-phosphate (S1P) receptor 1 expression, which in turn reduces S1P-induced [Ca2 +]i increases in renal mesangial cells by Alexander Koch; Anja Völzke; Bianca Puff; Kira Blankenbach; Dagmar Meyer zu Heringdorf; Andrea Huwiler; Josef Pfeilschifter (1634-1643).
We previously identified peroxisome proliferator-activated receptor gamma (PPARγ) agonists (thiazolidinediones, TZDs) as modulators of the sphingolipid metabolism in renal mesangial cells. TZDs upregulated sphingosine kinase 1 (SK-1) and increased the formation of intracellular sphingosine 1-phosphate (S1P), which in turn reduced the expression of pro-fibrotic connective tissue growth factor. Since S1P also acts as extracellular ligand at specific S1P receptors (S1PR, S1P1–5), we investigated here the effect of TZDs on S1PR expression in mesangial cells and evaluated the functional consequences by measuring S1P-induced increases in intracellular free Ca2 + concentration ([Ca2 +]i). Treatment with two different TZDs, troglitazone and rosiglitazone, enhanced S1P1 mRNA and protein expression in rat mesangial cells, whereas S1P2–5 expression levels were not altered. Upregulation of S1P1 mRNA upon TZD treatment was also detected in human mesangial cells and mouse glomeruli. PPARγ antagonism and promoter studies revealed that the TZD-dependent S1P1 mRNA induction involved a functional PPAR response element in the S1P1 promoter. Pharmacological approaches disclosed that S1P-induced [Ca2 +]i increases in rat mesangial cells were predominantly mediated by S1P2 and S1P3. Interestingly, the transcriptional upregulation of S1P1 by TZDs resulted in a reduction of S1P-induced [Ca2 +]i increases, which was reversed by the S1P1/3 antagonist VPC-23019, the protein kinase C (PKC) inhibitor PKC-412, and by S1P1 siRNA. These data suggest that PPARγ-dependent upregulation of S1P1 leads to an inhibition of S1P-induced Ca2 + signaling in a PKC-dependent manner. Overall, these results reveal that TZDs not only modulate intracellular S1P levels but also regulate S1PR signaling by increasing S1P1 expression in mesangial cells.Display Omitted
Keywords: Sphingosine 1-phosphate; S1P receptor; PPARγ; Calcium signaling; Mesangial cells;
Evidence for a role of CETP in HDL remodeling and cholesterol efflux: Role of cysteine 13 of CETP by Cyrille Maugeais; Anne Perez; Elisabeth von der Mark; Christine Magg; Philippe Pflieger; Eric J. Niesor (1644-1650).
Cholesteryl ester transfer protein (CETP), a key regulator of high-density lipoprotein (HDL) metabolism, induces HDL remodeling by transferring lipids between apolipoprotein B-containing lipoproteins and HDL, and/or by promoting lipid transfer between HDL subparticles. In this study, we investigated the mechanism as to how CETP induces the generation of lipid-poor particles (pre-β-HDL) from HDL, which increases ATP-binding cassette transporter 1-mediated cholesterol efflux. This CETP-dependent HDL remodeling is enhanced by the CETP modulator dalcetrapib both in plasma and isolated HDL. The interaction of dalcetrapib with cysteine 13 of CETP is required, since this effect was abolished when using mutant CETP in which cysteine 13 was substituted for a serine residue. Other thiol-containing compounds were identified as CETP modulators interacting with cysteine 13 of CETP. In order to mimic dalcetrapib-bound CETP, mutant CETP proteins were prepared by replacing cysteine 13 with the bulky amino acid tyrosine or tryptophan. The resultant mutants showed virtually no CETP-dependent lipid transfer activity but demonstrated preserved CETP-dependent pre-β-HDL generation. Overall, these data demonstrate that the two functions of CETP i.e., cholesteryl ester transfer and HDL remodeling can be uncoupled by interaction of thiol-containing compounds with cysteine 13 of CETP or by introducing large amino acid residues in place of cysteine 13.
Keywords: Dalcetrapib; Anacetrapib; Thiol; Cysteine 13; CETP; Pre-β-HDL;
Membrane binding properties of IRSp53-missing in metastasis domain (IMD) protein by Kinga Futó; Emőke Bódis; Laura M. Machesky; Miklós Nyitrai; Balázs Visegrády (1651-1655).
The 53-kDa insulin receptor substrate protein (IRSp53) organizes the actin cytoskeleton in response to stimulation of small GTPases, promoting the formation of cell protrusions such as filopodia and lamellipodia. IMD is the N-terminal 250 amino acid domain (IRSp53/MIM Homology Domain) of IRSp53 (also called I-BAR), which can bind to negatively charged lipid molecules. Overexpression of IMD induces filopodia formation in cells and purified IMD assembles finger-like protrusions in reconstituted lipid membranes. IMD was shown by several groups to bundle actin filaments, but other groups showed that it also binds to membranes. IMD binds to negatively charged lipid molecules with preference to clusters of PI(4,5)P2. Here, we performed a range of different in vitro fluorescence experiments to determine the binding properties of the IMD to phospholipids. We used different constructs of large unilamellar vesicles (LUVETs), containing neutral or negatively charged phospholipids. We found that IMD has a stronger binding interaction with negatively charged PI(4,5)P2 or PS lipids than PS/PC or neutral PC lipids. The equilibrium dissociation constant for the IMD–lipid interaction falls into the 78–170 μM range for all the lipids tested. The solvent accessibility of the fluorescence labels on the IMD during its binding to lipids is also reduced as the lipids become more negatively charged. Actin affects the IMD–lipid interaction, depending on its polymerization state. Monomeric actin partially disrupts the binding, while filamentous actin can further stabilize the IMD–lipid interaction.
Keywords: IRSp53; IMD; I-BAR; Actin; Fluorescence spectroscopy;