Unsaturated long-chain fatty acids inhibit the binding of oxidized low-density lipoproteins to a model CD36

Transmembrane protein CD36 binds multiple ligands, including oxidized low-density lipoproteins (oxLDLs) and long-chain fatty acids (LCFAs). Our aim was to determine whether LCFAs compete with oxLDLs for binding to CD36. We addressed this issue by examining the inhibitory effect of LCFAs against the binding of Alexa-fluor-labeled oxLDLs (AFL-oxLDL) to a synthetic peptide representing the oxLDL-binding site on CD36 (3S-CD36_150–168). All of the unsaturated LCFAs tested, inhibited the binding of AFL-oxLDL to 3S-CD36_150–168, albeit to varying degrees. For instance, the concentrations required for 50% inhibition of binding for oleic, linoleic, and α-linolenic acids were 0.25, 0.97, and 1.2?mM, respectively. None of the saturated LCFAs tested (e.g. stearic acid) exhibited inhibitory effects. These results suggest that at least unsaturated LCFAs can compete with oxLDLs for binding to CD36. The study also provides information on the structural requirements of LCFAs for inhibition of oxLDLs–CD36 binding.     

Distinct mechanisms for OxLDL uptake and cellular trafficking by class B scavenger receptors CD36 and SR-BI

Modified forms of LDL, including oxidized low density lipoprotein (OxLDL), contribute to macrophage lipid accumulation in the vessel wall. Despite the pathophysiological importance of uptake pathways for OxLDL, the molecular details of OxLDL endocytosis by macrophages are not well understood. Studies in vitro demonstrate that the class B scavenger receptor CD36 mediates macrophage uptake and degradation of OxLDL. Although the closely related scavenger receptor class B type I (SR-BI) binds OxLDL with high affinity, evidence that SR-BI plays a role in OxLDL metabolism is lacking. In this study, we directly compared OxLDL uptake and degradation by CD36 and SR-BI. Our results indicate that although CD36 and SR-BI internalize OxLDL, SR-BI mediates significantly less OxLDL degradation. Endocytosis of OxLDL by both SR-BI and CD36 is independent of caveolae, microtubules, and actin cytoskeleton. However, OxLDL uptake by CD36, but not SR-BI, is dependent on dynamin. The analysis of chimeric SR-BI/CD36 receptors shows that the CD36 C-terminal cytoplasmic tail is necessary and sufficient for dynamin-dependent OxLDL internalization by class B scavenger receptors. These findings indicate that different mechanisms are involved in OxLDL uptake by SR-BI and CD36, which may segregate these two structurally homologous receptors at the cell surface, leading to differences in intracellular trafficking and degradation.     

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) functions as an oligomer and oligomerization is dependent on receptor density

Lectin-like oxidized low-density lipoprotein (LDL) receptor (LOX-1) exists as a homodimer formed by an intermolecular disulfide bond. Although the dimer is the minimum structural unit of LOX-1 on cell membranes, LOX-1 can form larger noncovalent oligomeric complexes. But, the functional unit of LOX-1 is not known. We quantitatively analyzed the correlation between cyan fluorescent protein-tagged LOX-1 expression and the fluorescence-labeled ligand (DiD-AcLDL) binding ability on each cell. The results clearly indicate that there is a threshold level of expression that enables LOX-1 to bind ligand. Above this threshold level, the ability of LOX-1 to bind ligand was proportional to its level of expression. Using the membrane impermeable crosslinker BS(3), we detected oligomers (primarily hexamers) only on the cell lines that stably expressed LOX-1 above the threshold level. In contrast, little oligomer or ligand binding was detected in cell lines expressing LOX-1 below the threshold level. Moreover, oligomerization was independent of ligand binding. These results indicate that the functional unit of LOX-1 is an oligomer and that oligomerization of LOX-1 is dependent on the receptor density on the plasma membrane.     

Stabilin-1, a homeostatic scavenger receptor with multiple functions

The multifunctional scavenger receptor stabilin-1 (STAB1, FEEL-1, CLEVER-1, KIAA0246) was originally identified as the MS-1 antigen, expressed by sinusoidal endothelial cells in human spleen. Extensive histological studies revealed that stabilin-1 is also expressed by tissue macrophages and sinusoidal endothelial cells in the healthy organism; its expression on both macrophages and different subtypes of endothelial cells is induced during chronic inflammation and tumorigenesis. In vitro induction of stabilin-1 in macrophages requires the presence of glucocorticoids. Stabilin-1 is involved in two intracellular trafficking pathways: receptor mediated endocytosis and recycling; and shuttling between the endosomal compartment and trans-Golgi network (TGN). The latter intracellular pathway of stabilin-1 trafficking is mediated by GGAs, clathrin adaptors that interact with the DDSLL motif in the cytoplasmic tail of stabilin-1. When expressed by alternatively activated macrophages, stabilin-1 mediates the uptake and targeting for degradation of acLDL and SPARC, a regulator of tissue remodeling. Likewise, stabilin-1 in macrophages is involved in intracellular sorting and lysosomal delivery of the novel stabilin- 1-interacting chitinase-like protein (SI-CLP). Indirect evidence suggests that stabilin-1 is involved in adhesion and transmigration in various cell types (including tumor cells, leukocytes, and lymphocytes); however, its rapid recycling and scant level of surface expression argue against its universal role in cell adhesion. In summary, stabilin-1 is a homeostatic receptor which links signals from the extracellular environment to intracellular vesicular processes, creating a potential impact on the macrophage secretion profile.     

NMR structure of a concatemer of the first and second ligand-binding modules of the human low-density lipoprotein receptor

The ligand-binding domain of the human low-density lipoprotein receptor consists of seven modules, each of 40-45 residues. In the presence of calcium, these modules adopt a common polypeptide fold with three conserved disulfide bonds. A concatemer of the first and second modules (LB(1-2)) folds efficiently in the presence of calcium ions, forming the same disulfide connectivities as in the isolated modules. The three-dimensional structure of LB(1-2) has now been solved using two-dimensional 1H NMR spectroscopy and restrained molecular dynamics calculations. No intermodule nuclear Overhauser effects were observed, indicating the absence of persistent interaction between them. The near random-coil NH and H alpha chemical shifts and the low phi and psi angle order parameters of the four-residue linker suggest that it has considerable flexibility. The family of LB(1-2) structures superimposed well over LB1 or LB2, but not over both modules simultaneously. LB1 and LB2 have a similar pattern of calcium ligands, but the orientations of the indole rings of the tryptophan residues W23 and W66 differ, with the latter limiting solvent access to the calcium ion. From these studies, it appears that although most of the modules in the ligand-binding region of the receptor are joined by short segments, these linkers may impart considerable flexibility on this region.     

Nobiletin metabolite, 3′,4′-dihydroxy-5,6,7,8-tetramethoxyflavone,inhibits LDL oxidation and down-regulates scavenger receptor expression and activity in THP-1 cells

There is accumulating evidence that LDL oxidation is essential for atherogenesis and antioxidants that prevent oxidation may either decelerate or reduce atherogenesis. Current study focused on the effect and mechanism of 3′,4′-dihydroxy-5,6,7,8-tetramethoxyflavone (DTF), a major metabolite of nobiletin (NOB, a citrus polymethoxylated flavone) on atherogenesis. We found DTF had stronger inhibitory activity than α-tocopherol on inhibiting Cu²⁺-mediated LDL oxidation measured by thiobarbituric acid-reactive substances assay (TBARS), conjugated diene formation and electrophoretic mobility. Monocyte-to-macrophage differentiation plays a vital role in early atherogenesis. DTF (10–20 μM) dose-dependently attenuated differentiation along with the reduced gene expression of scavenger receptors, CD36 and SR-A, in both PMA- and oxidized low-density lipoprotein (oxLDL)-stimulated THP-1 monocytes. Furthermore, DTF treatment of monocytes and macrophages led to reduction of fluorescent DiI-acLDL and DiI-oxLDL uptake. In conclusion, at least three mechanisms are at work in parallel: DTF reduces LDL oxidation, attenuates monocyte differentiation into macrophage and blunts uptake of modified LDL by macrophage. The effect is different from that of NOB, from which DTF is derived. This study thus significantly enhanced our understanding on how DTF may be beneficial against atherogenesis.     

Metallothionein and a peptide modeled after metallothionein, EmtinB, induce neuronal differentiation and survival through binding to receptors of the low-density lipoprotein receptor family

Accumulating evidence suggests that metallothionein (MT)-I and -II promote neuronal survival and regeneration in vivo . The present study investigated the molecular mechanisms underlying the differentiation and survival-promoting effects of MT and a peptide modeled after MT, EmtinB. Both MT and EmtinB directly stimulated neurite outgrowth and promoted survival in vitro using primary cultures of cerebellar granule neurons. In addition, expression and surface localization of megalin, a known MT receptor, and the related lipoprotein receptor-related protein-1 (LRP) are demonstrated in cerebellar granule neurons. By means of surface plasmon resonance MT and EmtinB were found to bind to both megalin and LRP. The bindings were abrogated in the presence of receptor-associated protein-1, an antagonist of the low-density lipoprotein receptor family, which also inhibited MT- and EmtinB-induced neurite outgrowth and survival. MT-mediated neurite outgrowth was furthermore inhibited by an anti-megalin serum. EmtinB-mediated inhibition of apoptosis occurred without a reduction of caspase-3 activity, but was associated with reduced expression of the pro-apoptotic B-cell leukemia/lymphoma-2 interacting member of cell death (Bim_S). Finally, evidence is provided that MT and EmtinB activate extracellular signal-regulated kinase, protein kinase B, and cAMP response element binding protein. Altogether, these results strongly suggest that MT and EmtinB induce their neuronal effects through direct binding to surface receptors belonging to the low-density lipoprotein receptor family, such as megalin and LRP, thereby activating signal transduction pathways resulting in neurite outgrowth and survival.     

CD36 as a target to prevent cardiac lipotoxicity and insulin resistance

The fatty acid transporter and scavenger receptor CD36 is increasingly being implicated in the pathogenesis of insulin resistance and its progression towards type 2 diabetes and associated cardiovascular complications. The redistribution of CD36 from intracellular stores to the plasma membrane is one of the earliest changes occurring in the heart during diet induced obesity and insulin resistance. This elicits an increased rate of fatty acid uptake and enhanced incorporation into triacylglycerol stores and lipid intermediates to subsequently interfere with insulin-induced GLUT4 recruitment (i.e., insulin resistance). In the present paper we discuss the potential of CD36 to serve as a target to rectify abnormal myocardial fatty acid uptake rates in cardiac lipotoxic diseases. Two approaches are described: (i) immunochemical inhibition of CD36 present at the sarcolemma and (ii) interference with the subcellular recycling of CD36. Using in vitro model systems of high-fat diet induced insulin resistance, the results indicate the feasibility of using CD36 as a target for adaptation of cardiac metabolic substrate utilization. In conclusion, CD36 deserves further attention as a promising therapeutic target to redirect fatty acid fluxes in the body.     

Influence of class B scavenger receptors on cholesterol flux across the brush border membrane and intestinal absorption

To learn more about how the step of cholesterol uptake into the brush border membrane (BBM) of enterocytes influences overall cholesterol absorption, we measured cholesterol absorption 4 and 24 h after administration of an intragastric bolus of radioactive cholesterol in mice with scavenger receptor class B, type 1 (SR-BI) and/or cluster determinant 36 (CD36) deleted. The cholesterol absorption efficiency is unaltered by deletion of either one or both of the receptors. In vitro determinations of the cholesterol uptake specific activity of the BBM from the mice reveal that the scavenger receptors facilitate cholesterol uptake into the proximal BBM. It follows that cholesterol uptake into the BBM is not normally rate-limiting for the cholesterol absorption process in vivo; a subsequent step, such as NPC1L1-mediated transfer from the BBM into the interior of the enterocyte, is rate-limiting. The absorption of dietary cholesterol after 4 h in mice lacking SR-BI and/or CD36 and fed a high-fat/high-cholesterol diet is delayed to more distal regions of the small intestine. This effect probably arises because ATP binding cassette half transporters G5 and G8-mediated back flux of cholesterol from the BBM to the lumen of the small intestine limits absorption and causes the local cholesterol uptake facilitated by SR-BI and CD36 to become rate-limiting under this dietary condition.     

Adenosine A2A receptor activation supports an atheroprotective cholesterol balance in human macrophages and endothelial cells

The adenosine A_2A receptor (A_2AR) plays an important role in the regulation of inflammatory and immune responses. Our previous work has demonstrated that A_2AR agonists exhibit atheroprotective effects by increasing expression of reverse cholesterol transport proteins in cultured human macrophages. This study explores the impact of pharmacologic activation/inhibition and gene silencing of A_2AR on cholesterol homeostasis in both THP-1 human monocytes/macrophages and primary human aortic endothelial cells (HAEC).     

Upregulation of liver VLDL receptor and FAT/CD36 expression in LDLR-/- apoB100/100 mice fed trans-10,cis-12 conjugated linoleic acid

This study explores the mechanisms responsible for the fatty liver setup in mice fed trans-10,cis-12 conjugated linoleic acid (t10c12 CLA), hypothesizing that an induction of low density lipoprotein receptor (LDLR) expression is associated with lipid accumulation. To this end, the effects of t10c12 CLA treatment on lipid parameters, serum lipoproteins, and expression of liver lipid receptors were measured in LDLR(-/-) apoB(100/100) mice as a model of human familial hypercholesterolemia itself depleted of LDLR. Mice were fed t10c12 CLA over 2 or 4 weeks. We first observed that the treatment induced liver steatosis, even in the absence of LDLR. Mice treated for 2 weeks exhibited hypertriglyceridemia with high levels of VLDL and HDL, whereas a 4 week treatment inversely induced a reduction of serum triglycerides (TGs), essentially through a decrease in VLDL levels. In the absence of LDLR, the mRNA levels of other proteins, such as VLDL receptor, lipoprotein lipase, and fatty acid translocase, usually not expressed in the liver, were upregulated, suggesting their involvement in the steatosis setup and lipoprotein clearance. The data also suggest that the TG-lowering effect induced by t10c12 CLA treatment was attributable to both the reduction of circulating free fatty acids in response to the severe lipoatrophy and the high capacity of liver to clear off plasma lipids.     

Structural and dynamic differences of the estrogen receptor DNA-binding domain, binding as a dimer and as a monomer to DNA: molecular dynamics simulation studies

Molecular dynamics (MD) simulations of the estrogen receptor DNA-binding domain (ERDBD) as a dimer in complex with its DNA response element (ERE) show a significant difference in both structure and dynamics, compared to a MD simulation of monomeric ERDBD bound to its half-site response element (EREH). The C-terminal zinc binding domain (Zn_II), including a region (helix II) which is in a helical conformation in ERE-(ERDBD)₂, is considerably more flexible in EREH-ERDBD than in the dimeric complex. In EREH-ERDBD, all helical hydrogen bonds in helix II are broken and the entire Zn_II region is detached from a hydrogen bonding network that in ERE-(ERDBD)₂ connects to other parts of the protein as well as to the DNA. The regions that become flexible in EREH-ERDBD are identical to the regions where the NMR solution structure of free ERDBD is poorly ordered. This strongly suggests that dimerisation of ERDBD is required for ordering of the Zn_II region and that monomeric binding to DNA is not sufficient for the ordering. This contrasts to the glucocorticoid receptor DNA-binding domain (GRDBD) which has essentially the same mobility (uniform and limited), regardless of whether it is free as a monomer in solution, bound as a monomer to its half-site response element or in a dimeric complex with the full response element. The hydrogen bonding network that connects Zn_II with other parts of the protein and to DNA is almost identical in ERDBD and GRDBD. However, in GRDBD there is also a serine (in the N-terminal zinc coordinating region) with a central role in this network, connecting to the Zn_II region. This serine is replaced by a glycine in ERDBD and we suggest that this substitution is sufficient for destabilisation of the network, thus leading to a more flexible Zn_II region, which becomes ordered first upon forming a complex with another ERDBD and DNA. Received: 6 March 1998 / Revised version: 22 June 1998 / Accepted: 2 September 1998     

Putative alternative functions of human stefin B (cystatin B): binding to amyloid‐beta,membranes, and copper

We describe studies performed thus far on stefin B from the family of cystatins as a model protein for folding and amyloid fibril formation studies. We also briefly mention our studies on aggregation of some of the missense EPM1 mutants of stefin B in cells, which mimic additional pathological traits (gain in toxic function) in selected patients with EPM1 disease. We collected data on the reported interactors of stefin B and discuss several hypotheses of possible cytosolic alternative functions.     

Inhibition of mTOR down-regulates scavenger receptor,class B,type I (SR-BI) expression,reduces endothelial cell migration and impairs nitric oxide production

The mammalian target of rapamycin (mTOR) inhibiting drug rapamycin (Sirolimus) has severe side effects in patients including hyperlipidemia, an established risk factor for atherosclerosis. Recently, it was shown that rapamycin decreases hepatic LDL receptor (LDL-R) expression, which likely contributes to hypercholesterolemia. Scavenger receptor, class B, type I (SR-BI) is the major HDL receptor and consequently regulating HDL-cholesterol levels and the athero-protective effects of HDL. By using the mTOR inhibitor rapamycin, we show that SR-BI is down-regulated in human umbilical vein endothelial cells (HUVECs). This reduction of SR-BI protein as well as mRNA levels by about 50% did not alter HDL particle uptake or HDL-derived lipid transfer. However, rapamycin reduced HDL-induced activation of eNOS and stimulation of endothelial cell migration. The effects on cell migration could be counteracted by SR-BI overexpression, indicating that decreased SR-BI expression is in part responsible for the rapamycin-induced effects. We demonstrate that inhibition of mTOR leads to endothelial cell dysfunction and decreased SR-BI expression, which may contribute to atherogenesis during rapamycin treatment.     

Botulinum neurotoxins C, E and F bind gangliosides via a conserved binding site prior to stimulation-dependent uptake with botulinum neurotoxin F utilising the three isoforms of SV2 as second receptor

The high toxicity of clostridial neurotoxins primarily results from their specific binding and uptake into neurons. At motor neurons, the seven botulinum neurotoxin serotypes A–G (BoNT/A–G) inhibit acetylcholine release, leading to flaccid paralysis, while tetanus neurotoxin blocks neurotransmitter release in inhibitory neurons, resulting in spastic paralysis. Uptake of BoNT/A, B, E and G requires a dual interaction with gangliosides and the synaptic vesicle (SV) proteins synaptotagmin or SV2, whereas little is known about the entry mechanisms of the remaining serotypes. Here, we demonstrate that BoNT/F as wells depends on the presence of gangliosides, by employing phrenic nerve hemidiaphragm preparations derived from mice expressing GM3, GM2, GM1 and GD1a or only GM3. Subsequent site-directed mutagenesis based on homology models identified the ganglioside binding site at a conserved location in BoNT/E and F. Using the mice phrenic nerve hemidiaphragm assay as a physiological model system, cross-competition of full-length neurotoxin binding by recombinant binding fragments, plus accelerated neurotoxin uptake upon increased electrical stimulation, indicate that BoNT/F employs SV2 as protein receptor, whereas BoNT/C and D utilise different SV receptor structures. The co-precipitation of SV2A, B and C from Triton-solubilised SVs by BoNT/F underlines this conclusion.     

Identification of human low-density lipoprotein receptor as a novel target gene regulated by liver X receptor alpha

Liver X receptor alpha (LXRalpha) is a member of the nuclear receptor superfamily that is activated by oxysterols, and plays a pivotal role in regulating the metabolism, transport and uptake of cholesterol. Here, we demonstrate that LXRalpha also regulates the low-density lipoprotein receptor (LDLR) gene, which mediates the endocytic uptake of LDL cholesterol in the liver. An LXR agonist induced the expression of LDLR in cultured hepatoblastoma cells. Moreover, the LDLR promoter contained an LXR response element that was recognized by LXRalpha/RXRalpha (retinoid X receptor alpha) heterodimers in hepatoblastoma cells. These results suggest a novel pathway whereby LXRalpha might modulate cholesterol metabolism.