Cd36, a class B scavenger receptor, functions as a monomer to bind acetylated and oxidized low-density lipoproteins

Cd36 is a small-molecular-weight integral membrane protein expressed in a diverse, but select, range of cell types. It has an equally diverse range of ligands and physiological functions, which has implicated Cd36 in a number of diseases including insulin resistance, diabetes, and, most notably, atherosclerosis. The protein is reported to reside in detergent-resistant microdomains within the plasma membrane and to form homo- and hetero-intermolecular interactions. These data suggest that this class B scavenger receptor may gain functionality for ligand binding, and/or ligand internalization, by formation of protein complexes at the cell surface. Here, we have overexpressed Cd36 in insect cells, purified the recombinant protein to homogeneity, and analyzed its stability and solubility in a variety of nonionic and zwitterionic detergents. Octylglucoside conferred the greatest degree of stability, and by analytical ultracentrifugation we show that the protein is monomeric. A solid-phase ligand-binding assay demonstrated that the purified monomeric protein retains high affinity for acetylated and oxidized low-density lipoproteins. Therefore, no accessory proteins are required for interaction with ligand, and binding is a property of the monomeric fold of the protein. Thus, the highly purified and functional Cd36 should be suitable for crystallization in octylglucoside, and the in vitro ligand-binding assay represents a promising screen for identification of bioactive molecules targeting atherogenesis at the level of ligand binding.     

Nanoscale amphiphilic macromolecules as lipoprotein inhibitors: the role of charge and architecture

A series of novel amphiphilic macromolecules composed of alkyl chains as the hydrophobic block and poly(ethylene glycol) as the hydrophilic block were designed to inhibit highly oxidized low density lipoprotein (hoxLDL) uptake by synthesizing macromolecules with negatively charged moieties (ie, carboxylic acids) located in the two different blocks. The macromolecules have molecular weights around 5,500 g/mol, form micelles in aqueous solution with an average size of 20-35 nm, and display critical micelle concentration values as low as 10(-7) M. Their charge densities and hydrodynamic size in physiological buffer solutions correlated with the hydrophobic/ hydrophilic block location and quantity of the carboxylate groups. Generally, carboxylate groups located in the hydrophobic block destabilize micelle formation more than carboxylate groups in the hydrophilic block. Although all amphiphilic macromolecules inhibited unregulated uptake of hoxLDL by macrophages, inhibition efficiency was influenced by the quantity and location of the negatively charged-carboxylate on the macromolecules. Notably, negative charge is not the sole factor in reducing hoxLDL uptake. The combination of smaller size, micellar stability and charge density is critical for inhibiting hoxLDL uptake by macrophages.     

H2O2 but not $${\hbox{O}_{2}^{-}}$$ elevated by oxidized LDL enhances human aortic smooth muscle cell proliferation

The oxidation of low density lipoprotein (LDL) as a key event in atherosclerosis suggests that antioxidant interventions may reduce the risk of atherosclerosis. However, the better strategies among antioxidant remedies for atherosclerosis remains difficult to be determined. Here, we show that oxidized LDL increases the steady-state level of intracellular hydrogen peroxide through stimulating the protein expressions of Nox1 and Cu/Zn superoxide dismutase (SOD) in human aortic smooth muscle cells (SMCs). The intracellular content of hydrogen peroxide rather than superoxide is a key modulator for vascular SMC (VSMC) proliferation, implying that without co-expression of catalase, increased Cu/Zn-SOD activity alone may not be beneficial to reduce the growth of VSMC in an atherosclerotic plaque.     

Hitch-hiking between cells on lipoprotein particles

Cell surface proteins containing covalently linked lipids associate with specialized membrane domains. Morphogens like Hedgehog and Wnt use their lipid anchors to bind to lipoprotein particles and employ lipoproteins to travel through tissues. Removal of their lipid anchors or decreasing lipoprotein levels give rise to adverse Hedgehog and Wnt signaling. Some parasites can also transfer their glycosylphosphatidylinositol-anchored surface proteins to host lipoprotein particles. These antigen-loaded lipoproteins spread throughout the circulation, and probably hamper an adequate immune response by killing neutrophils. Together, these findings imply a widespread role for lipoproteins in intercellular transfer of lipid-anchored surface proteins, and may have various physiological consequences. Here, we discuss how lipid-modified proteins may be transferred to and from lipoproteins at the cellular level.     

Insights into the potential function and membrane organization of the TP0435 (Tp17) lipoprotein from Treponema pallidum derived from structural and biophysical analyses

The sexually transmitted disease syphilis is caused by the bacterial spirochete Treponema pallidum. This microorganism is genetically intractable, accounting for the large number of putative and undercharacterized members of the pathogen's proteome. In an effort to ascribe a function(s) to the TP0435 (Tp17) lipoprotein, we engineered a soluble variant of the protein (rTP0435) and determined its crystal structure at a resolution of 2.42 Å. The structure is characterized by an eight‐stranded β‐barrel protein with a shallow “basin” at one end of the barrel and an α‐helix stacked on the opposite end. Furthermore, there is a disulfide‐linked dimer of the protein in the asymmetric unit of the crystals. Solution hydrodynamic experiments established that purified rTP0435 is monomeric, but specifically forms the disulfide‐stabilized dimer observed in the crystal structure. The data herein, when considered with previous work on TP0435, imply plausible roles for the protein in either ligand binding, treponemal membrane architecture, and/or pathogenesis.     

Xanthine-based KMUP-1 improves HDL via PPARγ/SR-B1, LDL via LDLRs,and HSL via PKA/PKG for hepatic fat loss

The phosphodiesterase inhibitor (PDEI)/eNOS enhancer KMUP-1, targeting G-protein coupled receptors (GPCRs), improves dyslipidemia. We compared its lipid-lowering effects with simvastatin and explored hormone-sensitive lipase (HSL) translocation in hepatic fat loss. KMUP-1 HCl (1, 2.5, and 5 mg/kg/day) and simvastatin (5 mg/kg/day) were administered in C57BL/6J male mice fed a high-fat diet (HFD) by gavage for 8 weeks. KMUP-1 inhibited HFD-induced plasma/liver TG, total cholesterol, and LDL; increased HDL/3-hydroxy-3-methylglutaryl-CoA reductase (HMGR)/Rho kinase II (ROCK II)/PPARγ/ABCA1; and decreased liver and body weight. KMUP-1 HCl in drinking water (2.5 mg/200 ml tap water) for 1–14 or 8–14 weeks decreased HFD-induced liver and body weight and scavenger receptor class B type I expression and increased protein kinase A (PKA)/PKG/LDLRs/HSL expression and immunoreactivity. In HepG2 cells incubated with serum or exogenous mevalonate, KMUP-1 (10⁻⁷∼10⁻⁵ M) reversed HMGR expression by feedback regulation, colocalized expression of ABCA1/apolipoprotein A-I/LXRα/PPARγ, and reduced exogenous geranylgeranyl pyrophosphate/farnesyl pyrophosphate (FPP)-induced RhoA/ROCK II expression. A guanosine 3′,5′-cyclic monophosphate (cGMP) antagonist reversed KMUP-1-induced ROCK II reduction, indicating cGMP/eNOS involvement. KMUP-1 inceased PKG and LDLRs surrounded by LDL and restored oxidized LDL-induced PKA expresion. Unlike simvastatin, KMUP-1 could not inhibit ¹⁴C mevalonate formation. KMUP-1 could, but simvastatin could not, decrease ROCK II expression by exogenous FPP/CGPP. KMUP-1 improves HDL via PPARγ/LXRα/ABCA1/Apo-I expression and increases LDLRs/PKA/PKG/HSL expression and immunoreactivity, leading to TG hydrolysis to lower hepatic fat and body weight.     

Genetic meta-analysis of 15,901 African Americans identifies variation in EXOC3L1 is associated with HDL concentration

Meta-analyses of European populations has successfully identified genetic variants in over 150 loci associated with lipid levels, but results from additional ethnicities remain limited. Previously, we reported two novel lipid loci identified in a sample of 7,657 African Americans using a gene-centric array including 50,000 SNPs in 2,100 candidate genes. Initial discovery and follow-up of signals with P < 10⁻⁵ in additional African American samples confirmed CD36 and ICAM1. Using an additional 8,244 African American female samples from the Women’s Health Initiative SNP Health Association Resource genome-wide association study dataset, we further examined the previous meta-analyses results by attempting to replicate 20 additional putative lipid signals with P < 10⁻⁴. Replication confirmed rs868213, located in a splice donor region of exocyst complex component 3-like 1 (EXOC3L1) as a novel signal for HDL (additive allelic effect β = 0.02; P = 1.4 × 10⁻⁸; meta-analyses of discovery and replication). EXOC3L1 is strongly expressed in vascular endothelium and forms part of the exocyst complex, a key facilitator of the trafficking of lipid receptors. Increasing sample sizes for genetic studies in nonEuropean populations will continue to improve our understanding of lipid metabolism.     

Flexible origin of hydrocarbon/pheromone precursors in Drosophila melanogaster

In terrestrial insects, cuticular hydrocarbons (CHCs) provide protection from desiccation. Specific CHCs can also act as pheromones, which are important for successful mating. Oenocytes are abdominal cells thought to act as specialized units for CHC biogenesis that consists of long-chain fatty acid (LCFA) synthesis, optional desaturation(s), elongation to very long-chain fatty acids (VLCFAs), and removal of the carboxyl group. By investigating CHC biogenesis in Drosophila melanogaster, we showed that VLCFA synthesis takes place only within the oenocytes. Conversely, several pathways, which may compensate for one another, can feed the oenocyte pool of LCFAs, suggesting that this step is a critical node for regulating CHC synthesis. Importantly, flies deficient in LCFA synthesis sacrificed their triacylglycerol stores while maintaining some CHC production. Moreover, pheromone production was lower in adult flies that emerged from larvae that were fed excess dietary lipids, and their mating success was lower. Further, we showed that pheromone production in the oenocytes depends on lipid metabolism in the fat tissue and that fatty acid transport protein, a bipartite acyl-CoA synthase (ACS)/FA transporter, likely acts through its ACS domain in the oenocyte pathway of CHC biogenesis. Our study highlights the importance of environmental and physiological inputs in regulating LCFA synthesis to eventually control sexual communication in a polyphagous animal.     

The effect of phospholipid composition of reconstituted HDL on its cholesterol efflux and anti-inflammatory properties

The goal of this study was to understand how the reconstituted HDL (rHDL) phospholipid (PL) composition affects its cholesterol efflux and anti-inflammatory properties. An ApoA-I mimetic peptide, 5A, was combined with either SM or POPC. Both lipid formulations exhibited similar in vitro cholesterol efflux by ABCA1, but 5A-SM exhibited higher ABCG1- and SR-BI-mediated efflux relative to 5A-POPC (P < 0.05). Injection of both rHDLs in rats resulted in mobilization of plasma cholesterol, although the relative potency was 3-fold higher for the same doses of 5A-SM than for 5A-POPC. Formation of preβ HDL was observed following incubation of rHDLs with both human and rat plasma in vitro, with 5A-SM inducing a higher extent of preβ formation relative to 5A-POPC. Both rHDLs exhibited anti-inflammatory properties, but 5A-SM showed higher inhibition of TNF-α, IL-6, and IL-1β release than did 5A-POPC (P < 0.05). Both 5A-SM and 5A-POPC showed reduction in total plaque area in ApoE^−/− mice, but only 5A-SM showed a statistically significant reduction over placebo control and baseline (P < 0.01). The type of PL used to reconstitute peptide has significant influence on rHDL’s anti-inflammatory and anti-atherosclerosis properties.     

Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay

Quantitation of plasma lipopolysaccharides (LPSs) might be used to document Gram-negative bacterial infection. In the present work, LPS-derived 3-hydroxymyristate was extracted from plasma samples with an organic solvent, separated by reversed phase HPLC, and quantitated by MS/MS. This mass assay was combined with the limulus amebocyte lysate (LAL) bioassay to monitor neutralization of LPS activity in biological samples. The described HPLC/MS/MS method is a reliable, practical, accurate, and sensitive tool to quantitate LPS. The combination of the LAL and HPLC/MS/MS analyses provided new evidence for the intrinsic capacity of plasma lipoproteins and phospholipid transfer protein to neutralize the activity of LPS. In a subset of patients with systemic inflammatory response syndrome, with documented infection but with a negative plasma LAL test, significant amounts of LPS were measured by the HPLC/MS/MS method. Patients with the highest plasma LPS concentration were more severely ill. HPLC/MS/MS is a relevant method to quantitate endotoxin in a sample, to assess the efficacy of LPS neutralization, and to evaluate the proinflammatory potential of LPS in vivo.