Rapid characterization of disease-causing mutations in the low density lipoprotein receptor (LDL-R) gene by overexpression in COS cells

DEFF Research Database (Denmark)

Jensen, T G; Andresen, B S; Jensen, H K

1996-01-01

To characterize disease-causing mutations in the low density lipoprotein receptor (LDL-R) gene, COS cells are transfected with the mutant gene in an EBV-based expression vector and characterized by flow cytometry. Using antibodies against the LDL-receptor the amount of receptor protein on the cel...

The low-density lipoprotein receptor-related protein 1 and amyloid-β clearance in Alzheimer’s disease

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Takahisa eKanekiyo

2014-05-01

Full Text Available Accumulation and aggregation of amyloid-β (Aβ peptides in the brain trigger the development of progressive neurodegeneration and dementia associated with Alzheimer’s disease (AD. Perturbation in Aβ clearance, rather than Aβ production, is likely the cause of sporadic, late-onset AD, which accounts for the majority of AD cases. Since cellular uptake and subsequent degradation constitute a major Aβ clearance pathway, the receptor-mediated endocytosis of Aβ has been intensely investigated. Among Aβ receptors, the low-density lipoprotein receptor-related protein 1 (LRP1 is one of the most studied receptors. LRP1 is a large endocytic receptor for more than 40 ligands, including apolipoprotein E (apoE, α2-macroglobulin and Aβ. Emerging in vitro and in vivo evidence demonstrates that LRP1 is critically involved in brain Aβ clearance. LRP1 is highly expressed in a variety of cell types in the brain including neurons, vascular cells and glial cells, where LRP1 functions to maintain brain homeostasis and control Aβ metabolism. LRP1-mediated endocytosis regulates cellular Aβ uptake by binding to Aβ either directly or indirectly through its co-receptors or ligands. Furthermore, LRP1 regulates several signaling pathways, which also likely influences Aβ endocytic pathways. In this review, we discuss how LRP1 regulates the brain Aβ clearance and how this unique endocytic receptor participates in AD pathogenesis. Understanding of the mechanisms underlying LRP1-mediated Aβ clearance should enable the rational design of novel diagnostic and therapeutic strategies for AD.

Comparative studies of vertebrate scavenger receptor class B type 1: a high-density lipoprotein binding protein

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Holmes RS

2012-06-01

Full Text Available Roger S Holmes,1,2 Laura A Cox11Department of Genetics and Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, TX, USA; 2School of Biomolecular and Physical Sciences, Griffith University, Nathan, Queensland, AustraliaAbstract: Scavenger receptor class B type 1 protein (SCARB1 plays an essential role in cholesterol homeostasis and functions in binding high density lipoprotein cholesterol (HDL in liver and other tissues of the body. SCARB1 also functions in lymphocyte homeostasis and in the uptake of hepatitis C virus (HCV by the liver. A genetic deficiency of this protein results in autoimmune disorders and significant changes in blood cholesterol phenotype. Comparative SCARB1 amino acid sequences and structures and SCARB1 gene locations were examined using data from several vertebrate genome projects. Vertebrate SCARB1 sequences shared 50%–99% identity as compared with 28%–31% sequence identities with other CD36-like superfamily members, ie, SCARB2 and SCARB3 (also called CD36. At least eight N-glycosylation sites were conserved among most of the vertebrate SCARB1 proteins examined. Sequence alignments, key amino acid residues, and conserved predicted secondary structures were also studied, including: cytoplasmic, transmembrane, and exoplasmic sequences; conserved N-terminal and C-terminal transmembrane glycines which participate in oligomer formation; conserved cystine disulfides and a free SH residue which participates in lipid transport; carboxyl terminal PDZ-binding domain sequences (Ala507-Arg/Lys508-Leu509; and 30 conserved proline and 18 conserved glycine residues, which may contribute to short loop formation within the exoplasmic HDL-binding sequence. Vertebrate SCARB1 genes usually contained 12 coding exons. The human SCARB1 gene contained CpG islands, micro RNA binding sites, and several transcription factor binding sites (including PPARG which may contribute to the high level (13.7 times

Serum lipoproteins attenuate macrophage activation and Toll-Like Receptor stimulation by bacterial lipoproteins

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James Richard W

2010-09-01

Full Text Available Abstract Background Chlamydia trachomatis was previously shown to express a lipoprotein, the macrophage infectivity potentiator (Mip, exposed at the bacterial surface, and able to stimulate human primary monocytes/macrophages through Toll Like Receptor (TLR2/TLR1/TLR6, and CD14. In PMA-differentiated THP-1 cells the proinflammatory activity of Mip was significantly higher in the absence than in the presence of serum. The present study aims to investigate the ability of different serum factors to attenuate Mip proinflammatory activity in PMA-differentiated THP-1 cells and in primary human differentiated macrophages. The study was also extend to another lipoprotein, the Borrelia burgdorferi outer surface protein (OspA. The proinflammatory activity was studied through Tumor Necrosis Factor alpha (TNF-α and Interleukin (IL-8 release. Finally, TLR1/2 human embryonic kidney-293 (HEK-293 transfected cells were used to test the ability of the serum factors to inhibit Mip and OspA proinflammatory activity. Results In the absence of any serum and in the presence of 10% delipidated FBS, production of Mip-induced TNF-α and IL-8 in PMA-differentiated THP-1 cells were similar whereas they were significantly decreased in the presence of 10% FBS suggesting an inhibiting role of lipids present in FBS. In the presence of 10% human serum, the concentrations of TNF-α and IL-8 were 2 to 5 times lower than in the presence of 10% FBS suggesting the presence of more potent inhibitor(s in human serum than in FBS. Similar results were obtained in primary human differentiated macrophages. Different lipid components of human serum were then tested (total lipoproteins, HDL, LDL, VLDL, triglyceride emulsion, apolipoprotein (apoA-I, B, E2, and E3. The most efficient inhibitors were LDL, VLDL, and apoB that reduced the mean concentration of TNF-α release in Mip-induced macrophages to 24, 20, and 2%, respectively (p Conclusions These results demonstrated the ability of

Metabolism of lipoproteins by human fetal hepatocytes

International Nuclear Information System (INIS)

Carr, B.R.

1987-01-01

The rate of clearance of lipoproteins from plasma appears to play a role in the development of atherogenesis. The liver may account for as much as two thirds of the removal of low-density lipoprotein and one third of the clearance of high-density lipoprotein in certain animal species and humans, mainly by receptor-mediated pathways. The purpose of the present investigation was to determine if human fetal hepatocytes maintained in vitro take up and degrade lipoproteins. We first determined that the maximal binding capacity of iodine 125-iodo-LDL was approximately 300 ng of low-density lipoprotein protein/mg of membrane protein and an apparent dissociation constant of approximately 60 micrograms low-density lipoprotein protein/ml in membranes prepared from human fetal liver. We found that the maximal uptake of [ 125 I]iodo-LDL and [ 125 I]iodo-HDL by fetal hepatocytes occurred after 12 hours of incubation. Low-density lipoprotein uptake preceded the appearance of degradation products by 4 hours, and thereafter the degradation of low-density lipoprotein increased linearly for at least 24 hours. In contrast, high-density lipoprotein was not degraded to any extent by fetal hepatocytes. [ 125 I]Iodo-LDL uptake and degradation were inhibited more than 75% by preincubation with low-density lipoprotein but not significantly by high-density lipoprotein, whereas [ 125 I]iodo-HDL uptake was inhibited 70% by preincubation with high-density lipoprotein but not by low-density lipoprotein. In summary, human fetal hepatocytes take up and degrade low-density lipoprotein by a receptor-mediated process similar to that described for human extrahepatic tissues

Postprandial triglyceride-rich lipoproteins regulate perilipin-2 and perilipin-3 lipid-droplet-associated proteins in macrophages.

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Varela, Lourdes M; López, Sergio; Ortega-Gómez, Almudena; Bermúdez, Beatriz; Buers, Insa; Robenek, Horst; Muriana, Francisco J G; Abia, Rocío

2015-04-01

Lipid accumulation in macrophages contributes to atherosclerosis. Within macrophages, lipids are stored in lipid droplets (LDs); perilipin-2 and perilipin-3 are the main LD-associated proteins. Postprandial triglyceride (TG)-rich lipoproteins induce LD accumulation in macrophages. The role of postprandial lipoproteins in perilipin-2 and perilipin-3 regulation was studied. TG-rich lipoproteins (TRLs) induced the levels of intracellular TGs, LDs and perilipin-2 protein expression in THP-1 macrophages and in Apoe(-/-) mice bone-marrow-derived macrophages with low and high basal levels of TGs. Perilipin-3 was only synthesized in mice macrophages with low basal levels of TGs. The regulation was dependent on the fatty acid composition of the lipoproteins; monounsaturated and polyunsaturated fatty acids (PUFAs) more strongly attenuated these effects compared with saturated fatty acids. In THP-1 macrophages, immunofluorescence microscopy and freeze-fracture immunogold labeling indicated that the lipoproteins translocated perilipin-3 from the cytoplasm to the LD surface; only the lipoproteins that were rich in PUFAs suppressed this effect. Chemical inhibition showed that lipoproteins induced perilipin-2 protein expression through the peroxisome proliferator-activated nuclear receptor (PPAR) PPARα and PPARγ pathways. Overall, our data indicate that postprandial TRLs may be involved in atherosclerotic plaque formation through the regulation of perilipin-2 and perilipin-3 proteins in macrophages. Because the fatty acid composition of the lipoproteins is dependent on the type of fat consumed, the ingestion of olive oil, which is rich in monounsaturated fatty acids, and fish oil, which is rich in omega-3 fatty acids, can be considered a good nutritional strategy to reduce the risk of atherosclerosis by LD-associated proteins decrease. Copyright © 2015 Elsevier Inc. All rights reserved.

Experimental hypothyroidism modulates the expression of the low density lipoprotein receptor by the liver

International Nuclear Information System (INIS)

Scarabottolo, Lia; Trezzi, Ermanno; Roma, Paola; Catapano, A.L.

1986-01-01

The effect of exprimental hypothyroidism of the catabolism of plasma lipoproteins and on the expression of low density lipoprotein receptors by the liver was investigated in rats made hypothyroid by surgery. The animals developed mild hypercholesterolemia, mainly due to an increase of plasma low density lipoprotein, while other lipoprotein classes were only marginally affected. Kinetic studies using ( 125 I)LDL indicated that a decreased fractional catabolic rate of the lipoprotein was responsible for this finding in agreement with the in vitro observation of a reduced binding of lipoproteins to liver membranes from hyperthyroid rats and with the demonstrations, by ligand blotting analysis, of a decreasd expression of lipoprotein receptors in liver membranes. These data suggest that hypothyroidism affects lipoprotein distribution also by decreasing the catabolism of low density lipoproteins by the liver (author)

Cellular uptake of lipoproteins and Persistent Organic Compounds - An update and new data

DEFF Research Database (Denmark)

Hjelmborg, Philip Sebastian; Andreassen, Thomas Kjærgaard; Bonefeld-Jørgensen, Eva Cecilie

2008-01-01

including the pesticide DDT (p,p'-dichlorodiphenyltrichloroethane), and especially its metabolite DDE (p,p'-dichlorodiphenyldichloroethene), interacts with nuclear hormone receptors causing these to malfunction, which in turn results in a range of deleterious health effects in humans. The aim of the present...... study was to determine the role of lipoprotein receptors in mouse embryonic fibroblast (MEF) cells in conjunction with uptake of DDT-lipoprotein complexes from supplemented media in vitro. Uptake of DDT by MEF cells was investigated using MEF1 cells carrying the receptors LRP (low-density lipoprotein...... receptor-related protein) and LDLR (low density lipoprotein receptor) present and MEF4 cells with no LRP and LDLR expression. Cells were incubated together with the complex of LDL and [14C]DDT. The receptor function was further evaluated by adding the 40 kDa receptor-associated protein (RAP) which blocks...

Cellular uptake of lipoproteins and persistent organic compounds-An update and new data

International Nuclear Information System (INIS)

Hjelmborg, Philip Sebastian; Andreassen, Thomas Kjaergaard; Bonefeld-Jorgensen, Eva Cecilie

2008-01-01

There are a number of interactions related to the transport of lipophilic xenobiotic compounds in the blood stream of mammals. This paper will focus on the interactions between lipoproteins and persistent organic pollutants (POPs) and how these particles are taken up by cells. A number of POPs including the pesticide p,p'-dichlorodiphenyltrichloroethane (DDT), and especially its metabolite p,p'-dichlorodiphenyldichloroethene (DDE), interacts with nuclear hormone receptors causing these to malfunction, which in turn results in a range of deleterious health effects in humans. The aim of the present study was to determine the role of lipoprotein receptors in mouse embryonic fibroblast (MEF) cells in conjunction with uptake of DDT-lipoprotein complexes from supplemented media in vitro. Uptake of DDT by MEF cells was investigated using MEF1 cells carrying the receptors low-density lipoprotein receptor-related protein (LRP) and low-density lipoprotein receptor (LDLR) present and MEF4 cells with no LRP and LDLR expression. Cells were incubated together with the complex of low-density lipoproteins (LDL) and [ 14 C]DDT. The receptor function was further evaluated by adding the 40 kDa receptor-associated protein (RAP) which blocks receptor activity. The results showed that [ 14 C]DDT uptake was decreasing when the LDL concentration was increasing. There was no strong evidence for a receptor-mediated uptake of the [ 14 C]DDT-lipoprotein complex. To conclude, DDT travels in the blood stream and can cross cell membranes while being transported as a DDT-lipoprotein complex. The lipoproteins do not need receptors to cross cell membranes since passive diffusion constitutes a major passageway

Low-Density Lipoprotein Receptor-Related Protein 6 (LRP6 Is a Novel Nutritional Therapeutic Target for Hyperlipidemia, Non-Alcoholic Fatty Liver Disease, and Atherosclerosis

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Gwang-woong Go

2015-06-01

Full Text Available Low-density lipoprotein receptor-related protein 6 (LRP6 is a member of the low-density lipoprotein receptor family and has a unique structure, which facilitates its multiple functions as a co-receptor for Wnt/β-catenin signaling and as a ligand receptor for endocytosis. The role LRP6 plays in metabolic regulation, specifically in the nutrient-sensing pathway, has recently garnered considerable interest. Patients carrying an LRP6 mutation exhibit elevated levels of LDL cholesterol, triglycerides, and fasting glucose, which cooperatively constitute the risk factors of metabolic syndrome and atherosclerosis. Since the discovery of this mutation, the general role of LRP6 in lipid homeostasis, glucose metabolism, and atherosclerosis has been thoroughly researched. These studies have demonstrated that LRP6 plays a role in LDL receptor-mediated LDL uptake. In addition, when the LRP6 mutant impaired Wnt-LRP6 signaling, hyperlipidemia, non-alcoholic fatty liver disease, and atherosclerosis developed. LRP6 regulates lipid homeostasis and body fat mass via the nutrient-sensing mechanistic target of the rapamycin (mTOR pathway. Furthermore, the mutant LRP6 triggers atherosclerosis by activating platelet-derived growth factor (PDGF-dependent vascular smooth muscle cell differentiation. This review highlights the exceptional opportunities to study the pathophysiologic contributions of LRP6 to metabolic syndrome and cardiovascular diseases, which implicate LRP6 as a latent regulator of lipid metabolism and a novel therapeutic target for nutritional intervention.

A receptor-based biosensor for lipoprotein docking at the endothelial surface and vascular matrix.

Science.gov (United States)

Siegel, G; Malmsten, M; Klüssendorf, D; Michel, F

2001-12-01

Proteoheparan sulfate can be adsorbed to a methylated silica surface in a monomolecular layer via its transmembrane hydrophobic protein core domain. Due to electrostatic repulsion, its anionic glycosaminoglycan side chains are stretched out into the blood substitute solution, representing a receptor site for specific lipoprotein binding through basic amino acid-rich residues within their apolipoproteins. The binding process was studied by ellipsometric techniques showing that HDL has a high binding affinity to the receptor and a protective effect on interfacial heparan sulfate proteoglycan layers, with respect to LDL and Ca(2+) complexation. LDL was found to deposit strongly at the proteoheparan sulfate, particularly in the presence of Ca(2+), thus creating the complex formation "proteoglycan-low density lipoprotein-calcium". This ternary complex build-up may be interpreted as arteriosclerotic nanoplaque formation on the molecular level responsible for the arteriosclerotic primary lesion. On the other hand, HDL bound to heparan sulfate proteoglycan protected against LDL docking and completely suppressed calcification of the proteoglycan-lipoprotein complex. In addition, HDL and aqueous garlic extract were able to reduce the ternary complex deposition and to disintegrate HS-PG/LDL/Ca(2+) aggregates. Although much remains unclear regarding the mechanism of lipoprotein depositions at proteoglycan-coated surfaces, it seems clear that the use of such systems offers possibilities for investigating lipoprotein deposition at a "nanoscopic" level under close to physiological conditions. In particular, Ca(2+)-promoted LDL deposition and the protective effect of HDL, even at high Ca(2+) and LDL concentrations, agree well with previous clinical observations regarding risk and beneficial factors for early stages of atherosclerosis. Therefore, we believe that the system can be of some use in investigations, e.g. of the interplay between different lipoproteins in arteriosclerotic

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions.

Science.gov (United States)

Wang, Shengjun; Mao, Yang; Narimatsu, Yoshiki; Ye, Zilu; Tian, Weihua; Goth, Christoffer K; Lira-Navarrete, Erandi; Pedersen, Nis B; Benito-Vicente, Asier; Martin, Cesar; Uribe, Kepa B; Hurtado-Guerrero, Ramon; Christoffersen, Christina; Seidah, Nabil G; Nielsen, Rikke; Christensen, Erik I; Hansen, Lars; Bennett, Eric P; Vakhrushev, Sergey Y; Schjoldager, Katrine T; Clausen, Henrik

2018-05-11

The low-density lipoprotein receptor (LDLR) and related receptors are important for the transport of diverse biomolecules across cell membranes and barriers. Their functions are especially relevant for cholesterol homeostasis and diseases, including neurodegenerative and kidney disorders. Members of the LDLR-related protein family share LDLR class A (LA) repeats providing binding properties for lipoproteins and other biomolecules. We previously demonstrated that short linker regions between these LA repeats contain conserved O -glycan sites. Moreover, we found that O -glycan modifications at these sites are selectively controlled by the GalNAc-transferase isoform, GalNAc-T11. However, the effects of GalNAc-T11-mediated O -glycosylation on LDLR and related receptor localization and function are unknown. Here, we characterized O -glycosylation of LDLR-related proteins and identified conserved O -glycosylation sites in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of gene-edited isogenic cell line models, we demonstrate that GalNAc-T11-mediated LDLR and VLDLR O -glycosylation is not required for transport and cell-surface expression and stability of these receptors but markedly enhances LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O -glycosylation increased affinity for LDL by ∼5-fold. The molecular basis for this observation is currently unknown, but these findings open up new avenues for exploring the roles of LDLR-related proteins in disease. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Receptor-mediated endocytosis of low density lipoproteins in aortic endothelial cells

International Nuclear Information System (INIS)

Sanan, D.A.

1986-04-01

Lipoprotein binding and metabolism in actively-dividing (subconfluent) and quiescent (postconfluent) bovine aortic endothelial cells (ECs) were qualitatively investigated by fluorescence microscopy using dioctadecylindocarbocyanine-labelled lipoproteins and by indirect immunofluorescence microscopy. LDL and acetylated-LDL (AcLDL) were seen bound to the surfaces of subconfluent ECs (at 4 degrees C or at 37 degrees C), as a random distribution of punctate foci. ECs therefore closely resembled fibroblasts in the distribution of LDL receptors on their surfaces. No binding of LDL was seen on postconfluent EC surfaces by either direct or indirect fluorescence microscopy. The patterns of AcLDL binding on postconfluent ECs resembled those on subconfluent ECs. Intracellular LDL and AcLDL occurred as perinuclear accumulations of large fluorescent disc-shaped profiles in subconfluent ECs. These accumulations were shown to arise from surface-bound material by pulse-chase experiments. Intracellular LDL was absent in the majority of postconfluent ECs, while AcLDL accumulation was massive. 'Wounding' of cultures allowed simultaneous assessment of lipoprotein metabolism in quiescent and actively-dividing areas of the same culture. It is concluded that postconfluent quiescent bovine aortic ECs in vitro metabolise virtually no LDL via the LDL-receptor pathway due to a vanishingly low number of LDL receptors. This contrasts with the ability of postconfluent cells to metabolise relatively large amounts of AcLDL via a receptor-mediated mechanism. The significance of these conclusions is discussed with respect to the interaction of plasma lipoproteins with the endothelium in vivo. 301 refs

Sortilins: new players in lipoprotein metabolism

DEFF Research Database (Denmark)

Willnow, Thomas; Kjølby, Mads Fuglsang; Nykjær, Anders

2011-01-01

PURPOSE OF REVIEW: Sortilins are sorting receptors that direct proteins through secretory and endocytic pathways of the cell. Previously, these receptors have been shown to play important roles in regulating protein transport in neurons and to control neuronal viability and death in many diseases...... on the importance of sorting receptors in control of cellular and systemic lipoprotein metabolism and how altered trafficking pathways may represent a major risk factor for dyslipidemia and atherosclerosis in the human population....

Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein.

Science.gov (United States)

Andersen, O M; Petersen, H H; Jacobsen, C; Moestrup, S K; Etzerodt, M; Andreasen, P A; Thøgersen, H C

2001-07-01

The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.

Chlordecone, a mixed pregnane X receptor (PXR) and estrogen receptor alpha (ERα) agonist, alters cholesterol homeostasis and lipoprotein metabolism in C57BL/6 mice

International Nuclear Information System (INIS)

Lee, Junga; Scheri, Richard C.; Zhang Yuan; Curtis, Lawrence R.

2008-01-01

Chlordecone (CD) is one of many banned organochlorine (OC) insecticides that are widespread persistent organic pollutants. OC insecticides alter lipid homeostasis in rodents at doses that are not neurotoxic or carcinogenic. Pretreatment of mice or rats with CD altered tissue distribution of a subsequent dose of [ 14 C]CD or [ 14 C]cholesterol (CH). Nuclear receptors regulate expression of genes important in the homeostasis of CH and other lipids. In this study, we report that CD suppresses in vitro reporter systems for human liver X receptors (LXRs) and activates those for human farnesoid X receptor (FXR), pregnane X receptor (PXR) and estrogen receptor α (ERα) in a concentration-dependent manner (0-50 μM). Consistent with human PXR activation in vitro, three days after a single dose of CD (15 mg/kg) hepatic microsomal CYP3A11 protein increases in C57BL/6 mice. CD decreases hepatic CH ester content without altering total CH concentration. Apolipoprotein A-I (apoA-I) contents of hepatic lipoprotein-rich and microsomal fractions of CD-treated mice are higher than controls. There is a significant reduction in non-high density lipoprotein CH but not apolipoprotein B-48/100 (apoB-48/100) in plasma from CD-treated mice after a 4 h fast. At 14 days after 15 mg CD/kg apoA-I and apoB-100 proteins but not CYP3A11 protein in hepatic microsomes are similar to controls. This work indicates that altered CH homeostasis is a mode of OC insecticide action of relevance after a single dose. This at least partially explains altered CH tissue distribution in CD-pretreated mice

α-Tocopherol modulates the low density lipoprotein receptor of human HepG2 cells

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Bottema Cynthia DK

2003-05-01

Full Text Available Abstract The aim of this study was to determine the effects of vitamin E (α-tocopherol on the low density lipoprotein (LDL receptor, a cell surface protein which plays an important role in controlling blood cholesterol. Human HepG2 hepatoma cells were incubated for 24 hours with increasing amounts of α, δ, or γ-tocopherol. The LDL receptor binding activity, protein and mRNA, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA reductase mRNA, cell cholesterol and cell lathosterol were measured. The effect of α-tocopherol was biphasic. Up to a concentration of 50 μM, α-tocopherol progressively increased LDL receptor binding activity, protein and mRNA to maximum levels 2, 4 and 6-fold higher than control, respectively. The HMG-CoA reductase mRNA and the cell lathosterol concentration, indices of cholesterol synthesis, were also increased by 40% over control by treatment with 50 μM α-tocopherol. The cell cholesterol concentration was decreased by 20% compared to control at 50 μM α-tocopherol. However, at α-tocopherol concentrations higher than 50 μM, the LDL receptor binding activity, protein and mRNA, the HMG-CoA reductase mRNA and the cell lathosterol and cholesterol concentrations all returned to control levels. The biphasic effect on the LDL receptor was specific for α-tocopherol in that δ and γ-tocopherol suppressed LDL receptor binding activity, protein and mRNA at all concentrations tested despite the cells incorporating similar amounts of the three homologues. In conclusion, α-tocopherol, exhibits a specific, concentration-dependent and biphasic "up then down" effect on the LDL receptor of HepG2 cells which appears to be at the level of gene transcription. Cholesterol synthesis appears to be similarly affected and the cell cholesterol concentration may mediate these effects.

Low density lipoprotein induces upregulation of vasoconstrictive endothelin type B receptor expression

DEFF Research Database (Denmark)

Xu, Cang-Bao; Zheng, Jian-Pu; Zhang, Wei

2014-01-01

Vasoconstrictive endothelin type B (ET(B)) receptors promote vasospasm and ischemic cerebro- and cardiovascular diseases. The present study was designed to examine if low density lipoprotein (LDL) induces upregulation of vasoconstrictive ET(B) receptor expression and if extracellular signal...

LDL Receptor-Related Protein-1 (LRP1 Regulates Cholesterol Accumulation in Macrophages.

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Anna P Lillis

Full Text Available Within the circulation, cholesterol is transported by lipoprotein particles and is taken up by cells when these particles associate with cellular receptors. In macrophages, excessive lipoprotein particle uptake leads to foam cell formation, which is an early event in the development of atherosclerosis. Currently, mechanisms responsible for foam cell formation are incompletely understood. To date, several macrophage receptors have been identified that contribute to the uptake of modified forms of lipoproteins leading to foam cell formation, but the in vivo contribution of the LDL receptor-related protein 1 (LRP1 to this process is not known [corrected]. To investigate the role of LRP1 in cholesterol accumulation in macrophages, we generated mice with a selective deletion of LRP1 in macrophages on an LDL receptor (LDLR-deficient background (macLRP1-/-. After feeding mice a high fat diet for 11 weeks, peritoneal macrophages isolated from Lrp+/+ mice contained significantly higher levels of total cholesterol than those from macLRP1-/- mice. Further analysis revealed that this was due to increased levels of cholesterol esters. Interestingly, macLRP1-/- mice displayed elevated plasma cholesterol and triglyceride levels resulting from accumulation of large, triglyceride-rich lipoprotein particles in the circulation. This increase did not result from an increase in hepatic VLDL biosynthesis, but rather results from a defect in catabolism of triglyceride-rich lipoprotein particles in macLRP1-/- mice. These studies reveal an important in vivo contribution of macrophage LRP1 to cholesterol homeostasis.

Loss of Macrophage Low-Density Lipoprotein Receptor-Related Protein 1 Confers Resistance to the Antiatherogenic Effects of Tumor Necrosis Factor-α Inhibition.

Science.gov (United States)

Zhu, Lin; Giunzioni, Ilaria; Tavori, Hagai; Covarrubias, Roman; Ding, Lei; Zhang, Youmin; Ormseth, Michelle; Major, Amy S; Stafford, John M; Linton, MacRae F; Fazio, Sergio

2016-08-01

Antiatherosclerotic effects of tumor necrosis factor-α (TNF-α) blockade in patients with systemic inflammatory states are not conclusively demonstrated, which suggests that effects depend on the cause of inflammation. Macrophage LRP1 (low-density lipoprotein receptor-related protein 1) and apoE contribute to inflammation through different pathways. We studied the antiatherosclerosis effects of TNF-α blockade in hyperlipidemic mice lacking either LRP1 (MΦLRP1(-/-)) or apoE from macrophages. Lethally irradiated low-density lipoprotein receptor (LDLR)(-/-) mice were reconstituted with bone marrow from either wild-type, MΦLRP1(-/-), apoE(-/-) or apoE(-/-)/MΦLRP1(-/-)(DKO) mice, and then treated with the TNF-α inhibitor adalimumab while fed a Western-type diet. Adalimumab reduced plasma TNF-α concentration, suppressed blood ly6C(hi) monocyte levels and their migration into the lesion, and reduced lesion cellularity and inflammation in both wild-type→LDLR(-/-) and apoE(-/-)→LDLR(-/-) mice. Overall, adalimumab reduced lesion burden by 52% to 57% in these mice. Adalimumab reduced TNF-α and blood ly6C(hi) monocyte levels in MΦLRP1(-/-)→LDLR(-/-) and DKO→LDLR(-/-) mice, but it did not suppress ly6C(hi) monocyte migration into the lesion or atherosclerosis progression. Our results show that TNF-α blockade exerts antiatherosclerotic effects that are dependent on the presence of macrophage LRP1. © 2016 American Heart Association, Inc.

Lipoprotein Nanoplatform for Targeted Delivery of Diagnostic and Therapeutic Agents

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Jerry D. Glickson

2008-03-01

Full Text Available Low-density lipoprotein (LDL provides a highly versatile natural nanoplatform for delivery of visible or near-infrared fluorescent optical and magnetic resonance imaging (MRI contrast agents and photodynamic therapy and chemotherapeutic agents to normal and neoplastic cells that overexpress low-density lipoprotein receptors (LDLRs. Extension to other lipoproteins ranging in diameter from about 10 nm (high-density lipoprotein [HDL] to over a micron (chylomicrons is feasible. Loading of contrast or therapeutic agents onto or into these particles has been achieved by protein loading (covalent attachment to protein side chains, surface loading (intercalation into the phospholipid monolayer, and core loading (extraction and reconstitution of the triglyceride/cholesterol ester core. Core and surface loading of LDL have been used for delivery of optical imaging agents to tumor cells in vivo and in culture. Surface loading was used for delivery of gadolinium-bis-stearylamide contrast agents for in vivo MRI detection in tumor-bearing mice. Chlorin and phthalocyanine near-infrared photodynamic therapy agents (≤ 400/LDL have been attached by core loading. Protein loading was used to reroute the LDL from its natural receptor (LDLR to folate receptors and could be used to target other receptors. A semisynthetic nanoparticle has been constructed by coating magnetite iron oxide nanoparticles with carboxylated cholesterol and overlaying a monolayer of phospholipid to which apolipoprotein A1 or E was adsorbed for targeting HDL or adsorbing synthetic amphipathic helical peptides ltargeting LDL or folate receptors. These particles can be used for in situ loading of magnetite into cells for MRI-monitored cell tracking or gene expression.

The low-density lipoprotein receptor-related protein 10 is a negative regulator of the canonical Wnt/β-catenin signaling pathway

International Nuclear Information System (INIS)

Jeong, Young-Hee; Sekiya, Manami; Hirata, Michiko; Ye, Mingjuan; Yamagishi, Azumi; Lee, Sang-Mi; Kang, Man-Jong; Hosoda, Akemi; Fukumura, Tomoe; Kim, Dong-Ho; Saeki, Shigeru

2010-01-01

Wnt signaling pathways play fundamental roles in the differentiation, proliferation and functions of many cells as well as developmental, growth, and homeostatic processes in animals. Low-density lipoprotein receptor (LDLR)-related protein (LRP) 5 and LRP6 serve as coreceptors of Wnt proteins together with Frizzled receptors, triggering activation of canonical Wnt/β-catenin signaling. Here, we found that LRP10, a new member of the LDLR gene family, inhibits the canonical Wnt/β-catenin signaling pathway. The β-catenin/T cell factor (TCF) transcriptional activity in HEK293 cells was activated by transfection with Wnt3a or LRP6, which was then inhibited by co-transfection with LRP10. Deletion of the extracellular domain of LRP10 negated its inhibitory effect. The inhibitory effect of LRP10 was consistently conserved in HEK293 cells even when GSK3β phosphorylation was inhibited by incubation with lithium chloride and co-transfection with constitutively active S33Y-mutated β-catenin. Nuclear β-catenin accumulation was unaffected by LRP10. The present studies suggest that LRP10 may interfere with the formation of the β-catenin/TCF complex and/or its binding to target DNA in the nucleus, and that the extracellular domain of LRP10 is critical for inhibition of the canonical Wnt/β-catenin signaling pathway.

Lipoprotein receptor LRP1 regulates leptin signaling and energy homeostasis in the adult central nervous system.

Science.gov (United States)

Liu, Qiang; Zhang, Juan; Zerbinatti, Celina; Zhan, Yan; Kolber, Benedict J; Herz, Joachim; Muglia, Louis J; Bu, Guojun

2011-01-11

Obesity is a growing epidemic characterized by excess fat storage in adipocytes. Although lipoprotein receptors play important roles in lipid uptake, their role in controlling food intake and obesity is not known. Here we show that the lipoprotein receptor LRP1 regulates leptin signaling and energy homeostasis. Conditional deletion of the Lrp1 gene in the brain resulted in an obese phenotype characterized by increased food intake, decreased energy consumption, and decreased leptin signaling. LRP1 directly binds to leptin and the leptin receptor complex and is required for leptin receptor phosphorylation and Stat3 activation. We further showed that deletion of the Lrp1 gene specifically in the hypothalamus by Cre lentivirus injection is sufficient to trigger accelerated weight gain. Together, our results demonstrate that the lipoprotein receptor LRP1, which is critical in lipid metabolism, also regulates food intake and energy homeostasis in the adult central nervous system.

Inhibition of lectin-like oxidized low-density lipoprotein receptor-1 reduces cardiac fibroblast proliferation by suppressing GATA Binding Protein 4

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Liu, Bin; Liu, Ning-Ning; Liu, Wei-Hua; Zhang, Shuang-Wei; Zhang, Jing-Zhi; Li, Ai-Qun [Department of Cardiology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Guangzhou Institute of Cardiovascular Disease, Guangzhou (China); Liu, Shi-Ming, E-mail: gzliushiming@126.com [Department of Cardiology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Guangzhou Institute of Cardiovascular Disease, Guangzhou (China)

2016-07-08

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and GATA Binding Protein 4 (GATA4) are important for the growth of cardiac fibroblasts (CFs). When deregulated, LOX-1 and GATA4 can cause cardiac remodeling. In the present study, we found novel evidence that GATA4 was required for the LOX-1 regulation of CF proliferation. The inhibition of LOX-1 by RNA interference LOX-1 lentivirus resulted in the loss of PI3K/Akt activation and GATA4 protein expression. The overexpression of LOX-1 by lentivirus rescued CF proliferation, PI3K/Akt activation, and GATA4 protein expression. Moreover, GATA4 overexpression enhanced CF proliferation with LOX-1 inhibition. We also found that the inhibition of PI3K/Akt activation by LY294002, a PI3K inhibitor, reduced cell proliferation and protein level of GATA4. In summary, GATA4 may play an important role in the LOX-1 and PI3K/Akt regulation of CF proliferation. -- Highlights: •GATA4 is regulated by LOX-1 signaling in CFs. •GATA4 is involved in LOX-1 regulating CF proliferation. •GATA4 is regulated by PI3K/Akt signaling in CFs.

Lipoprotein receptor LRP1 regulates leptin signaling and energy homeostasis in the adult central nervous system.

Directory of Open Access Journals (Sweden)

Qiang Liu

2011-01-01

Full Text Available Obesity is a growing epidemic characterized by excess fat storage in adipocytes. Although lipoprotein receptors play important roles in lipid uptake, their role in controlling food intake and obesity is not known. Here we show that the lipoprotein receptor LRP1 regulates leptin signaling and energy homeostasis. Conditional deletion of the Lrp1 gene in the brain resulted in an obese phenotype characterized by increased food intake, decreased energy consumption, and decreased leptin signaling. LRP1 directly binds to leptin and the leptin receptor complex and is required for leptin receptor phosphorylation and Stat3 activation. We further showed that deletion of the Lrp1 gene specifically in the hypothalamus by Cre lentivirus injection is sufficient to trigger accelerated weight gain. Together, our results demonstrate that the lipoprotein receptor LRP1, which is critical in lipid metabolism, also regulates food intake and energy homeostasis in the adult central nervous system.

Mitogen-activated protein kinase signaling pathways promote low-density lipoprotein receptor-related protein 1-mediated internalization of beta-amyloid protein in primary cortical neurons.

Science.gov (United States)

Yang, Wei-Na; Ma, Kai-Ge; Qian, Yi-Hua; Zhang, Jian-Shui; Feng, Gai-Feng; Shi, Li-Li; Zhang, Zhi-Chao; Liu, Zhao-Hui

2015-07-01

Mounting evidence suggests that the pathological hallmarks of Alzheimer's disease (AD) are caused by the intraneuronal accumulation of beta-amyloid protein (Aβ). Reuptake of extracellular Aβ is believed to contribute significantly to the intraneuronal Aβ pool in the early stages of AD. Published reports have claimed that the low-density lipoprotein receptor-related protein 1 (LRP1) mediates Aβ1-42 uptake and lysosomal trafficking in GT1-7 neuronal cells and mouse embryonic fibroblast non-neuronal cells. However, there is no direct evidence supporting the role of LRP1 in Aβ internalization in primary neurons. Our recent study indicated that p38 MAPK and ERK1/2 signaling pathways are involved in regulating α7 nicotinic acetylcholine receptor (α7nAChR)-mediated Aβ1-42 uptake in SH-SY5Y cells. This study was designed to explore the regulation of MAPK signaling pathways on LRP1-mediated Aβ internalization in neurons. We found that extracellular Aβ1-42 oligomers could be internalized into endosomes/lysosomes and mitochondria in cortical neurons. Aβ1-42 and LRP1 were also found co-localized in neurons during Aβ1-42 internalization, and they could form Aβ1-42-LRP1 complex. Knockdown of LRP1 expression significantly decreased neuronal Aβ1-42 internalization. Finally, we identified that p38 MAPK and ERK1/2 signaling pathways regulated the internalization of Aβ1-42 via LRP1. Therefore, these results demonstrated that LRP1, p38 MAPK and ERK1/2 mediated the internalization of Aβ1-42 in neurons and provided evidence that blockade of LRP1 or inhibitions of MAPK signaling pathways might be a potential approach to lowering brain Aβ levels and served a potential therapeutic target for AD. Copyright © 2015 Elsevier Ltd. All rights reserved.

Low density lipoprotein receptors: preliminary results on 'in vivo' study

International Nuclear Information System (INIS)

Lupattelli, G.; Virgolini, I.; Li, S.R.; Sinzinger, H.

1991-01-01

Plasmatic levels of low density lipoproteins (LDL) are regulated by the receptor pathway and most LDL receptor are located in the liver. A receptor defect due to genetic mutations of the LDL receptor gene is the cause of familial hypercholesterolemia (F.H.), a disease characterized by high cholesterol levels and premature atherosclerosis. Injections of autologous radiolabelled LDL, followed by hepatic scintiscanning, can be used to obtain 'in vivo' quantification of hepatic receptor activity, both in normal and hypercholesterolemic patients. In this study we observe no hepatic increase of radioactivity in patients affected by F.H., confirming the liver receptor defect. Scintigraphy is a non-invasive technique which can be used to diagnose this disease and to monitor the efficiacy of hypolipidemic therapy. (Authors)

The majority of lipoprotein lipase in plasma is bound to remnant lipoproteins: A new definition of remnant lipoproteins.

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Sato, Koichi; Okajima, Fumikazu; Miyashita, Kazuya; Imamura, Shigeyuki; Kobayashi, Junji; Stanhope, Kimber L; Havel, Peter J; Machida, Tetsuo; Sumino, Hiroyuki; Murakami, Masami; Schaefer, Ernst; Nakajima, Katsuyuki

2016-10-01

Lipoprotein lipase (LPL) is a multifunctional protein and a key enzyme involved in the regulation of lipoprotein metabolism. We determined the lipoproteins to which LPL is bound in the pre-heparin and post-heparin plasma. Tetrahydrolipstatin (THL), a potent inhibitor of serine lipases, was used to block the lipolytic activity of LPL, thereby preventing changes in the plasma lipoproteins due to ex vivo lipolysis. Gel filtration was performed to obtain the LPL elution profiles in plasma and the isolated remnant lipoproteins (RLP). When ex vivo lipolytic activity was inhibited by THL in the post-heparin plasma, majority of the LPL was found in the VLDL elution range, specifically in the RLP as inactive dimers. However, in the absence of THL, most of the LPL was found in the HDL elution range as active dimers. Furthermore, majority of the LPL in the pre-heparin plasma was found in the RLP as inactive form, with broadly diffused lipoprotein profiles in the presence and absence of THL. It is suggested that during lipolysis in vivo, the endothelial bound LPL dimers generates RLP, forming circulating RLP-LPL complexes in an inactive form that subsequently binds and initiates receptor-mediated catabolism. Copyright © 2016 Elsevier B.V. All rights reserved.

Transfer of plasma lipoprotein components and of plasma proteins into aortas of cholesterol-fed rabbits. Molecular size as a determinant of plasma lipoprotein influx

International Nuclear Information System (INIS)

Stender, S.; Zilversmit, D.B.

1981-01-01

The arterial influx of esterified and free cholesterol from low density lipoproteins and very low density lipoproteins in 20 hypercholesterolemic rabbits was measured simultaneously by the use of lipoproteins labeled in vivo with [ 3 H]- and [ 14 C]-cholesterol. The simultaneous arterial influx of either [ 3 H]-leucine-labeled very low density lipoproteins, low density lipoproteins, high density lipoproteins, or plasma proteins was also measured in each rabbit. The arterial influx was calculated as intimal clearance, i.e., the influx of a given fraction divided by its plasma concentration. The intimal clearance of low density lipoprotein esterified cholesterol was equal to that for the apolipoproteins of that fraction, which is compatible with an arterial influx of intact low density lipoprotein molecules. The intimal clearance of very low density apolipoprotein or cholesteryl ester was less than that for low density lipoprotein, whereas high density lipoprotein and albumin clearances exceeded low density lipoprotein clearance by 1.5- to 3-fold. The intimal clearances of plasma proteins, high density, low density, and very low density lipoproteins decreased linearly with the logarithm of the macromolecular diameter. This indicates that the arterial influx of three plasma lipoprotein fractions and of plasma proteins proceeds by similar mechanisms. Apparently the relative intimal clearances of lipoproteins are more dependent on their size relative to pores or vesicular diameters at the plasma-artery interface than on specific interactions between lipoproteins and the arterial intimal surface

Tissue-type plasminogen activator-binding RNA aptamers inhibiting low-density lipoprotein receptor family-mediated internalisation.

Science.gov (United States)

Bjerregaard, Nils; Bøtkjær, Kenneth A; Helsen, Nicky; Andreasen, Peter A; Dupont, Daniel M

2015-07-01

Recombinant tissue-type plasminogen activator (tPA, trade name Alteplase), currently the only drug approved by the US Food and Drug Administration and the European Medicines Agency for the treatment of cerebral ischaemic stroke, has been implicated in a number of adverse effects reportedly mediated by interactions with the low-density lipoprotein (LDL) family receptors, including neuronal cell death and an increased risk of cerebral haemorrhage. The tissue-type plasminogen activator is the principal initiator of thrombolysis in human physiology, an effect that is mediated directly via localised activation of the plasmin zymogen plasminogen at the surface of fibrin clots in the vascular lumen. Here, we sought to identify a ligand to tPA capable of inhibiting the relevant LDL family receptors without interfering with the fibrinolytic activity of tPA. Systematic evolution of ligands by exponential enrichment (SELEX) was employed to isolate tPA-binding RNA aptamers, which were characterised in biochemical assays of tPA association to low density lipoprotein receptor-related protein-1 (LRP-1, an LDL receptor family member); tPA-mediated in vitro and ex vivo clot lysis; and tPA-mediated plasminogen activation in the absence and presence of a stimulating soluble fibrin fragment. Two aptamers, K18 and K32, had minimal effects on clot lysis, but were able to efficiently inhibit tPA-LRP-1 association and LDL receptor family-mediated endocytosis in human vascular endothelial cells and astrocytes. These observations suggest that coadministration alongside tPA may be a viable strategy to improve the safety of thrombolytic treatment of cerebral ischaemic stroke by restricting tPA activity to the vascular lumen.

Regulation of low density lipoprotein receptor function in a human hepatoma cell line

International Nuclear Information System (INIS)

Leichtner, A.M.; Krieger, M.; Schwartz, A.L.

1984-01-01

Low density lipoprotein (LDL) processing was investigated in a human hepatoma-derived cell line, Hep G2. Hep G2 cells bound, internalized and degraded LDL via a saturable, high affinity pathway similar to that present in other mammalian cells. Although 80% of the uptake and degradation of 125 I-LDL was inhibited by 40-fold excess native LDL, the same concentration of methylated LDL, which cannot bind to LDL receptors, had virtually no effect on processing. When added at low concentrations, the lysosomotropic agent, chloroquine, inhibited degradation without affecting the rate of lipoprotein internalization. Receptor activity was decreased 60% by preincubation of the cells in medium containing a source of cholesterol (LDL or unesterified cholesterol) and increased 1.7-fold by preincubation with compactin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. The Hep G2 cell line may prove a useful system both for the further study of hepatic lipoprotein metabolism and for the evaluation of new antihypercholesterolemic agents

Effects of 1,2-cyclohexanedione modification on the metabolism of very low density lipoprotein apolipoprotein B: potential role of receptors in intermediate density lipoprotein catabolism

International Nuclear Information System (INIS)

Packard, C.J.; Boag, D.E.; Clegg, R.; Bedford, D.; Shepherd, J.

1985-01-01

The conversion of very low density (VLDL) to low density lipoproteins (LDL) is a two-step process. The first step is mediated by lipoprotein lipase, but the mechanism responsible for the second is obscure. In this study we examined the possible involvement of receptors at this stage. Apolipoprotein B (apoB)-containing lipoproteins were separated into three fractions, VLDL (Sf 100-400), an intermediate fraction IDL (Sf 12-100), and LDL (Sf 0-12). Autologous 125I-labeled VLDL and 131I-labeled 1,2-cyclohexanedione-modified VLDL were injected into the plasma of four normal subjects and the rate of transfer of apoB radioactivity was followed through IDL to LDL. Modification did not affect VLDL to IDL conversion. Thereafter, however, the catabolism of modified apoB in IDL was retarded and its appearance in LDL was delayed. Hence, functional arginine residues (and by implication, receptors) are required in this process. Confirmation of this was obtained by injecting 125I-labeled IDL and 131I-labeled cyclohexanedione-treated IDL into two additional subjects. Again, IDL metabolism was delayed by approximately 50% as a result of the modification. These data are consistent with the view that receptors are involved in the metabolism of intermediate density lipoprotein

Low-Density Lipoprotein Receptor–Related Protein-1 Is a Therapeutic Target in Acute Myocardial Infarction

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Stefano Toldo, PhD

2017-10-01

Full Text Available Low-density lipoprotein receptor–related protein-1 (LRP1 is a ubiquitous membrane receptor functioning as a scavenger and regulatory receptor, inducing anti-inflammatory and prosurvival signals. Based on the known structure–activity of the LRP1 receptor binding site, the authors synthesized a small peptide (SP16. SP16 induced a >50% reduction in infarct size (p < 0.001 and preservation of left ventricular systolic function (p < 0.001, and treatment with an LRP1 blocking antibody eliminated the protective effects of SP16. In conclusion, LRP1 activation with SP16 given within 30 min of reperfusion during experimental acute myocardial infarction leads to a cardioprotective signal reducing infarct size and preservation of cardiac systolic function.

Redefining the essential trafficking pathway for outer membrane lipoproteins.

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Grabowicz, Marcin; Silhavy, Thomas J

2017-05-02

The outer membrane (OM) of Gram-negative bacteria is a permeability barrier and an intrinsic antibiotic resistance factor. Lipoproteins are OM components that function in cell wall synthesis, diverse secretion systems, and antibiotic efflux pumps. Moreover, each of the essential OM machines that assemble the barrier requires one or more lipoproteins. This dependence is thought to explain the essentiality of the periplasmic chaperone LolA and its OM receptor LolB that traffic lipoproteins to the OM. However, we show that in strains lacking substrates that are toxic when mislocalized, both LolA and LolB can be completely bypassed by activating an envelope stress response without compromising trafficking of essential lipoproteins. We identify the Cpx stress response as a monitor of lipoprotein trafficking tasked with protecting the cell from mislocalized lipoproteins. Moreover, our findings reveal that an alternate trafficking pathway exists that can, under certain conditions, bypass the functions of LolA and LolB, implying that these proteins do not perform any truly essential mechanistic steps in lipoprotein trafficking. Instead, these proteins' key function is to prevent lethal accumulation of mislocalized lipoproteins.

Existence of B/E and E receptors on Hep-G2 cells: a study using colloidal gold- and 125I-labeled lipoproteins

International Nuclear Information System (INIS)

Hesz, A.; Ingolic, E.; Krempler, F.; Kostner, G.M.

1987-01-01

The presence of specific receptors for apolipoprotein B (low-density lipoproteins) and apolipoprotein E (HDL-E) on Hep-G2 cells and human skin fibroblasts was studied by chemical methods and by electron microscopy using a differential gold labeling technique. Fibroblasts bound both types of lipoproteins to one and the same receptor (B/E receptor) as deduced from competition experiments with HDL-E and LDL. Labeled HDL-E, on the other hand, was only partially displaced by cold LDL but was completely displaced by unlabeled HDL-E. Scatchard analysis of lipoprotein binding to Hep-G2 cells revealed an approx 10 times higher binding affinity of apoE-containing lipoproteins as compared to apoB-containing ones. No differences between apoE- or apoB-containing lipoproteins with respect to the morphology of cell binding and intracellular processing were observed. The results are compatible with the concept that Hep-G2 cells possess two kinds of receptors, one specific for apoB- and apoE-containing lipoproteins (B/E receptor) and another specific for apoE only. From these studies we conclude that Hep-G2 cells may serve as a suitable model for studying the lipoprotein metabolism in the liver

Sorting of bacterial lipoproteins to the outer membrane by the Lol system.

Science.gov (United States)

Narita, Shin-ichiro; Tokuda, Hajime

2010-01-01

Bacterial lipoproteins comprise a subset of membrane proteins with a lipid-modified cysteine residue at their amino termini through which they are anchored to the membrane. In Gram-negative bacteria, lipoproteins are localized on either the inner or the outer membrane. The Lol system is responsible for the transport of lipoproteins to the outer membrane.The Lol system comprises an inner-membrane ABC transporter LolCDE complex, a periplasmic carrier protein, LolA, and an outer membrane receptor protein, LolB. Lipoproteins are synthesized as precursors in the cytosol and then translocated across the inner membrane by the Sec translocon to the outer leaflet of the inner membrane, where lipoprotein precursors are processed to mature lipoproteins. The LolCDE complex then mediates the release of outer membrane-specific lipoproteins from the inner membrane while the inner membrane-specific lipoproteins possessing Asp at position 2 are not released by LolCDE because it functions as a LolCDE avoidance signal, causing the retention of these lipoproteins in the inner membrane. A water-soluble lipoprotein-LolA complex is formed as a result of the release reaction mediated by LolCDE. This complex traverses the hydrophilic periplasm to reach the outer membrane, where LolB accepts a lipoprotein from LolA and then catalyzes its incorporation into the inner leaflet of the outer membrane.

The p38 mitogen-activated protein kinase signaling pathway is involved in regulating low-density lipoprotein receptor-related protein 1-mediated β-amyloid protein internalization in mouse brain.

Science.gov (United States)

Ma, Kai-Ge; Lv, Jia; Hu, Xiao-Dan; Shi, Li-Li; Chang, Ke-Wei; Chen, Xin-Lin; Qian, Yi-Hua; Yang, Wei-Na; Qu, Qiu-Min

2016-07-01

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. Recently, increasing evidence suggests that intracellular β-amyloid protein (Aβ) alone plays a pivotal role in the progression of AD. Therefore, understanding the signaling pathway and proteins that control Aβ internalization may provide new insight for regulating Aβ levels. In the present study, the regulation of Aβ internalization by p38 mitogen-activated protein kinases (MAPK) through low-density lipoprotein receptor-related protein 1 (LRP1) was analyzed in vivo. The data derived from this investigation revealed that Aβ1-42 were internalized by neurons and astrocytes in mouse brain, and were largely deposited in mitochondria and lysosomes, with some also being found in the endoplasmic reticulum. Aβ1-42-LRP1 complex was formed during Aβ1-42 internalization, and the p38 MAPK signaling pathway was activated by Aβ1-42 via LRP1. Aβ1-42 and LRP1 were co- localized in the cells of parietal cortex and hippocampus. Furthermore, the level of LRP1-mRNA and LRP1 protein involved in Aβ1-42 internalization in mouse brain. The results of this investigation demonstrated that Aβ1-42 induced an LRP1-dependent pathway that related to the activation of p38 MAPK resulting in internalization of Aβ1-42. These results provide evidence supporting a key role for the p38 MAPK signaling pathway which is involved in the regulation of Aβ1-42 internalization in the parietal cortex and hippocampus of mouse through LRP1 in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

One precursor, three apolipoproteins: the relationship between two crustacean lipoproteins, the large discoidal lipoprotein and the high density lipoprotein/β-glucan binding protein.

Science.gov (United States)

Stieb, Stefanie; Roth, Ziv; Dal Magro, Christina; Fischer, Sabine; Butz, Eric; Sagi, Amir; Khalaila, Isam; Lieb, Bernhard; Schenk, Sven; Hoeger, Ulrich

2014-12-01

The novel discoidal lipoprotein (dLp) recently detected in the crayfish, differs from other crustacean lipoproteins in its large size, apoprotein composition and high lipid binding capacity, We identified the dLp sequence by transcriptome analyses of the hepatopancreas and mass spectrometry. Further de novo assembly of the NGS data followed by BLAST searches using the sequence of the high density lipoprotein/1-glucan binding protein (HDL-BGBP) of Astacus leptodactylus as query revealed a putative precursor molecule with an open reading frame of 14.7 kb and a deduced primary structure of 4889 amino acids. The presence of an N-terminal lipid bind- ing domain and a DUF 1943 domain suggests the relationship with the large lipid transfer proteins. Two-putative dibasic furin cleavage sites were identified bordering the sequence of the HDL-BGBP. When subjected to mass spectroscopic analyses, tryptic peptides of the large apoprotein of dLp matched the N-terminal part of the precursor, while the peptides obtained for its small apoprotein matched the C-terminal part. Repeating the analysis in the prawn Macrobrachium rosenbergii revealed a similar protein with identical domain architecture suggesting that our findings do not represent an isolated instance. Our results indicate that the above three apolipoproteins (i.e HDL-BGBP and both the large and the small subunit of dLp) are translated as a large precursor. Cleavage at the furin type sites releases two subunits forming a heterodimeric dLP particle, while the remaining part forms an HDL-BGBP whose relationship with other lipoproteins as well as specific functions are yet to be elucidated.

Activation of lipoprotein lipase by lipoprotein fractions of human serum.

Science.gov (United States)

Bier, D M; Havel, R J

1970-11-01

Triglycerides in fat emulsions are hydrolyzed by lipoprotein lipase only when they are "activated" by serum lipoproteins. The contribution of different lipoprotein fractions to hydrolysis of triglycerides in soybean oil emulsion was assessed by determining the quantity of lipoprotein fraction required to give half-maximal hydrolysis. Most of the activator property of whole serum from normolipidemic, postabsorptive subjects was in high density lipoproteins. Low density lipoproteins and serum from which all lipoprotein classes were removed had little or no activity. Also, little activator was present in guinea pig serum or in very low density poor serum from an individual with lecithin:cholesterol acyltransferase deficiency, both of which are deficient in high density lipoproteins. Human very low density lipoproteins are potent activators and are much more active than predicted from their content of high density lipoprotein-protein. Per unit weight of protein, very low density lipoproteins had 13 times the activity of high density lipoproteins. These observations suggest that one or more of the major apoproteins of very low density lipoproteins, present as a minor constituent of high density lipoproteins, may be required for the activation process.

Pneumococcal lipoproteins involved in bacterial fitness, virulence, and immune evasion.

Science.gov (United States)

Kohler, Sylvia; Voß, Franziska; Gómez Mejia, Alejandro; Brown, Jeremy S; Hammerschmidt, Sven

2016-11-01

Streptococcus pneumoniae (pneumococcus) has evolved sophisticated strategies to survive in several niches within the human body either as a harmless commensal or as a serious pathogen causing a variety of diseases. The dynamic interaction between pneumococci and resident host cells during colonization of the upper respiratory tract and at the site of infection is critical for bacterial survival and the development of disease. Pneumococcal lipoproteins are peripherally anchored membrane proteins and have pivotal roles in bacterial fitness including envelope stability, cell division, nutrient acquisition, signal transduction, transport (as substrate-binding proteins of ABC transporter systems), resistance to oxidative stress and antibiotics, and protein folding. In addition, lipoproteins are directly involved in virulence-associated processes such as adhesion, colonization, and persistence through immune evasion. Conversely, lipoproteins are also targets for the host response both as ligands for toll-like receptors and as targets for acquired antibodies. This review summarizes the multifaceted roles of selected pneumococcal lipoproteins and how this knowledge can be exploited to combat pneumococcal infections. © 2016 Federation of European Biochemical Societies.

PCSK9 and triglyceride-rich lipoprotein metabolism.

Science.gov (United States)

Druce, I; Abujrad, H; Ooi, T C

2015-07-20

Pro-protein convertase subtilisin-kexin 9 (PCSK9) is known to affect low-density lipoprotein (LDL) metabolism, but there are indications from several lines of research that it may also influence the metabolism of other lipoproteins, especially triglyceride-rich lipoproteins (TRL). This review summarizes the current data on this possible role of PCSK9. A link between PCSK9 and TRL has been suggested through the demonstration of (1) a correlation between plasma PCSK9 and triglyceride (TG) levels in health and disease, (2) a correlation between plasma PCSK9 and markers of carbohydrate metabolism, which is closely related to TG metabolism, (3) an effect of TG-lowering fibrate therapy on plasma PCSK9 levels, (4) an effect of PCSK9 on postprandial lipemia, (5) an effect of PCSK9 on adipose tissue biology, (6) an effect of PCSK9 on apolipoprotein B production from the liver and intestines, (7) an effect of PCSK9 on receptors other than low density lipoprotein receptor (LDLR) that are involved in TRL metabolism, and (8) an effect of anti-PCSK9 therapy on serum TG levels. The underlying mechanisms are unclear but starting to emerge. © 2015 the Journal of Biomedical Research. All rights reserved.

Cholesterol efflux from THP-1 macrophages is impaired by the fatty acid component from lipoprotein hydrolysis by lipoprotein lipase

International Nuclear Information System (INIS)

Yang, Yanbo; Thyagarajan, Narmadaa; Coady, Breanne M.; Brown, Robert J.

2014-01-01

Highlights: • Lipoprotein hydrolysis products were produced by lipoprotein lipase. • Hydrolysis products lowers expression of macrophage cholesterol transporters. • Hydrolysis products reduces expression of select nuclear receptors. • Fatty acid products lowers cholesterol transporters and select nuclear receptors. • Fatty acid products reduces cholesterol efflux from macrophages. - Abstract: Lipoprotein lipase (LPL) is an extracellular lipase that primarily hydrolyzes triglycerides within circulating lipoproteins. Macrophage LPL contributes to atherogenesis, but the mechanisms behind it are poorly understood. We hypothesized that the products of lipoprotein hydrolysis generated by LPL promote atherogenesis by inhibiting the cholesterol efflux ability by macrophages. To test this hypothesis, we treated human THP-1 macrophages with total lipoproteins that were hydrolyzed by LPL and we found significantly reduced transcript levels for the cholesterol transporters ATP binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor BI. These decreases were likely due to significant reductions for the nuclear receptors liver-X-receptor-α, peroxisome proliferator activated receptor (PPAR)-α, and PPAR-γ. We prepared a mixture of free fatty acids (FFA) that represented the ratios of FFA species within lipoprotein hydrolysis products, and we found that the FFA mixture also significantly reduced cholesterol transporters and nuclear receptors. Finally, we tested the efflux of cholesterol from THP-1 macrophages to apolipoprotein A-I, and we found that the treatment of THP-1 macrophages with the FFA mixture significantly attenuated cholesterol efflux. Overall, these data show that the FFA component of lipoprotein hydrolysis products generated by LPL may promote atherogenesis by inhibiting cholesterol efflux, which partially explains the pro-atherogenic role of macrophage LPL

Cholesterol efflux from THP-1 macrophages is impaired by the fatty acid component from lipoprotein hydrolysis by lipoprotein lipase

Energy Technology Data Exchange (ETDEWEB)

Yang, Yanbo; Thyagarajan, Narmadaa; Coady, Breanne M.; Brown, Robert J., E-mail: rbrown@mun.ca

2014-09-05

Highlights: • Lipoprotein hydrolysis products were produced by lipoprotein lipase. • Hydrolysis products lowers expression of macrophage cholesterol transporters. • Hydrolysis products reduces expression of select nuclear receptors. • Fatty acid products lowers cholesterol transporters and select nuclear receptors. • Fatty acid products reduces cholesterol efflux from macrophages. - Abstract: Lipoprotein lipase (LPL) is an extracellular lipase that primarily hydrolyzes triglycerides within circulating lipoproteins. Macrophage LPL contributes to atherogenesis, but the mechanisms behind it are poorly understood. We hypothesized that the products of lipoprotein hydrolysis generated by LPL promote atherogenesis by inhibiting the cholesterol efflux ability by macrophages. To test this hypothesis, we treated human THP-1 macrophages with total lipoproteins that were hydrolyzed by LPL and we found significantly reduced transcript levels for the cholesterol transporters ATP binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor BI. These decreases were likely due to significant reductions for the nuclear receptors liver-X-receptor-α, peroxisome proliferator activated receptor (PPAR)-α, and PPAR-γ. We prepared a mixture of free fatty acids (FFA) that represented the ratios of FFA species within lipoprotein hydrolysis products, and we found that the FFA mixture also significantly reduced cholesterol transporters and nuclear receptors. Finally, we tested the efflux of cholesterol from THP-1 macrophages to apolipoprotein A-I, and we found that the treatment of THP-1 macrophages with the FFA mixture significantly attenuated cholesterol efflux. Overall, these data show that the FFA component of lipoprotein hydrolysis products generated by LPL may promote atherogenesis by inhibiting cholesterol efflux, which partially explains the pro-atherogenic role of macrophage LPL.

Recombinant Lipoproteins as Novel Vaccines with Intrinsic Adjuvant.

Science.gov (United States)

Chong, Pele; Huang, Jui-Hsin; Leng, Chih-Hsiang; Liu, Shih-Jen; Chen, Hsin-Wei

2015-01-01

A core platform technology for high production of recombinant lipoproteins with built-in immunostimulator for novel subunit vaccine development has been established. This platform technology has the following advantages: (1) easily convert antigen into lipidated recombinant protein using a fusion sequence containing lipobox and express high level (50-150mg/L) in Escherichia coli; (2) a robust high-yield up- and downstream bioprocess for lipoprotein production is successfully developed to devoid endotoxin contamination; (3) the lipid moiety of recombinant lipoproteins, which is identical to that of bacterial lipoproteins is recognized as danger signals by the immune system (Toll-like receptor 2 agonist), so both innate and adaptive immune responses can be induced by lipoproteins; and (4) successfully demonstrate the feasibility and safety of this core platform technology in meningococcal group B subunit vaccine, dengue subunit vaccine, novel subunit vaccine against Clostridium difficile-associated diseases, and HPV-based immunotherapeutic vaccines in animal model studies. © 2015 Elsevier Inc. All rights reserved.

Distinct functional domains contribute to degradation of the low density lipoprotein receptor (LDLR) by the E3 ubiquitin ligase inducible Degrader of the LDLR (IDOL)

NARCIS (Netherlands)

Sorrentino, Vincenzo; Scheer, Lilith; Santos, Ana; Reits, Eric; Bleijlevens, Boris; Zelcer, Noam

2011-01-01

We recently identified the liver X receptor-regulated E3 ubiquitin ligase inducible degrader of the LDL receptor (IDOL) as a modulator of lipoprotein metabolism. Acting as an E3 ubiquitin ligase, IDOL triggers ubiquitination and subsequent degradation of the low density lipoprotein receptor (LDLR).

Receptor-mediated uptake of low density lipoprotein stimulates bile acid synthesis by cultured rat hepatocytes

International Nuclear Information System (INIS)

Junker, L.H.; Davis, R.A.

1989-01-01

The cellular mechanisms responsible for the lipoprotein-mediated stimulation of bile acid synthesis in cultured rat hepatocytes were investigated. Adding 280 micrograms/ml of cholesterol in the form of human or rat low density lipoprotein (LDL) to the culture medium increased bile acid synthesis by 1.8- and 1.6-fold, respectively. As a result of the uptake of LDL, the synthesis of [14C]cholesterol from [2-14C]acetate was decreased and cellular cholesteryl ester mass was increased. Further studies demonstrated that rat apoE-free LDL and apoE-rich high density lipoprotein (HDL) both stimulated bile acid synthesis 1.5-fold, as well as inhibited the formation of [14C]cholesterol from [2-14C]acetate. Reductive methylation of LDL blocked the inhibition of cholesterol synthesis, as well as the stimulation of bile acid synthesis, suggesting that these processes require receptor-mediated uptake. To identify the receptors responsible, competitive binding studies using 125I-labeled apoE-free LDL and 125I-labeled apoE-rich HDL were performed. Both apoE-free LDL and apoE-rich HDL displayed an equal ability to compete for binding of the other, suggesting that a receptor or a group of receptors that recognizes both apolipoproteins is involved. Additional studies show that hepatocytes from cholestyramine-treated rats displayed 2.2- and 3.4-fold increases in the binding of apoE-free LDL and apoE-rich HDL, respectively. These data show for the first time that receptor-mediated uptake of LDL by the liver is intimately linked to processes activating bile acid synthesis

Redefining the essential trafficking pathway for outer membrane lipoproteins

Science.gov (United States)

Grabowicz, Marcin; Silhavy, Thomas J.

2017-01-01

The outer membrane (OM) of Gram-negative bacteria is a permeability barrier and an intrinsic antibiotic resistance factor. Lipoproteins are OM components that function in cell wall synthesis, diverse secretion systems, and antibiotic efflux pumps. Moreover, each of the essential OM machines that assemble the barrier requires one or more lipoproteins. This dependence is thought to explain the essentiality of the periplasmic chaperone LolA and its OM receptor LolB that traffic lipoproteins to the OM. However, we show that in strains lacking substrates that are toxic when mislocalized, both LolA and LolB can be completely bypassed by activating an envelope stress response without compromising trafficking of essential lipoproteins. We identify the Cpx stress response as a monitor of lipoprotein trafficking tasked with protecting the cell from mislocalized lipoproteins. Moreover, our findings reveal that an alternate trafficking pathway exists that can, under certain conditions, bypass the functions of LolA and LolB, implying that these proteins do not perform any truly essential mechanistic steps in lipoprotein trafficking. Instead, these proteins’ key function is to prevent lethal accumulation of mislocalized lipoproteins. PMID:28416660

Streptococcus gordonii induces nitric oxide production through its lipoproteins stimulating Toll-like receptor 2 in murine macrophages.

Science.gov (United States)

Kim, Hyun Young; Baik, Jung Eun; Ahn, Ki Bum; Seo, Ho Seong; Yun, Cheol-Heui; Han, Seung Hyun

2017-02-01

Streptococcus gordonii, a Gram-positive commensal in the oral cavity, is an opportunistic pathogen that can cause endodontic and systemic infections resulting in infective endocarditis. Lipoteichoic acid (LTA) and lipoprotein are major virulence factors of Gram-positive bacteria that are preferentially recognized by Toll-like receptor 2 (TLR2) on immune cells. In the present study, we investigated the effect of S. gordonii LTA and lipoprotein on the production of the representative inflammatory mediator nitric oxide (NO) by the mouse macrophages. Heat-killed S. gordonii wild-type and an LTA-deficient mutant (ΔltaS) but not a lipoprotein-deficient mutant (Δlgt) induced NO production in mouse primary macrophages and the cell line, RAW 264.7. S. gordonii wild-type and ΔltaS also induced the expression of inducible NO synthase (iNOS) at the mRNA and protein levels. In contrast, the Δlgt mutant showed little effect under the same condition. Furthermore, S. gordonii wild-type and ΔltaS induced NF-κB activation, STAT1 phosphorylation, and IFN-β expression, which are important for the induction of iNOS gene expression, with little activation by Δlgt. S. gordonii wild-type and ΔltaS showed an increased adherence and internalization to RAW 264.7 cells compared to Δlgt. In addition, S. gordonii wild-type and ΔltaS, but not Δlgt, substantially increased TLR2 activation while none of these induced NO production in TLR2-deficient macrophages. Triton X-114-extracted lipoproteins from S. gordonii were sufficient to induce NO production. Collectively, we suggest that lipoprotein is an essential cell wall component of S. gordonii to induce NO production in macrophages through TLR2 triggering NF-κB and STAT1 activation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cholesterol efflux from THP-1 macrophages is impaired by the fatty acid component from lipoprotein hydrolysis by lipoprotein lipase.

Science.gov (United States)

Yang, Yanbo; Thyagarajan, Narmadaa; Coady, Breanne M; Brown, Robert J

2014-09-05

Lipoprotein lipase (LPL) is an extracellular lipase that primarily hydrolyzes triglycerides within circulating lipoproteins. Macrophage LPL contributes to atherogenesis, but the mechanisms behind it are poorly understood. We hypothesized that the products of lipoprotein hydrolysis generated by LPL promote atherogenesis by inhibiting the cholesterol efflux ability by macrophages. To test this hypothesis, we treated human THP-1 macrophages with total lipoproteins that were hydrolyzed by LPL and we found significantly reduced transcript levels for the cholesterol transporters ATP binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor BI. These decreases were likely due to significant reductions for the nuclear receptors liver-X-receptor-α, peroxisome proliferator activated receptor (PPAR)-α, and PPAR-γ. We prepared a mixture of free fatty acids (FFA) that represented the ratios of FFA species within lipoprotein hydrolysis products, and we found that the FFA mixture also significantly reduced cholesterol transporters and nuclear receptors. Finally, we tested the efflux of cholesterol from THP-1 macrophages to apolipoprotein A-I, and we found that the treatment of THP-1 macrophages with the FFA mixture significantly attenuated cholesterol efflux. Overall, these data show that the FFA component of lipoprotein hydrolysis products generated by LPL may promote atherogenesis by inhibiting cholesterol efflux, which partially explains the pro-atherogenic role of macrophage LPL. Copyright © 2014 Elsevier Inc. All rights reserved.

Lipoprotein-a

Science.gov (United States)

... this page: //medlineplus.gov/ency/article/007262.htm Lipoprotein-a To use the sharing features on this page, please enable JavaScript. Lipoproteins are molecules made of proteins and fat. They ...

Secretion of Bacterial Lipoproteins: Through the Cytoplasmic Membrane, the Periplasm and Beyond

Science.gov (United States)

Zückert, Wolfram R.

2014-01-01

Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., grampositive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporterlike LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the “+2 rule”. Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation

Secretion of bacterial lipoproteins: through the cytoplasmic membrane, the periplasm and beyond.

Science.gov (United States)

Zückert, Wolfram R

2014-08-01

Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., gram-positive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporter-like LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the "+2 rule". Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation

Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.

Directory of Open Access Journals (Sweden)

Yingying Cai

Full Text Available Family B G protein-coupled receptors (GPCRs play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1 receptor (GLP1R, whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.

Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.

Science.gov (United States)

Cai, Yingying; Liu, Yuting; Culhane, Kelly J; DeVree, Brian T; Yang, Yang; Sunahara, Roger K; Yan, Elsa C Y

2017-01-01

Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.

Regulation of low-density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase expression by Zingiber officinale in the liver of high-fat diet-fed rats.

Science.gov (United States)

Nammi, Srinivas; Kim, Moon S; Gavande, Navnath S; Li, George Q; Roufogalis, Basil D

2010-05-01

Zingiber officinale has been used to control lipid disorders and reported to possess remarkable cholesterol-lowering activity in experimental hyperlipidaemia. In the present study, the effect of a characterized and standardized extract of Zingiber officinale on the hepatic lipid levels as well as on the hepatic mRNA and protein expression of low-density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was investigated in a high-fat diet-fed rat model. Rats were treated with an ethanol extract of Zingiber officinale (400 mg/kg) extract along with a high-fat diet for 6 weeks. The extract of Zingiber officinale significantly decreased hepatic triglyceride and tended to decrease hepatic cholesterol levels when administered over 6 weeks to the rats fed a high-fat diet. We found that in parallel, the extract up-regulated both LDL receptor mRNA and protein level and down-regulated HMG-CoA reductase protein expression in the liver of these rats. The metabolic control of body lipid homeostasis is in part due to enhanced cholesterol biosynthesis and reduced expression of LDL receptor sites following long-term consumption of high-fat diets. The present results show restoration of transcriptional and post-transcriptional changes in low-density lipoprotein and HMG CoA reductase by Zingiber officinale administration with a high-fat diet and provide a rational explanation for the effect of ginger in the treatment of hyperlipidaemia.

Association of peroxisome proliferator-activated receptor single-nucleotide polymorphisms and gene-gene interactions with the lipoprotein(a)

Institute of Scientific and Technical Information of China (English)

解惠坚

2014-01-01

Objective To examine the associations of 10 singlenucleotide polymorphisms(SNPs)in peroxisome proliferator-activated receptor(PPARs)gene with lipoprotein(a)level,and to investigate if there is gene-gene interaction among the SNPs on lipoprotein(a)level.Methods Totally 644 subjects(234 men and 410 women)were enrolled from Prevention of Multiple Metabolic Disorders and Metabolic Syndrome Study Cohort,which was an urban community survey study conducted in Jiangsu province.Ten SNPs in PPARα(rs135539,rs4253778,

Small-Molecule Inhibitors of Gram-Negative Lipoprotein Trafficking Discovered by Phenotypic Screening

Science.gov (United States)

Fleming, Paul R.; MacCormack, Kathleen; McLaughlin, Robert E.; Whiteaker, James D.; Narita, Shin-ichiro; Mori, Makiko; Tokuda, Hajime; Miller, Alita A.

2015-01-01

In Gram-negative bacteria, lipoproteins are transported to the outer membrane by the Lol system. In this process, lipoproteins are released from the inner membrane by the ABC transporter LolCDE and passed to LolA, a diffusible periplasmic molecular chaperone. Lipoproteins are then transferred to the outer membrane receptor protein, LolB, for insertion in the outer membrane. Here we describe the discovery and characterization of novel pyridineimidazole compounds that inhibit this process. Escherichia coli mutants resistant to the pyridineimidazoles show no cross-resistance to other classes of antibiotics and map to either the LolC or LolE protein of the LolCDE transporter complex. The pyridineimidazoles were shown to inhibit the LolA-dependent release of the lipoprotein Lpp from E. coli spheroplasts. These results combined with bacterial cytological profiling are consistent with LolCDE-mediated disruption of lipoprotein targeting to the outer membrane as the mode of action of these pyridineimidazoles. The pyridineimidazoles are the first reported inhibitors of the LolCDE complex, a target which has never been exploited for therapeutic intervention. These compounds open the door to further interrogation of the outer membrane lipoprotein transport pathway as a target for antimicrobial therapy. PMID:25583975

Small-molecule inhibitors of gram-negative lipoprotein trafficking discovered by phenotypic screening.

Science.gov (United States)

McLeod, Sarah M; Fleming, Paul R; MacCormack, Kathleen; McLaughlin, Robert E; Whiteaker, James D; Narita, Shin-Ichiro; Mori, Makiko; Tokuda, Hajime; Miller, Alita A

2015-03-01

In Gram-negative bacteria, lipoproteins are transported to the outer membrane by the Lol system. In this process, lipoproteins are released from the inner membrane by the ABC transporter LolCDE and passed to LolA, a diffusible periplasmic molecular chaperone. Lipoproteins are then transferred to the outer membrane receptor protein, LolB, for insertion in the outer membrane. Here we describe the discovery and characterization of novel pyridineimidazole compounds that inhibit this process. Escherichia coli mutants resistant to the pyridineimidazoles show no cross-resistance to other classes of antibiotics and map to either the LolC or LolE protein of the LolCDE transporter complex. The pyridineimidazoles were shown to inhibit the LolA-dependent release of the lipoprotein Lpp from E. coli spheroplasts. These results combined with bacterial cytological profiling are consistent with LolCDE-mediated disruption of lipoprotein targeting to the outer membrane as the mode of action of these pyridineimidazoles. The pyridineimidazoles are the first reported inhibitors of the LolCDE complex, a target which has never been exploited for therapeutic intervention. These compounds open the door to further interrogation of the outer membrane lipoprotein transport pathway as a target for antimicrobial therapy. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Human plasma phospholipid transfer protein increases the antiatherogenic potential of high density lipoproteins in transgenic mice

NARCIS (Netherlands)

M.J. van Haperen (Rien); A. van Tol (Arie); P. Vermeulen; M. Jauhiainen; T. van Gent (Teus); P.M. van den Berg (Paul); S. Ehnholm (Sonja); A.W.M. van der Kamp (Arthur); M.P.G. de Crom (Rini); F.G. Grosveld (Frank)

2000-01-01

textabstractPlasma phospholipid transfer protein (PLTP) transfers phospholipids between lipoprotein particles and alters high density lipoprotein (HDL) subfraction patterns in vitro, but its physiological function is poorly understood. Transgenic mice that overexpress

Serum amyloid A stimulates macrophage foam cell formation via lectin-like oxidized low-density lipoprotein receptor 1 upregulation

International Nuclear Information System (INIS)

Lee, Ha Young; Kim, Sang Doo; Baek, Suk-Hwan; Choi, Joon Hyuk; Cho, Kyung-Hyun; Zabel, Brian A.; Bae, Yoe-Sik

2013-01-01

Highlights: ► SAA induced macrophage foam cell formation. ► SAA stimulated upregulation of lectin-like oxidized low-density lipoprotein receptor 1 (LOX1). ► SAA-induced LOX1 expression and foam cell formation is mediated by JNK/NF-κB signaling. ► HDL-conjugated SAA also stimulates foam cell formation via LOX1 upregulation. ► The finding reveals a novel mechanism of action of SAA in the pathogenesis of atherosclerosis. -- Abstract: Elevated levels of serum amyloid A (SAA) is a risk factor for cardiovascular diseases, however, the role of SAA in the pathophysiology of atherosclerosis remains unclear. Here we show that SAA induced macrophage foam cell formation. SAA-stimulated foam cell formation was mediated by c-jun N-terminal kinase (JNK) signaling. Moreover, both SAA and SAA-conjugated high density lipoprotein stimulated the expression of the important scavenger receptor lectin-like oxidized low-density lipoprotein receptor 1 (LOX1) via nuclear factor-κB (NF-κB). A LOX1 antagonist carrageenan significantly blocked SAA-induced foam cell formation, indicating that SAA promotes foam cell formation via LOX1 expression. Our findings therefore suggest that SAA stimulates foam cell formation via LOX1 induction, and thus likely contributes to atherogenesis

Serum amyloid A stimulates macrophage foam cell formation via lectin-like oxidized low-density lipoprotein receptor 1 upregulation

Energy Technology Data Exchange (ETDEWEB)

Lee, Ha Young, E-mail: hayoung@skku.edu [Department of Biological Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Mitochondria Hub Regulation Center, Dong-A University, Busan 602-714 (Korea, Republic of); Kim, Sang Doo [Department of Biological Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Baek, Suk-Hwan [Department of Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, Daegu 705-717 (Korea, Republic of); Choi, Joon Hyuk [Department of Pathology, College of Medicine, Yeungnam University, Daegu 705-717 (Korea, Republic of); Cho, Kyung-Hyun [School of Biotechnology, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Zabel, Brian A. [Palo Alto Institute for Research and Education, Veterans Affairs Hospital, Palo Alto, CA 94304 (United States); Bae, Yoe-Sik, E-mail: yoesik@skku.edu [Department of Biological Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Mitochondria Hub Regulation Center, Dong-A University, Busan 602-714 (Korea, Republic of); Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul 135-710 (Korea, Republic of)

2013-03-29

Highlights: ► SAA induced macrophage foam cell formation. ► SAA stimulated upregulation of lectin-like oxidized low-density lipoprotein receptor 1 (LOX1). ► SAA-induced LOX1 expression and foam cell formation is mediated by JNK/NF-κB signaling. ► HDL-conjugated SAA also stimulates foam cell formation via LOX1 upregulation. ► The finding reveals a novel mechanism of action of SAA in the pathogenesis of atherosclerosis. -- Abstract: Elevated levels of serum amyloid A (SAA) is a risk factor for cardiovascular diseases, however, the role of SAA in the pathophysiology of atherosclerosis remains unclear. Here we show that SAA induced macrophage foam cell formation. SAA-stimulated foam cell formation was mediated by c-jun N-terminal kinase (JNK) signaling. Moreover, both SAA and SAA-conjugated high density lipoprotein stimulated the expression of the important scavenger receptor lectin-like oxidized low-density lipoprotein receptor 1 (LOX1) via nuclear factor-κB (NF-κB). A LOX1 antagonist carrageenan significantly blocked SAA-induced foam cell formation, indicating that SAA promotes foam cell formation via LOX1 expression. Our findings therefore suggest that SAA stimulates foam cell formation via LOX1 induction, and thus likely contributes to atherogenesis.

Deletion in the first cysteine-rich repeat of low density lipoprotein receptor impairs its transport but not lipoprotein binding in fibroblasts from a subject with familial hypercholesterolemia

International Nuclear Information System (INIS)

Leitersdorf, E.; Hobbs, H.H.; Fourie, A.M.; Jacobs, M.; Van Der Westhuyzen, D.R.; Coetzee, G.A.

1988-01-01

The ligand-binding domain of the low density lipoprotein (LDL) receptor is composed of seven cysteine-rich repeats, each ∼ 40 amino acids long. Previous studies showed that if the first repeat of the ligand-binding domain (encoded by exon 2) is deleted, the receptor fails to bind an anti-LDL receptor monoclonal antibody (IgG-C7) but continues to bind LDL with high affinity. Cultured fibroblasts from a Black South African Xhosa patient (TT) with the clinical syndrome of homozygous familial hypercholesterolemia demonstrated high-affinity cell-surface binding of 125 I-labeled LDL but not 125 I-labeled IgG-C7. previous haplotype analysis, using 10 restriction fragment length polymorphic sites, suggested that the patient inherited two identical LDL receptor alleles. The polymerase chain reaction technique was used to selectively amplify exon 2 of the LDL receptor gene from this patient. Sequence analysis of the amplified fragment disclosed a deletion of six base pairs that removes two amino acids, aspartic acid and glycine, from the first cysteine-rich ligand binding repeat. The mutation creates a new Pst I restriction site that can be used to detect the deletion. The existence of this mutant allele confirms that the epitope of IgG-C7 is located in the first cysteine-rich repeat and that this repeat is not necessary for LDL binding. The mutant gene produced a normally sized 120-kilodalton LDL receptor precursor protein that matured to the 160-kilodalton form at less than one-fourth the normal rate

Peroxisome Proliferator Activated Receptors and Lipoprotein Metabolism

NARCIS (Netherlands)

Kersten, A.H.

2008-01-01

Plasma lipoproteins are responsible for carrying triglycerides and cholesterol in the blood and ensuring their delivery to target organs. Regulation of lipoprotein metabolism takes place at numerous levels including via changes in gene transcription. An important group of transcription factors that

Dietary fatty acids regulate hepatic low density lipoprotein (LDL) transport by altering LDL receptor protein and mRNA levels.

Science.gov (United States)

Horton, J D; Cuthbert, J A; Spady, D K

1993-01-01

The concentration of LDL in plasma is strongly influenced by the amount and the type of lipid in the diet. Recent studies in the hamster have shown that dietary fatty acids differentially affect circulating LDL levels primarily by altering receptor-dependent LDL uptake in the liver. To investigate the mechanistic basis of this effect, rates of receptor-dependent LDL transport in the liver were correlated with LDL receptor protein and mRNA levels in hamsters fed safflower oil or coconut oil and varying amounts of cholesterol. Hepatic LDL receptor activity was significantly lower in animals fed coconut oil than in animals fed safflower oil at all levels of cholesterol intake (26, 53, and 61% lower at cholesterol intakes of 0, 0.06, and 0.12%, respectively). These fatty acid-induced changes in hepatic LDL receptor activity were accompanied by parallel changes in hepatic LDL receptor protein and mRNA levels, suggesting that dietary fatty acids regulate the LDL receptor pathway largely at the mRNA level. Images PMID:8349814

Activated α2 -Macroglobulin Induces Mesenchymal Cellular Migration Of Raw264.7 Cells Through Low-Density Lipoprotein Receptor-Related Protein 1.

Science.gov (United States)

Ferrer, Darío G; Dato, Virginia Actis; Fincati, Javier R Jaldín; Lorenc, Valeria E; Sánchez, María C; Chiabrando, Gustavo A

2017-07-01

Distinct modes of cell migration contribute to diverse types of cell movements. The mesenchymal mode is characterized by a multistep cycle of membrane protrusion, the formation of focal adhesion, and the stabilization at the leading edge associated with the degradation of extracellular matrix (ECM) components and with regulated extracellular proteolysis. Both α 2 -Macroglobulin (α 2 M) and its receptor, low density lipoprotein receptor-related protein 1 (LRP1), play important roles in inflammatory processes, by controlling the extracellular activity of several proteases. The binding of the active form of α 2 M (α 2 M*) to LRP1 can also activate different signaling pathways in macrophages, thus inducing extracellular matrix metalloproteinase-9 (MMP-9) activation and cellular proliferation. In the present study, we investigated whether the α 2 M*/LRP1 interaction induces cellular migration of the macrophage-derived cell line, Raw264.7. By using the wound-scratch migration assay and confocal microscopy, we demonstrate that α 2 M* induces LRP1-mediated mesenchymal cellular migration. This migration exhibits the production of enlarged cellular protrusions, MT1-MMP distribution to these leading edge protrusions, actin polymerization, focal adhesion formation, and increased intracellular LRP1/β1-integrin colocalization. Moreover, the presence of calphostin-C blocked the α 2 M*-stimulated cellular protrusions, suggesting that the PKC activation is involved in the cellular motility of Raw264.7 cells. These findings could constitute a therapeutic target for inflammatory processes with deleterious consequences for human health, such as rheumatoid arthritis, atherosclerosis and cancer. J. Cell. Biochem. 118: 1810-1818, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Lipoprotein profiles in human heterozygote carriers of a functional mutation P297S in scavenger receptor class B1

NARCIS (Netherlands)

Ljunggren, Stefan A.; Levels, Johannes H. M.; Hovingh, Kees; Holleboom, Adriaan G.; Vergeer, Menno; Argyri, Letta; Gkolfinopoulou, Christina; Chroni, Angeliki; Sierts, Jeroen A.; Kastelein, John J.; Kuivenhoven, Jan Albert; Lindahl, Mats; Karlsson, Helen

2015-01-01

The scavenger receptor class B type 1 (SR-B1) is an important HDL receptor involved in cholesterol uptake and efflux, but its physiological role in human lipoprotein metabolism is not fully understood. Heterozygous carriers of the SR-B1(P297S) mutation are characterized by increased HDL cholesterol

Lipoprotein(a)

DEFF Research Database (Denmark)

Langsted, Anne; Kamstrup, Pia R; Nordestgaard, Børge G

2014-01-01

OBJECTIVE: There are no recommendations in guidelines on measuring lipoprotein(a) in the fasting or nonfasting state, or on the influence of inflammation. We tested the hypotheses that lipoprotein(a) levels change only minimally in response to normal food intake, and to inflammation. Also, we...... tested whether normal food intake or inflammation influenced lipoprotein(a)'s ability to predict ischemic heart disease. METHODS: We studied 34 829 individuals from the Danish general population using the Copenhagen General Population Study and the Copenhagen City Heart Study. RESULTS: Lipoprotein......(a) levels did not change in response to normal food intake: median fasting levels were 17.3 mg/dL, while median levels at 3-4 h since last meal were 19.4 mg/dL(p = 0.38). Lipoprotein(a) levels increased minimally with increasing levels of C-reactive protein(CRP): median lipoprotein(a) levels at CRP

Learning from biology: synthetic lipoproteins for drug delivery.

Science.gov (United States)

Huang, Huang; Cruz, William; Chen, Juan; Zheng, Gang

2015-01-01

Synthetic lipoproteins represent a relevant tool for targeted delivery of biological/chemical agents (chemotherapeutics, siRNAs, photosensitizers, and imaging contrast agents) into various cell types. These nanoparticles offer a number of advantages for drugs delivery over their native counterparts while retaining their natural characteristics and biological functions. Their ultra-small size (lipoprotein receptors, i.e., low-density lipoprotein receptor (LDLR) and Scavenger receptor class B member 1 (SRB1) that are found in a number of pathological conditions (e.g., cancer, atherosclerosis), make them superior delivery strategies when compared with other nanoparticle systems. We review the various approaches that have been developed for the generation of synthetic lipoproteins and their respective applications in vitro and in vivo. More specifically, we summarize the approaches employed to address the limitation on use of reconstituted lipoproteins by means of natural or recombinant apolipoproteins, as well as apolipoprotein mimetic molecules. Finally, we provide an overview of the advantages and disadvantages of these approaches and discuss future perspectives for clinical translation of these nanoparticles. © 2014 Wiley Periodicals, Inc.

In LDL receptor-deficient mice, catabolism of remnant lipoproteins requires a high level of apoE but is inhibited by excess apoE

NARCIS (Netherlands)

Dijk, K.W. van; Vlijmen, B.J.M. van; Hof, H.B. van 't; Zee, A. van der; Santamarina-Fojo, S.; Berkel, T.J.C. van; Havekes, L.M.; Hofker, M.H.

1999-01-01

To investigate the quantitative requirement for apolipoprotein (apo) E in the clearance of lipoproteins via the non-low density lipoprotein (LDL) receptor mediated pathway, human APOE was overexpressed at various levels in the livers of mice deficient for both the endogenous Apoe and Ldlr genes

Biogenesis and Membrane Targeting of Lipoproteins.

Science.gov (United States)

Narita, Shin-Ichiro; Tokuda, Hajime

2010-09-01

Bacterial lipoproteins represent a unique class of membrane proteins, which are anchored to membranes through triacyl chains attached to the amino-terminal cysteine. They are involved in various functions localized in cell envelope. Escherichia coli possesses more than 90 species of lipoproteins, most of which are localized in the outer membrane, with others being in the inner membrane. All lipoproteins are synthesized in the cytoplasm with an N-terminal signal peptide, translocated across the inner membrane by the Sec translocon to the periplasmic surface of the inner membrane, and converted to mature lipoproteins through sequential reactions catalyzed by three lipoprotein-processing enzymes: Lgt, LspA, and Lnt. The sorting of lipoproteins to the outer membrane requires a system comprising five Lol proteins. An ATP-binding cassette transporter, LolCDE, initiates the sorting by mediating the detachment of lipoproteins from the inner membrane. Formation of the LolA-lipoprotein complex is coupled to this LolCDE-dependent release reaction. LolA accommodates the amino-terminal acyl chain of lipoproteins in its hydrophobic cavity, thereby generating a hydrophilic complex that can traverse the periplasmic space by diffusion. Lipoproteins are then transferred to LolB on the outer membrane and anchored to the inner leaflet of the outer membrane by the action of LolB. In contrast, since LolCDE does not recognize lipoproteins possessing Asp at position +2, these lipoproteins remain anchored to the inner membrane. Genes for Lol proteins are widely conserved among gram-negative bacteria, and Lol-mediated outer membrane targeting of lipoproteins is considered to be the general lipoprotein localization mechanism.

Ectodomains of the LDL receptor-related proteins LRP1b and LRP4 have anchorage independent functions in vivo.

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Martin F Dietrich

2010-04-01

Full Text Available The low-density lipoprotein (LDL receptor gene family is a highly conserved group of membrane receptors with diverse functions in developmental processes, lipoprotein trafficking, and cell signaling. The low-density lipoprotein (LDL receptor-related protein 1b (LRP1B was reported to be deleted in several types of human malignancies, including non-small cell lung cancer. Our group has previously reported that a distal extracellular truncation of murine Lrp1b that is predicted to secrete the entire intact extracellular domain (ECD is fully viable with no apparent phenotype.Here, we have used a gene targeting approach to create two mouse lines carrying internally rearranged exons of Lrp1b that are predicted to truncate the protein closer to the N-terminus and to prevent normal trafficking through the secretary pathway. Both mutations result in early embryonic lethality, but, as expected from the restricted expression pattern of LRP1b in vivo, loss of Lrp1b does not cause cellular lethality as homozygous Lrp1b-deficient blastocysts can be propagated normally in culture. This is similar to findings for another LDL receptor family member, Lrp4. We provide in vitro evidence that Lrp4 undergoes regulated intramembraneous processing through metalloproteases and gamma-secretase cleavage. We further demonstrate negative regulation of the Wnt signaling pathway by the soluble extracellular domain.Our results underline a crucial role for Lrp1b in development. The expression in mice of truncated alleles of Lrp1b and Lrp4 with deletions of the transmembrane and intracellular domains leads to release of the extracellular domain into the extracellular space, which is sufficient to confer viability. In contrast, null mutations are embryonically (Lrp1b or perinatally (Lrp4 lethal. These findings suggest that the extracellular domains of both proteins may function as a scavenger for signaling ligands or signal modulators in the extracellular space, thereby

Lipoprotein complex formation

International Nuclear Information System (INIS)

Musliner, T.A.; Krauss, R.M.

1987-01-01

Transfers of lipids and proteins between different lipoproteins are known to occur in the course of their metabolism. It is likely that these transfers take place during transient physical associations between lipoprotein particles, but the nature and chemical basis for such interactions are poorly understood. The fact that lipid and apolipoprotein movements are particularly prevalent during the intravascular lipolysis of triglyceride-rich lipoproteins suggested to us that lipolysis products accumulating on these particles might promote physical binding with other lipoproteins. To test this hypothesis, we studied interactions between very low-density, low density, and high-density lipoproteins in the setting of partial lipolysis by bovine milk lipoprotein lipase in the presence of limited unesterified fatty acid acceptor. 2 figs., 1 tab

Lipoprotein profile, lipoprotein-associated phospholipase A2 and cardiovascular risk in hemodialysis patients.

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Rolla, Roberta; De Mauri, Andreana; Valsesia, Ambra; Vidali, Matteo; Chiarinotti, Doriana; Bellomo, Giorgio

2015-12-01

Cardiovascular disease is the leading cause of morbidity and mortality in hemodialysis patients; the increased risk of cardiovascular disease is due to accelerated atherosclerosis, inflammation and impaired lipoprotein metabolism. We aimed to evaluate lipoprotein-associated phospholipase A2 (Lp-PLA2) and some pro-inflammatory aspects of the lipoprotein profile in dialyzed patients in order to evaluate the relationship with the accelerated atherosclerosis and vascular accidents. In 102 dialysis patients and 40 non-uremic controls, we investigated the lipoprotein plasma profile, high sensitivity C-reactive protein (CRP), ceruloplasmin and serum amyloid A protein (SAA), and followed patients for 1 year to analyze the risk of acute cardiovascular events. Total cholesterol, low-density lipoprotein and high-density lipoprotein plasma levels were significantly lower in uremic patients than controls, whereas CRP, SAA, ceruloplasmin, Lp-PLA2 and their ratio with apolipoprotein A1 were significantly higher. Patients with Lp-PLA2 levels >194 nmol/min/ml had more acute cardiovascular events than patients with lower values. Our results show that in dialysis subjects: (1) low-density lipoproteins show a more atherogenic phenotype than in the general population; (2) high-density lipoproteins are less anti-inflammatory; (3) Lp-PLA2 could potentially be used to evaluate cardiovascular risk.

Bacterial lipoproteins; biogenesis, sorting and quality control.

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Narita, Shin-Ichiro; Tokuda, Hajime

2017-11-01

Bacterial lipoproteins are a subset of membrane proteins localized on either leaflet of the lipid bilayer. These proteins are anchored to membranes through their N-terminal lipid moiety attached to a conserved Cys. Since the protein moiety of most lipoproteins is hydrophilic, they are expected to play various roles in a hydrophilic environment outside the cytoplasmic membrane. Gram-negative bacteria such as Escherichia coli possess an outer membrane, to which most lipoproteins are sorted. The Lol pathway plays a central role in the sorting of lipoproteins to the outer membrane after lipoprotein precursors are processed to mature forms in the cytoplasmic membrane. Most lipoproteins are anchored to the inner leaflet of the outer membrane with their protein moiety in the periplasm. However, recent studies indicated that some lipoproteins further undergo topology change in the outer membrane, and play critical roles in the biogenesis and quality control of the outer membrane. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop. Copyright © 2016 Elsevier B.V. All rights reserved.

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) in sickle cell disease vasculopathy

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Chen, Mingyi; Qiu, Hong; Lin, Xin; Nam, David; Ogbu-Nwobodo, Lucy; Archibald, Hannah; Joslin, Amelia; Wun, Ted; Sawamura, Tatsuya; Green, Ralph

2017-01-01

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is an endothelial receptor for oxidized LDL. Increased expression of LOX-1 has been demonstrated in atherosclerotic lesions and diabetic vasculopathy. In this study, we investigate the expression of LOX-1 receptor in sickle cell disease (SCD) vasculopathy. Expression of LOX-1 in brain vascular endothelium is markedly increased and LOX-1 gene expression is upregulated in cultured human brain microvascular endothelial cells by incubation with SCD erythrocytes. Also, the level of circulating soluble LOX-1 concentration is elevated in the plasma of SCD patients. Increased LOX-1 expression in endothelial cells is potentially involved in the pathogenesis of SCD vasculopathy. Soluble LOX-1 concentration in SCD may provide a novel biomarker for risk stratification of sickle cell vascular complications. PMID:27519944

Roles of the Protruding Loop of Factor B Essential for the Localization of Lipoproteins (LolB) in the Anchoring of Bacterial Triacylated Proteins to the Outer Membrane*

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Hayashi, Yumi; Tsurumizu, Ryoji; Tsukahara, Jun; Takeda, Kazuki; Narita, Shin-ichiro; Mori, Makiko; Miki, Kunio; Tokuda, Hajime

2014-01-01

The Lol system comprising five Lol proteins, LolA through LolE, sorts Escherichia coli lipoproteins to outer membranes. The LolCDE complex, an ATP binding cassette transporter in inner membranes, releases outer membrane-specific lipoproteins in an ATP-dependent manner, causing formation of the LolA-lipoprotein complex in the periplasm. LolA transports lipoproteins through the periplasm to LolB on outer membranes. LolB is itself a lipoprotein anchored to outer membranes, although the membrane anchor is functionally dispensable. LolB then localizes lipoproteins to outer membranes through largely unknown mechanisms. The crystal structure of LolB is similar to that of LolA, and it possesses a hydrophobic cavity that accommodates acyl chains of lipoproteins. To elucidate the molecular function of LolB, a periplasmic version of LolB, mLolB, was mutagenized at various conserved residues. Despite the lack of acyl chains, most defective mutants were insoluble. However, a derivative with glutamate in place of leucine 68 was soluble and unable to localize lipoproteins to outer membranes. This leucine is present in a loop protruding from mLolB into an aqueous environment, and no analogous loop is present in LolA. Thus, leucine 68 was replaced with other residues. Replacement by acidic, but not hydrophobic, residues generated for the first time mLolB derivatives that can accept but cannot localize lipoproteins to outer membranes. Moreover, deletion of the leucine with neighboring residues impaired the lipoprotein receptor activity. Based on these observations, the roles of the protruding loop of LolB in the last step of lipoprotein sorting are discussed. PMID:24569999

Roles of the protruding loop of factor B essential for the localization of lipoproteins (LolB) in the anchoring of bacterial triacylated proteins to the outer membrane.

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Hayashi, Yumi; Tsurumizu, Ryoji; Tsukahara, Jun; Takeda, Kazuki; Narita, Shin-ichiro; Mori, Makiko; Miki, Kunio; Tokuda, Hajime

2014-04-11

The Lol system comprising five Lol proteins, LolA through LolE, sorts Escherichia coli lipoproteins to outer membranes. The LolCDE complex, an ATP binding cassette transporter in inner membranes, releases outer membrane-specific lipoproteins in an ATP-dependent manner, causing formation of the LolA-lipoprotein complex in the periplasm. LolA transports lipoproteins through the periplasm to LolB on outer membranes. LolB is itself a lipoprotein anchored to outer membranes, although the membrane anchor is functionally dispensable. LolB then localizes lipoproteins to outer membranes through largely unknown mechanisms. The crystal structure of LolB is similar to that of LolA, and it possesses a hydrophobic cavity that accommodates acyl chains of lipoproteins. To elucidate the molecular function of LolB, a periplasmic version of LolB, mLolB, was mutagenized at various conserved residues. Despite the lack of acyl chains, most defective mutants were insoluble. However, a derivative with glutamate in place of leucine 68 was soluble and unable to localize lipoproteins to outer membranes. This leucine is present in a loop protruding from mLolB into an aqueous environment, and no analogous loop is present in LolA. Thus, leucine 68 was replaced with other residues. Replacement by acidic, but not hydrophobic, residues generated for the first time mLolB derivatives that can accept but cannot localize lipoproteins to outer membranes. Moreover, deletion of the leucine with neighboring residues impaired the lipoprotein receptor activity. Based on these observations, the roles of the protruding loop of LolB in the last step of lipoprotein sorting are discussed.

Familial defective apolipoprotein B-100: low density lipoproteins with abnormal receptor binding

International Nuclear Information System (INIS)

Innerarity, T.L.; Weisgraber, K.H.; Arnold, K.S.; Mahley, R.W.; Krauss, R.M.; Vega, G.L.; Grundy, S.M.

1987-01-01

Previous in vivo turnover studies suggested that retarded clearance of low density lipoproteins (LDL) from the plasma of some hypercholesterolemic patients is due to LDL with defective receptor binding. The present study examined this postulate directly by receptor binding experiments. The LDL from a hypercholesterolemic patient (G.R.) displayed a reduced ability to bind to the LDL receptors on normal human fibroblasts. The G.R. LDL possessed 32% of normal receptor binding activity. Likewise, the G.R. LDL were much less effective than normal LDL in competing with 125 I-labeled normal LDL for cellular uptake and degradation and in stimulating intracellular cholesteryl ester synthesis. The defect in LDL binding appears to be due to a genetic abnormality of apolipoprotein B-100: two brothers of the proband possess LDL defective in receptor binding, whereas a third brother and the proband's son have normally binding LDL. Further, the defect in receptor binding does not appear to be associated wit an abnormal lipid composition or structure of the LDL. Normal and abnormal LDL subpopulations were partially separated from plasma of two subjects by density-gradient ultracentrifugation, a finding consistent with the presence of a normal and a mutant allele. The affected family members appear to be heterozygous for this disorder, which has been designated familial defective apolipoprotein B-100. These studies indicate that the defective receptor binding results in inefficient clearance of LDL and the hypercholesterolemia observed in these patients

Low density lipoprotein uptake by an endothelial-smooth muscle cell bilayer

International Nuclear Information System (INIS)

Alexander, J.J.; Miguel, R.; Graham, D.

1991-01-01

To study the interaction of endothelial and smooth muscle cells, and the means by which such interaction may affect lipid permeability of the arterial wall, cell bilayers were established by use of a transwell culture system. After confluent growth of both cell types had been achieved, iodine 125 bound to low-density lipoprotein (10 ng protein/ml) was added to the media of the upper well. After a 3-hour incubation period, the iodine 125-bound low-density lipoprotein content of the upper and lower media demonstrated an impedance to lipoprotein movement across the endothelial cell monolayer as compared to the bare porous polycarbonate filter of the transwell (p less than 10(-6)). The presence of smooth muscle cells in the bottom well significantly enhanced the permeability of the endothelial cell layer (p less than 10(-60)). This effect remained unchanged over a 9-day time course. Membrane binding and cellular uptake of low-density lipoprotein by endothelial cells was not altered by smooth muscle cells, indicating that this change in permeability could not be easily attributed to changes in receptor-mediated transport or transcytosis. Membrane binding (p less than 0.02) and cellular uptake (p less than 10(-6)) of low-density lipoprotein by smooth muscle cells in the bilayer, when adjusted for counts available in the smooth muscle cell media, were both reduced in the early incubation period as compared to isolated smooth muscle cells. The disproportionate reduction in uptake as compared to binding would suggest that this was not entirely a receptor-dependent process

β-Amyloid promotes accumulation of lipid peroxides by inhibiting CD36-mediated clearance of oxidized lipoproteins

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Khan Tayeba

2004-11-01

lipoprotein binding and its subsequent degradation via CD36, but not SRA, and this was independent of Aβ-CD36-signaling. Furthermore, Aβ treatment decreased CD36, but not SRA, mRNA and protein, thereby reducing cell surface expression of this oxLDL receptor. Conclusions Together, these data demonstrate that in the presence of β-amyloid, CD36-mediated clearance of oxidized lipoproteins is abrogated, which would promote the extracellular accumulation of these pro-inflammatory lipids and perpetuate lipid peroxidation.

Potential cellular receptors involved in hepatitis C virus entry into cells

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Muellhaupt Beat

2005-04-01

Full Text Available Abstract Hepatitis C virus (HCV infects hepatocytes and leads to permanent, severe liver damage. Since the genomic sequence of HCV was determined, progress has been made towards understanding the functions of the HCV-encoded proteins and identifying the cellular receptor(s responsible for adsorption and penetration of the virus particle into the target cells. Several cellular receptors for HCV have been proposed, all of which are associated with lipid and lipoprotein metabolism. This article reviews the cellular receptors for HCV and suggests a general model for HCV entry into cells, in which lipoproteins play a crucial role.

Acetaldehyde binding increases the catabolism of rat serum low-density lipoproteins

International Nuclear Information System (INIS)

Savolainen, M.J.; Baraona, E.; Lieber, C.S.

1987-01-01

Acetaldehyde was found to form adducts with rat serum lipoproteins. The binding of [ 14 C]acetaldehyde to lipoproteins was studied at low concentrations which are known to exist during ethanol oxidation. The amount of lipoprotein adducts was a linear function of acetaldehyde concentration up to 250 μM. Incubation of rat plasma low-density lipoproteins (LDL) with 200 μM acetaldehyde increased the disappearance rate of the 3 H-label from the cholesterol ester moiety of LDL injected into normal rats. The data show that even low concentrations of acetaldehyde are capable of affecting LDL metabolism. These findings may provide an explanation for the low concentrations of serum LDL in alcoholics. The alcohol-induced hyperlipidemia includes either a lack of increase or a decrease in the low-density lipoprotein (LDL) concentration, but the underlying mechanism is not known. It has been shown previously, that the acetylation of lysine residues of LDL apoprotein (apoB) by acetanhydride leads to rapid uptake of LDL particles by macrophages through a non-LDL receptor pathway. Since acetaldehyde, the first toxic metabolite of ethanol, is a chemically reactive compound capable of binding to proteins, they tested whether acetaldehyde forms adducts with serum lipoproteins and subsequently alters the catabolism of LDL. 19 references, 2 figures, 1 table

Antagonism of Secreted PCSK9 Increases Low Density Lipoprotein Receptor Expression in HepG2 Cells

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McNutt, Markey C.; Kwon, Hyock Joo; Chen, Chiyuan; Chen, Justin R.; Horton, Jay D.; Lagace, Thomas A.; (USMC); (UTSMC)

2009-07-10

PCSK9 is a secreted protein that degrades low density lipoprotein receptors (LDLRs) in liver by binding to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR. It is not known whether PCSK9 causes degradation of LDLRs within the secretory pathway or following secretion and reuptake via endocytosis. Here we show that a mutation in the LDLR EGF-A domain associated with familial hypercholesterolemia, H306Y, results in increased sensitivity to exogenous PCSK9-mediated cellular degradation because of enhanced PCSK9 binding affinity. The crystal structure of the PCSK9-EGF-A(H306Y) complex shows that Tyr-306 forms a hydrogen bond with Asp-374 in PCSK9 at neutral pH, which strengthens the interaction with PCSK9. To block secreted PCSK9 activity, LDLR (H306Y) subfragments were added to the medium of HepG2 cells stably overexpressing wild-type PCSK9 or gain-of-function PCSK9 mutants associated with hypercholesterolemia (D374Y or S127R). These subfragments blocked secreted PCSK9 binding to cell surface LDLRs and resulted in the recovery of LDLR levels to those of control cells. We conclude that PCSK9 acts primarily as a secreted factor to cause LDLR degradation. These studies support the concept that pharmacological inhibition of the PCSK9-LDLR interaction extracellularly will increase hepatic LDLR expression and lower plasma low density lipoprotein levels.

Lower Squalene Epoxidase and Higher Scavenger Receptor Class B Type 1 Protein Levels Are Involved in Reduced Serum Cholesterol Levels in Stroke-Prone Spontaneously Hypertensive Rats.

Science.gov (United States)

Michihara, Akihiro; Mido, Mayuko; Matsuoka, Hiroshi; Mizutani, Yurika

2015-01-01

A lower serum cholesterol level was recently shown to be one of the causes of stroke in an epidemiological study. Spontaneously hypertensive rats stroke-prone (SHRSP) have lower serum cholesterol levels than normotensive Wistar-Kyoto rats (WKY). To elucidate the mechanisms responsible for the lower serum cholesterol levels in SHRSP, we determined whether the amounts of cholesterol biosynthetic enzymes or the receptor and transporter involved in cholesterol uptake and efflux in the liver were altered in SHRSP. When the mRNA levels of seven cholesterol biosynthetic enzymes were measured using real-time polymerase chain reaction (PCR), farnesyl pyrophosphate synthase and squalene epoxidase (SQE) levels in the liver of SHRSP were significantly lower than those in WKY. SQE protein levels were significantly reduced in tissues other than the brain of SHRSP. No significant differences were observed in low-density lipoprotein (LDL) receptor (uptake of serum LDL-cholesterol) or ATP-binding cassette transporter A1 (efflux of cholesterol from the liver/formation of high-density lipoprotein (HDL)) protein levels in the liver and testis between SHRSP and WKY, whereas scavenger receptor class B type 1 (SRB1: uptake of serum HDL-cholesterol) protein levels were higher in the livers of SHRSP. These results indicated that the lower protein levels of SQE and higher protein levels of SRB1 in the liver were involved in the reduced serum cholesterol levels in SHRSP.

Outer membrane lipoprotein biogenesis: Lol is not the end.

Science.gov (United States)

Konovalova, Anna; Silhavy, Thomas J

2015-10-05

Bacterial lipoproteins are lipid-anchored proteins that contain acyl groups covalently attached to the N-terminal cysteine residue of the mature protein. Lipoproteins are synthesized in precursor form with an N-terminal signal sequence (SS) that targets translocation across the cytoplasmic or inner membrane (IM). Lipid modification and SS processing take place at the periplasmic face of the IM. Outer membrane (OM) lipoproteins take the localization of lipoproteins (Lol) export pathway, which ends with the insertion of the N-terminal lipid moiety into the inner leaflet of the OM. For many lipoproteins, the biogenesis pathway ends here. We provide examples of lipoproteins that adopt complex topologies in the OM that include transmembrane and surface-exposed domains. Biogenesis of such lipoproteins requires additional steps beyond the Lol pathway. In at least one case, lipoprotein sequences reach the cell surface by being threaded through the lumen of a beta-barrel protein in an assembly reaction that requires the heteropentomeric Bam complex. The inability to predict surface exposure reinforces the importance of experimental verification of lipoprotein topology and we will discuss some of the methods used to study OM protein topology. © 2015 The Author(s).

Scavenger receptor-mediated recognition of maleyl bovine plasma albumin and the demaleylated protein in human monocyte macrophages

International Nuclear Information System (INIS)

Haberland, M.E.; Fogelman, A.M.

1985-01-01

Maleyl bovine plasma albumin competed on an equimolar basis with malondialdehyde low density lipoprotein (LDL) in suppressing the lysosomal hydrolysis of 125 I-labeled malondialdehyde LDL mediated by the scavenger receptor of human monocyte macrophages. Maleyl bovine plasma albumin, in which 94% of the amino groups were modified, exhibited an anodic mobility in agarose electrophoresis 1.7 times that of the native protein. Incubation of maleyl bovine plasma albumin at pH 3.5 regenerated the free amino groups and restored the protein to the same electrophoretic mobility as native albumin. Although ligands recognized by the scavenger receptor typically are anionic, the authors propose that addition of new negative charge achieved by maleylation, rather than directly forming the receptor binding site(s), induces conformational changes in albumin as a prerequisite to expression of the recognition domain(s). They conclude that the primary sequence of albumin, rather than addition of new negative charge, provides the recognition determinant(s) essential for interaction of maleyl bovine plasma albumin with the scavenger receptor

Impaired low-density lipoprotein receptor activity in chronic B-lymphocytic leukaemia cells

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Juliusson, G.; Vitols, S.

1988-01-01

Cellular degradation of /sup 125/I-labelled low-density lipoprotein (LDL) was analysed in freshly isolated blood mononuclear cells from 26 patients with chronic B-lymphocytic leukaemia (CLL) and 8 healthy subjects, and in cells following 1,2 and 3 d of culture in medium containing 10% human lipoprotein-deficient serum (LPDS). Fresh CLL cells had lower LDL degradation rates than mononuclear cells from healthy subjects (p < 0.01). The LDL degradation rates increased during culture (p < 0.001), but to a lesser degree in CLL cells than in normal blood mononuclear cells (p < 0.001). The cellular degradation rate of /sup 125/I-LDL was markedly inhibited by an excess of unlabelled LDL, indicating that most of the /sup 125/I-LDL that was degraded had been internalized following binding to the LPDS-induced LDL degradation of CLL cells and the thymidine uptake in CLL cell cultures with (r = 0.70, p < 0.001) and without (r = 0.59, p < 0.01) the B cell mitogen, Epstein-Barr virus. The results indicate that LDL receptors might be involved in the regulation of CLL cell proliferation.

Serum and urinary lipoproteins in the human nephrotic syndrome: evidence for renal catabolism of lipoproteins

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Shore, V.G.; Forte, T.; Licht, H.; Lewis, S.B.

1982-03-01

The urinary excretion of lipoproteins and the possibility of catabolic alterations on glomerular filtration were investigated in four nephrotic subjects difering in etiology, serum lipoprotein profile, and 24 hr urinary output of protein and lipids. The apolipoproteins and lipoproteins of urine were compared with those of serum with respect to distribution profile, physical properties, and composition. As expected from molecular sieving effects during glomerular filtration, the urinary HDL were more abundant than the lower density lipoproteins even when the plasma LDL was elevated markedly. Intact apolipoproteins were not found in the concentrated urinary fraction isolated by ultrafiltration between the limits of 10/sup 4/ and 5 x 10/sup 4/ daltons. On the basis of immunoreactivity, gel electrophoresis, and amino acid composition, apolipoproteins B and AI are the major and minor proteins, respectively, of urinary LDL, and apo B is the major protein of the urinary IDL and VLDL. Apolipoproteins AI, AII, CI, CIII, and possibly AIV were isolated from the urinary HDL. As much as 20% of the protein moiety of the urinary HDL appeared to be large apolipoprotien fragments with molecular weights and isoelectric points similar to those of apo CII and apo CIII. The lower density classes of urinary lipoproteins also appeared to have lost apo E and apo C's and to have undergone partial proteolysis.

Inhibitory Effects of North American Wild Rice on Monocyte Adhesion and Inflammatory Modulators in Low-Density Lipoprotein Receptor-Knockout Mice.

Science.gov (United States)

Moghadasian, Mohammed H; Zhao, Ruozhi; Ghazawwi, Nora; Le, Khuong; Apea-Bah, Franklin B; Beta, Trust; Shen, Garry X

2017-10-18

The present study examined the effects of wild rice on monocyte adhesion, inflammatory and fibrinolytic mediators in low-density lipoprotein receptor-knockout (LDLr-KO) mice. Male LDLr-KO mice received a cholesterol (0.06%, w/w)-supplemented diet with or without white or wild rice (60%, w/w) for 20 weeks. White rice significantly increased monocyte adhesion and abundances of monocyte chemoattractant protein-1, tissue necrosis factor-α, intracellular cell adhesion molecule-1, plasminogen activator inhibitor-1, urokinase plasminogen activator (uPA), and uPA receptor in aortae and hearts of LDLr-KO mice compared to the control diet. Wild rice inhibited monocyte adhesion to the aorta, atherosclerosis, and abundances of the inflammatory and fibrinolytic regulators in the cardiovascular tissue of LDLr-KO mice compared to white rice. White or wild rice did not significantly alter the levels of cholesterol, triglycerides, or antioxidant enzymes in plasma. The anti-atherosclerotic effect of wild rice may result from its inhibition on monocyte adhesion and inflammatory modulators in LDLr-KO mice.

Alcohol-extracted, but not intact, dietary soy protein lowers lipoprotein(a) markedly

DEFF Research Database (Denmark)

Meinertz, Hans; Nilausen, Karin; Hilden, Jørgen

2002-01-01

We previously found that dietary soy protein produces higher lipoprotein(a) [Lp(a)] plasma concentrations than does casein. This study tested the hypothesis that soy protein contains Lp(a)-raising alcohol-removable components. Twelve normolipidemic women and men consumed, in a crossover design......, liquid-formula diets containing casein, soy protein, or alcohol-extracted soy protein. Dietary periods of 32 days were separated by washout periods on self-selected diets. Fasting lipid and Lp(a) levels were measured throughout. Median Lp(a) concentration was >2-fold greater after 28 to 32 days on a soy...... protein diet than after an extracted soy protein diet (Psoy protein diets were virtually identical. Women and men responded similarly. When the switch was made from a self-selected to a soy protein diet, median Lp(a) concentration increased 16...

Prediction of lipoprotein signal peptides in Gram-negative bacteria.

Science.gov (United States)

Juncker, Agnieszka S; Willenbrock, Hanni; Von Heijne, Gunnar; Brunak, Søren; Nielsen, Henrik; Krogh, Anders

2003-08-01

A method to predict lipoprotein signal peptides in Gram-negative Eubacteria, LipoP, has been developed. The hidden Markov model (HMM) was able to distinguish between lipoproteins (SPaseII-cleaved proteins), SPaseI-cleaved proteins, cytoplasmic proteins, and transmembrane proteins. This predictor was able to predict 96.8% of the lipoproteins correctly with only 0.3% false positives in a set of SPaseI-cleaved, cytoplasmic, and transmembrane proteins. The results obtained were significantly better than those of previously developed methods. Even though Gram-positive lipoprotein signal peptides differ from Gram-negatives, the HMM was able to identify 92.9% of the lipoproteins included in a Gram-positive test set. A genome search was carried out for 12 Gram-negative genomes and one Gram-positive genome. The results for Escherichia coli K12 were compared with new experimental data, and the predictions by the HMM agree well with the experimentally verified lipoproteins. A neural network-based predictor was developed for comparison, and it gave very similar results. LipoP is available as a Web server at www.cbs.dtu.dk/services/LipoP/.

Prediction of lipoprotein signal peptides in Gram-negative bacteria

DEFF Research Database (Denmark)

Juncker, Agnieszka; Willenbrock, Hanni; Von Heijne, G.

2003-01-01

A method to predict lipoprotein signal peptides in Gram-negative Eubacteria, LipoP, has been developed. The hidden Markov model (HMM) was able to distinguish between lipoproteins (SPaseII-cleaved proteins), SPaseI-cleaved proteins, cytoplasmic proteins, and transmembrane proteins. This predictor ...

Identification and Localization of Myxococcus xanthus Porins and Lipoproteins

Science.gov (United States)

Bhat, Swapna; Zhu, Xiang; Patel, Ricky P.; Orlando, Ron; Shimkets, Lawrence J.

2011-01-01

Myxococcus xanthus DK1622 contains inner (IM) and outer membranes (OM) separated by a peptidoglycan layer. Integral membrane, β-barrel proteins are found exclusively in the OM where they form pores allowing the passage of nutrients, waste products and signals. One porin, Oar, is required for intercellular communication of the C-signal. An oar mutant produces CsgA but is unable to ripple or stimulate csgA mutants to develop suggesting that it is the channel for C-signaling. Six prediction programs were evaluated for their ability to identify β-barrel proteins. No program was reliable unless the predicted proteins were first parsed using Signal P, Lipo P and TMHMM, after which TMBETA-SVM and TMBETADISC-RBF identified β-barrel proteins most accurately. 228 β-barrel proteins were predicted from among 7331 protein coding regions, representing 3.1% of total genes. Sucrose density gradients were used to separate vegetative cell IM and OM fractions, and LC-MS/MS of OM proteins identified 54 β-barrel proteins. Another class of membrane proteins, the lipoproteins, are anchored in the membrane via a lipid moiety at the N-terminus. 44 OM proteins identified by LC-MS/MS were predicted lipoproteins. Lipoproteins are distributed between the IM, OM and ECM according to an N-terminal sorting sequence that varies among species. Sequence analysis revealed conservation of alanine at the +7 position of mature ECM lipoproteins, lysine at the +2 position of IM lipoproteins, and no noticable conservation within the OM lipoproteins. Site directed mutagenesis and immuno transmission electron microscopy showed that alanine at the +7 position is essential for sorting of the lipoprotein FibA into the ECM. FibA appears at normal levels in the ECM even when a +2 lysine is added to the signal sequence. These results suggest that ECM proteins have a unique method of secretion. It is now possible to target lipoproteins to specific IM, OM and ECM locations by manipulating the amino acid

Impaired LDL receptor-related protein 1 translocation correlates with improved dyslipidemia and atherosclerosis in apoE-deficient mice.

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Philip L S M Gordts

Full Text Available OBJECTIVE: Determination of the in vivo significance of LDL receptor-related protein 1 (LRP1 dysfunction on lipid metabolism and atherosclerosis development in absence of its main ligand apoE. METHODS AND RESULTS: LRP1 knock-in mice carrying an inactivating mutation in the NPxYxxL motif were crossed with apoE-deficient mice. In the absence of apoE, relative to LRP1 wild-type animals, LRP1 mutated mice showed an increased clearance of postprandial lipids despite a compromised LRP1 endocytosis rate and inefficient insulin-mediated translocation of the receptor to the plasma membrane, likely due to inefficient slow recycling of the mutated receptor. Postprandial lipoprotein improvement was explained by increased hepatic clearance of triglyceride-rich remnant lipoproteins and accompanied by a compensatory 1.6-fold upregulation of LDLR expression in hepatocytes. One year-old apoE-deficient mice having the dysfunctional LRP1 revealed a 3-fold decrease in spontaneous atherosclerosis development and a 2-fold reduction in LDL-cholesterol levels. CONCLUSION: These findings demonstrate that the NPxYxxL motif in LRP1 is important for insulin-mediated translocation and slow perinuclear endosomal recycling. These LRP1 impairments correlated with reduced atherogenesis and cholesterol levels in apoE-deficient mice, likely via compensatory LDLR upregulation.

Increased cell motility and invasion upon knockdown of lipolysis stimulated lipoprotein receptor (LSR) in SW780 bladder cancer cells

DEFF Research Database (Denmark)

Herbsleb, Malene; Birkenkamp-Demtroder, Karin; Thykjaer, Thomas

2008-01-01

Mechanisms underlying the malignant development in bladder cancer are still not well understood. Lipolysis stimulated lipoprotein receptor (LSR) has previously been found to be upregulated by P53. Furthermore, we have previously found LSR to be differentially expressed in bladder cancer. Here we...... investigated the role of LSR in bladder cancer....

Identification of a point mutation in growth factor repeat C of the low density lipoprotein-receptor gene in a patient with homozygous familial hypercholesterolemia

International Nuclear Information System (INIS)

Soutar, A.K.; Knight, B.L.; Patel, D.D.

1989-01-01

The coding region of the low density lipoprotein (LDL)-receptor gene from a patient (MM) with homozygous familial hypercholesterolemia (FH) has been sequenced from six overlapping 500-base-pair amplified fragments of the cDNA from cultured skin fibroblasts. Two separate single nucleotide base changes from the normal sequence were detected. The first involved substitution of guanine for adenine in the third position of the codon for amino acid residue Cys-27 and did not affect the protein sequence. The second mutation was substitution of thymine for cytosine in the DNA for the codon for amino acid residue 664, changing the codon from CCG (proline) to CTG (leucine) and introducing a new site for the restriction enzyme PstI. MM is a true homozygote with two identical genes, and the mutation cosegregated with clinically diagnosed FH in his family in which first cousin marriages occurred frequently. LDL receptors in MM's skin fibroblasts bind less LDL than normal and with reduced affinity. Thus this naturally occurring single point mutation affects both intracellular transport of the protein and ligand binding and occurs in growth factor-like repeat C, a region that has not previously been found to influence LDL binding

Structures and Interactions of Proteins in the Brain

DEFF Research Database (Denmark)

Nielsen, Lau Dalby

The protein low density lipoprotein receptor related protein 1 (LRP1) plays multiple roles in the biology of amyloid β peptide (Aβ) and Alzheimer’s disease. LRP1 is very important for clearance of Aβ both in the brain and by facilitating Aβ export over the blood brain barrier. In spite of the app......The protein low density lipoprotein receptor related protein 1 (LRP1) plays multiple roles in the biology of amyloid β peptide (Aβ) and Alzheimer’s disease. LRP1 is very important for clearance of Aβ both in the brain and by facilitating Aβ export over the blood brain barrier. In spite...... coding for Arc protein has been domesticated from the same branch of genes that has given rise to retroviruses. We show that even despite the large evolutional distance between Arc and retroviruses. Despite large evolutionary distance Arc still self-assemble into higher order structures that resembles...

Oxidized low-density lipoproteins may induce expression of monocyte chemotactic protein-3 in atherosclerotic plaques

International Nuclear Information System (INIS)

Jang, Moon Kyoo; Kim, Ji Young; Jeoung, Nam Ho; Kang, Mi Ae; Choi, Myung-Sook; Oh, Goo Taeg; Nam, Kyung Tak; Lee, Won-Ha; Park, Yong Bok

2004-01-01

Genes induced or suppressed by oxidized low-density lipoproteins (oxLDL) in human monocytic THP-1 cells were searched using the differential display reverse transcriptase polymerase chain reaction. One of the differentially expressed (up-regulated) cDNA fragments was found to contain sequences corresponding to monocyte chemotactic protein-3 (MCP-3). The stimulatory effect of the oxLDL on the expression of MCP-3 mRNA was both time- and dose-dependent. Treatment with GF109203X and genistein, inhibitors of protein kinase C and tyrosine kinase, respectively, had no effect on the induction of MCP-3 mRNA by oxLDL, while treatment with cycloheximide inhibited the induction. The induction was reproduced by the lipid components in oxLDL such as 9-HODE and 13-HODE, which are known to activate the peroxisome proliferator-activated receptor γ (PPARγ). Introduction of an endogenous PPARγ ligand, 15d-PGJ2, in the culture of THP-1 cells resulted in the induction of MCP-3 gene expression. Furthermore, analyses of human atherosclerotic plaques revealed that the expressional pattern of MCP-3 in the regions of neointimal and necrotic core overlapped with that of PPARγ. These results suggest that oxLDL delivers its signal for MCP-3 expression via PPARγ, which may be further related to the atherogenesis

Release of endothelial cell lipoprotein lipase by plasma lipoproteins and free fatty acids

International Nuclear Information System (INIS)

Saxena, U.; Witte, L.D.; Goldberg, I.J.

1989-01-01

Lipoprotein lipase (LPL) bound to the lumenal surface of vascular endothelial cells is responsible for the hydrolysis of triglycerides in plasma lipoproteins. Studies were performed to investigate whether human plasma lipoproteins and/or free fatty acids would release LPL which was bound to endothelial cells. Purified bovine milk LPL was incubated with cultured porcine aortic endothelial cells resulting in the association of enzyme activity with the cells. When the cells were then incubated with media containing chylomicrons or very low density lipoproteins (VLDL), a concentration-dependent decrease in the cell-associated LPL enzymatic activity was observed. In contrast, incubation with media containing low density lipoproteins or high density lipoproteins produced a much smaller decrease in the cell-associated enzymatic activity. The addition of increasing molar ratios of oleic acid:bovine serum albumin to the media also reduced enzyme activity associated with the endothelial cells. To determine whether the decrease in LPL activity was due to release of the enzyme from the cells or inactivation of the enzyme, studies were performed utilizing radioiodinated bovine LPL. Radiolabeled LPL protein was released from endothelial cells by chylomicrons, VLDL, and by free fatty acids (i.e. oleic acid bound to bovine serum albumin). The release of radiolabeled LPL by VLDL correlated with the generation of free fatty acids from the hydrolysis of VLDL triglyceride by LPL bound to the cells. Inhibition of LPL enzymatic activity by use of a specific monoclonal antibody, reduced the extent of release of 125 I-LPL from the endothelial cells by the added VLDL. These results demonstrated that LPL enzymatic activity and protein were removed from endothelial cells by triglyceride-rich lipoproteins (chylomicrons and VLDL) and oleic acid

Functional relevance of G-protein-coupled-receptor-associated proteins, exemplified by receptor-activity-modifying proteins (RAMPs).

Science.gov (United States)

Fischer, J A; Muff, R; Born, W

2002-08-01

The calcitonin (CT) receptor (CTR) and the CTR-like receptor (CRLR) are close relatives within the type II family of G-protein-coupled receptors, demonstrating sequence identity of 50%. Unlike the interaction between CT and CTR, receptors for the related hormones and neuropeptides amylin, CT-gene-related peptide (CGRP) and adrenomedullin (AM) require one of three accessory receptor-activity-modifying proteins (RAMPs) for ligand recognition. An amylin/CGRP receptor is revealed when CTR is co-expressed with RAMP1. When complexed with RAMP3, CTR interacts with amylin alone. CRLR, initially classed as an orphan receptor, is a CGRP receptor when co-expressed with RAMP1. The same receptor is specific for AM in the presence of RAMP2. Together with human RAMP3, CRLR defines an AM receptor, and with mouse RAMP3 it is a low-affinity CGRP/AM receptor. CTR-RAMP1, antagonized preferentially by salmon CT-(8-32) and not by CGRP-(8-37), and CRLR-RAMP1, antagonized by CGRP-(8-37), are two CGRP receptor isotypes. Thus amylin and CGRP interact specifically with heterodimeric complexes between CTR and RAMP1 or RAMP3, and CGRP and AM interact with complexes between CRLR and RAMP1, RAMP2 or RAMP3.

Effect of soy protein on swine intestinal lipoproteins

International Nuclear Information System (INIS)

Ho, H.T.

1987-01-01

Hypocholesterolemic effect of soy protein appears to be the result of reduced cholesterol absorption and enhanced cholesterol excretion. The objective of this study is to delineate the underlying mechanism of soy protein effect on cholesterol absorption. At the end of a 5-week soy-protein or casein diet, swine were subjected to cannulation of mesenteric lymph duct under halothane anesthesia. A single dose of 250 μCi [ 14 C]-cholesterol and 10 mCi [ 3 H]-leucine was infused into the upper jejunum two hours after one-fifth of daily food was given. Then lymph was collected hourly for three hours and the lipoprotein fractions were separated by ultracentrifugation. SDS-PAGE (5%) was used to measure the concentrations of individual apoproteins by densitometric scanning. The three-hour lymphatic transport of cholesterol in casein-fed swine was significantly higher than in those fed soy protein. Triglyceride transports were similar in two groups. The [ 3 H]-leucine incorporation study revealed that transport of apo B-48 bore a significant positive relationship to transport of cholesterol in both chylomicron and VLDL fractions of mesenteric lymph. A greater apo B-48 secretion with higher specific activity was probably responsible for the greater transport of cholesterol in chylomicrons in casein-fed swine. On the other hand, the lesser cholesterol transport in chylomicrons in soy protein-fed swine was probably caused by lower apo B-48 secretion. Similarly, the transport of lymph VLDL cholesterol in swine fed casein or soy protein paralleled the amount of accompanying apo B-48. Dietary proteins probably influence the intestinal synthesis of apo B-48 which in turn affects cholesterol transport into the lymphatics

Accumulation of native and methylated low density lipoproteins by healing rabbit arterial wall

International Nuclear Information System (INIS)

Fischman, A.J.; Lees, A.M.; Lees, R.S.; Barlai-Kovach, M.; Strauss, H.W.

1987-01-01

To determine whether healing arterial wall accumulation of low density lipoproteins (LDL) is mediated by the high affinity LDL receptor, normocholesterolemic rabbits were injected with 125 I-LDL, /sup 99m/Tc-LDL, or the reductively methylated analogs of these compounds ( 125 I-MeLDL, /sup 99m/Tc-MeLDL), 1 month after balloon catheter deendothelialization of the abdominal aorta. If the mechanism of accumulation requires interaction with the LDL receptor, reductively methylated lipoproteins which do not bind to the receptor should not accumulate in healing arterial wall. Twenty-four hours after injection of labelled lipoproteins, each animal was injected with Evans blue dye, in order to distinguish reendothelialized from deendothelialized aorta. One hour after dye injection, the aorta was fixed, removed, divided into abdominal (ballooned) and thoracic (unballooned) regions and counted. For all lipoprotein preparations, there were three to four times as many counts in the abdominal as in the thoracic aorta. En face autoradiographs were made of the aortas that had been exposed to 125 I-labelled lipoproteins. In the autoradiographs, the areas of the lowest activity corresponded to the centers of healing endothelial islands. The most intense radioactivity for both lipoproteins occurred in the region of the leading edge of the endothelial islands where active endothelial regeneration was in progress. The overall distribution of native and MeLDL accumulation was the same. The results suggest that low density lipoproteins are accumulated in areas of active endothelial regeneration by a mechanism that does not involve the high affinity LDL receptor

[A history and review of cholesterol ester transfer protein inhibitors and their contribution to the understanding of the physiology and pathophysiology of high density lipoprotein].

Science.gov (United States)

Corral, Pablo; Schreier, Laura

2014-01-01

There is irrefutable evidence that statins reduce the risk of cardiovascular events in a magnitude proportional to the intensity of the decrease in cholesterol transport by the low density lipoproteins. Despite this great advance there is still a residual risk of cardiovascular events. For this reason, an increase in the levels of high density lipoprotein is considered in order to boost the main action of this lipoprotein, which is reverse cholesterol transport. Distinct classes of evidence (epidemiological, genetic, and pathophysiological) show that the inhibition and/or modulation of cholesterol ester transfer protein increases plasma high density lipoprotein-cholesterol levels. The main reason for presenting this review is to look at the physiology of cholesterol ester transfer protein, its interrelationship with high density lipoproteins, and to give an update on the development of different cholesterol ester transfer protein inhibitor/modulator molecules. Copyright © 2013 Elsevier España, S.L. y SEA. All rights reserved.

Classifying lipoproteins based on their polar profiles.

Science.gov (United States)

Polanco, Carlos; Castañón-González, Jorge Alberto; Buhse, Thomas; Uversky, Vladimir N; Amkie, Rafael Zonana

2016-01-01

The lipoproteins are an important group of cargo proteins known for their unique capability to transport lipids. By applying the Polarity index algorithm, which has a metric that only considers the polar profile of the linear sequences of the lipoprotein group, we obtained an analytical and structural differentiation of all the lipoproteins found in UniProt Database. Also, the functional groups of lipoproteins, and particularly of the set of lipoproteins relevant to atherosclerosis, were analyzed with the same method to reveal their structural preference, and the results of Polarity index analysis were verified by an alternate test, the Cumulative Distribution Function algorithm, applied to the same groups of lipoproteins.

Farnesoid X receptor up-regulates expression of Lipid transfer inhibitor protein in liver cells and mice

Energy Technology Data Exchange (ETDEWEB)

Li, Liangpeng [Department of Biochemistry and Molecular Biology, College of Basic Medical Science, Third Military Medical University, Chongqing 400038 (China); Liu, Hong [Department of Hematology, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Peng, Jiahe; Wang, Yongchao; Zhang, Yan; Dong, Jinyu; Liu, Xiaohua; Guo, Dongmei [Department of Biochemistry and Molecular Biology, College of Basic Medical Science, Third Military Medical University, Chongqing 400038 (China); Jiang, Yu, E-mail: yujiang61@gmail.com [Department of Biochemistry and Molecular Biology, College of Basic Medical Science, Third Military Medical University, Chongqing 400038 (China)

2013-11-29

Highlights: •FXR up-regulates apoF. •It binds to ER1 element. •It activates apoF gene promoter. -- Abstract: Apolipoprotein F is a component protein mainly secreted by liver and resides on several lipoprotein classes. It can inhibit lipids transfer between different lipoproteins. FXR is a member of the nuclear receptor superfamily which is also highly expressed in the liver. It modulates bile acids synthesis and lipids metabolism by transcriptional regulation. We aimed to determine whether apoF can be regulated by FXR. The FXR agonist Chenodeoxycholic acid (CDCA) and GW4064 both can activate the expression of apoF in liver cell lines and in C57/BL6 mouse liver. This is dependent on the binding of FXR to the FXR element ER1 (−2904 to −2892 bp) in the apoF gene promoter. Taken together, we have identified apoF as likely another target gene of FXR.

G-protein coupling of cannabinoid receptors

International Nuclear Information System (INIS)

Glass, M.

2001-01-01

Full text: Since the cloning of the cannabinoid CB1 and CB2 receptors in the early 1990's extensive research has focused on understanding their signal transduction pathways. While it has been known for sometime that both receptors can couple to intracellular signalling via pertussis toxin sensitive G-proteins (Gi/Go), the specificity and kinetics of these interactions have only recently been elucidated. We have developed an in situ reconstitution approach to investigating receptor-G-protein interactions. This approach involves chaotropic extraction of receptor containing membranes in order to inactivate or remove endogenous G-proteins. Recombinant or isolated brain G-proteins can then be added back to the receptors, and their activation monitored through the binding of [ 35 S]-GTPγS. This technique has been utilised for an extensive study of cannabinoid receptor mediated activation of G-proteins. In these studies we have established that CB1 couples with high affinity to both Gi and Go type G-proteins. In contrast, CB2 couples strongly to Gi, but has a very low affinity for Go. This finding correlated well with the previous findings that while CB1 and CB2 both couple to the inhibition of adenylate cyclase, CB1 but not CB2 could also inhibit calcium channels. We then examined the ability of a range of cannabinoid agonists to activate the Gi and Go via CB1. Conventional receptor theory suggests that a receptor is either active or inactive with regard to a G-protein and that the active receptor activates all relevant G-proteins equally. However, in this study we found that agonists could produce different degrees of activation, depending on which G-protein was present. Further studies have compared the ability of the two endocannabinoids to drive the activation of Gi or Go. These studies show that agonists can induce multiple forms of activated receptor that differ in their ability to catalyse the activation of Gi or Go. The ability of an agonist to drive a receptor

Lactoferrin binding protein B - a bi-functional bacterial receptor protein.

Directory of Open Access Journals (Sweden)

Nicholas K H Ostan

2017-03-01

Full Text Available Lactoferrin binding protein B (LbpB is a bi-lobed outer membrane-bound lipoprotein that comprises part of the lactoferrin (Lf receptor complex in Neisseria meningitidis and other Gram-negative pathogens. Recent studies have demonstrated that LbpB plays a role in protecting the bacteria from cationic antimicrobial peptides due to large regions rich in anionic residues in the C-terminal lobe. Relative to its homolog, transferrin-binding protein B (TbpB, there currently is little evidence for its role in iron acquisition and relatively little structural and biophysical information on its interaction with Lf. In this study, a combination of crosslinking and deuterium exchange coupled to mass spectrometry, information-driven computational docking, bio-layer interferometry, and site-directed mutagenesis was used to probe LbpB:hLf complexes. The formation of a 1:1 complex of iron-loaded Lf and LbpB involves an interaction between the Lf C-lobe and LbpB N-lobe, comparable to TbpB, consistent with a potential role in iron acquisition. The Lf N-lobe is also capable of binding to negatively charged regions of the LbpB C-lobe and possibly other sites such that a variety of higher order complexes are formed. Our results are consistent with LbpB serving dual roles focused primarily on iron acquisition when exposed to limited levels of iron-loaded Lf on the mucosal surface and effectively binding apo Lf when exposed to high levels at sites of inflammation.

Plasma lipoprotein(a) levels in patients with homozygous autosomal dominant hypercholesterolemia

NARCIS (Netherlands)

Sjouke, B.; Yahya, R.; Tanck, M.W.T.; Defesche, J.C.; Graaf, J. de; Wiegman, A.; Kastelein, J.J.; Mulder, M.T.; Hovingh, G.K.; Roeters van Lennep, J.E.

2017-01-01

BACKGROUND: Patients with autosomal dominant hypercholesterolemia (ADH), caused by mutations in either low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), or proprotein convertase subtilisin-kexin type 9 (PCSK9) are characterized by high low-density lipoprotein cholesterol levels and

Plasma lipoprotein(a) levels in patients with homozygous autosomal dominant hypercholesterolemia

NARCIS (Netherlands)

Sjouke, Barbara; Yahya, Reyhana; Tanck, Michael W. T.; Defesche, Joep C.; de Graaf, Jacqueline; Wiegman, Albert; Kastelein, John J. P.; Mulder, Monique T.; Hovingh, G. Kees; Roeters van Lennep, Jeanine E.

2017-01-01

Patients with autosomal dominant hypercholesterolemia (ADH), caused by mutations in either low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), or proprotein convertase subtilisin-kexin type 9 (PCSK9) are characterized by high low-density lipoprotein cholesterol levels and in some

Flow-mediated lipoprotein retention; Pregnancy-associated plasma protein-A inhibition in atherosclerosis and its release during myocardial ischemia

DEFF Research Database (Denmark)

Steffensen, Lasse Bach

, which states that retention of lipoproteins is the key initiating event of atherosclerosis development. Pregnancy-associated plasma protein-A (PAPP-A) regulates the insulin-like growth factor (IGF) system by proteolytic cleavage of a subset of IGF-binding proteins. Studies in mice have established PAPP...

Androgen and FSH synergistically stimulate lipoprotein degradation and utilization by ovary granulosa cells

International Nuclear Information System (INIS)

Schreiber, J.R.; Nakamura, K.; Schmit, V.; Weinstein, D.B.

1984-01-01

Androgen can directly modulate the induction of steroidogenic enzymes by FSH (follicle stimulating hormone) in ovary granulosa cells. In studies of its mechanism of action, the authors examined the androgen effect on granulosa cell interaction with lipoproteins, the physiologic source of cholesterol. After granulosa cells were cultured for 48 hours with and without androgen and/or FSH, the cells were incubated for 24 hours with 125 I-lipoproteins [human high density lipoprotein (HDL), rat HDL, or human low density lipoprotein (LDL)]. The media were then analyzed for lipoprotein protein coat degradation products (mainly 125 I-monoiodotyrosine) and progestin [mainly 20 alpha-dihydroprogesterone (20 alpha-DHP)]. In the absence of FSH and androgen, 2 X 10(5) granulosa cells degraded basal levels of all three lipoproteins, but produced no measurable 20 alpha-DHP. The addition of 10(-7) M androstenedione (A), testosterone (T), or 5 alpha-dihydrotestosterone (DHT) had no effect on lipoprotein protein degradation or 20 alpha-DHP production. FSH alone stimulated lipoprotein protein degradation by 50 to 300% while the addition of androgen synergistically augmented the FSH-stimulated 20 alpha-DHP production as well as protein coat degradation of all three lipoproteins. DHT and T were both effective, indicating that androgens themselves, and not estrogen products, were responsible for the effect on lipoprotein protein degradation and 20 alpha-DHP production

Lipoproteins comprise at least 10 different classes in rats, each of which contains a unique set of proteins as the primary component.

Science.gov (United States)

Konishi, Tomokazu; Takahashi, Yoko

2018-01-01

Although lipoproteins are conventionally separated into a few classes using density gradient centrifugation, there may be a much higher number of physical classes that differ in origin or phase. Comprehensive knowledge of the classes of lipoproteins is rather limited, which hinders both the study of their functions and the identification of the primary causes of related diseases. This study aims to determine the number of classes of lipoproteins that can be practically distinguishable and identify the differences between them. We separated rat serum samples by gel filtration. The elution was continuously monitored for triglyceride (TG), cholesterol, and protein, and fractionated for further SDS-PAGE and immunological detection of apoprotein A-I (ApoA1) and apoprotein B (ApoB). The elution patterns were analyzed using a parsimonious method, i.e., the estimation of the least number of classes. Ten classes were recognized that contained different amounts of TG and cholesterol, as well as a unique protein content. Each of the classes contained much more protein than that observed previously, especially in low-density lipoproteins (LDL) classes. In particular, two major antiproteases formed complexes with specific classes of LDL; because these classes exclusively carry cholesterol and antiproteases, they may lead to the progression of atheroma by supplying materials that enlarge fatty streaks and protecting thrombi from enzymatic digestion. The separated classes may have specific biological functions. The attribution of protein species to certain classes will help understand the functions. A distinction among lipoprotein classes may provide important information in the field of vascular pathology.

The Acylation State of Surface Lipoproteins of Mollicute Acholeplasma laidlawii*

Science.gov (United States)

Serebryakova, Marina V.; Demina, Irina A.; Galyamina, Maria A.; Kondratov, Ilya G.; Ladygina, Valentina G.; Govorun, Vadim M.

2011-01-01

Acylation of the N-terminal Cys residue is an essential, ubiquitous, and uniquely bacterial posttranslational modification that allows anchoring of proteins to the lipid membrane. In Gram-negative bacteria, acylation proceeds through three sequential steps requiring lipoprotein diacylglyceryltransferase, lipoprotein signal peptidase, and finally lipoprotein N-acyltransferase. The apparent lack of genes coding for recognizable homologs of lipoprotein N-acyltransferase in Gram-positive bacteria and Mollicutes suggests that the final step of the protein acylation process may be absent in these organisms. In this work, we monitored the acylation state of eight major lipoproteins of the mollicute Acholeplasma laidlawii using a combination of standard two-dimensional gel electrophoresis protein separation, blotting to nitrocellulose membranes, and MALDI-MS identification of modified N-terminal tryptic peptides. We show that for each A. laidlawii lipoprotein studied a third fatty acid in an amide linkage on the N-terminal Cys residue is present, whereas diacylated species were not detected. The result thus proves that A. laidlawii encodes a lipoprotein N-acyltransferase activity. We hypothesize that N-acyltransferases encoded by genes non-homologous to N-acyltransferases of Gram-negative bacteria are also present in other mollicutes and Gram-positive bacteria. PMID:21540185

Large-scale preparation of the homogeneous LolA–lipoprotein complex and efficient in vitro transfer of lipoproteins to the outer membrane in a LolB-dependent manner

OpenAIRE

Watanabe, Shoji; Oguchi, Yuki; Yokota, Naoko; Tokuda, Hajime

2007-01-01

An ATP-binding cassette transporter LolCDE complex of Escherichia coli releases lipoproteins destined to the outer membrane from the inner membrane as a complex with a periplasmic chaperone, LolA. Interaction of the LolA–lipoprotein complex with an outer membrane receptor, LolB, then causes localization of lipoproteins to the outer membrane. As far as examined, formation of the LolA–lipoprotein complex strictly depends on ATP hydrolysis by the LolCDE complex in the presence of LolA. It has be...

Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2.

Science.gov (United States)

Li, Qiong; Kumar, Ashok; Gui, Jian-Fang; Yu, Fu-Shin X

2008-05-01

Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-kappaB, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-kappaB activation in HUCL cells after lipoprotein lipase treatment and in cell lines expressing TLR4 or TLR9, but not TLR2, indicating lipoprotein nature of the extracts. saLP induced the up-regulation of a variety of inflammatory cytokines and chemokines (IL-6, IL-8, ICAM-1), antimicrobial molecules (hBD-2, LL-37, and iNOS), and homeostasis genes (Mn-SOD) at both the mRNA level and protein level. Similar inflammatory response to saLP was also observed in primarily cultured HCECs using the production of IL-6 as readout. Moreover, TLR2 neutralizing antibody blocked the saLP-induced secretion of IL-6, IL-8 and hBD2 in HUCL cells. Our findings suggest that saLP activates TLR2 and triggers innate immune response in the cornea to S. aureus infection via production of proinflammatory cytokines and defense molecules.

Essential oil of Pinus koraiensis leaves exerts antihyperlipidemic effects via up-regulation of low-density lipoprotein receptor and inhibition of acyl-coenzyme A: cholesterol acyltransferase.

Science.gov (United States)

Kim, Ji-Hyun; Lee, Hyo-Jung; Jeong, Soo-Jin; Lee, Min-Ho; Kim, Sung-Hoon

2012-09-01

Hyperlipidemia is an important factor to induce metabolic syndrome such as obesity, diabetes and cardiovascular diseases. Recently, some antihyperlipidemic agents from herbal medicines have been in the spotlight in the medical science field. Thus, the present study evaluated the antihyperlipidemic activities of the essential oil from the leaves of Pinus koraiensis SIEB (EOPK) that has been used as a folk remedy for heart disease. The reverse transcription polymerase chain reaction (RT-PCR) revealed that EOPK up-regulated low density lipoprotein receptor (LDLR) at the mRNA level as well as negatively suppressed the expression of sterol regulatory element-binding protein (SREBP)-1c, SREBP-2, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), fatty acid synthase (FAS) and glycerol-3-phosphate acyltransferase (GPAT) involved in lipid metabolism in HepG2 cells. Also, western blotting showed that EOPK activated LDLR and attenuated the expression of FAS at the protein level in the cells. Consistently, EOPK significantly inhibited the level of human acylcoenzyme A: cholesterol acyltransferase (hACAT)1 and 2 and reduced the low-density lipoprotein (LDL) oxidation activity. Furthermore, chromatography-mass spectrometry (GC-MS) analysis showed that EOPK, an essential oil mixture, contained camphene (21.11%), d-limonene (21.01%), α-pinene (16.74%) and borneol (11.52%). Overall, the findings suggest that EOPK can be a potent pharmaceutical agent for the prevention and treatment of hyperlipidemia. Copyright © 2012 John Wiley & Sons, Ltd.

Lectin-like oxidized low-density lipoprotein receptor-1 promotes endothelial dysfunction in LDL receptor knockout background.

Science.gov (United States)

Hofmann, Anja; Brunssen, Coy; Poitz, David M; Langbein, Heike; Strasser, Ruth H; Henle, Thomas; Ravens, Ursula; Morawietz, Henning

2017-11-01

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is the major receptor for oxidized LDL in endothelial cells. LOX-1 is highly expressed in atherosclerotic plaques. The impact of LOX-1 on development of endothelial dysfunction in large vessels in absence or presence of atherosclerosis-prone conditions has not been studied to date. Mice with endothelial cell-specific LOX-1 overexpression (bLOX-1tg) were analyzed. Wild-type (WT) mice served as controls. In addition, bLOX-1tg mice were crossed with LDL receptor knockout (Ldlr -/- ) mice. All mice were fed a western-type diet (WD) or control diet (CD) for 20 weeks. Afterwards, endothelial function was analyzed ex vivo in thoracic aortas using a Mulvany myograph. WD induced hypertriglyceridemia (bLOX-1tg: 1.6-fold; WT: 1.4-fold) and hypercholesterolemia (P LDL-cholesterol (∼9-fold) compared to WT and bLOX-1tg mice on WD. Endothelial function in response to WD was impaired in bLOX-1tg/Ldlr -/- mice (Eff max : 56.7 ± 23.0%) compared to WT (Eff max : 88.2 ± 15.8%, P < 0.001), bLOX-1tg (Eff max : 76.7 ± 12.9%, P < 0.05) and Ldlr -/- mice (Eff max : 70.1 ± 13.1%, P < 0.05). No differences between WT, bLOX-1tg and Ldlr -/- mice were detectable when comparing all genotypes. Endothelial LOX-1 overexpression in an atherosclerosis-prone background impairs endothelial function, proving its importance in the development of atherosclerosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Lipoprotein metabolism in familial hypercholesterolemia: Serial assessment using a one-step ultracentrifugation method

Directory of Open Access Journals (Sweden)

Hayato Tada

2015-04-01

Full Text Available Objectives: It is well known that familial hypercholesterolemia (FH is a common inherited disorder that can markedly elevate the level of plasma LDL cholesterol. However, little data exists regarding the clinical impact of the plasma triglyceride (TG-rich lipoprotein fraction, including VLDL and IDL, in FH. Thus, we assessed the hypothesis that the mutations in the LDL receptor modulate lipoprotein metabolism other than the LDL fraction. Design and methods: We investigated plasma lipoprotein with a one-step ultracentrifugation method for 146 controls (mean age=61.4±17.1 yr, mean LDL cholesterol=92.7±61.2 mg/dl, 213 heterozygous mutation-determined FH subjects (mean age=46.0±18.0 yr, mean LDL cholesterol=225.1±61.2 mg/dl, and 16 homozygous/compound heterozygous mutation-determined FH subjects (mean age=26.9±17.1 yr, mean LDL cholesterol=428.6±86.1 mg/dl. In addition, we evaluated cholesterol/TG ratio in each lipoprotein fraction separated by ultracentrifugation. Results: In addition to total cholesterol and LDL cholesterol levels, VLDL cholesterol (19.5±10.4, 25.2±19.3, 29.5±21.4 mg/dl, respectively and IDL cholesterol (8.3±3.7, 16.8±11.5, 40.0±37.3 mg/dl, respectively exhibited a tri-model distribution according to their status in LDL receptor mutation(s. Moreover, the ratios of cholesterol/TG of each lipoprotein fraction increased significantly in heterozygous FH and homozygous/compound heterozygous FH groups, compared with that of controls, suggesting that the abnormality in LDL receptor modulates the quality as well as the quantity of each lipoprotein fraction. Conclusions: Our results indicate that cholesterol in TG-rich lipoproteins, including VLDL and IDL, are significantly higher in FH subjects, revealing a tri-modal distribution according to the number of LDL receptor mutations. Keywords: LDL cholesterol, Familial hypercholesterolemia, Ultracentrifugation, Lipoprotein

Changes in plasma low-density lipoprotein (LDL)- and high-density lipoprotein cholesterol in hypo- and hyperthyroid patients are related to changes in free thyroxine, not to polymorphisms in LDL receptor or cholesterol ester transfer protein genes

NARCIS (Netherlands)

Diekman, M. J.; Anghelescu, N.; Endert, E.; Bakker, O.; Wiersinga, W. M.

2000-01-01

Thyroid function disorders lead to changes in lipoprotein metabolism. Both plasma low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) increase in hypothyroidism and decrease in hyperthyroidism. Changes in LDL-C relate to altered clearance of LDL particles

Association of polymorphisms in low-density lipoprotein receptor- related protein 5 gene with bone mineral density in postmenopausal Chinese women

Institute of Scientific and Technical Information of China (English)

Zhen-lin ZHANG; Yue-juan QIN; Jin-wei HE; Qi-ren HUANG; Miao LI; Yun-qiu HU; Yu-juan LIU

2005-01-01

Aim: To investigate the possible association of Q89R, N740N and A1330V polymorphisms in low-density lipoprotein receptor-related protein 5 (LRP5) gene with bone mineral density (BMD) in postmenopausal Chinese women. Methods: Q89R,N740N and A1330V genotypes were determined by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) in 647 unrelated healthy postmenopausal Han Chinese women aged 43-76 years in Shanghai. BMD at lumbar spine 1-4 and the left proximal femur including the femoral neck, trochanter and Ward's triangle were measured by dual-energy X-ray absorptionmetry in all subjects. Results: The distribution of the Q89R, N740N and A1330V genotypes in this population was as follows: QQ 80.5%, QR 18.7%, and RR 0.8%; TT 66.9%,TC 31.1%, and CC 2.0%; AA 68.0%, AV 29.7%, and VV 2.3%. The frequencies of the Q89R, N740N and A1330V genotypes and alleles did not deviate from the HardyWeinberg equilibrium. We found that the Q89R and A1330V polymorphisms were in linkage disequilibrium in our population (x2= 13.50, P＜0.01). Both before and after adjusting for age, years since menopause, height, and weight, the Q89R or N740N genotypes were significantly associated with BMD at the femoral neck (P＜0.05).Subjects with the Q89R QQ genotype or the N740N TT genotype had a significantly higher BMD at the femoral neck, compared with those with the QR/RR or TC/CC genotypes, respectively. No significant association was found between A1330V polymorphism and BMD at any site. Conclusion: Our findings suggest that the LRP5 gene is a candidate for the genetic determination of BMD in postmenopausal Chinese women.

Ultrasound-targeted microbubble destruction improves the low density lipoprotein receptor gene expression in HepG2 cells

International Nuclear Information System (INIS)

Guo Dongping; Li Xiaoyu; Sun, Ping; Tang Yibo; Chen Xiuying; Chen Qi; Fan Leming; Zang Bin; Shao Lizheng; Li Xiaorong

2006-01-01

Ultrasound-targeted microbubble destruction had been employed in gene delivery and promised great potential. Liver has unique features that make it attractive for gene therapy. However, it poses formidable obstacles to hepatocyte-specific gene delivery. This study was designed to test the efficiency of therapeutic gene transfer and expression mediated by ultrasound/microbubble strategy in HepG 2 cell line. Air-filled albumin microbubbles were prepared and mixed with plasmid DNA encoding low density lipoprotein receptor (LDLR) and green fluorescent protein. The mixture of the DNA and microbubbles was administer to cultured HepG 2 cells under variable ultrasound conditions. Transfection rate of the transferred gene and cell viability were assessed by FACS analysis, confocal laser scanning microscopy, Western blot analysis and Trypan blue staining. The result demonstrated that microbubbles with ultrasound irradiation can significantly elevate exogenous LDLR gene expression and the expressed LDLRs were functional and active to uptake their ligands. We conclude that ultrasound-targeted microbubble destruction has the potential to promote safe and efficient LDLR gene transfer into hepatocytes. With further refinement, it may represent an effective nonviral avenue of gene therapy for liver-involved genetic diseases

Germinated Brown Rice Attenuates Atherosclerosis and Vascular Inflammation in Low-Density Lipoprotein Receptor-Knockout Mice.

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Zhao, Ruozhi; Ghazzawi, Nora; Wu, Jiansu; Le, Khuong; Li, Chunyang; Moghadasian, Mohammed H; Siow, Yaw L; Apea-Bah, Franklin B; Beta, Trust; Yin, Zhengfeng; Shen, Garry X

2018-05-02

The present study investigates the impact of germinated brown rice (GBR) on atherosclerosis and the underlying mechanism in low-density lipoprotein receptor-knockout (LDLr-KO) mice. The intensity of atherosclerosis in aortas of LDLr-KO mice receiving diet supplemented with 60% GBR (weight/weight) was significantly less than that in mice fed with 60% white rice (WR) or control diet ( p mice fed with WR diet was significantly more than that from mice receiving the control diet ( p mice in comparison to the WR diet ( p mice compared to WR. The anti-atherosclerotic effect of GBR in LDLr-KO mice at least in part results from its anti-inflammatory activity.

A high-fat diet and the threonine-encoding allele (Thr54) polymorphism of fatty acid–binding protein 2 reduce plasma triglyceride–rich lipoproteins

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The Thr54 allele of the fatty acid binding protein 2 (FABP2) DNA polymorphism is associated with increased triglyceride-rich lipoproteins and insulin resistance. We investigated whether the triglyceride-rich lipoprotein response to diets of varied fat content is affected by the fatty acid binding pr...

Analyzing the molecular mechanism of lipoprotein localization in Brucella.

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Goolab, Shivani; Roth, Robyn L; van Heerden, Henriette; Crampton, Michael C

2015-01-01

Bacterial lipoproteins possess diverse structure and functionality, ranging from bacterial physiology to pathogenic processes. As such many lipoproteins, originating from Brucella are exploited as potential vaccines to countermeasure brucellosis infection in the host. These membrane proteins are translocated from the cytoplasm to the cell membrane where they are anchored peripherally by a multifaceted targeting mechanism. Although much research has focused on the identification and classification of Brucella lipoproteins and their potential use as vaccine candidates for the treatment of Brucellosis, the underlying route for the translocation of these lipoproteins to the outer surface of the Brucella (and other pathogens) outer membrane (OM) remains mostly unknown. This is partly due to the complexity of the organism and evasive tactics used to escape the host immune system, the variation in biological structure and activity of lipoproteins, combined with the complex nature of the translocation machinery. The biosynthetic pathway of Brucella lipoproteins involves a distinct secretion system aiding translocation from the cytoplasm, where they are modified by lipidation, sorted by the lipoprotein localization machinery pathway and thereafter equipped for export to the OM. Surface localized lipoproteins in Brucella may employ a lipoprotein flippase or the β-barrel assembly complex for translocation. This review provides an overview of the characterized Brucella OM proteins that form part of the OM, including a handful of other characterized bacterial lipoproteins and their mechanisms of translocation. Lipoprotein localization pathways in gram negative bacteria will be used as a model to identify gaps in Brucella lipoprotein localization and infer a potential pathway. Of particular interest are the dual topology lipoproteins identified in Escherichia coli and Haemophilus influenza. The localization and topology of these lipoproteins from other gram negative bacteria

Analyzing the molecular mechanism of lipoprotein localization in Brucella

Science.gov (United States)

Goolab, Shivani; Roth, Robyn L.; van Heerden, Henriette; Crampton, Michael C.

2015-01-01

Bacterial lipoproteins possess diverse structure and functionality, ranging from bacterial physiology to pathogenic processes. As such many lipoproteins, originating from Brucella are exploited as potential vaccines to countermeasure brucellosis infection in the host. These membrane proteins are translocated from the cytoplasm to the cell membrane where they are anchored peripherally by a multifaceted targeting mechanism. Although much research has focused on the identification and classification of Brucella lipoproteins and their potential use as vaccine candidates for the treatment of Brucellosis, the underlying route for the translocation of these lipoproteins to the outer surface of the Brucella (and other pathogens) outer membrane (OM) remains mostly unknown. This is partly due to the complexity of the organism and evasive tactics used to escape the host immune system, the variation in biological structure and activity of lipoproteins, combined with the complex nature of the translocation machinery. The biosynthetic pathway of Brucella lipoproteins involves a distinct secretion system aiding translocation from the cytoplasm, where they are modified by lipidation, sorted by the lipoprotein localization machinery pathway and thereafter equipped for export to the OM. Surface localized lipoproteins in Brucella may employ a lipoprotein flippase or the β-barrel assembly complex for translocation. This review provides an overview of the characterized Brucella OM proteins that form part of the OM, including a handful of other characterized bacterial lipoproteins and their mechanisms of translocation. Lipoprotein localization pathways in gram negative bacteria will be used as a model to identify gaps in Brucella lipoprotein localization and infer a potential pathway. Of particular interest are the dual topology lipoproteins identified in Escherichia coli and Haemophilus influenza. The localization and topology of these lipoproteins from other gram negative bacteria

Chimeric RXFP1 and RXFP2 receptors highlight the similar mechanism of activation utilizing their N-terminal low density lipoprotein class A modules

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Shoni eBruell

2013-11-01

Full Text Available Relaxin family peptide (RXFP receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low density lipoprotein type A (LDLa module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are potentially involved in hydrophobic interactions with the receptor to drive activation. RXFP2 shares two out of three of the residues implicated, suggesting that the two LDLa modules could be interchanged without adversely affecting activity. However, in 2007 it was shown that a chimera consisting of the RXFP1 receptor with its LDLa swapped for that of RXFP2 did not signal. We noticed this construct also contained the RXFP2 region linking the LDLa to the leucine-rich repeats. We therefore constructed chimeric RXFP1 and RXFP2 receptors with their LDLa modules swapped immediately C-terminally to the final cysteine residue of the module, retaining the native linker. In addition, we exchanged the TM domains of the chimeras to explore if matching the LDLa module with the TM domain of its native receptor altered activity. All of the chimeras were expressed at the surface of HEK293T cells with ligand binding profiles similar to the wild-type receptors. Importantly, as predicted, ligand binding was able to induce cAMP based signalling. Chimeras of RXFP1 with the LDLa of RXFP2 demonstrated reduced H2 relaxin potency with the pairing of the RXFP2 TM with the RXFP2 LDLa necessary for full ligand efficacy. In contrast the ligand mediated potencies and efficacies on the RXFP2 chimeras were similar suggesting the RXFP1 LDLa module has similar efficacy on the RXFP2 TM domain. Our studies demonstrate the LDLa modules of RXFP1 and RXFP2 modulate receptor activation via a

Comparative transcriptional analysis of Bacillus subtilis cells overproducing either secreted proteins, lipoproteins or membrane proteins

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Marciniak Bogumiła C

2012-05-01

Full Text Available Abstract Background Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe status, its genetic accessibility and its capacity to grow in large fermentations. However, production of heterologous proteins still faces limitations. Results This study aimed at the identification of bottlenecks in secretory protein production by analyzing the response of B. subtilis at the transcriptome level to overproduction of eight secretory proteins of endogenous and heterologous origin and with different subcellular or extracellular destination: secreted proteins (NprE and XynA of B. subtilis, Usp45 of Lactococcus lactis, TEM-1 β-lactamase of Escherichia coli, membrane proteins (LmrA of L. lactis and XylP of Lactobacillus pentosus and lipoproteins (MntA and YcdH of B. subtilis. Responses specific for proteins with a common localization as well as more general stress responses were observed. The latter include upregulation of genes encoding intracellular stress proteins (groES/EL, CtsR regulated genes. Specific responses include upregulation of the liaIHGFSR operon under Usp45 and TEM-1 β-lactamase overproduction; cssRS, htrA and htrB under all secreted proteins overproduction; sigW and SigW-regulated genes mainly under membrane proteins overproduction; and ykrL (encoding an HtpX homologue specifically under membrane proteins overproduction. Conclusions The results give better insights into B. subtilis responses to protein overproduction stress and provide potential targets for genetic engineering in order to further improve B. subtilis as a protein production host.

Protein Connectivity in Chemotaxis Receptor Complexes.

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Stephan Eismann

2015-12-01

Full Text Available The chemotaxis sensory system allows bacteria such as Escherichia coli to swim towards nutrients and away from repellents. The underlying pathway is remarkably sensitive in detecting chemical gradients over a wide range of ambient concentrations. Interactions among receptors, which are predominantly clustered at the cell poles, are crucial to this sensitivity. Although it has been suggested that the kinase CheA and the adapter protein CheW are integral for receptor connectivity, the exact coupling mechanism remains unclear. Here, we present a statistical-mechanics approach to model the receptor linkage mechanism itself, building on nanodisc and electron cryotomography experiments. Specifically, we investigate how the sensing behavior of mixed receptor clusters is affected by variations in the expression levels of CheA and CheW at a constant receptor density in the membrane. Our model compares favorably with dose-response curves from in vivo Förster resonance energy transfer (FRET measurements, demonstrating that the receptor-methylation level has only minor effects on receptor cooperativity. Importantly, our model provides an explanation for the non-intuitive conclusion that the receptor cooperativity decreases with increasing levels of CheA, a core signaling protein associated with the receptors, whereas the receptor cooperativity increases with increasing levels of CheW, a key adapter protein. Finally, we propose an evolutionary advantage as explanation for the recently suggested CheW-only linker structures.

Self-assembling peptide and protein nanodiscs for studies of membrane proteins

DEFF Research Database (Denmark)

Midtgaard, Søren Roi

Particles containing both lipids and proteins (so-called lipoproteins) are vital to study. They are selfassembling particles that, in the human body, are responsible for the transport of lipids and cholesterol. Due to the increasing problems of obesity and related illnesses in the world, obtaining...... more knowledge about the cholesterol and lipid metabolism is paramount. As an example, in 2012, cardiovascular disease was still the main cause of death in the U.S. This means that the study of lipoproteins is not only of pure academic interest but vital to current world problems. Another reason...... of proteins encoded by the human genome. G-protein coupled receptors mediate the majority of hormone and neurotransmitter signals as well as being responsible for perception of light, smell and taste in the human body, and a number of Nobel prizes has been awarded based on their study. Structural...

The role of lipolysis stimulated lipoprotein receptor in breast cancer and directing breast cancer cell behavior.

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Denise K Reaves

Full Text Available The claudin-low molecular subtype of breast cancer is of particular interest for clinically the majority of these tumors are poor prognosis, triple negative, invasive ductal carcinomas. Claudin-low tumors are characterized by cancer stem cell-like features and low expression of cell junction and adhesion proteins. Herein, we sought to define the role of lipolysis stimulated lipoprotein receptor (LSR in breast cancer and cancer cell behavior as LSR was recently correlated with tumor-initiating features. We show that LSR was expressed in epithelium, endothelium, and stromal cells within the healthy breast tissue, as well as in tumor epithelium. In primary breast tumor bioposies, LSR expression was significantly correlated with invasive ductal carcinomas compared to invasive lobular carcinomas, as well as ERα positive tumors and breast cancer cell lines. LSR levels were significantly reduced in claudin-low breast cancer cell lines and functional studies illustrated that re-introduction of LSR into a claudin-low cell line suppressed the EMT phenotype and reduced individual cell migration. However, our data suggest that LSR may promote collective cell migration. Re-introduction of LSR in claudin-low breast cancer cell lines reestablished tight junction protein expression and correlated with transepithelial electrical resistance, thereby reverting claudin-low lines to other intrinsic molecular subtypes. Moreover, overexpression of LSR altered gene expression of pathways involved in transformation and tumorigenesis as well as enhanced proliferation and survival in anchorage independent conditions, highlighting that reestablishment of LSR signaling promotes aggressive/tumor initiating cell behaviors. Collectively, these data highlight a direct role for LSR in driving aggressive breast cancer behavior.

Scavenger Receptor Structure and Function in Health and Disease

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Izma Abdul Zani

2015-05-01

Full Text Available Scavenger receptors (SRs are a ‘superfamily’ of membrane-bound receptors that were initially thought to bind and internalize modified low-density lipoprotein (LDL, though it is currently known to bind to a variety of ligands including endogenous proteins and pathogens. New family of SRs and their properties have been identified in recent years, and have now been classified into 10 eukaryote families, defined as Classes A-J. These receptors are classified according to their sequences, although in each class they are further classified based in the variations of the sequence. Their ability to bind a range of ligands is reflected on the biological functions such as clearance of modified lipoproteins and pathogens. SR members regulate pathophysiological states including atherosclerosis, pathogen infections, immune surveillance, and cancer. Here, we review our current understanding of SR structure and function implicated in health and disease.

In vivo regulation of scavenger receptor BI and the selective uptake of high density lipoprotein cholesteryl esters in rat liver parenchymal and Kupffer cells

NARCIS (Netherlands)

Fluiter, K.; van der Westhuijzen, D. R.; van Berkel, T. J.

1998-01-01

High density lipoprotein cholesteryl esters (HDL-CE) are selectively taken up by liver parenchymal cells without parallel apolipoprotein uptake. This selective uptake route forms an important step in the so-called reverse cholesterol transport. Scavenger receptor BI (SR-BI) is the only known HDL

ELEVATED CHOLESTERYL ESTER TRANSFER PROTEIN-ACTIVITY IN IDDM MEN WHO SMOKE - POSSIBLE FACTOR FOR UNFAVORABLE LIPOPROTEIN PROFILE

NARCIS (Netherlands)

DULLAART, RPF; GROENER, JEM; DIKKESCHEI, BD; ERKELENS, DW; DOORENBOS, H

Objectives: To determine the effect of cigarette smoking on the activity of cholesteryl ester transfer protein (CETP) and high-density (HDL), low-density (LDL), and very-low-density (VLDL) lipoproteins in insulin-dependent diabetic (IDDM) men with microvascular complications. Research Design and

Characterization of the Pseudomonas aeruginosa Lol system as a lipoprotein sorting mechanism.

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Tanaka, Shin-Ya; Narita, Shin-Ichiro; Tokuda, Hajime

2007-05-04

Escherichia coli lipoproteins are localized to either the inner or the outer membrane depending on the residue that is present next to the N-terminal acylated Cys. Asp at position 2 causes the retention of lipoproteins in the inner membrane. In contrast, the accompanying study (9) revealed that the residues at positions 3 and 4 determine the membrane specificity of lipoproteins in Pseudomonas aeruginosa. Since the five Lol proteins involved in the sorting of E. coli lipoproteins are conserved in P. aeruginosa, we examined whether or not the Lol proteins of P. aeruginosa are also involved in lipoprotein sorting but utilize different signals. The genes encoding LolCDE, LolA, and LolB homologues were cloned and expressed. The LolCDE homologue thus purified was reconstituted into proteoliposomes with lipoproteins. When incubated in the presence of ATP and a LolA homologue, the reconstituted LolCDE homologue released lipoproteins, leading to the formation of a LolA-lipoprotein complex. Lipoproteins were then incorporated into the outer membrane depending on a LolB homologue. As revealed in vivo, lipoproteins with Lys and Ser at positions 3 and 4, respectively, remained in proteoliposomes. On the other hand, E. coli LolCDE released lipoproteins with this signal and transferred them to LolA of not only E. coli but also P. aeruginosa. These results indicate that Lol proteins are responsible for the sorting of lipoproteins to the outer membrane of P. aeruginosa, as in the case of E. coli, but respond differently to inner membrane retention signals.

Gonadal cell surface receptor for plasma retinol-binding protein

International Nuclear Information System (INIS)

Krishna Bhat, M.; Cama, H.R.

1979-01-01

A specific membrane receptor for plasma retinol-binding protein has been demonstrated in testicular cells. Prealbumin-2 did not show any specific binding to the membrane. The affinity of retinol-binding protein for receptor drastically decreases upon delivery of retinol and the retinol-binding protein does not enter the cell. The mechanism of delivery of retinol to the target cell by plasma retinol-binding protein has been investigated. The process involves two steps; direct binding of retinol-binding protein to the receptor and uptake of retinol by the target cell with a concomitant drastic reduction in the affinity of the retinol-binding protein to the receptor. Probably the second step of the process needs a cytosolic factor, possibly the cellular retinol-binding protein or an enzyme. The binding of retinol-binding protein to the receptor is saturable and reversible. The interaction shows a Ksub(d) value of 2.1x10 -10 . The specific binding of a retinol-binding protein with great affinity has been employed in the development of a method for radioassay of the receptor. The receptor level of the gonadal cell has been found to vary with the stage of differentiation. The receptor concentrations in 11-week-old birds and adult birds are comparable. Testosterone treatment of 11-week-old birds produced a substantial increase in the receptor concentration over control, while the protein content increased marginally, indicating that, probably, synthesis of the receptor is specifcally induced by testosterone during spermatogenesis, and the concentration of receptor is relatively higher before the formation of the acrosome. (Auth.)

Pyrene-Labeled Amphiphiles: Dynamic And Structural Probes Of Membranes And Lipoproteins

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Pownall, Henry J.; Homan, Reynold; Massey, John B.

1987-01-01

Lipids and proteins are important functional and structural components of living organisms. Although proteins are frequently found as soluble components of plasma or the cell cytoplasm, many lipids are much less soluble and separate into complex assemblies that usually contain proteins. Cell membranes and plasma lipoproteins' are two important macro-molecular assemblies that contain both lipids and proteins. Cell membranes are composed of a variety of lipids and proteins that form an insoluble bilayer array that has relatively little curvature over distances of several nm. Plasma lipoproteins are different in that they are much smaller, water-soluble, and have highly curved surfaces. A model of a high density lipoprotein (HDL) is shown in Figure 1. This model (d - 10 nm) contains a surface of polar lipids and proteins that surrounds a small core of insoluble lipids, mostly triglycerides and cholesteryl esters. The low density (LDL) (d - 25 nm) and very low density (VLDL) (d 90 nm) lipoproteins have similar architectures, except the former has a cholesteryl ester core and the latter a core that is almost exclusively triglyceride (Figure 1). The surface proteins of HDL are amphiphilic and water soluble; the single protein of LDL is insoluble, whereas VLDL contains both soluble and insoluble proteins. The primary structures of all of these proteins are known.

The alpha-fetoprotein third domain receptor binding fragment: in search of scavenger and associated receptor targets.

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Mizejewski, G J

2015-01-01

Recent studies have demonstrated that the carboxyterminal third domain of alpha-fetoprotein (AFP-CD) binds with various ligands and receptors. Reports within the last decade have established that AFP-CD contains a large fragment of amino acids that interact with several different receptor types. Using computer software specifically designed to identify protein-to-protein interaction at amino acid sequence docking sites, the computer searches identified several types of scavenger-associated receptors and their amino acid sequence locations on the AFP-CD polypeptide chain. The scavenger receptors (SRs) identified were CD36, CD163, Stabilin, SSC5D, SRB1 and SREC; the SR-associated receptors included the mannose, low-density lipoprotein receptors, the asialoglycoprotein receptor, and the receptor for advanced glycation endproducts (RAGE). Interestingly, some SR interaction sites were localized on the AFP-derived Growth Inhibitory Peptide (GIP) segment at amino acids #480-500. Following the detection studies, a structural subdomain analysis of both the receptor and the AFP-CD revealed the presence of epidermal growth factor (EGF) repeats, extracellular matrix-like protein regions, amino acid-rich motifs and dimerization subdomains. For the first time, it was reported that EGF-like sequence repeats were identified on each of the three domains of AFP. Thereafter, the localization of receptors on specific cell types were reviewed and their functions were discussed.

Cholesteryl ester transfer protein decreases high-density lipoprotein and severely aggravates atherosclerosis in APOE*3-Leiden mice

NARCIS (Netherlands)

Westerterp, M.; Hoogt, C.C. van der; Haan, W. de; Offerman, E.H.; Dallinga-Thie, G.M.; Jukema, J.W.; Havekes, L.M.; Rensen, P.C.N.

2006-01-01

OBJECTIVE - The role of cholesteryl ester transfer protein (CETP) in the development of atherosclerosis is still undergoing debate. Therefore, we evaluated the effect of human CETP expression on atherosclerosis in APOE*3-Leiden (E3L) mice with a humanized lipoprotein profile. METHODS AND RESULTS -

Streptococcus gordonii lipoproteins induce IL-8 in human periodontal ligament cells.

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Kim, A Reum; Ahn, Ki Bum; Kim, Hyun Young; Seo, Ho Seong; Kum, Kee-Yeon; Yun, Cheol-Heui; Han, Seung Hyun

2017-11-01

Streptococcus gordonii, a Gram-positive oral bacterium, is a life-threatening pathogen that causes infective endocarditis. It is frequently isolated from the periapical lesions of patients with apical periodontitis and has thus been implicated in inflammatory responses. However, little is known about the virulence factors of S. gordonii responsible for the induction of inflammatory responses in the periapical areas. Here, we investigated the role of S. gordonii cell wall-associated virulence factors on interleukin (IL)-8 induction in human periodontal ligament (PDL) cells using ethanol-inactivated wild-type S. gordonii, a lipoteichoic acid (LTA)-deficient mutant (ΔltaS), and a lipoprotein-deficient mutant (Δlgt). Wild-type S. gordonii induced IL-8 expression at both the protein and mRNA levels in human PDL cells in a dose- and time-dependent manner. A transient transfection and reporter gene assay demonstrated that wild-type S. gordonii activated Toll-like receptor 2 (TLR2). Additionally, IL-8 production induced by wild-type S. gordonii was substantially inhibited by anti-TLR2-neutralizing antibodies. Both wild-type S. gordonii and the ΔltaS mutant induced IL-8 production; however, this response was not observed when cells were stimulated with the Δlgt mutant. Interestingly, lipoproteins purified from S. gordonii induced IL-8 production, whereas purified LTA did not. In addition, purified lipoproteins stimulated TLR2 more potently than LTA. Furthermore, S. gordonii-induced IL-8 expression was specifically inhibited by blocking p38 kinase, while lipoprotein-induced IL-8 expression was inhibited by blocking p38 kinase, ERK, or JNK. Of particular note, exogenous addition of purified S. gordonii lipoproteins enhanced Δlgt-induced IL-8 production in human PDL cells to an extent similar to that induced by the wild-type strain. Collectively, these results suggest that lipoproteins are an important component of S. gordonii for the induction of IL-8 production in human

Effect of Porphyromonas gingivalis infection on post-transcriptional regulation of the low-density lipoprotein receptor in mice

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Miyazawa Haruna

2012-09-01

Full Text Available Abstract Background Periodontal disease is suggested to increase the risk of atherothrombotic disease by inducing dyslipidemia. Recently, we demonstrated that proprotein convertase subtilisin/kexin type 9 (PCSK9, which is known to play a critical role in the regulation of circulating low-density lipoprotein (LDL cholesterol levels, is elevated in periodontitis patients. However, the underlying mechanisms of elevation of PCSK9 in periodontitis patients are largely unknown. Here, we explored whether Porphyromonas gingivalis, a representative periodontopathic bacterium, -induced inflammatory response regulates serum PCSK9 and cholesterol levels using animal models. Methods We infected C57BL/6 mice intraperitoneally with Porphyromonas gingivalis, a representative strain of periodontopathic bacteria, and evaluated serum PCSK9 levels and the serum lipid profile. PCSK9 and LDL receptor (LDLR gene and protein expression, as well as liver X receptors (Lxrs, inducible degrader of the LDLR (Idol, and sterol regulatory element binding transcription factor (Srebf2 gene expression, were examined in the liver. Results P. gingivalis infection induced a significant elevation of serum PCSK9 levels and a concomitant elevation of total and LDL cholesterol compared with sham-infected mice. The LDL cholesterol levels were significantly correlated with PCSK9 levels. Expression of the Pcsk9, Ldlr, and Srebf2 genes was upregulated in the livers of the P. gingivalis-infected mice compared with the sham-infected mice. Although Pcsk9 gene expression is known to be positively regulated by sterol regulatory element binding protein (SREBP2 (human homologue of Srebf2, whereas Srebf2 is negatively regulated by cholesterol, the elevated expression of Srebf2 found in the infected mice is thought to be mediated by P. gingivalis infection. Conclusions P. gingivalis infection upregulates PCSK9 production via upregulation of Srebf2, independent of cholesterol levels. Further studies

Wolbachia lipoproteins: abundance, localisation and serology of Wolbachia peptidoglycan associated lipoprotein and the Type IV Secretion System component, VirB6 from Brugia malayi and Aedes albopictus.

Science.gov (United States)

Voronin, Denis; Guimarães, Ana F; Molyneux, Gemma R; Johnston, Kelly L; Ford, Louise; Taylor, Mark J

2014-10-06

Lipoproteins are the major agonists of Wolbachia-dependent inflammatory pathogenesis in filariasis and a validated target for drug discovery. Here we characterise the abundance, localisation and serology of the Wolbachia lipoproteins: Wolbachia peptidoglycan associated lipoprotein and the Type IV Secretion System component, VirB6. We used proteomics to confirm lipoprotein presence and relative abundance; fractionation, immunoblotting and confocal and electron immuno-microscopy for localisation and ELISA for serological analysis. Proteomic analysis of Brugia malayi adult female protein extracts confirmed the presence of two lipoproteins, previously predicted through bioinformatics: Wolbachia peptidoglycan associated lipoprotein (wBmPAL) and the Type IV Secretion System component, VirB6 (wBmVirB6). wBmPAL was among the most abundant Wolbachia proteins present in an extract of adult female worms with wBmVirB6 only detected at a much lower abundance. This differential abundance was reflected in the immunogold-labelling, which showed wBmPAL localised at numerous sites within the bacterial membranes, whereas wBmVirB6 was present as a single cluster on each bacterial cell and also located within the bacterial membranes. Immunoblotting of fractionated extracts confirmed the localisation of wBmPAL to membranes and its absence from cytosolic fractions of C6/36 mosquito cells infected with wAlbB. In whole worm mounts, antibody labelling of both lipoproteins were associated with Wolbachia. Serological analysis showed that both proteins were immunogenic and raised antibody responses in the majority of individuals infected with Wuchereria bancrofti. Two Wolbachia lipoproteins, wBmPAL and wBmVirB6, are present in extracts of Brugia malayi with wBmPAL among the most abundant of Wolbachia proteins. Both lipoproteins localised to bacterial membranes with wBmVirB6 present as a single cluster suggesting a single Type IV Secretory System on each Wolbachia cell.

A comparison of the effects of 2 doses of soy protein or casein on serum lipids, serum lipoproteins, and plasma total homocysteine in hypercholesterolemic subjects.

Science.gov (United States)

Tonstad, Serena; Smerud, Knut; Høie, Lars

2002-07-01

Studies have shown that soy protein reduces some atherogenic lipid and lipoprotein concentrations, although lipoprotein(a) concentrations may be increased. The dose response of soy protein has not been established; neither has its effect on plasma total homocysteine. Our objective was to evaluate the effect of 2 doses of soy protein on lipid, lipoprotein, and homocysteine concentrations. Four to 24 wk after being instructed to consume a lipid-lowering diet, 130 men and women with LDL-cholesterol concentrations > or = 4 mmol/L were studied during a parallel group trial in which 4 interventions were assigned randomly. Thirty grams isolated soy protein (ISP) and 10 g cotyledon fiber or 50 g ISP and 16.6 g cotyledon fiber or equivalent doses of casein and cellulose were consumed daily as a beverage for 16 wk. When the 2 groups who consumed ISP were compared with the 2 groups who consumed casein, the differences in the net changes from baseline to week 16 in the concentrations of LDL cholesterol and plasma total homocysteine were -0.26 mmol/L (95% CI: -0.43, -0.09 mmol/L; P = 0.01) and -0.8 micromol/L (-1.4, -0.2 micromol/L; P = 0.005), respectively. The effect of the ISP dose was not significant. There were no significant differences between the 2 ISP and the 2 casein groups in changes in lipoprotein(a), HDL-cholesterol, or triacylglycerol concentrations. Adding 30-50 g soy protein/d to a lipid-lowering diet significantly reduced LDL-cholesterol concentrations without increasing lipoprotein(a) concentrations. Plasma total homocysteine concentrations also decreased, suggesting a novel, possibly antiatherosclerotic effect.

G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs).

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Li, Lingyong; Homan, Kristoff T; Vishnivetskiy, Sergey A; Manglik, Aashish; Tesmer, John J G; Gurevich, Vsevolod V; Gurevich, Eugenia V

2015-04-24

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in homologous desensitization of GPCRs. It is widely assumed that most GRKs selectively phosphorylate only active GPCRs. Here, we show that although this seems to be the case for the GRK2/3 subfamily, GRK5/6 effectively phosphorylate inactive forms of several GPCRs, including β2-adrenergic and M2 muscarinic receptors, which are commonly used as representative models for GPCRs. Agonist-independent GPCR phosphorylation cannot be explained by constitutive activity of the receptor or membrane association of the GRK, suggesting that it is an inherent ability of GRK5/6. Importantly, phosphorylation of the inactive β2-adrenergic receptor enhanced its interactions with arrestins. Arrestin-3 was able to discriminate between phosphorylation of the same receptor by GRK2 and GRK5, demonstrating preference for the latter. Arrestin recruitment to inactive phosphorylated GPCRs suggests that not only agonist activation but also the complement of GRKs in the cell regulate formation of the arrestin-receptor complex and thereby G protein-independent signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Lipoprotein distribution and serum concentrations of 7α-hydroxy-4-cholesten-3-one and bile acids: effects of monogenic disturbances in high-density lipoprotein metabolism

DEFF Research Database (Denmark)

Steiner, Carine; Holleboom, Adriaan G; Karuna, Ratna

2012-01-01

BA (bile acid) formation is considered an important final step in RCT (reverse cholesterol transport). HDL (high-density lipoprotein) has been reported to transport BAs. We therefore investigated the effects of monogenic disturbances in human HDL metabolism on serum concentrations and lipoprotein...... concentrations of conjugated and secondary BAs differed between heterozygous carriers of SCARB1 (scavenger receptor class B1) mutations and unaffected individuals (P...

Inactivation of lipoprotein lipase occurs on the surface of THP-1 macrophages where oligomers of angiopoietin-like protein 4 are formed

International Nuclear Information System (INIS)

Makoveichuk, Elena; Sukonina, Valentina; Kroupa, Olessia; Thulin, Petra; Ehrenborg, Ewa; Olivecrona, Thomas; Olivecrona, Gunilla

2012-01-01

Highlights: ► Lipoprotein lipase (LPL) activity is controlled by ANGPTL4 in THP-1 macrophages. ► Both LPL and ANGPTL4 bind to THP-1 macrophages in a heparin-releasable fashion. ► Only monomers of ANGPTL4 are present within THP-1 macrophages. ► Covalent oligomers of ANGPTL4 appear on cell surface and in medium. ► Inactivation of LPL coincide with ANGPTL4 oligomer formation on cell surfaces. -- Abstract: Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins causing release of fatty acids for metabolic purposes in muscles and adipose tissue. LPL in macrophages in the artery wall may, however, promote foam cell formation and atherosclerosis. Angiopoietin-like protein (ANGPTL) 4 inactivates LPL and ANGPTL4 expression is controlled by peroxisome proliferator-activated receptors (PPAR). The mechanisms for inactivation of LPL by ANGPTL4 was studied in THP-1 macrophages where active LPL is associated with cell surfaces in a heparin-releasable form, while LPL in the culture medium is mostly inactive. The PPARδ agonist GW501516 had no effect on LPL mRNA, but increased ANGPTL4 mRNA and caused a marked reduction of the heparin-releasable LPL activity concomitantly with accumulation of inactive, monomeric LPL in the medium. Intracellular ANGPTL4 was monomeric, while dimers and tetramers of ANGPTL4 were present in the heparin-releasable fraction and medium. GW501516 caused an increase in the amount of ANGPTL4 oligomers on the cell surface that paralleled the decrease in LPL activity. Actinomycin D blocked the effects of GW501516 on ANGPTL4 oligomer formation and prevented the inactivation of LPL. Antibodies against ANGPTL4 interfered with the inactivation of LPL. We conclude that inactivation of LPL in THP-1 macrophages primarily occurs on the cell surface where oligomers of ANGPTL4 are formed.

A Phospholipidomic Analysis of All Defined Human Plasma Lipoproteins

Science.gov (United States)

Dashti, Monireh; Kulik, Willem; Hoek, Frans; Veerman, Enno C.; Peppelenbosch, Maikel P.; Rezaee, Farhad

2011-01-01

Since plasma lipoproteins contain both protein and phospholipid components, either may be involved in processes such as atherosclerosis. In this study the identification of plasma lipoprotein-associated phospholipids, which is essential for understanding these processes at the molecular level, are performed. LC-ESI/MS, LC-ESI-MS/MS and High Performance Thin Layer Chromatography (HPTLC) analysis of different lipoprotein fractions collected from pooled plasma revealed the presence of phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyeline (SM) only on lipoproteins and phosphatidylcholine (PC), Lyso-PC on both lipoproteins and plasma lipoprotein free fraction (PLFF). Cardiolipin, phosphatidylglycerol (PG) and Phosphatidylserine (PS) were observed neither in the lipoprotein fractions nor in PLFF. All three approaches led to the same results regarding phospholipids occurrence in plasma lipoproteins and PLFF. A high abundancy of PE and SM was observed in VLDL and LDL fractions respectively. This study provides for the first time the knowledge about the phospholipid composition of all defined plasma lipoproteins. PMID:22355656

Deficiency of Lipoprotein Lipase in Neurons Decreases AMPA Receptor Phosphorylation and Leads to Neurobehavioral Abnormalities in Mice.

Directory of Open Access Journals (Sweden)

Tian Yu

Full Text Available Alterations in lipid metabolism have been found in several neurodegenerative disorders, including Alzheimer's disease. Lipoprotein lipase (LPL hydrolyzes triacylglycerides in lipoproteins and regulates lipid metabolism in multiple organs and tissues, including the central nervous system (CNS. Though many brain regions express LPL, the functions of this lipase in the CNS remain largely unknown. We developed mice with neuron-specific LPL deficiency that became obese on chow by 16 wks in homozygous mutant mice (NEXLPL-/- and 10 mo in heterozygous mice (NEXLPL+/-. In the present study, we show that 21 mo NEXLPL+/- mice display substantial cognitive function decline including poorer learning and memory, and increased anxiety with no difference in general motor activities and exploratory behavior. These neurobehavioral abnormalities are associated with a reduction in the 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl propanoic acid (AMPA receptor subunit GluA1 and its phosphorylation, without any alterations in amyloid β accumulation. Importantly, a marked deficit in omega-3 and omega-6 polyunsaturated fatty acids (PUFA in the hippocampus precedes the development of the neurobehavioral phenotype of NEXLPL+/- mice. And, a diet supplemented with n-3 PUFA can improve the learning and memory of NEXLPL+/- mice at both 10 mo and 21 mo of age. We interpret these findings to indicate that LPL regulates the availability of PUFA in the CNS and, this in turn, impacts the strength of synaptic plasticity in the brain of aging mice through the modification of AMPA receptor and its phosphorylation.

Surface-Exposed Lipoproteins: An Emerging Secretion Phenomenon in Gram-Negative Bacteria.

Science.gov (United States)

Wilson, Marlena M; Bernstein, Harris D

2016-03-01

Bacterial lipoproteins are hydrophilic proteins that are anchored to a cell membrane by N-terminally linked fatty acids. It is widely believed that nearly all lipoproteins produced by Gram-negative bacteria are either retained in the inner membrane (IM) or transferred to the inner leaflet of the outer membrane (OM). Lipoproteins that are exposed on the cell surface have also been reported but are generally considered to be rare. Results from a variety of recent studies, however, now suggest that the prevalence of surface-exposed lipoproteins has been underestimated. In this review we describe the evidence that the surface exposure of lipoproteins in Gram-negative bacteria is a widespread phenomenon and discuss possible mechanisms by which these proteins might be transported across the OM. Published by Elsevier Ltd.

Preparation and Characterization of Stable α-Synuclein Lipoprotein Particles.

Science.gov (United States)

Eichmann, Cédric; Campioni, Silvia; Kowal, Julia; Maslennikov, Innokentiy; Gerez, Juan; Liu, Xiaoxia; Verasdonck, Joeri; Nespovitaya, Nadezhda; Choe, Senyon; Meier, Beat H; Picotti, Paola; Rizo, Josep; Stahlberg, Henning; Riek, Roland

2016-04-15

Multiple neurodegenerative diseases are caused by the aggregation of the human α-Synuclein (α-Syn) protein. α-Syn possesses high structural plasticity and the capability of interacting with membranes. Both features are not only essential for its physiological function but also play a role in the aggregation process. Recently it has been proposed that α-Syn is able to form lipid-protein particles reminiscent of high-density lipoproteins. Here, we present a method to obtain a stable and homogeneous population of nanometer-sized particles composed of α-Syn and anionic phospholipids. These particles are called α-Syn lipoprotein (nano)particles to indicate their relationship to high-density lipoproteins formed by human apolipoproteins in vivo and of in vitro self-assembling phospholipid bilayer nanodiscs. Structural investigations of the α-Syn lipoprotein particles by circular dichroism (CD) and magic angle solid-state nuclear magnetic resonance (MAS SS-NMR) spectroscopy establish that α-Syn adopts a helical secondary structure within these particles. Based on cryo-electron microscopy (cryo-EM) and dynamic light scattering (DLS) α-Syn lipoprotein particles have a defined size with a diameter of ∼23 nm. Chemical cross-linking in combination with solution-state NMR and multiangle static light scattering (MALS) of α-Syn particles reveal a high-order protein-lipid entity composed of ∼8-10 α-Syn molecules. The close resemblance in size between cross-linked in vitro-derived α-Syn lipoprotein particles and a cross-linked species of endogenous α-Syn from SH-SY5Y human neuroblastoma cells indicates a potential functional relevance of α-Syn lipoprotein nanoparticles. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of lipoproteins in human and canine cerebrospinal fluid (CSF)

International Nuclear Information System (INIS)

Pitas, R.E.; Weisgraber, K.H.; Boyles, J.K.; Lee, S.; Mahley, R.W.

1986-01-01

Previously the authors demonstrated that rat brain astrocytes in vitro synthesize and secrete apo-E and possess apo-B,E(LDL) receptors. The apo-E secreted by astrocytes and apo-E in rat brain extracts differed from serum apo-E in two respects. Brain apo-E had a higher apparent molecular weight and a higher percentage of more acidic isoforms. To characterize further the apo-E within the central nervous system, apo-E in human and canine CSF was investigated. Compared to plasma apo-E, CSF apo-E had a higher apparent M/sub r/ and a higher percentage of acidic isoforms which were sialylated, as shown by neuraminidase digestion. The apo-E in human CSF was approx.5-10% of the plasma level. In CSF 60-80% of the apo-E was in lipoproteins with d = 1.09-1.15. The remainder of the apo-E was in the d > 1.21 fraction. Human CSF lipoproteins were primarily spherical (110-190 A) while canine CSF lipoproteins were a mixture of discs (205 x 65 A) while canine CSF lipoproteins were a mixture of discs (205 x 65 A) and spheres (100-150 A). The CSF also contained apo-AI in the d = 1.09-1.15 g/ml fraction. Human CSF lipoproteins containing both apo-E and apo-AI were isolated on an anti-apo-E affinity column, suggesting that apo-E and AI occurred in the same particles. The CSF apo-E-containing lipoproteins competed for binding of 125 I-LDL to the apo-B,E(LDL) receptor. There was no detectable apo-B in CSF. These data suggest that CSF lipoproteins might transport lipid and regulate lipid homeostasis within the brain

A Phospholipidomic Analysis of All Defined Human Plasma Lipoproteins

NARCIS (Netherlands)

Dashti, Monireh; Kulik, Willem; Hoek, Frans; Veerman, Enno C.; Peppelenbosch, Maikel P.; Rezaee, Farhad

2011-01-01

Since plasma lipoproteins contain both protein and phospholipid components, either may be involved in processes such as atherosclerosis. In this study the identification of plasma lipoprotein-associated phospholipids, which is essential for understanding these processes at the molecular level, are

A phospholipidomic analysis of all defined human plasma lipoproteins

NARCIS (Netherlands)

Dashti, Monireh; Kulik, Willem; Hoek, Frans; Veerman, Enno C.; Peppelenbosch, Maikel P.; Rezaee, Farhad

2011-01-01

Since plasma lipoproteins contain both protein and phospholipid components, either may be involved in processes such as atherosclerosis. In this study the identification of plasma lipoprotein-associated phospholipids, which is essential for understanding these processes at the molecular level, are

Lipophorin Receptor: The Insect Lipoprotein Receptor

Indian Academy of Sciences (India)

IAS Admin

Director of ... function of the Lp is to deliver lipids throughout the insect body for metabolism ... Lipid is used as a major energy source for development as well as other metabolic .... LpR4 receptor variant was expressed exclusively in the brain and.

Low-Density Lipoprotein Modified by Myeloperoxidase in Inflammatory Pathways and Clinical Studies

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Cédric Delporte

2013-01-01

Full Text Available Oxidation of low-density lipoprotein (LDL has a key role in atherogenesis. Among the different models of oxidation that have been studied, the one using myeloperoxidase (MPO is thought to be more physiopathologically relevant. Apolipoprotein B-100 is the unique protein of LDL and is the major target of MPO. Furthermore, MPO rapidly adsorbs at the surface of LDL, promoting oxidation of amino acid residues and formation of oxidized lipoproteins that are commonly named Mox-LDL. The latter is not recognized by the LDL receptor and is accumulated by macrophages. In the context of atherogenesis, Mox-LDL accumulates in macrophages leading to foam cell formation. Furthermore, Mox-LDL seems to have specific effects and triggers inflammation. Indeed, those oxidized lipoproteins activate endothelial cells and monocytes/macrophages and induce proinflammatory molecules such as TNFα and IL-8. Mox-LDL may also inhibit fibrinolysis mediated via endothelial cells and consecutively increase the risk of thrombus formation. Finally, Mox-LDL has been involved in the physiopathology of several diseases linked to atherosclerosis such as kidney failure and consequent hemodialysis therapy, erectile dysfunction, and sleep restriction. All these issues show that the investigations of MPO-dependent LDL oxidation are of importance to better understand the inflammatory context of atherosclerosis.

Influence of impaired lipoprotein biogenesis on surface and exoproteome of Streptococcus pneumoniae.

Science.gov (United States)

Pribyl, Thomas; Moche, Martin; Dreisbach, Annette; Bijlsma, Jetta J E; Saleh, Malek; Abdullah, Mohammed R; Hecker, Michael; van Dijl, Jan Maarten; Becher, Dörte; Hammerschmidt, Sven

2014-02-07

Surface proteins are important for the fitness and virulence of the Gram-positive pathogen Streptococcus pneumoniae. They are crucial for interaction of the pathogen with its human host during infection. Therefore, the analysis of the pneumococcal surface proteome is an important task that requires powerful tools. In this study, two different methods, an optimized biotinylation approach and shaving with trypsin beads, were applied to study the pneumococcal surface proteome and to identify surface-exposed protein domains, respectively. The identification of nearly 95% of the predicted lipoproteins and 75% of the predicted sortase substrates reflects the high coverage of the two classical surface protein classes accomplished in this study. Furthermore, the biotinylation approach was applied to study the impact of an impaired lipoprotein maturation pathway on the cell envelope proteome and exoproteome. Loss of the lipoprotein diacylglyceryl transferase Lgt leads to striking changes in the lipoprotein distribution. Many lipoproteins disappear from the surface proteome and accumulate in the exoproteome. Further insights into lipoprotein processing in pneumococci are provided by immunoblot analyses of bacterial lysates and corresponding supernatant fractions. Taken together, the first comprehensive overview of the pneumococcal surface and exoproteome is presented, and a model for lipoprotein processing in S. pneumoniae is proposed.

Influence of medium components on the expression of recombinant lipoproteins in Escherichia coli.

Science.gov (United States)

Tseng, Chi-Ling; Leng, Chih-Hsiang

2012-02-01

Bacterial lipoproteins are crucial antigens for protective immunity against bacterial pathogens. Expression of exogenous lipoproteins in Escherichia coli at high levels is thought to be an extremely difficult endeavor because it frequently results in incomplete or absent lipid modification. Previously, we identified a fusion sequence (D1) from a Neisseria meningitidis lipoprotein that induced a non-lipidated protein, E3 (the domain III of the dengue virus envelope protein), to become lipidated. However, without optimizing the growth conditions, some of the D1-fusion proteins were not lipidated. Here, we report the influence of medium components on the expression of recombinant lipoproteins in E. coli. For high-level expression of mature lipoproteins in the C43 (DE3) strain, M9 medium was better than M63 and the rich medium. Furthermore, we analyzed the influence of other media factors (including nitrogen and carbon sources, phosphate, ferrous ions, calcium, magnesium, and pH) on the levels of lipoprotein expression. The results showed that excess nitrogen sources and phosphate in M9 medium could increase the amount of immature lipoproteins, and glucose was a better carbon source than glycerol for expressing mature lipoproteins. We also found that lipoproteins tended to be completely processed in the alkaline environment, even in the nutrient-rich medium. Additional constructs expressing different immunogens or lipid signal peptides as targets were also utilized, demonstrating that these targets could be expressed as completely mature lipoproteins in the M9 medium but not in the rich medium. Our results provide the useful information for expressing mature exogenous lipoproteins in E. coli.

Receptor-mediated endocytosis and intracellular trafficking of insulin and low-density lipoprotein by retinal vascular endothelial cells.

Science.gov (United States)

Stitt, A W; Anderson, H R; Gardiner, T A; Bailie, J R; Archer, D B

1994-08-01

The authors investigated the receptor-mediated endocytosis (RME) and intracellular trafficking of insulin and low-density lipoprotein (LDL) in cultured retinal vascular endothelial cells (RVECs). Low-density lipoprotein and insulin were conjugated to 10 nm colloidal gold, and these ligands were added to cultured bovine RVECs for 20 minutes at 4 degrees C. The cultures were then warmed to 37 degrees C and fixed after incubation times between 30 seconds and 1 hour. Control cells were incubated with unconjugated gold colloid at times and concentrations similar to those of the ligands. Additional control cells were exposed to several concentrations of anti-insulin receptor antibody or a saturating solution of unconjugated insulin before incubation with gold insulin. Using transmission electron microscopy, insulin gold and LDL gold were both observed at various stages of RME. Insulin-gold particles were first seen to bind to the apical plasma membrane (PM) before clustering in clathrin-coated pits and internalization in coated vesicles. Gold was later visualized in uncoated cytoplasmic vesicles, corresponding to early endosomes and multivesicular bodies (MVBs) or late endosomes. In several instances, localized regions of the limiting membrane of the MVBs appeared coated, a feature of endosomal membranes not previously described. After RME at the apical PM and passage through the endosomal system, the greater part of both insulin- and LDL-gold conjugates was seen to accumulate in large lysosome-like compartments. However, a small but significant proportion of the internalized ligands was transcytosed and released as discrete membrane-associated quanta at the basal cell surface. The uptake of LDL gold was greatly increased in highly vacuolated, late-passage RVECs. In controls, anti-insulin receptor antibody and excess unconjugated insulin caused up to 89% inhibition in gold-insulin binding and internalization. These results illustrate the internalization and intracellular

L-Cysteine-induced up-regulation of the low-density lipoprotein receptor is mediated via a transforming growth factor-alpha signalling pathway.

Science.gov (United States)

Tanaka, Yuma; Shimada, Masaya; Nagaoka, Satoshi

2014-02-14

Sulphur-containing amino acids regulate plasma cholesterol levels in animals and humans. However, their mechanism of action remains unclear. Low-density lipoprotein receptor (LDLR) plays an important role in cholesterol metabolism. We therefore investigated the effects of sulphur-containing amino acids on the expression of LDLR in hepatocytes. HepG2 cells were cultured in Dulbecco's Modified Eagle's Medium with or without sulphur-containing amino acids and cysteine-containing compounds. We found that L-cysteine increased LDLR mRNA and enhanced LDLR gene promoter activity through the extracellular-signal-related kinase and p38 mitogen-activated protein kinase signalling pathways in HepG2 cells. Moreover, we observed that L-cysteine stimulated the release of transforming growth factor-alpha (TGF-α) and that TGF-α increased the LDLR mRNA levels. This study provides a report of the L-cysteine mediated up-regulation of the LDLR expression via TGF-α signalling pathway. Our findings provide insights into cholesterol homeostasis and amino acid signalling. Copyright © 2014 Elsevier Inc. All rights reserved.

A bovine papillomavirus-1 based vector restores the function of the low-density lipoprotein receptor in the receptor-deficient CHO-ldlA7 cell line

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Ustav Mart

2002-04-01

Full Text Available Abstract Background The rationale of using bovine papillomavirus-1 (BPV-1 derived vectors in gene therapy protocols lies in their episomal maintenance at intermediate to high copy number, and stable, high-level expression of the gene products. We constructed the BPV-1 based vector harbouring the human low-density lipoprotein receptor (LDLR gene cDNA and tested its ability to restore the function of the LDLR in the receptor-deficient cell line CHO-ldlA7. Results The introduced vector p3.7LDL produced functionally active LDL receptors in the receptor-deficient cell line CHO-ldlA7 during the 32-week period of observation as determined by the internalisation assay with the labelled LDL particles. Conclusion Bovine papillomavirus type-1 (BPV-1-derived vectors could be suitable for gene therapy due to their episomal maintenance at intermediate to high copy number and stable, high-level expression of the gene products. The constructed BPV-1 based vector p3.7LDL produced functionally active LDL receptors in the LDLR-deficient cell line CHO-ldlA7 during the 32-week period of observation. In vivo experiments should reveal, whether 1–5% transfection efficiency obtained in the current work is sufficient to bring about detectable and clinically significant lowering of the amount of circulating LDL cholesterol particles.

Identification of the receptor scavenging hemopexin-heme complexes

DEFF Research Database (Denmark)

Hvidberg, Vibeke; Maniecki, Maciej B; Jacobsen, Christian

2005-01-01

and is suggested to facilitate cellular heme metabolism. Using a ligand-affinity approach, we purified the human hemopexin-heme receptor and identified it as the low-density lipoprotein receptor-related protein (LRP)/CD91, a receptor expressed in several cell types including macrophages, hepatocytes, neurons......, and syncytiotrophoblasts. Binding experiments, including Biacore analysis, showed that hemopexin-heme complex formation elicits the high receptor affinity. Uptake studies of radio-labeled hemopexin-heme complex in LRP/CD91-expressing COS cells and confocal microscopy of the cellular processing of fluorescent hemopexin......-heme complexes are removed by a receptor-mediated pathway showing striking similarities to the CD163-mediated haptoglobin-hemoglobin clearance in macrophages. Furthermore, the data indicate a hitherto unknown role of LRP/CD91 in inflammation....

Binding of digitoxin and digoxin to normal human β-lipoproteins

International Nuclear Information System (INIS)

Brock, A.

1976-01-01

The binding of digitoxin and digoxin to purified β-lipoprotein, obtained from pooled normal human serum, was studied under equilibrium conditions. Even with as high concentrations of unbound digitoxin or digoxin as 4 μmol/l, the preparations of β-lipoproteins, containing cholesterol 1.98-3.95 mmol/l, showed no signs of saturation. The binding affinity of digitoxin was about ten times as high as that of digoxin. Gel filtration chromatography, performed on native serum after addition of 3 H-digitoxin or 3 H-digoxin, showed a minor fraction of the cardiac glycosides to be associated with te protein fraction of highest molecular weight. This phenomenon disappeared after precipitation of the β-lipoproteins. In clinical relations the contribution of protein-bound digitoxin caused by the lipoprotein interaction is immaterial compared to that caused by the albumin interaction. (author)

Radiochemical and immunohistochemical detection of low density lipoprotein surface binding by lymphocytes

International Nuclear Information System (INIS)

Melzner, I.; Hambitzer, R.; Haferkamp, O.

1983-01-01

Human peripheral blood lymphocytes bind and take up low density lipoprotein (LDL) by receptor-mediated endocytosis. The binding of LDL was determiend by incubation with 125 I-LDL and an immunohistochemical assay. By both techniques a diminished rate of binding was found when cells were freshly isolated from the blood, but increased 5 to 10 fold when lymphocytes were incubated in lipoprotein-deficient medium for 72 hours. In addition, it was shown immunohistochemically that only few ceels showed an LDL-dependent fluorescent labelling: approximately 5 to 10 % of the freshly isolated lymphocytes and 40 to 50 % of the cells incubated for 72 hours under lipoprotein-free conditions. The present data indicate that not only the high affinity LDL receptor described by Goldstein and Braun may be involved in the uptake of cholesterol by lymphocytes, but also other binding sites, which may have immunological function in some lymphocyte subpopulations. (author)

Genetic spectrum of low density lipoprotein receptor gene variations in South Indian population.

Science.gov (United States)

ArulJothi, K N; Suruthi Abirami, B; Devi, Arikketh

2018-03-01

Low density lipoprotein receptor (LDLR) is a membrane bound receptor maintaining cholesterol homeostasis along with Apolipoprotein B (APOB), Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) and other genes of lipid metabolism. Any pathogenic variation in these genes alters the function of the receptor and leads to Familial Hypercholesterolemia (FH) and other cardiovascular diseases. This study was aimed at screening the LDLR, APOB and PCSK9 genes in Hypercholesterolemic patients to define the genetic spectrum of FH in Indian population. Familial Hypercholesterolemia patients (n=78) of South Indian Tamil population with LDL cholesterol and Total cholesterol levels above 4.9mmol/l and 7.5mmol/l with family history of Myocardial infarction were involved. DNA was isolated by organic extraction method from blood samples and LDLR, APOB and PCSK9 gene exons were amplified using primers that cover exon-intron boundaries. The amplicons were screened using High Resolution Melt (HRM) Analysis and the screened samples were sequenced after purification. This study reports 20 variations in South Indian population for the first time. In this set of variations 9 are novel variations which are reported for the first time, 11 were reported in other studies also. The in silico analysis for all the variations detected in this study were done to predict the probabilistic effect in pathogenicity of FH. This study adds 9 novel variations and 11 recurrent variations to the spectrum of LDLR gene mutations in Indian population. All these variations are reported for the first time in Indian population. This spectrum of variations was different from the variations of previous Indian reports. Copyright © 2017 Elsevier B.V. All rights reserved.

In vitro studies on the distribution of probucol among human plasma lipoproteins

International Nuclear Information System (INIS)

Urien, S.; Riant, P.; Albengres, E.; Brioude, R.; Tillement, J.P.

1984-01-01

The role of human plasma lipoproteins as carriers in the blood transport of the cholesterol-lowering and water-insoluble drug, probucol, was investigated in in vitro studies. [ 14 C]Probucol was incubated in whole human blood, a serum pool, individual diluted sera, and isolated protein and lipoprotein fractions. In whole blood, about 90% partitioned in plasma. Following ultracentrifugal fractionation of the serum, it was found that less than 5% distributed in the d greater than 1.20 protein fraction (albumin-rich fraction) and more than 95% in the lipoprotein fractions. The distribution of probucol in the lipoprotein fractions correlated with the lipoprotein total lipid volume under saturation conditions (incubation of isolated lipoprotein fractions) as well as nonsaturation conditions (fractionation of serum exposed to [ 14 C]probucol). Incubation of the albumin-rich fraction and of apolipoproteins originating from the isolated lipoprotein fractions showed that they account for a negligible part in the interaction of probucol with blood components. The probucol uptake of individual sera was shown to be correlated to the lipid content of the serum. When probucol was incubated in erythrocyte suspensions containing variable amounts of lipoproteins, probucol partitioned less in erythrocytes as the lipoprotein concentration increased in the suspension

Genomic determinants of triglyceride and cholesterol distribution into lipoprotein fractions in the rat.

Directory of Open Access Journals (Sweden)

Miloslava Hodúlová

Full Text Available The plasma profile of major lipoprotein classes and its subdivision into particular fractions plays a crucial role in the pathogenesis of atherosclerosis and is a major predictor of coronary artery disease. Our aim was to identify genomic determinants of triglyceride and cholesterol distribution into lipoprotein fractions and lipoprotein particle sizes in the recombinant inbred rat set PXO, in which alleles of two rat models of the metabolic syndrome (SHR and PD inbred strains segregate together with those from Brown Norway rat strain. Adult male rats of 15 PXO strains (n = 8-13/strain and two progenitor strains SHR-Lx (n = 13 and BXH2/Cub (n = 18 were subjected to one-week of high-sucrose diet feeding. We performed association analyses of triglyceride (TG and cholesterol (C concentrations in 20 lipoprotein fractions and the size of major classes of lipoprotein particles utilizing 704 polymorphic microsatellite markers, the genome-wide significance was validated by 2,000 permutations per trait. Subsequent in silico focusing of the identified quantitative trait loci was completed using a map of over 20,000 single nucleotide polymorphisms. In most of the phenotypes we identified substantial gradient among the strains (e.g. VLDL-TG from 5.6 to 66.7 mg/dl. We have identified 14 loci (encompassing 1 to 65 genes on rat chromosomes 3, 4, 7, 8, 11 and 12 showing suggestive or significant association to one or more of the studied traits. PXO strains carrying the SHR allele displayed significantly higher values of the linked traits except for LDL-TG and adiposity index. Cholesterol concentrations in large, medium and very small LDL particles were significantly associated to a haplotype block spanning part of a single gene, low density lipoprotein receptor-related protein 1B (Lrp1b. Using genome-wide association we have identified new genetic determinants of triglyceride and cholesterol distribution into lipoprotein fractions in the recombinant

Regulation of hepatic peroxisome proliferator-activated receptor alpha expression but not adiponectin by dietary protein in finishing pigs.

Science.gov (United States)

Weber, T E; Kerr, B J; Spurlock, M E

2008-10-01

Soy protein regulates adiponectin and peroxisome proliferator-activated receptor alpha (PPARalpha) in some species, but the effect of dietary soy protein on adiponectin and PPARalpha in the pig has not been studied. Therefore, the objective of this study was to determine whether soya bean meal reduction or replacement influences serum adiponectin, adiponectin mRNA, serum metabolites and the expression of PPARalpha and other genes involved in lipid deposition. Thirty-three pigs (11 pigs per treatment) were subjected to one of three dietary treatments: (i) reduced crude protein (CP) diet containing soya bean meal (RCP-Soy), (ii) high CP diet containing soya bean meal (HCP-Soy) or (iii) high CP diet with corn gluten meal replacing soya bean meal (HCP-CGM) for 35 days. Dietary treatment had no effect on overall growth performance, feed intake or measures of body composition. There was no effect of dietary treatment on serum adiponectin or leptin. Dietary treatment did not affect the abundance of the mRNAs for adiponectin, PPARalpha, PPARgamma2, lipoprotein lipase or fatty acid synthase in adipose tissue. The mRNA expression of PPARalpha, PPARgamma2, lipoprotein lipase or fatty acid synthetase in loin muscle was not affected by dietary treatment. In liver tissue, the relative abundance of PPARalpha mRNA was greater (p Soy diets when compared to pigs fed RCP-Soy or HCP-CGM diets. Hepatic mRNA expression of acyl-CoA oxidase or fatty acid synthase was not affected by dietary treatment. Western blot analysis indicated that hepatic PPARalpha protein levels were decreased (p Soy diets when compared to pigs fed the HCP-Soy diets. These data suggest that increasing the soy protein content of swine diets increases hepatic expression of PPARalpha without associated changes in body composition.

Lipoproteins from Clostridium perfringens and their protective efficacy in mouse model.

Science.gov (United States)

Dwivedi, Pratistha; Alam, Syed Imteyaz; Kumar, Om; Kumar, Ravi Bhushan

2015-08-01

Clostridium perfringens is an obligately anaerobic rod-shaped bacterium and etiological agent for several diseases in humans and animals. The pathogen has been listed as Validated Biological Agent and warrants development of medical countermeasures. The homologs of some of the lipoproteins identified from various fractions of C. perfringens in our previous studies were observed to be virulence determinants in other pathogenic bacteria. Three putative virulence associated lipoproteins; polysaccharide deacetylase family protein, probable ion-uptake ABC transporter, and a putative lipoprotein of no known function are reported here with respect to their immuno-protective potentials. The three proteins were over expressed and purified to near homogeneity. The lipoproteins were shown to be exposed on the C. perfringens surface and, hence, accessible to antibodies and potentially visible to the host immune system. Immunization of mice with purified recombinant proteins elicited protective immunity against challenge with C. perfringens in mouse gas gangrene model. Distribution and relationship of orthologous proteins across other bacterial select agents especially among the members of Firmicutes, was carried out to look for conserved antigenic determinants. Copyright © 2015 Elsevier B.V. All rights reserved.

The fibrate drug gemfibrozil disrupts lipoprotein metabolism in rainbow trout

International Nuclear Information System (INIS)

Prindiville, John S.; Mennigen, Jan A.; Zamora, Jake M.; Moon, Thomas W.; Weber, Jean-Michel

2011-01-01

Gemfibrozil (GEM) is a fibrate drug consistently found in effluents from sewage treatment plants. This study characterizes the pharmacological effects of GEM on the plasma lipoproteins of rainbow trout (Oncorhynchus mykiss). Our goals were to quantify the impact of the drug on: 1) lipid constituents of lipoproteins (phospholipids (PL), triacylglycerol (TAG), and cholesterol), 2) lipoprotein classes (high, low and very low density lipoproteins), and 3) fatty acid composition of lipoproteins. Potential mechanisms of GEM action were investigated by measuring lipoprotein lipase activity (LPL) and the hepatic gene expression of LPL and of the peroxisome proliferator-activated receptor (PPAR) α, β, and γ isoforms. GEM treatment resulted in decreased plasma lipoprotein levels (- 29%) and a reduced size of all lipoprotein classes (lower PL:TAG ratios). However, the increase in HDL-cholesterol elicited by GEM in humans failed to be observed in trout. Therefore, HDL-cholesterol cannot be used to assess the impact of the drug on fish. GEM also modified lipoprotein composition by reducing the abundance of long-chain n-3 fatty acids, thereby potentially reducing the nutritional quality of exposed fish. The relative gene expression of LPL was increased, but the activity of the enzyme was not, and we found no evidence for the activation of PPAR pathways. The depressing effects of GEM on fish lipoproteins demonstrated here may be a concern in view of the widespread presence of fibrates in aquatic environments. Work is needed to test whether exposure to environmental concentrations of these drugs jeopardizes the capacity of fish for reproduction, temperature acclimation or migratory behaviors.

The G protein-coupled receptors deorphanization landscape.

Science.gov (United States)

Laschet, Céline; Dupuis, Nadine; Hanson, Julien

2018-07-01

G protein-coupled receptors (GPCRs) are usually highlighted as being both the largest family of membrane proteins and the most productive source of drug targets. However, most of the GPCRs are understudied and hence cannot be used immediately for innovative therapeutic strategies. Besides, there are still around 100 orphan receptors, with no described endogenous ligand and no clearly defined function. The race to discover new ligands for these elusive receptors seems to be less intense than before. Here, we present an update of the various strategies employed to assign a function to these receptors and to discover new ligands. We focus on the recent advances in the identification of endogenous ligands with a detailed description of newly deorphanized receptors. Replication being a key parameter in these endeavors, we also discuss the latest controversies about problematic ligand-receptor pairings. In this context, we propose several recommendations in order to strengthen the reporting of new ligand-receptor pairs. Copyright © 2018 Elsevier Inc. All rights reserved.

EFFECT OF ADIPOSITY ON PLASMA-LIPID TRANSFER PROTEIN ACTIVITIES - A POSSIBLE LINK BETWEEN INSULIN-RESISTANCE AND HIGH-DENSITY-LIPOPROTEIN METABOLISM

NARCIS (Netherlands)

DULLAART, RPF; SLUITER, WJ; DIKKESCHEI, LD; HOOGENBERG, K; VANTOL, A

The mechanisms responsible for the decreased high density lipoprotein (HDL) cholesterol levels associated with obesity and insulin resistance are not well understood. Lecithin: cholesterol acyltransferase (LCAT) and cholesterol ester transfer protein (CETP) are key factors in the esterification of

Nuclear magnetic resonance studies of lipoproteins

International Nuclear Information System (INIS)

Hamilton, J.A.; Morrisett, J.D.

1986-01-01

Several nuclei in lipoproteins are magnetically active and are thus potential NMR probes of lipoprotein structure. Table I lists the magnetic isotopes preset in the covalent structures of the molecular constituents of lipoproteins: lipids, proteins, and carbohydrates. Every type of nucleus that is part of the endogenous structure of these molecules has at least one magnetic isotope. Each magnetic nucleus represents an intrinsic and completely nonperturbing probe (when at the natural abundance level) of local molecular motion and magnetic environment. The NMR experiment itself is also nonperturbing and nondestructive. Table I also lists for each nucleus its nuclear spin, its natural isotopic abundance, its sensitivity, and its resonance frequency at two commonly employed magnetic in the low field range (21.14 kG or 2.11 Tesla) and the other in the high field range (47.0 kG or 4.70 Tesla). Of the nuclei listed in Table I, /sup 1/H, /sup 13/C, and /sup 31/P have been the primary ones studied in lipoproteins. The general advantages and disadvantages afforded by these and other nuclei as probes of lipoprotein structure are discussed. /sup 13/C NMR spectroscopy, the method which has had the most extensive application (and probably has the greatest future potential) to lipoproteins, is treated in greatest detail, but many of the principles described apply to other nuclei as well

Are oxidized low-density lipoprotein and C-reactive protein markers of atherosclerosis in nephrotic children?

OpenAIRE

Rybi-Szumińska, A.; Wasilewska, A.; Michaluk-Skutnik, J.; Osipiuk-Remża, B.; Fiłonowicz, R.; Zając, M.

2014-01-01

Background Lipid disorders are known to be linked to disturbance in oxidative reactions and play an important role in the progression and complications of idiopathic nephrotic syndrome (INS). Aims The aim of this study was to assess oxidized low-density lipoprotein (oxLDL), high-sensitive C-reactive protein (hs-CRP) serum concentrations and other parameters of lipid metabolism in children with INS during relapse and remission of proteinuria. Methods The examination was performed on 23 childre...

Redefining the essential trafficking pathway for outer membrane lipoproteins

OpenAIRE

Grabowicz, Marcin; Silhavy, Thomas J.

2017-01-01

In Gram-negative bacteria, most lipoproteins synthesized in the inner membrane (IM) are trafficked to the outer membrane (OM). The Lol pathway is the trafficking paradigm: LolCDE releases lipoproteins from the IM; LolA shuttles them between membranes to LolB in the OM. Several OM lipoproteins are essential for viability. In apparent concordance, the Lol proteins are each essential in wild-type cells. However, we show that Escherichia coli grows well without LolA and LolB in the absence of one...

Lipoprotein-associated phospholipase A2 distribution among lipoproteins differs in type 1 diabetes.

Science.gov (United States)

Jarvie, Jennifer L; Wang, Hong; Kinney, Gregory L; Snell-Bergeon, Janet; Hokanson, John E; Eckel, Robert H

2016-01-01

LpPLA2 mass and activity have been variably related to cardiovascular disease risk, and the distribution of LpPLA2 in patients with type 1 diabetes (T1D), wherein cardiovascular disease risk is high despite normal or higher levels of high-density lipoprotein (HDL) cholesterol, is unknown. To determine whether there are differences in the distribution of LpPLA2 mass and activity across lipoproteins and their association with coronary artery calcium (CAC) in patients with T1D. Men with T1D (n = 19) not on statins, with and without CAC progression, and men without diabetes matched for HDL cholesterol (n = 25) had lipoproteins separated by fast protein liquid chromatography. Both LpPLA2 mass and activity were found within low-density lipoprotein (LDL) and HDL pools with more LpPLA2 mass being associated with HDL (54% vs 44%; P-value lipoprotein subfractions was observed between all groups, and there was no relationship between LpPLA2 activity or mass and its distribution and CAC score progression in healthy or T1D men. LpPLA2 is found in both LDL and HDL and is distributed differently in men with T1D without any relationship to CAC score progression. Copyright © 2016 National Lipid Association. Published by Elsevier Inc. All rights reserved.

Inactivation of lipoprotein lipase occurs on the surface of THP-1 macrophages where oligomers of angiopoietin-like protein 4 are formed

Energy Technology Data Exchange (ETDEWEB)

Makoveichuk, Elena; Sukonina, Valentina; Kroupa, Olessia [Department of Medical Biosciences, Physiological Chemistry Umea University, SE-901 87 Umea (Sweden); Thulin, Petra; Ehrenborg, Ewa [Atherosclerosis Research Unit, Department of Medicine, Karolinska Institutet, SE-171 76 Stockholm (Sweden); Olivecrona, Thomas [Department of Medical Biosciences, Physiological Chemistry Umea University, SE-901 87 Umea (Sweden); Olivecrona, Gunilla, E-mail: Gunilla.Olivecrona@medbio.umu.se [Department of Medical Biosciences, Physiological Chemistry Umea University, SE-901 87 Umea (Sweden)

2012-08-24

Highlights: Black-Right-Pointing-Pointer Lipoprotein lipase (LPL) activity is controlled by ANGPTL4 in THP-1 macrophages. Black-Right-Pointing-Pointer Both LPL and ANGPTL4 bind to THP-1 macrophages in a heparin-releasable fashion. Black-Right-Pointing-Pointer Only monomers of ANGPTL4 are present within THP-1 macrophages. Black-Right-Pointing-Pointer Covalent oligomers of ANGPTL4 appear on cell surface and in medium. Black-Right-Pointing-Pointer Inactivation of LPL coincide with ANGPTL4 oligomer formation on cell surfaces. -- Abstract: Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins causing release of fatty acids for metabolic purposes in muscles and adipose tissue. LPL in macrophages in the artery wall may, however, promote foam cell formation and atherosclerosis. Angiopoietin-like protein (ANGPTL) 4 inactivates LPL and ANGPTL4 expression is controlled by peroxisome proliferator-activated receptors (PPAR). The mechanisms for inactivation of LPL by ANGPTL4 was studied in THP-1 macrophages where active LPL is associated with cell surfaces in a heparin-releasable form, while LPL in the culture medium is mostly inactive. The PPAR{delta} agonist GW501516 had no effect on LPL mRNA, but increased ANGPTL4 mRNA and caused a marked reduction of the heparin-releasable LPL activity concomitantly with accumulation of inactive, monomeric LPL in the medium. Intracellular ANGPTL4 was monomeric, while dimers and tetramers of ANGPTL4 were present in the heparin-releasable fraction and medium. GW501516 caused an increase in the amount of ANGPTL4 oligomers on the cell surface that paralleled the decrease in LPL activity. Actinomycin D blocked the effects of GW501516 on ANGPTL4 oligomer formation and prevented the inactivation of LPL. Antibodies against ANGPTL4 interfered with the inactivation of LPL. We conclude that inactivation of LPL in THP-1 macrophages primarily occurs on the cell surface where oligomers of ANGPTL4 are formed.

Receptor targeting of hemoglobin mediated by the haptoglobins

DEFF Research Database (Denmark)

Nielsen, Marianne Jensby; Moestrup, Søren Kragh

2009-01-01

Haptoglobin, the haptoglobin-hemoglobin receptor CD163, and the heme oxygenase-1 are proteins with a well-established function in the clearance and metabolism of "free" hemoglobin released during intravascular hemolysis. This scavenging system counteracts the potentially harmful oxidative and NO......-scavenging effects associated with "free" hemoglobin, and, furthermore, elicits an anti-inflammatory response. In the late primate evolution, haptoglobin variants with distinct functions have arisen, including haptoglobin polymers and the haptoglobin-related protein. The latter associates with a subspecies of high......-density lipoprotein (HDL) particles playing a crucial role in the innate immunity against certain trypanosome parasites. Recent studies have elucidated this fairly sophisticated immune defense mechanism that takes advantage of a trypanosomal haptoglobin-hemoglobin receptor evolved to supply the parasite with heme...

The evolution of the class A scavenger receptors

Directory of Open Access Journals (Sweden)

Whelan Fiona J

2012-11-01

Full Text Available Abstract Background The class A scavenger receptors are a subclass of a diverse family of proteins defined based on their ability to bind modified lipoproteins. The 5 members of this family are strikingly variable in their protein structure and function, raising the question as to whether it is appropriate to group them as a family based on their ligand binding abilities. Results To investigate these relationships, we defined the domain architecture of each of the 5 members followed by collecting and annotating class A scavenger receptor mRNA and amino acid sequences from publicly available databases. Phylogenetic analyses, sequence alignments, and permutation tests revealed a common evolutionary ancestry of these proteins, indicating that they form a protein family. We postulate that 4 distinct gene duplication events and subsequent domain fusions, internal repeats, and deletions are responsible for the diverse protein structures and functions of this family. Despite variation in domain structure, there are highly conserved regions across all 5 members, indicating the possibility that these regions may represent key conserved functional motifs. Conclusions We have shown with significant evidence that the 5 members of the class A scavenger receptors form a protein family. We have indicated that these receptors have a common origin which may provide insight into future functional work with these proteins.

EST Table: DC540266 [KAIKOcDNA[Archive

Lifescience Database Archive (English)

Full Text Available ar to Low density lipoprotein receptor adapter protein 1 (Autosomal recessive hypercholesterolemia protein) ... similar to Low density lipoprotein receptor adapter protein 1 (Autosomal recessive hypercholesterolemia protein) isoform 1 [Tribolium castaneum] FS929848 dpe- ...

Characterization of Two Metal Binding Lipoproteins as Vaccine Candidates for Enterococcal Infections.

Science.gov (United States)

Romero-Saavedra, Felipe; Laverde, Diana; Budin-Verneuil, Aurélie; Muller, Cécile; Bernay, Benoit; Benachour, Abdellah; Hartke, Axel; Huebner, Johannes

2015-01-01

Enterococcus faecium and faecalis are Gram-positive opportunistic pathogens that have become leading causes of nosocomial infections over the last decades. Especially multidrug resistant enterococci have become a challenging clinical problem worldwide. Therefore, new treatment options are needed and the identification of alternative targets for vaccine development has emerged as a feasible alternative to fight the infections caused by these pathogens. We extrapolate the transcriptomic data from a mice peritonitis infection model in E. faecalis to identify putative up-regulated surface proteins under infection conditions in E. faecium. After the bionformatic analyses two metal binding lipoproteins were identified to have a high homology (>72%) between the two species, the manganese ABC transporter substrate-binding lipoprotein (PsaAfm,) and the zinc ABC transporter substrate-binding lipoprotein (AdcAfm). These candidate lipoproteins were overexpressed in Escherichia coli and purified. The recombinant proteins were used to produce rabbit polyclonal antibodies that were able to induce specific opsonic antibodies that mediated killing of the homologous strain E. faecium E155 as well as clinical strains E. faecium E1162, Enterococcus faecalis 12030, type 2 and type 5. Mice were passively immunized with the antibodies raised against recombinant lipoproteins, showing significant reduction of colony counts in mice livers after the bacterial challenge and demonstrating the efficacy of these metal binding lipoproteins as promising vaccine candidates to treat infections caused by these enterococcal pathogens. Overall, our results demonstrate that these two metal binding lipoproteins elicited specific, opsonic and protective antibodies, with an extensive cross-reactivity and serotype-independent coverage among these two important nocosomial pathogens. Pointing these two protein antigens as promising immunogens, that can be used as single components or as carrier proteins

EST Table: FS936166 [KAIKOcDNA[Archive

Lifescience Database Archive (English)

Full Text Available ar to Low density lipoprotein receptor adapter protein 1 (Autosomal recessive hypercholesterolemia protein) ... similar to Low density lipoprotein receptor adapter protein 1 (Autosomal recessive hypercholesterolemia protein) isoform 1 [Tribolium castaneum] FS929848 fwgP ...

A high-density lipoprotein-mediated drug delivery system.

Science.gov (United States)

Mo, Zhong-Cheng; Ren, Kun; Liu, Xing; Tang, Zhen-Li; Yi, Guang-Hui

2016-11-15

High-density lipoprotein (HDL) is a comparatively dense and small lipoprotein that can carry lipids as a multifunctional aggregate in plasma. Several studies have shown that increasing the levels or improving the functionality of HDL is a promising target for treating a wide variety of diseases. Among lipoproteins, HDL particles possess unique physicochemical properties, including naturally synthesized physiological components, amphipathic apolipoproteins, lipid-loading and hydrophobic agent-incorporating characteristics, specific protein-protein interactions, heterogeneity, nanoparticles, and smaller size. Recently, the feasibility and superiority of using HDL particles as drug delivery vehicles have been of great interest. In this review, we summarize the structure, constituents, biogenesis, remodeling, and reconstitution of HDL drug delivery systems, focusing on their delivery capability, characteristics, applications, manufacturing, and drug-loading and drug-targeting characteristics. Finally, the future prospects are presented regarding the clinical application and challenges of using HDL as a pharmacodelivery carrier. Copyright © 2016 Elsevier B.V. All rights reserved.

In vitro and in vivo Analysis of the Binding of the C Terminus of the HDL Receptor Scavenger Receptor Class B type I (SR-BI) to the PDZ1 Domain of its Cytoplasmic Adaptor Protein PDZK1

Energy Technology Data Exchange (ETDEWEB)

O Kocher; G Birrane; K Tsukamoto; S Fenske; A Yesilaltay; R Pal; K Daniels; J Ladias; M Krieger

2011-12-31

The PDZ1 domain of the four PDZ domain-containing protein PDZK1 has been reported to bind the C terminus of the HDL receptor scavenger receptor class B, type I (SR-BI), and to control hepatic SR-BI expression and function. We generated wild-type (WT) and mutant murine PDZ1 domains, the mutants bearing single amino acid substitutions in their carboxylate binding loop (Lys(14)-Xaa(4)-Asn(19)-Tyr-Gly-Phe-Phe-Leu(24)), and measured their binding affinity for a 7-residue peptide corresponding to the C terminus of SR-BI ((503)VLQEAKL(509)). The Y20A and G21Y substitutions abrogated all binding activity. Surprisingly, binding affinities (K(d)) of the K14A and F22A mutants were 3.2 and 4.0 ?M, respectively, similar to 2.6 ?M measured for the WT PDZ1. To understand these findings, we determined the high resolution structure of WT PDZ1 bound to a 5-residue sequence from the C-terminal SR-BI ((505)QEAKL(509)) using x-ray crystallography. In addition, we incorporated the K14A and Y20A substitutions into full-length PDZK1 liver-specific transgenes and expressed them in WT and PDZK1 knock-out mice. In WT mice, the transgenes did not alter endogenous hepatic SR-BI protein expression (intracellular distribution or amount) or lipoprotein metabolism (total plasma cholesterol, lipoprotein size distribution). In PDZK1 knock-out mice, as expected, the K14A mutant behaved like wild-type PDZK1 and completely corrected their hepatic SR-BI and plasma lipoprotein abnormalities. Unexpectedly, the 10-20-fold overexpressed Y20A mutant also substantially, but not completely, corrected these abnormalities. The results suggest that there may be an additional site(s) within PDZK1 that bind(s) SR-BI and mediate(s) productive SR-BI-PDZK1 interaction previously attributed exclusively to the canonical binding of the C-terminal SR-BI to PDZ1.

In vitro production of beta-very low density lipoproteins and small, dense low density lipoproteins in mildly hypertriglyceridemic plasma: role of activities of lecithin:cholester acyltransferase, cholesterylester transfer proteins and lipoprotein lipase.

Science.gov (United States)

Chung, B H; Segrest, J P; Franklin, F

1998-12-01

As a model for the formation of beta-very low density lipoproteins (VLDL) and small, dense LDL by the intraplasma metabolic activities in vivo, lipoproteins in fresh plasma were interacted in vitro with endogenous lecithin:cholesterol acyltransferase (LCAT) and cholesterylester transfer proteins (CETP) and subsequently with purified lipoprotein lipase (LpL). The LCAT and CETP reactions in a mildly hypertriglyceridemic (HTG) plasma at 37 degrees C for 18 h resulted in (1) esterification of about 45% plasma unesterified cholesterol (UC), (2) a marked increase in cholesterylester (CE) (+129%) and a decrease in triglyceride (TG) (-45%) in VLDL, and (3) a marked increase of TG (+ 341%) with a small net decrease of CE (-3.6%) in LDL, causing a significant alteration in the TG/CE of VLDL (from 8.0 to 1.9) and of LDL (from 0.20 to 0.93). The LDL in LCAT and CETP-reacted plasma is larger and more buoyant than that in control plasma. In vitro lipolysis of control and LCAT and CETP-reacted plasma by LpL, which hydrolyzed >90% of VLDL-TG and about 50-60% of LDL-TG, converted most of VLDL in control plasma (>85%) but less than half (40%) of VLDL in LCAT and CETP-reacted plasma into the IDL-LDL density fraction and transformed the large, buoyant LDL in the LCAT and CETP-reacted plasma into particles smaller and denser than those in the control plasma. The remnants that accumulated in the VLDL density region of the postlipolysis LCAT and CETP-reacted plasma contained apo B-100 and E but little or no detectable apo Cs and consisted of particles having pre-beta and beta-electrophoretic mobilities. The inhibition of LCAT during incubation of plasma, which lessened the extent of alteration in VLDL and LDL core lipids, increased the extent of lipolytic removal of VLDL from the VLDL density region but lowered the extent of alteration in the size and density of LDL. The LCAT, CETP and/or LpL-mediated alterations in the density of LDL in normolipidemic fasting plasma were less pronounced

Common low-density lipoprotein receptor p.G116S variant has a large effect on plasma low-density lipoprotein cholesterol in circumpolar inuit populations.

Science.gov (United States)

Dubé, Joseph B; Wang, Jian; Cao, Henian; McIntyre, Adam D; Johansen, Christopher T; Hopkins, Scarlett E; Stringer, Randa; Hosseinzadeh, Siyavash; Kennedy, Brooke A; Ban, Matthew R; Young, T Kue; Connelly, Philip W; Dewailly, Eric; Bjerregaard, Peter; Boyer, Bert B; Hegele, Robert A

2015-02-01

Inuit are considered to be vulnerable to cardiovascular disease because their lifestyles are becoming more Westernized. During sequence analysis of Inuit individuals at extremes of lipid traits, we identified 2 nonsynonymous variants in low-density lipoprotein receptor (LDLR), namely p.G116S and p.R730W. Genotyping these variants in 3324 Inuit from Alaska, Canada, and Greenland showed they were common, with allele frequencies 10% to 15%. Only p.G116S was associated with dyslipidemia: the increase in LDL cholesterol was 0.54 mmol/L (20.9 mg/dL) per allele (P=5.6×10(-49)), which was >3× larger than the largest effect sizes seen with other common variants in other populations. Carriers of p.G116S had a 3.02-fold increased risk of hypercholesterolemia (95% confidence interval, 2.34-3.90; P=1.7×10(-17)), but did not have classical familial hypercholesterolemia. In vitro, p.G116S showed 60% reduced ligand-binding activity compared with wild-type receptor. In contrast, p.R730W was associated with neither LDL cholesterol level nor altered in vitro activity. LDLR p.G116S is thus unique: a common dysfunctional variant in Inuit whose large effect on LDL cholesterol may have public health implications. © 2014 American Heart Association, Inc.

Caenorhabditis elegans reveals a FxNPxY-independent low-density lipoprotein receptor internalization mechanism mediated by epsin1

Science.gov (United States)

Kang, Yuan-Lin; Yochem, John; Bell, Leslie; Sorensen, Erika B.; Chen, Lihsia; Conner, Sean D.

2013-01-01

Low-density lipoprotein receptor (LDLR) internalization clears cholesterol-laden LDL particles from circulation in humans. Defects in clathrin-dependent LDLR endocytosis promote elevated serum cholesterol levels and can lead to atherosclerosis. However, our understanding of the mechanisms that control LDLR uptake remains incomplete. To identify factors critical to LDLR uptake, we pursued a genome-wide RNA interference screen using Caenorhabditis elegans LRP-1/megalin as a model for LDLR transport. In doing so, we discovered an unanticipated requirement for the clathrin-binding endocytic adaptor epsin1 in LDLR endocytosis. Epsin1 depletion reduced LDLR internalization rates in mammalian cells, similar to the reduction observed following clathrin depletion. Genetic and biochemical analyses of epsin in C. elegans and mammalian cells uncovered a requirement for the ubiquitin-interaction motif (UIM) as critical for receptor transport. As the epsin UIM promotes the internalization of some ubiquitinated receptors, we predicted LDLR ubiquitination as necessary for endocytosis. However, engineered ubiquitination-impaired LDLR mutants showed modest internalization defects that were further enhanced with epsin1 depletion, demonstrating epsin1-mediated LDLR endocytosis is independent of receptor ubiquitination. Finally, we provide evidence that epsin1-mediated LDLR uptake occurs independently of either of the two documented internalization motifs (FxNPxY or HIC) encoded within the LDLR cytoplasmic tail, indicating an additional internalization mechanism for LDLR. PMID:23242996

Selective uptake of a toxic lipophilic anthracycline derivative by the low-density lipoprotein receptor pathway in cultured fibroblasts

International Nuclear Information System (INIS)

Vitols, S.G.; Masquelier, M.; Peterson, C.O.

1985-01-01

N-(N-Retinoyl)-L-leucyldoxorubicin 14-linoleate (r11-DOX), a new lipophilic derivative of doxorubicin, was synthesized and incorporated into low-density lipoprotein (LDL). The drug-LDL complex contained 100- 200 drug molecules/LDL particle. When cultured normal human fibroblasts were incubated with 125 I-LDL-incorporated drug, there was a perfect correlation between the cellular uptake plus degradation of 125 I-LDL and the cellular drug accumulation. The presence of excess native LDL inhibited the cellular uptake and degradation of 125 I-LDL and the drug accumulation to the same extent. In contrast, methylated LDL, which does not bind to the LDL receptor, did not alter the cellular uptake and degradation of 125 I-LDL nor did it alter the drug accumulation. When LDL receptor negative fibroblasts from a patient with the homozygous form of familial hypercholesterolemia were incubated with the drug- 125 I-LDL complex, cellular drug accumulation was very low. The drug-LDL complex inhibited the growth of cultured normal human fibroblasts. The drug incorporated into methylated LDL was much less toxic. These findings suggest that r11-DOX incorporated into LDL is delivered to cells selectively by the LDL receptor pathway. This might be of value in the treatment of leukemia, since it has been previously found that leukemic cells exhibit higher LDL receptor activity than white blood cells and bone marrow cells from healthy subjects

EST Table: BY921544 [KAIKOcDNA[Archive

Lifescience Database Archive (English)

Full Text Available ar to Low density lipoprotein receptor adapter protein 1 (Autosomal recessive hypercholesterolemia protein) ... similar to Low density lipoprotein receptor adapter protein 1 (Autosomal recessive hypercholesterolemia protein) isoform 1 [Tribolium castaneum] FS929848 ovS0 ...

Structure of the LDL receptor extracellular domain at endosomalpH

Energy Technology Data Exchange (ETDEWEB)

Rudenko, Gabby; Henry, Lisa; Henderson, Keith; Ichtchenko,Konstantin; Brown, Michael S.; Goldstein, Joseph L.; Deisenhofer, Johann

2002-09-05

The structure of the low-density lipoprotein receptor extracellular portion has been determined. The document proposes a mechanism for the release of lipoprotein in the endosome. Without this release, the mechanism of receptor recycling cannot function.

Oligomerization of G protein-coupled receptors: computational methods.

Science.gov (United States)

Selent, J; Kaczor, A A

2011-01-01

Recent research has unveiled the complexity of mechanisms involved in G protein-coupled receptor (GPCR) functioning in which receptor dimerization/oligomerization may play an important role. Although the first high-resolution X-ray structure for a likely functional chemokine receptor dimer has been deposited in the Protein Data Bank, the interactions and mechanisms of dimer formation are not yet fully understood. In this respect, computational methods play a key role for predicting accurate GPCR complexes. This review outlines computational approaches focusing on sequence- and structure-based methodologies as well as discusses their advantages and limitations. Sequence-based approaches that search for possible protein-protein interfaces in GPCR complexes have been applied with success in several studies, but did not yield always consistent results. Structure-based methodologies are a potent complement to sequence-based approaches. For instance, protein-protein docking is a valuable method especially when guided by experimental constraints. Some disadvantages like limited receptor flexibility and non-consideration of the membrane environment have to be taken into account. Molecular dynamics simulation can overcome these drawbacks giving a detailed description of conformational changes in a native-like membrane. Successful prediction of GPCR complexes using computational approaches combined with experimental efforts may help to understand the role of dimeric/oligomeric GPCR complexes for fine-tuning receptor signaling. Moreover, since such GPCR complexes have attracted interest as potential drug target for diverse diseases, unveiling molecular determinants of dimerization/oligomerization can provide important implications for drug discovery.

Protective effect of the silkworm protein 30Kc6 on human vascular endothelial cells damaged by oxidized low density lipoprotein (Ox-LDL.

Directory of Open Access Journals (Sweden)

Wei Yu

Full Text Available Although the 30K family proteins are important anti-apoptotic molecules in silkworm hemolymph, the underlying mechanism remains to be investigated. This is especially the case in human vascular endothelial cells (HUVECs. In this study, a 30K protein, 30Kc6, was successfully expressed and purified using the Bac-to-Bac baculovirus expression system in silkworm cells. Furthermore, the 30Kc6 expressed in Escherichia coli was used to generate a polyclonal antibody. Western blot analysis revealed that the antibody could react specifically with the purified 30Kc6 expressed in silkworm cells. The In vitro cell apoptosis model of HUVEC that was induced by oxidized low density lipoprotein (Ox-LDL and in vivo atherosclerosis rabbit model were constructed and were employed to analyze the protective effects of the silkworm protein 30Kc6 on these models. The results demonstrated that the silkworm protein 30Kc6 significantly enhanced the cell viability in HUVEC cells treated with Ox-LDL, decreased the degree of DNA fragmentation and markedly reduced the level of 8-isoprostane. This could be indicative of the silkworm protein 30Kc6 antagonizing the Ox-LDL-induced cell apoptosis by inhibiting the intracellular reactive oxygen species (ROS generation. Furthermore, Ox-LDL activated the cell mitogen activated protein kinases (MAPK, especially JNK and p38. As demonstrated with Western analysis, 30Kc6 inhibited Ox-LDL-induced cell apoptosis in HUVEC cells by preventing the MAPK signaling pathways. In vivo data have demonstrated that oral feeding of the silkworm protein 30Kc6 dramatically improved the conditions of the atherosclerotic rabbits by decreasing serum levels of total triglyceride (TG, high density lipoprotein cholesterol (HDL-C, low density lipoprotein cholesterol (LDL-C and total cholesterol (TC. Furthermore, 30Kc6 alleviated the extent of lesions in aorta and liver in the atherosclerotic rabbits. These data are not only helpful in understanding the anti

Electrophoretic demonstration of glycoproteins, lipoproteins, and phosphoproteins in human and bovine enamel

DEFF Research Database (Denmark)

Kirkeby, S; Moe, D; Bøg-Hansen, T C

1990-01-01

Enamel proteins from fully mineralized human molars and from bovine tooth germs were separated by electrophoresis. The gels were stained for detection of glycoproteins, lipoproteins, and phosphoproteins. Glycoproteins were shown by periodic acid-Schiff staining and lectin blotting. In mature human...... enamel a number of high molecular weight proteins could be demonstrated after ethylenediaminetetra-acetic acid demineralization and subsequent Triton X-100 extraction. These proteins are suggested to be lipoproteins. Phosphoproteins could only be visualized in enamel matrix from the tooth germs....

Receptor Activity-modifying Protein-directed G Protein Signaling Specificity for the Calcitonin Gene-related Peptide Family of Receptors.

Science.gov (United States)

Weston, Cathryn; Winfield, Ian; Harris, Matthew; Hodgson, Rose; Shah, Archna; Dowell, Simon J; Mobarec, Juan Carlos; Woodlock, David A; Reynolds, Christopher A; Poyner, David R; Watkins, Harriet A; Ladds, Graham

2016-10-14

The calcitonin gene-related peptide (CGRP) family of G protein-coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in a Gα s -mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gα s and Gα q but also identify a Gα i component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMP-dependent signaling bias among the Gα s , Gα i , and Gα q/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.

Science.gov (United States)

Miao, Yinglong; McCammon, J Andrew

2018-03-20

Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

Lipoproteins and lipoprotein mimetics for imaging and drug delivery.

Science.gov (United States)

Thaxton, C Shad; Rink, Jonathan S; Naha, Pratap C; Cormode, David P

2016-11-15

Lipoproteins are a set of natural nanoparticles whose main role is the transport of fats within the body. While much work has been done to develop synthetic nanocarriers to deliver drugs or contrast media, natural nanoparticles such as lipoproteins represent appealing alternatives. Lipoproteins are biocompatible, biodegradable, non-immunogenic and are naturally targeted to some disease sites. Lipoproteins can be modified to act as contrast agents in many ways, such as by insertion of gold cores to provide contrast for computed tomography. They can be loaded with drugs, nucleic acids, photosensitizers or boron to act as therapeutics. Attachment of ligands can re-route lipoproteins to new targets. These attributes render lipoproteins attractive and versatile delivery vehicles. In this review we will provide background on lipoproteins, then survey their roles as contrast agents, in drug and nucleic acid delivery, as well as in photodynamic therapy and boron neutron capture therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

Genes encoding two lipoproteins in the leuS-dacA region of the Escherichia coli chromosome

International Nuclear Information System (INIS)

Takase, I.; Ishino, F.; Wachi, M.; Kamata, H.; Doi, M.; Asoh, S.; Matsuzawa, H.; Ohta, T.; Matsuhashi, M.

1987-01-01

The coding of two rare lipoproteins by two genes, rlpA and rlpB, located in the leuS-dacA region (15 min) on the Escherichia coli chromosome was demonstrated by expression of subcloned genes in a maxicell system. The formation of these two proteins was inhibited by globomycin, which is an inhibitor of the signal peptidase for the known lipoproteins of E. coli. In each case, this inhibition was accompanied by formation of a new protein, which showed a slightly lower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and which we suppose to be a prolipoprotein with an N-terminal signal peptide sequence similar to those of the bacterial major lipoproteins and lysis proteins of some bacteriocins. The incorporation of 3 H-labeled palmitate and glycerol into the two lipoproteins was also observed. Sequencing of DNA showed that the two lipoprotein genes contained sequences that could code for signal peptide sequences of 17 amino acids (rlpA lipoprotein) and 18 amino acids (rlpB lipoprotein). The deduced sequences of the mature peptides consisted of 345 amino acids (M/sub r/ 35,615, rlpA lipoprotein) and 175 amino acids (M/sub r/ 19,445, rlpB lipoprotein), with an N-terminal cysteine to which thioglyceride and N-fatty acyl residues may be attached. These two lioproteins may be important in duplication of the cells

Increased transvascular lipoprotein transport in diabetes

DEFF Research Database (Denmark)

Jensen, Jan Skov; Feldt-Rasmussen, Bo; Borch-Johnsen, Knut

2005-01-01

CONTEXT: Diabetes is associated with a highly increased risk of atherosclerosis, especially if hypertension or albuminuria is present. OBJECTIVE: We hypothesized that the increased transvascular lipoprotein transport in diabetes may be further accelerated if hypertension or albuminuria is present...... of transvascular transport. RESULTS: Transvascular LDL transport was 1.8 (1.6-2.0), 2.3 (2.0-2.6), and 2.6 (1.3-4.0)%/[h x (liter/m2)] in healthy controls, diabetic controls, and diabetes patients with systolic hypertension or albuminuria, respectively (P = 0.013; F = 4.5; df =2; ANOVA). These differences most...... likely were not caused by altered hepatic LDL receptor expression, glycosylation of LDL, small LDL size, or medicine use. CONCLUSIONS: Transvascular LDL transport is increased in patients with diabetes mellitus, especially if systolic hypertension or albuminuria is present. Accordingly, lipoprotein flux...

SOCS proteins in regulation of receptor tyrosine kinase signaling

DEFF Research Database (Denmark)

Kazi, Julhash U.; Kabir, Nuzhat N.; Flores Morales, Amilcar

2014-01-01

Receptor tyrosine kinases (RTKs) are a family of cell surface receptors that play critical roles in signal transduction from extracellular stimuli. Many in this family of kinases are overexpressed or mutated in human malignancies and thus became an attractive drug target for cancer treatment....... The signaling mediated by RTKs must be tightly regulated by interacting proteins including protein-tyrosine phosphatases and ubiquitin ligases. The suppressors of cytokine signaling (SOCS) family proteins are well-known negative regulators of cytokine receptors signaling consisting of eight structurally similar...

Real-Time G-Protein-Coupled Receptor Imaging to Understand and Quantify Receptor Dynamics

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María S. Aymerich

2011-01-01

Full Text Available Understanding the trafficking of G-protein-coupled receptors (GPCRs and their regulation by agonists and antagonists is fundamental to develop more effective drugs. Optical methods using fluorescent-tagged receptors and spinning disk confocal microscopy are useful tools to investigate membrane receptor dynamics in living cells. The aim of this study was to develop a method to characterize receptor dynamics using this system which offers the advantage of very fast image acquisition with minimal cell perturbation. However, in short-term assays photobleaching was still a problem. Thus, we developed a procedure to perform a photobleaching-corrected image analysis. A study of short-term dynamics of the long isoform of the dopamine type 2 receptor revealed an agonist-induced increase in the mobile fraction of receptors with a rate of movement of 0.08 μm/s For long-term assays, the ratio between the relative fluorescence intensity at the cell surface versus that in the intracellular compartment indicated that receptor internalization only occurred in cells co-expressing G protein-coupled receptor kinase 2. These results indicate that the lateral movement of receptors and receptor internalization are not directly coupled. Thus, we believe that live imaging of GPCRs using spinning disk confocal image analysis constitutes a powerful tool to study of receptor dynamics.

Scavenger Receptors and Their Potential as Therapeutic Targets in the Treatment of Cardiovascular Disease

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Sam L. Stephen

2010-01-01

Full Text Available Scavenger receptors act as membrane-bound and soluble proteins that bind to macromolecular complexes and pathogens. This diverse supergroup of proteins mediates binding to modified lipoprotein particles which regulate the initiation and progression of atherosclerotic plaques. In vascular tissues, scavenger receptors are implicated in regulating intracellular signaling, lipid accumulation, foam cell development, and cellular apoptosis or necrosis linked to the pathophysiology of atherosclerosis. One approach is using gene therapy to modulate scavenger receptor function in atherosclerosis. Ectopic expression of membrane-bound scavenger receptors using viral vectors can modify lipid profiles and reduce the incidence of atherosclerosis. Alternatively, expression of soluble scavenger receptors can also block plaque initiation and progression. Inhibition of scavenger receptor expression using a combined gene therapy and RNA interference strategy also holds promise for long-term therapy. Here we review our current understanding of the gene delivery by viral vectors to cells and tissues in gene therapy strategies and its application to the modulation of scavenger receptor function in atherosclerosis.

Structural basis for receptor recognition of vitamin-B(12)-intrinsic factor complexes

DEFF Research Database (Denmark)

Andersen, Christian Brix Folsted; Madsen, Mette; Storm, Tina

2010-01-01

Cobalamin (Cbl, vitamin B(12)) is a bacterial organic compound and an essential coenzyme in mammals, which take it up from the diet. This occurs by the combined action of the gastric intrinsic factor (IF) and the ileal endocytic cubam receptor formed by the 460-kilodalton (kDa) protein cubilin...... and the 45-kDa transmembrane protein amnionless. Loss of function of any of these proteins ultimately leads to Cbl deficiency in man. Here we present the crystal structure of the complex between IF-Cbl and the cubilin IF-Cbl-binding-region (CUB(5-8)) determined at 3.3 A resolution. The structure provides...... of how Cbl indirectly induces ligand-receptor coupling. Finally, the comparison of Ca(2+)-binding CUB domains and the low-density lipoprotein (LDL) receptor-type A modules suggests that the electrostatic pairing of a basic ligand arginine/lysine residue with Ca(2+)-coordinating acidic aspartates...

Effects of human low and high density lipoproteins on the binding of rat intermediate density lipoproteins to rat liver membranes

International Nuclear Information System (INIS)

Brissette, L.; Nol, S.P.

1986-01-01

Upon incubation with rat liver membranes, radioiodinated rat intermediate density lipoproteins (IDL) interacted with at least two binding sites having a low and a high affinity as demonstrated by the curvilinear Scatchard plots obtained from the specific binding data. The purpose of our work was to identify the nature of these binding sites. Human low density lipoproteins (LDL), contain apolipoprotein B only, and human high density lipoproteins (HDL3), containing neither apolipoprotein B nor E, were both capable of decreasing the specific binding of rat 125 I-IDL. The Scatchard analysis clearly revealed that only the low affinity component was affected by the addition of these human lipoproteins. In fact, the low affinity binding component gradually decreased as the amount of human LDL or HDL3 increased in the binding assay. At a 200-fold excess of human LDL or HDL3, the low affinity binding was totally masked, and the Scatchard plot of the specific 125 I-IDL binding became linear. Only the high affinity binding component was left, enabling a precise measurement of its binding parameters. In a series of competitive displacement experiments in which the binding assay contained a 200-fold excess of human LDL or HDL3, only unlabeled rat IDL effectively displaced the binding of rat 125 I-IDL. We conclude that the low affinity binding of rat IDL to rat liver membranes is due to weak interactions with unspecified lipoprotein binding sites. The camouflage of these sites by human lipoproteins makes possible the study of IDL binding to the high affinity component which likely represents the combined effect of IDL binding to both the remnant and the LDL receptors

Sort1, encoded by the cardiovascular risk locus 1p13.3, is a regulator of hepatic lipoprotein export

DEFF Research Database (Denmark)

Kjølby, Mads Fuglsang; Andersen, Olav Michael; Breiderhoff, Tilman

2010-01-01

of lipoproteins from the liver and ameliorates hypercholesterolemia and atherosclerotic lesion formation in LDL receptor-deficient animals. In contrast, sortilin overexpression stimulates hepatic release of lipoproteins and increases plasma LDL levels. Our data have uncovered a regulatory pathway in hepatic...... lipoprotein export and suggest a molecular explanation for the cardiovascular risk being associated with 1p13.3. Udgivelsesdato: september 8...

Bio F1B hamster: a unique animal model with reduced lipoprotein lipase activity to investigate nutrient mediated regulation of lipoprotein metabolism

Directory of Open Access Journals (Sweden)

Cornish Marion L

2007-12-01

Full Text Available Abstract Background Bio F1B hamster is an inbred hybrid strain that is highly susceptible to diet-induced atherosclerosis. We previously reported that feeding a high fat fish oil diet to Bio F1B hamster caused severe hyperlipidaemia. In this study we compared the effects of various diets in the Bio F1B hamster and the Golden Syrian hamster, which is an outbred hamster strain to investigate whether genetic background plays an important role in dietary fat mediated regulation of lipoprotein metabolism. We further investigated the mechanisms behind diet-induced hyperlipidaemia in F1B hamster. Methods The Bio F1B and Golden Syrian hamsters, 8 weeks old, were fed high fat diets rich in either monounsaturated fatty acids, an n-6: n-3 ratio of 5 or a fish oil diet for 4 weeks. Animals were fasted overnight and blood and tissue samples were collected. Plasma was fractionated into various lipoprotein fractions and assayed for triacylglycerol and cholesterol concentrations. Plasma lipoprotein lipase activity was measured using radioisotope method. Microsomal triglyceride transfer protein activity was measured in the liver and intestine. Plasma apolipoproteinB48, -B100 and apolipoprotein E was measured using Western blots. Two-way ANOVA was used to determine the effect of diet type and animal strain. Results The fish oil fed F1B hamsters showed milky plasma after a 14-hour fast. Fish oil feeding caused accumulation of apolipoproteinB48 containing lipoprotein particles suggesting hindrance of triglyceride-rich lipoprotein clearance. There was no significant effect of diet or strain on hepatic or intestinal microsomal triglyceride transfer protein activity indicating that hyperlipidaemia is not due to an increase in the assembly or secretion of lipoprotein particles. F1B hamsters showed significantly reduced levels of lipoprotein lipase activity, which was inhibited by fish oil feeding. Conclusion Evidence is presented for the first time that alterations in

An unusual protein kinase phosphorylates the chemotactic receptor of Dictystelium discoideum

International Nuclear Information System (INIS)

Meier, K.; Klein, C.

1988-01-01

The authors report the cAMP-dependent phosphorylation of the chemotactic receptor of Dictyostelium discoideum in partially purified plasma membranes. The protein kinase responsible for receptor phosphorylation is associated with this fraction and preferentially phosphorylates the ligand-occupied form of the receptor. 8-Azido[ 32 P]cAMP labeling of the cell surface has shown that the cAMP receptor exists in two forms. A 45-kDa protein is predominant on unstimulated cells. cAMP stimulation results in an increased receptor phosphorylation such that the receptor migrates on NaDodSO 4 /PAGE as a 47-kDa protein. Phosphorylation of the chemotactic receptor is not detected in membrane preparations unless cAMP is added to the incubation mixture. Only under those conditions is the phosphorylated 47-kDa form observed. The requirement for cAMP reflects the fact that the kinase involved preferentially uses the ligand-occupied receptor as a substrate. In vitro phosphorylation of the receptor does not involve tyrosine residues. The enzyme does not appear to be a cAMP- or cGMP-dependent protein kinase nor is it sensitive to guanine nucleotides, Ca 2+ /calmodulin, Ca 2+ /phospholipid, or EGTA. Similarities with the β-adrenergic receptor protein kinase are discussed

EST Table: FS929848 [KAIKOcDNA[Archive

Lifescience Database Archive (English)

Full Text Available DICTED: similar to Low density lipoprotein receptor adapter protein 1 (Autosomal recessive hypercholesterolemia...1| PREDICTED: similar to Low density lipoprotein receptor adapter protein 1 (Autosomal recessive hypercholesterolemia protein) isoform 1 [Tribolium castaneum] FS929848 fwgP ...

DMPD: LPS-binding proteins and receptors. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 9665271 LPS-binding proteins and receptors. Fenton MJ, Golenbock DT. J Leukoc Biol.... 1998 Jul;64(1):25-32. (.png) (.svg) (.html) (.csml) Show LPS-binding proteins and receptors. PubmedID 9665271 Title LPS-binding prot...eins and receptors. Authors Fenton MJ, Golenbock DT. Publication J Leukoc Biol. 199

Reduced beta-adrenergic receptor activation decreases G-protein expression and beta-adrenergic receptor kinase activity in porcine heart.

OpenAIRE

Ping, P; Gelzer-Bell, R; Roth, D A; Kiel, D; Insel, P A; Hammond, H K

1995-01-01

To determine whether beta-adrenergic receptor agonist activation influences guanosine 5'-triphosphate-binding protein (G-protein) expression and beta-adrenergic receptor kinase activity in the heart, we examined the effects of chronic beta 1-adrenergic receptor antagonist treatment (bisoprolol, 0.2 mg/kg per d i.v., 35 d) on components of the myocardial beta-adrenergic receptor-G-protein-adenylyl cyclase pathway in porcine myocardium. Three novel alterations in cardiac adrenergic signaling as...

Low density lipoproteins mediated nanoplatforms for cancer targeting

International Nuclear Information System (INIS)

Jain, Anupriya; Jain, Keerti; Kesharwani, Prashant; Jain, Narendra K.

2013-01-01

Chemotherapy is a foremost remedial approach for the treatment of localized and metastasized tumors. In order to explore new treatment modalities for cancer, it is important to identify qualitative or quantitative differences in metabolic processes between normal and malignant cells. One such difference may be that of increased receptor-mediated cellular uptake of low density lipoproteins (LDLs) by cancer cells. Lipoproteins in general and specifically LDL are ideal candidates for loading and delivering cancer therapeutic and diagnostic agents due to their biocompatibility. By mimicking the endogenous shape and structure of lipoproteins, the reconstituted lipoproteins can remain in circulation for an extended period of time, while largely evading the reticuloendothelial cells in the body’s defenses. In this account, we review the field of low density inspired nanoparticles in relation to the delivery of cancer imaging and therapeutic agents. LDL has instinctive cancer targeting potential and has been used to incorporate various lipophillic molecules to transport them to tumors. Nature’s method of rerouting LDL provides a strategy to extend the cancer targeting potential of lipoproteins far off its constricted purview. In this review, we have discussed the various aspects of LDL including its role in cancer imaging and chemotherapy in retrospect and prospect and current efforts aimed to further improve the delivery efficacy of LDL–drug complexes with reduced chances of drug resistance leading to optimal drug delivery. This review provides a strong support for the concept of using LDL as a drug carrier

Low density lipoproteins mediated nanoplatforms for cancer targeting

Energy Technology Data Exchange (ETDEWEB)

Jain, Anupriya; Jain, Keerti; Kesharwani, Prashant, E-mail: prashant_pharmacy04@rediffmail.com; Jain, Narendra K., E-mail: jnarendr@yahoo.co.in [Dr. H. S. Gour University, Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences (India)

2013-09-15

Chemotherapy is a foremost remedial approach for the treatment of localized and metastasized tumors. In order to explore new treatment modalities for cancer, it is important to identify qualitative or quantitative differences in metabolic processes between normal and malignant cells. One such difference may be that of increased receptor-mediated cellular uptake of low density lipoproteins (LDLs) by cancer cells. Lipoproteins in general and specifically LDL are ideal candidates for loading and delivering cancer therapeutic and diagnostic agents due to their biocompatibility. By mimicking the endogenous shape and structure of lipoproteins, the reconstituted lipoproteins can remain in circulation for an extended period of time, while largely evading the reticuloendothelial cells in the body's defenses. In this account, we review the field of low density inspired nanoparticles in relation to the delivery of cancer imaging and therapeutic agents. LDL has instinctive cancer targeting potential and has been used to incorporate various lipophillic molecules to transport them to tumors. Nature's method of rerouting LDL provides a strategy to extend the cancer targeting potential of lipoproteins far off its constricted purview. In this review, we have discussed the various aspects of LDL including its role in cancer imaging and chemotherapy in retrospect and prospect and current efforts aimed to further improve the delivery efficacy of LDL-drug complexes with reduced chances of drug resistance leading to optimal drug delivery. This review provides a strong support for the concept of using LDL as a drug carrier.

Low density lipoproteins mediated nanoplatforms for cancer targeting

Science.gov (United States)

Jain, Anupriya; Jain, Keerti; Kesharwani, Prashant; Jain, Narendra K.

2013-09-01

Chemotherapy is a foremost remedial approach for the treatment of localized and metastasized tumors. In order to explore new treatment modalities for cancer, it is important to identify qualitative or quantitative differences in metabolic processes between normal and malignant cells. One such difference may be that of increased receptor-mediated cellular uptake of low density lipoproteins (LDLs) by cancer cells. Lipoproteins in general and specifically LDL are ideal candidates for loading and delivering cancer therapeutic and diagnostic agents due to their biocompatibility. By mimicking the endogenous shape and structure of lipoproteins, the reconstituted lipoproteins can remain in circulation for an extended period of time, while largely evading the reticuloendothelial cells in the body's defenses. In this account, we review the field of low density inspired nanoparticles in relation to the delivery of cancer imaging and therapeutic agents. LDL has instinctive cancer targeting potential and has been used to incorporate various lipophillic molecules to transport them to tumors. Nature's method of rerouting LDL provides a strategy to extend the cancer targeting potential of lipoproteins far off its constricted purview. In this review, we have discussed the various aspects of LDL including its role in cancer imaging and chemotherapy in retrospect and prospect and current efforts aimed to further improve the delivery efficacy of LDL-drug complexes with reduced chances of drug resistance leading to optimal drug delivery. This review provides a strong support for the concept of using LDL as a drug carrier.

Does protein binding modulate the effect of angiotensin II receptor antagonists?

Directory of Open Access Journals (Sweden)

Marc P Maillard

2001-03-01

Full Text Available IntroductionAngiotensin II AT 1-receptor antagonists are highly bound to plasma proteins (≥ 99%. With some antagonists, such as DuP-532, the protein binding was such that no efficacy of the drug could be demonstrated clinically. Whether protein binding interferes with the efficacy of other antagonists is not known. We have therefore investigated in vitro how plasma proteins may affect the antagonistic effect of different AT1-receptor antagonists.MethodsA radio-receptor binding assay was used to analyse the interaction between proteins and the ability of various angiotensin II (Ang II antagonists to block AT1-receptors. In addition, the Biacore technology, a new technique which enables the real-time monitoring of binding events between two molecules, was used to evaluate the dissociation rate constants of five AT1-receptor antagonists from human serum albumin.ResultsThe in vitro AT 1-antagonistic effects of different Ang II receptor antagonists were differentially affected by the presence of human plasma, with rightward shifts of the IC50 ranging from one to several orders of magnitude. The importance of the shift correlates with the dissociation rate constants of these drugs from albumin. Our experiments also show that the way that AT1-receptor antagonists bind to proteins differs from one compound to another. These results suggest that the interaction with plasma proteins appears to modulate the efficacy of some Ang II antagonists.ConclusionAlthough the high binding level of Ang II receptor antagonist to plasma proteins appears to be a feature common to this class of compounds, the kinetics and characteristics of this binding is of great importance. With some antagonists, protein binding interferes markedly with their efficacy to block AT1-receptors.

Oxidized low-density lipoproteins upregulate proline oxidase to initiate ROS-dependent autophagy.

Science.gov (United States)

Zabirnyk, Olga; Liu, Wei; Khalil, Shadi; Sharma, Anit; Phang, James M

2010-03-01

Epidemiological studies showed that high levels of oxidized low-density lipoproteins (oxLDLs) are associated with increased cancer risk. We examined the direct effect of physiologic concentrations oxLDL on cancer cells. OxLDLs were cytotoxic and activate both apoptosis and autophagy. OxLDLs have ligands for peroxisome proliferator-activated receptor gamma and upregulated proline oxidase (POX) through this nuclear receptor. We identified 7-ketocholesterol (7KC) as a main component responsible for the latter. To elucidate the role of POX in oxLDL-mediated cytotoxicity, we knocked down POX via small interfering RNA and found that this (i) further reduced viability of cancer cells treated with oxLDL; (ii) decreased oxLDL-associated reactive oxygen species generation; (iii) decreased autophagy measured via beclin-1 protein level and light-chain 3 protein (LC3)-I into LC3-II conversion. Using POX-expressing cell model, we established that single POX overexpression was sufficient to activate autophagy. Thus, it led to autophagosomes accumulation and increased conversion of LC3-I into LC3-II. Moreover, beclin-1 gene expression was directly dependent on POX catalytic activity, namely the generation of POX-dependent superoxide. We conclude that POX is critical in the cellular response to the noxious effects of oxLDL by activating protective autophagy.

Nanobody-Enabled Reverse Pharmacology on G-Protein-Coupled Receptors.

Science.gov (United States)

Pardon, Els; Betti, Cecilia; Laeremans, Toon; Chevillard, Florent; Guillemyn, Karel; Kolb, Peter; Ballet, Steven; Steyaert, Jan

2018-05-04

The conformational complexity of transmembrane signaling of G-protein-coupled receptors (GPCRs) is a central hurdle for the design of screens for receptor agonists. In their basal states, GPCRs have lower affinities for agonists compared to their G-protein-bound active state conformations. Moreover, different agonists can stabilize distinct active receptor conformations and do not uniformly activate all cellular signaling pathways linked to a given receptor (agonist bias). Comparative fragment screens were performed on a β 2 -adrenoreceptor-nanobody fusion locked in its active-state conformation by a G-protein-mimicking nanobody, and the same receptor in its basal-state conformation. This simple biophysical assay allowed the identification and ranking of multiple novel agonists and permitted classification of the efficacy of each hit in agonist, antagonist, or inverse agonist categories, thereby opening doors to nanobody-enabled reverse pharmacology. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Current guidelines for high-density lipoprotein cholesterol in therapy and future directions

Directory of Open Access Journals (Sweden)

Subedi BH

2014-04-01

Full Text Available Bishnu H Subedi,1,2 Parag H Joshi,1 Steven R Jones,1 Seth S Martin,1 Michael J Blaha,1 Erin D Michos1 1Johns Hopkins Ciccarone Center for the Prevention of Heart Disease, 2Greater Baltimore Medical Center, Baltimore, MD, USA Abstract: Many studies have suggested that a significant risk factor for atherosclerotic cardiovascular disease (ASCVD is low high-density lipoprotein cholesterol (HDL-C. Therefore, increasing HDL-C with therapeutic agents has been considered an attractive strategy. In the prestatin era, fibrates and niacin monotherapy, which cause modest increases in HDL-C, reduced ASCVD events. Since their introduction, statins have become the cornerstone of lipoprotein therapy, the benefits of which are primarily attributed to decrease in low-density lipoprotein cholesterol. Findings from several randomized trials involving niacin or cholesteryl ester transfer protein inhibitors have challenged the concept that a quantitative elevation of plasma HDL-C will uniformly translate into ASCVD benefits. Consequently, the HDL, or more correctly, HDL-C hypothesis has become more controversial. There are no clear guidelines thus far for targeting HDL-C or HDL due to lack of solid outcomes data for HDL specific therapies. HDL-C levels are only one marker of HDL out of its several structural or functional properties. Novel approaches are ongoing in developing and assessing agents that closely mimic the structure of natural HDL or replicate its various functions, for example, reverse cholesterol transport, vasodilation, anti-inflammation, or inhibition of platelet aggregation. Potential new approaches like HDL infusions, delipidated HDL, liver X receptor agonists, Apo A-I upregulators, Apo A mimetics, and gene therapy are in early phase trials. This review will outline current therapies and describe future directions for HDL therapeutics. Keywords: high-density lipoprotein, lipids, cholesterol, atherosclerosis, cardiovascular disease, therapy

Characterization of a family of gamma-ray-induced CHO mutants demonstrates that the ldlA locus is diploid and encodes the low-density lipoprotein receptor

International Nuclear Information System (INIS)

Sege, R.D.; Kozarsky, K.F.; Krieger, M.

1986-01-01

The ldlA locus is one of four Chinese hamster ovary (CHO) cell loci which are known to be required for the synthesis of functional low-density lipoprotein (LDL) receptors. Previous studies have suggested that the ldlA locus is diploid and encodes the LDL receptor. To confirm this assignment, we have isolated a partial genomic clone of the Chinese hamster LDL receptor gene and used this and other nucleic acid and antibody probes to study a family of ldlA mutants isolated after gamma-irradiation. Our analysis suggests that there are two LDL receptor alleles in wild-type CHO cells. Each of the three mutants isolated after gamma-irradiation had detectable deletions affecting one of the two LDL receptor alleles. One of the mutants also had a disruption of the remaining allele, resulting in the synthesis of an abnormal receptor precursor which was not subject to Golgi-associated posttranslational glycoprotein processing. The correlation of changes in the expression, structure, and function of LDL receptors with deletions in the LDL receptor genes in these mutants directly demonstrated that the ldlA locus in CHO cells is diploid and encodes the LDL receptor. In addition, our analysis suggests that CHO cells in culture may contain a partial LDL receptor pseudogene

Structural studies on lipoprotein lipase

International Nuclear Information System (INIS)

Socorro, L.

1985-01-01

The structure of lipoprotein lipase is not known. The lack of information on its primary sequence has been due to the inability of preparing it in homogeneous and stable form. This research has focused on the structural characterization of lipoprotein lipase. The first approach taken was to develop a purification method using bovine milk and affinity chromatography on heparin-Sepharose. The protein obtained was a heterogeneous peak with the activity shifted towards the trailing edge fractions. These fractions only presented a 55 Kdalton band on polyacrylamide gel electrophoresis. Monoclonal antibodies against this band detected an endogenous, phenyl methane sulfonyl fluoride-sensitive protease responsible for the presence of lower molecular weight fragments. The second approach was to label the lipoprotein lipase with a radioactive, active site, directed probe. After incubation and affinity chromatography a complex [ 3 H]inhibitor enzyme was isolated with a stoichiometry of 1.00 +/- 0.2. The complex was digested with CNBr and the insoluble peptides at low ionic strength (>90% [ 3 H]dpm) were used for further purification. Differential extraction of the [ 3 H]-peptide, digestion with S. aureus V8 protease, and high performance liquid chromatography yielded a hexapeptide with a composition consistent with the consensus sequence of the active site peptides of many serine-esterase. This and the kinetic data imply this being the mechanism of action for lipoprotein lipase

Remnant lipoproteins

DEFF Research Database (Denmark)

Varbo, Anette; Nordestgaard, Børge G.

2017-01-01

Purpose of review: To review recent advances in the field of remnant lipoproteins and remnant cholesterol with a focus on cardiovascular disease risk. Recent findings: In line with previous years' research, current observational, genetic, and mechanistic studies find remnant lipoproteins (defined...... of cardiovascular disease risk reduction through remnant lipoprotein lowering are under way....

Receptor oligomerization in family B1 of G-protein-coupled receptors

DEFF Research Database (Denmark)

Roed, Sarah Norklit; Ørgaard, Anne; Jørgensen, Rasmus

2012-01-01

, the glucagon receptor, and the receptors for parathyroid hormone (PTHR1 and PTHR2). The dysregulation of several family B1 receptors is involved in diseases, such as diabetes, chronic inflammation, and osteoporosis which underlines the pathophysiological importance of this GPCR subfamily. In spite of this......, investigation of family B1 receptor oligomerization and especially its pharmacological importance is still at an early stage. Even though GPCR oligomerization is a well-established phenomenon, there is a need for more investigations providing a direct link between these interactions and receptor functionality......The superfamily of the seven transmembrane G-protein-coupled receptors (7TM/GPCRs) is the largest family of membrane-associated receptors. GPCRs are involved in the pathophysiology of numerous human diseases, and they constitute an estimated 30-40% of all drug targets. During the last two decades...

Sorting of an integral outer membrane protein via the lipoprotein-specific Lol pathway and a dedicated lipoprotein pilotin.

Science.gov (United States)

Collin, Séverine; Guilvout, Ingrid; Nickerson, Nicholas N; Pugsley, Anthony P

2011-05-01

The lipoprotein PulS is a dedicated chaperone that is required to target the secretin PulD to the outer membrane in Klebsiella or Escherichia coli, and to protect it from proteolysis. Here, we present indirect evidence that PulD protomers do not assemble into the secretin dodecamer before they reach the outer membrane, and that PulS reaches the outer membrane in a soluble heterodimer with the general lipoprotein chaperone LolA. However, we could not find any direct evidence for PulD protomer association with the PulS-LolA heterodimer. Instead, in cells producing PulD and a permanently locked PulS-LolA dimer (in which LolA carries an R43L substitution that prevents lipoprotein transfer to LolB in the outer membrane), LolAR43L was found in the inner membrane, probably still associated with PulS bound to PulD that had been incorrectly targeted because of the LolAR43L substitution. It is speculated that PulD protomers normally cross the periplasm together with PulS bound to LolA but when the latter cannot be separated (due to the mutation in lolA), the PulD protomers form dodecamers that insert into the inner membrane. © 2011 Blackwell Publishing Ltd.

Polymorphisms of the low-density lipoprotein receptor gene in Brazilian individuals with heterozygous familial hypercholesterolemia

Directory of Open Access Journals (Sweden)

L.A. Salazar

2000-11-01

Full Text Available Familial hypercholesterolemia (FH is a metabolic disorder inherited as an autosomal dominant trait characterized by an increased plasma low-density lipoprotein (LDL level. The disease is caused by several different mutations in the LDL receptor gene. Although early identification of individuals carrying the defective gene could be useful in reducing the risk of atherosclerosis and myocardial infarction, the techniques available for determining the number of the functional LDL receptor molecules are difficult to carry out and expensive. Polymorphisms associated with this gene may be used for unequivocal diagnosis of FH in several populations. The aim of our study was to evaluate the genotype distribution and relative allele frequencies of three polymorphisms of the LDL receptor gene, HincII1773 (exon 12, AvaII (exon 13 and PvuII (intron 15, in 50 unrelated Brazilian individuals with a diagnosis of heterozygous FH and in 130 normolipidemic controls. Genomic DNA was extracted from blood leukocytes by a modified salting-out method. The polymorphisms were detected by PCR-RFLP. The FH subjects showed a higher frequency of A+A+ (AvaII, H+H+ (HincII1773 and P1P1 (PvuII homozygous genotypes when compared to the control group (P<0.05. In addition, FH probands presented a high frequency of A+ (0.58, H+ (0.61 and P1 (0.78 alleles when compared to normolipidemic individuals (0.45, 0.45 and 0.64, respectively. The strong association observed between these alleles and FH suggests that AvaII, HincII1773 and PvuII polymorphisms could be useful to monitor the inheritance of FH in Brazilian families.

Pharmacological characterization of receptor-activity-modifying proteins (RAMPs) and the human calcitonin receptor.

Science.gov (United States)

Armour, S L; Foord, S; Kenakin, T; Chen, W J

1999-12-01

Receptor-activity-modifying proteins (RAMPs) are a family of single transmembrane domain proteins shown to be important for the transport and ligand specificity of the calcitonin gene-related peptide (CGRP) receptor. In this report, we describe the analysis of pharmacological properties of the human calcitonin receptor (hCTR) coexpressed with different RAMPs with the use of the Xenopus laevis melanophore expression system. We show that coexpression of RAMP3 with human calcitonin receptor changed the relative potency of hCTR to human calcitonin (hCAL) and rat amylin. RAMP1 and RAMP2, in contrast, had little effect on the change of hCTR potency to hCAL or rat amylin. When coexpressed with RAMP3, hCTR reversed the relative potency by a 3.5-fold loss in sensitivity to hCAL and a 19-fold increase in sensitivity to rat amylin. AC66, an inverse agonist, produced apparent simple competitive antagonism of hCAL and rat amylin, as indicated by linear Schild regressions. The potency of AC66 was changed in the blockade of rat amylin but not hCAL responses with RAMP3 coexpression. The mean pK(B) for AC66 to hCAL was 9.4 +/- 0.3 without RAMP3 and 9.45 +/- 0.07 with RAMP3. For the antagonism of AC66 to rat amylin, the pK(B) was 9.25 +/- 0.15 without RAMP3 and 8.2 +/- 0.35 with RAMP3. The finding suggests that RAMP3 might modify the active states of calcitonin receptor in such a way as to create a new receptor phenotype that is "amylin-like." Irrespective of the physiological association of the new receptor species, the finding that a coexpressed membrane protein can completely change agonist and antagonist affinities for a receptor raises implications for screening in recombinant receptor systems.

[Alterations in the protein content and dysfunction of high-density lipoproteins from hyperhomocysteinemic mice].

Science.gov (United States)

Julve, Josep; Errico, Teresa Laura; Chen, Xiangyu; Santos, David; Freixa, Júlia; Porcel, Inmaculada; Cubero, Esther; Escolà-Gil, Joan Carles; Blanco-Vaca, Francisco

2013-01-01

The aim of this study was to evaluate the proteic changes in high-density lipoproteins (HDL) induced by methionine-induced hyperhomocysteinemia in mice and its relationship with two of their main antiatherogenic properties. The oral administration of methionine resulted in an elevation (~8 times) in the plasma concentration of homocysteine. Hyperhomocysteinemia was inversely correlated with the plasma concentration of HDL cholesterol and its main protein component of HDL, apolipoprotein (apo) A-I, respectively. The cholesterol efflux in vivo from macrophages to HDL was decreased in hyperhomocysteinemic mice compared with the control mice. However, the reverse cholesterol transport from macrophages to feces remained unchanged. On the other hand, the ability of HDL from hyperhomocysteinemic mice to prevent the oxidative modification of low-density lipoproteins (LDL) was found decreased and associated with a concomitant reduction in the plasma activity of paraoxonase-1 (PON1) and the plasma concentration of apoA-I, and with a relative reduction in the apoA-IV content (~1.5 times) in the hyperhomocysteinemic HDL, respectively. The decrease in the ability of HDL from hyperhomocysteinemic mice to prevent LDL from oxidation was associated with a decrease in the apoA-I, PON1 and apoA-IV. Copyright © 2013 Elsevier España, S.L. and SEA. All rights reserved.

A haptoglobin-hemoglobin receptor conveys innate immunity to Trypanosoma brucei in humans

DEFF Research Database (Denmark)

Vanhollebeke, Benoit; De Muylder, Géraldine; Nielsen, Marianne J

2008-01-01

The protozoan parasite Trypanosoma brucei is lysed by apolipoprotein L-I, a component of human high-density lipoprotein (HDL) particles that are also characterized by the presence of haptoglobin-related protein. We report that this process is mediated by a parasite glycoprotein receptor, which...... binds the haptoglobin-hemoglobin complex with high affinity for the uptake and incorporation of heme into intracellular hemoproteins. In mice, this receptor was required for optimal parasite growth and the resistance of parasites to the oxidative burst by host macrophages. In humans, the trypanosome...... immunity against the parasite....

Structure-based drug design for G protein-coupled receptors.

Science.gov (United States)

Congreve, Miles; Dias, João M; Marshall, Fiona H

2014-01-01

Our understanding of the structural biology of G protein-coupled receptors has undergone a transformation over the past 5 years. New protein-ligand complexes are described almost monthly in high profile journals. Appreciation of how small molecules and natural ligands bind to their receptors has the potential to impact enormously how medicinal chemists approach this major class of receptor targets. An outline of the key topics in this field and some recent examples of structure- and fragment-based drug design are described. A table is presented with example views of each G protein-coupled receptor for which there is a published X-ray structure, including interactions with small molecule antagonists, partial and full agonists. The possible implications of these new data for drug design are discussed. © 2014 Elsevier B.V. All rights reserved.

Protein-induced satiation and the calcium-sensing receptor

Directory of Open Access Journals (Sweden)

Ojha U

2018-03-01

Full Text Available Utkarsh Ojha Faculty of Medicine, Imperial College School of Medicine, Imperial College London, London, UK Abstract: Obesity is a major global health issue. High-protein diets have been shown to be associated with weight loss and satiety. The precise mechanism by which protein-rich diets promote weight loss remains unclear. Evidence suggests amino acids, formed as a consequence of protein digestion, are sensed by specific receptors on L-cells in the gastrointestinal (GI tract. These L-cells respond by secreting gut hormones that subsequently induce satiety. In recent years, the calcium-sensing receptor has been identified in several cells of the GI tract, including L-cells, and suggested to sense specific amino acids. This review evaluates the evidence for protein-rich diets in inducing weight loss and how the calcium-sensing receptor may be implicated in this phenomenon. Commandeering the mechanisms by which elements of a protein-rich diet suppress appetite may provide another successful avenue for developing anti-obesity drugs. Keywords: amino acids, energy regulation, obesity therapy, glucagon-like-peptide-1, peptide YY

Triglyceride-rich lipoprotein regulates APOB48 receptor gene expression in human THP-1 monocytes and macrophages.

Science.gov (United States)

Bermudez, Beatriz; Lopez, Sergio; Varela, Lourdes M; Ortega, Almudena; Pacheco, Yolanda M; Moreda, Wenceslao; Moreno-Luna, Rafael; Abia, Rocio; Muriana, Francisco J G

2012-02-01

The postprandial metabolism of dietary fats implies that the production of TG-rich lipoproteins (TRL) contributes to the progression of plaque development. TRL and their remnants cause rapid receptor-mediated monocyte/macrophage lipid engorgement via the cell surface apoB48 receptor (apoB48R). However, the mechanistic basis for apoB48 receptor (APOB48R) regulation by postprandial TRL in monocytes and macrophages is not well established. In this study, we investigated the effects of postprandial TRL from healthy volunteers on the expression of APOB48R mRNA and lipid uptake in human THP-1 monocytes and THP-1-derived macrophages. The expression of APOB48R mRNA was upregulated in THP-1 monocytes, but downregulated in THP-1-derived macrophages when treated with postprandial TRL (P < 0.05), in a dose- and time-dependent manner. TG and free cholesterol were dramatically increased in THP-1-derived macrophages (140 and 50%, respectively; P < 0.05) and in THP-1 monocytes (160 and 95%, respectively; P < 0.05). This lipid accumulation was severely decreased (~50%; P < 0.05) in THP-1-derived macrophages by small interfering RNA (siRNA) targeting of APOB48R. Using PPAR and retinoid X receptor (RXR) agonists, antagonists, and siRNA, our data indicate that PPARα, PPARγ, and RXRα are involved in postprandial TRL-induced APOB48R transcriptional regulation. Co-incubation with acyl-CoA synthetase or acyl-CoA:cholesterol acyltransferase inhibitors potentiated the effects of postprandial TRL on the expression of APOB48R mRNA in THP-1 monocytes and THP-1-derived macrophages. Our findings collectively suggest that APOB48R represents a molecular target of postprandial TRL via PPAR-dependent pathways in human THP-1 monocytes and macrophages and advance a potentially important link between postprandial metabolism of dietary fats and atherogenesis.

Trichoderma G protein-coupled receptors: functional characterisation of a cAMP receptor-like protein from Trichoderma atroviride.

Science.gov (United States)

Brunner, Kurt; Omann, Markus; Pucher, Marion E; Delic, Marizela; Lehner, Sylvia M; Domnanich, Patrick; Kratochwill, Klaus; Druzhinina, Irina; Denk, Dagmar; Zeilinger, Susanne

2008-12-01

Galpha subunits act to regulate vegetative growth, conidiation, and the mycoparasitic response in Trichoderma atroviride. To extend our knowledge on G protein signalling, we analysed G protein-coupled receptors (GPCRs). As the genome sequence of T. atroviride is not publicly available yet, we carried out an in silico exploration of the genome database of the close relative T. reesei. Twenty genes encoding putative GPCRs distributed over eight classes and additional 35 proteins similar to the Magnaporthe grisea PTH11 receptor were identified. Subsequently, four T. atroviride GPCR-encoding genes were isolated and affiliated to the cAMP receptor-like family by phylogenetic and topological analyses. All four genes showed lowest expression on glycerol and highest mRNA levels upon carbon starvation. Transcription of gpr3 and gpr4 responded to exogenously added cAMP and the shift from liquid to solid media. gpr3 mRNA levels also responded to the presence of fungal hyphae or cellulose membranes. Further characterisation of mutants bearing a gpr1-silencing construct revealed that Gpr1 is essential for vegetative growth, conidiation and conidial germination. Four genes encoding the first GPCRs described in Trichoderma were isolated and their expression characterized. At least one of these GPCRs is important for several cellular processes, supporting the fundamental role of G protein signalling in this fungus.

Modulation of β-catenin signaling by glucagon receptor activation.

Directory of Open Access Journals (Sweden)

Jiyuan Ke

Full Text Available The glucagon receptor (GCGR is a member of the class B G protein-coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA pathway, activation of GCGR also induced β-catenin stabilization and activated β-catenin-mediated transcription. Activation of β-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R and glucagon-like peptide 1 (GLP-1R receptors. Since low-density-lipoprotein receptor-related protein 5 (Lrp5 is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced β-catenin stabilization and TCF promoter-mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1 or by expression of the Lrp5 extracellular domain blocked glucagon-induced β-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and β-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations.

Hepatitis C virus E2 protein promotes human hepatoma cell proliferation through the MAPK/ERK signaling pathway via cellular receptors

International Nuclear Information System (INIS)

Zhao Lanjuan; Wang Lu; Ren Hao; Cao Jie; Li Li; Ke Jinshan; Qi Zhongtian

2005-01-01

Dysregulation of mitogen-activated protein kinase (MAPK) signaling pathways by various viruses has been shown to be responsible for viral pathogenicity. The molecular mechanism by which hepatitis C virus (HCV) infection caused human liver diseases has been investigated on the basis of abnormal intracellular signal events. Current data are very limited involved in transmembrane signal transduction triggered by HCV E2 protein. Here we explored regulation of the MAPK/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway by E2 expressed in Chinese hamster oval cells. In human hepatoma Huh-7 cells, E2 specifically activated the MAPK/ERK pathway including downstream transcription factor ATF-2 and greatly promoted cell proliferation. CD81 and low density lipoprotein receptor (LDLR) on the cell surface mediated binding of E2 to Huh-7 cells. The MAPK/ERK activation and cell proliferation driven by E2 were suppressed by blockage of CD81 as well as LDLR. Furthermore, pretreatment with an upstream kinase MEK1/2 inhibitor U0126 also impaired the MAPK/ERK activation and cell proliferation induced by E2. Our results suggest that the MAPK/ERK signaling pathway triggered by HCV E2 via its receptors maintains survival and growth of target cells

G protein-coupled receptor mutations and human genetic disease.

Science.gov (United States)

Thompson, Miles D; Hendy, Geoffrey N; Percy, Maire E; Bichet, Daniel G; Cole, David E C

2014-01-01

Genetic variations in G protein-coupled receptor genes (GPCRs) disrupt GPCR function in a wide variety of human genetic diseases. In vitro strategies and animal models have been used to identify the molecular pathologies underlying naturally occurring GPCR mutations. Inactive, overactive, or constitutively active receptors have been identified that result in pathology. These receptor variants may alter ligand binding, G protein coupling, receptor desensitization and receptor recycling. Receptor systems discussed include rhodopsin, thyrotropin, parathyroid hormone, melanocortin, follicle-stimulating hormone (FSH), luteinizing hormone, gonadotropin-releasing hormone (GNRHR), adrenocorticotropic hormone, vasopressin, endothelin-β, purinergic, and the G protein associated with asthma (GPRA or neuropeptide S receptor 1 (NPSR1)). The role of activating and inactivating calcium-sensing receptor (CaSR) mutations is discussed in detail with respect to familial hypocalciuric hypercalcemia (FHH) and autosomal dominant hypocalemia (ADH). The CASR mutations have been associated with epilepsy. Diseases caused by the genetic disruption of GPCR functions are discussed in the context of their potential to be selectively targeted by drugs that rescue altered receptors. Examples of drugs developed as a result of targeting GPCRs mutated in disease include: calcimimetics and calcilytics, therapeutics targeting melanocortin receptors in obesity, interventions that alter GNRHR loss from the cell surface in idiopathic hypogonadotropic hypogonadism and novel drugs that might rescue the P2RY12 receptor congenital bleeding phenotype. De-orphanization projects have identified novel disease-associated receptors, such as NPSR1 and GPR35. The identification of variants in these receptors provides genetic reagents useful in drug screens. Discussion of the variety of GPCRs that are disrupted in monogenic Mendelian disorders provides the basis for examining the significance of common

The Journey of Lipoproteins Through the Cell: One Birthplace, Multiple Destinations.

Science.gov (United States)

Szewczyk, J; Collet, J-F

2016-01-01

Bacterial lipoproteins are a very diverse group of proteins characterized by the presence of an N-terminal lipid moiety that serves as a membrane anchor. Lipoproteins have a wide variety of crucial functions, ranging from envelope biogenesis to stress response. In Gram-negative bacteria, lipoproteins can be targeted to various destinations in the cell, including the periplasmic side of the cytoplasmic or outer membrane, the cell surface or the external milieu. The sorting mechanisms have been studied in detail in Escherichia coli, but exceptions to the rules established in this model bacterium exist in other bacteria. In this chapter, we will present the current knowledge on lipoprotein sorting in the cell. Our particular focus will be on the surface-exposed lipoproteins that appear to be much more common than previously assumed. We will discuss the different targeting strategies, provide numerous examples of surface-exposed lipoproteins and discuss the techniques used to assess their surface exposure. © 2016 Elsevier Ltd All rights reserved.

Specific deletion of LDL receptor-related protein on macrophages has skewed in vivo effects on cytokine production by invariant natural killer T cells.

Directory of Open Access Journals (Sweden)

Roman Covarrubias

Full Text Available Expression of molecules involved in lipid homeostasis such as the low density lipoprotein receptor (LDLr on antigen presenting cells (APCs has been shown to enhance invariant natural killer T (iNKT cell function. However, the contribution to iNKT cell activation by other lipoprotein receptors with shared structural and ligand binding properties to the LDLr has not been described. In this study, we investigated whether a structurally related receptor to the LDLr, known as LDL receptor-related protein (LRP, plays a role in iNKT cell activation. We found that, unlike the LDLr which is highly expressed on all immune cells, the LRP was preferentially expressed at high levels on F4/80+ macrophages (MΦ. We also show that CD169+ MΦs, known to present antigen to iNKT cells, exhibited increased expression of LRP compared to CD169- MΦs. To test the contribution of MΦ LRP to iNKT cell activation we used a mouse model of MΦ LRP conditional knockout (LRP-cKO. LRP-cKO MΦs pulsed with glycolipid alpha-galactosylceramide (αGC elicited normal IL-2 secretion by iNKT hybridoma and in vivo challenge of LRP-cKO mice led to normal IFN-γ, but blunted IL-4 response in both serum and intracellular expression by iNKT cells. Flow cytometric analyses show similar levels of MHC class-I like molecule CD1d on LRP-cKO MΦs and normal glycolipid uptake. Survey of the iNKT cell compartment in LRP-cKO mice revealed intact numbers and percentages and no homeostatic disruption as evidenced by the absence of programmed death-1 and Ly-49 surface receptors. Mixed bone marrow chimeras showed that the inability iNKT cells to make IL-4 is cell extrinsic and can be rescued in the presence of wild type APCs. Collectively, these data demonstrate that, although MΦ LRP may not be necessary for IFN-γ responses, it can contribute to iNKT cell activation by enhancing early IL-4 secretion.

Induction of bacterial lipoprotein tolerance is associated with suppression of toll-like receptor 2 expression.

LENUS (Irish Health Repository)

Wang, Jiang Huai

2012-02-03

Tolerance to bacterial cell wall components including lipopolysaccharide (LPS) may represent an essential regulatory mechanism during bacterial infection. Two members of the Toll-like receptor (TLR) family, TLR2 and TLR4, recognize the specific pattern of bacterial cell wall components. TLR4 has been found to be responsible for LPS tolerance. However, the role of TLR2 in bacterial lipoprotein (BLP) tolerance and LPS tolerance is unclear. Pretreatment of human THP-1 monocytic cells with a synthetic bacterial lipopeptide induced tolerance to a second BLP challenge with diminished tumor necrosis factor-alpha and interleukin-6 production, termed BLP tolerance. Furthermore, BLP-tolerized THP-1 cells no longer responded to LPS stimulation, indicating a cross-tolerance to LPS. Induction of BLP tolerance was CD14-independent, as THP-1 cells that lack membrane-bound CD14 developed tolerance both in serum-free conditions and in the presence of a specific CD14 blocking monoclonal antibody (MEM-18). Pre-exposure of THP-1 cells to BLP suppressed mitogen-activated protein kinase phosphorylation and nuclear factor-kappaB activation in response to subsequent BLP and LPS stimulation, which is comparable with that found in LPS-tolerized cells, indicating that BLP tolerance and LPS tolerance may share similar intracellular pathways. However, BLP strongly enhanced TLR2 expression in non-tolerized THP-1 cells, whereas LPS stimulation had no effect. Furthermore, a specific TLR2 blocking monoclonal antibody (2392) attenuated BLP-induced, but not LPS-induced, tumor necrosis factor-alpha and interleukin-6 production, indicating BLP rather than LPS as a ligand for TLR2 engagement and activation. More importantly, pretreatment of THP-1 cells with BLP strongly inhibited TLR2 activation in response to subsequent BLP stimulation. In contrast, LPS tolerance did not prevent BLP-induced TLR2 overexpression. These results demonstrate that BLP tolerance develops through down-regulation of TLR2

Constitutive dimerization of the G-protein coupled receptor, neurotensin receptor 1, reconstituted into phospholipid bilayers.

Science.gov (United States)

Harding, Peter J; Attrill, Helen; Boehringer, Jonas; Ross, Simon; Wadhams, George H; Smith, Eleanor; Armitage, Judith P; Watts, Anthony

2009-02-01

Neurotensin receptor 1 (NTS1), a Family A G-protein coupled receptor (GPCR), was expressed in Escherichia coli as a fusion with the fluorescent proteins eCFP or eYFP. A fluorophore-tagged receptor was used to study the multimerization of NTS1 in detergent solution and in brain polar lipid bilayers, using fluorescence resonance energy transfer (FRET). A detergent-solubilized receptor was unable to form FRET-competent complexes at concentrations of up to 200 nM, suggesting that the receptor is monomeric in this environment. When reconstituted into a model membrane system at low receptor density, the observed FRET was independent of agonist binding, suggesting constitutive multimer formation. In competition studies, decreased FRET in the presence of untagged NTS1 excludes the possibility of fluorescent protein-induced interactions. A simulation of the experimental data indicates that NTS1 exists predominantly as a homodimer, rather than as higher-order multimers. These observations suggest that, in common with several other Family A GPCRs, NTS1 forms a constitutive dimer in lipid bilayers, stabilized through receptor-receptor interactions in the absence of other cellular signaling components. Therefore, this work demonstrates that well-characterized model membrane systems are useful tools for the study of GPCR multimerization, allowing fine control over system composition and complexity, provided that rigorous control experiments are performed.

Getting a Handle on Neuropharmacology by Targeting Receptor-Associated Proteins.

Science.gov (United States)

Maher, Michael P; Matta, Jose A; Gu, Shenyan; Seierstad, Mark; Bredt, David S

2017-12-06

Targeted therapy for neuropsychiatric disorders requires selective modulation of dysfunctional neuronal pathways. Receptors relevant to CNS disorders typically have associated proteins discretely expressed in specific neuronal pathways; these accessory proteins provide a new dimension for drug discovery. Recent studies show that targeting a TARP auxiliary subunit of AMPA receptors selectively modulates neuronal excitability in specific forebrain pathways relevant to epilepsy. Other medicinally important ion channels, gated by glutamate, γ-aminobutyric acid (GABA), and acetylcholine, also have associated proteins, which may be druggable. This emerging pharmacology of receptor-associated proteins provides a new approach for improving drug efficacy while mitigating side effects. Copyright © 2017 Elsevier Inc. All rights reserved.

The association of metabotropic glutamate receptor type 5 with the neuronal Ca2+-binding protein 2 modulates receptor function.

Science.gov (United States)

Canela, Laia; Fernández-Dueñas, Víctor; Albergaria, Catarina; Watanabe, Masahiko; Lluís, Carme; Mallol, Josefa; Canela, Enric I; Franco, Rafael; Luján, Rafael; Ciruela, Francisco

2009-10-01

Metabotropic glutamate (mGlu) receptors mediate in part the CNS effects of glutamate. These receptors interact with a large array of intracellular proteins in which the final role is to regulate receptor function. Here, using co-immunoprecipitation and pull-down experiments we showed a close and specific interaction between mGlu(5) receptor and NECAB2 in both transfected human embryonic kidney cells and rat hippocampus. Interestingly, in pull-down experiments increasing concentrations of calcium drastically reduced the ability of these two proteins to interact, suggesting that NECAB2 binds to mGlu(5) receptor in a calcium-regulated manner. Immunoelectron microscopy detection of NECAB2 and mGlu(5) receptor in the rat hippocampal formation indicated that both proteins are codistributed in the same subcellular compartment of pyramidal cells. In addition, the NECAB2/mGlu(5) receptor interaction regulated mGlu(5b)-mediated activation of both inositol phosphate accumulation and the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Overall, these findings indicate that NECAB2 by its physical interaction with mGlu(5b) receptor modulates receptor function.

Ligand binding to G protein-coupled receptors in tethered cell membranes

DEFF Research Database (Denmark)

Martinez, Karen L.; Meyer, Bruno H.; Hovius, Ruud

2003-01-01

for the surface immobilization of membrane proteins was developed using the prototypic seven transmembrane neurokinin-1 receptor. The receptor was expressed as a biotinylated protein in mammalian cells. Membranes from cell homogenates were selectively immobilized on glass surfaces covered with streptavidin. TIRF...... measurements showed that a fluorescent agonist binds to the receptor on the sensor surface with similar affinity as to the receptor in live cells. This approach offers the possibility to investigate minute amounts of membrane protein in an active form and in its native environment without purification....

Model of mouth-to-mouth transfer of bacterial lipoproteins through inner membrane LolC, periplasmic LolA, and outer membrane LolB

OpenAIRE

Okuda, Suguru; Tokuda, Hajime

2009-01-01

Outer membrane-specific lipoproteins in Escherichia coli are released from the inner membrane by an ATP-binding cassette transporter, the LolCDE complex, which causes the formation of a soluble complex with a periplasmic molecular chaperone, LolA. LolA then transports lipoproteins to the outer membrane where an outer membrane receptor, LolB, incorporates lipoproteins into the outer membrane. The molecular mechanisms underlying the Lol-dependent lipoprotein sorting have been clarified in detai...

Guanine nucleotide regulatory protein co-purifies with the D2-dopamine receptor

International Nuclear Information System (INIS)

Senogles, S.E.; Caron, M.G.

1986-01-01

The D 2 -dopamine receptor from bovine anterior pituitary was purified ∼1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with 3 H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D 2 receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 μM NPA. 35 S-GTPγS binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D 2 -dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D 2 -dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes

Cloning of human genes encoding novel G protein-coupled receptors

Energy Technology Data Exchange (ETDEWEB)

Marchese, A.; Docherty, J.M.; Heiber, M. [Univ. of Toronto, (Canada)] [and others

1994-10-01

We report the isolation and characterization of several novel human genes encoding G protein-coupled receptors. Each of the receptors contained the familiar seven transmembrane topography and most closely resembled peptide binding receptors. Gene GPR1 encoded a receptor protein that is intronless in the coding region and that shared identity (43% in the transmembrane regions) with the opioid receptors. Northern blot analysis revealed that GPR1 transcripts were expressed in the human hippocampus, and the gene was localized to chromosome 15q21.6. Gene GPR2 encoded a protein that most closely resembled an interleukin-8 receptor (51% in the transmembrane regions), and this gene, not expressed in the six brain regions examined, was localized to chromosome 17q2.1-q21.3. A third gene, GPR3, showed identity (56% in the transmembrane regions) with a previously characterized cDNA clone from rat and was localized to chromosome 1p35-p36.1. 31 refs., 5 figs., 1 tab.

Glycation and glycoxidation of low-density lipoproteins by glucose and low-molecular mass aldehydes. Formation of modified and oxidized particles

DEFF Research Database (Denmark)

Knott, Heather M; Brown, Bronwyn E; Davies, Michael Jonathan

2003-01-01

by the adduction of glucose or species derived from glucose, such as low-molecular mass aldehydes, to proteins. These reactions can be nonoxidative (glycation) or oxidative (glycoxidation) and result in the conversion of low-density lipoproteins (LDL) to a form that is recognized by the scavenger receptors...... with glucose. These processes are rapid and unaffected by low concentrations of copper ions. In contrast, lipid and protein oxidation are slow processes and occur to a limited extent in the absence of added copper ions. No evidence was obtained for the stimulation of lipid or protein oxidation by glucose...... or methylglyoxal in the presence of copper ions, whereas glycolaldehyde stimulated such reactions to a modest extent. These results suggest that the earliest significant events in this system are metal ion-independent glycation (modification) of the protein component of LDL, whilst oxidative events (glycoxidation...

Synthetic Nano-Low Density Lipoprotein as Targeted Drug DeliveryVehicle for Glioblastoma Multiforme

Energy Technology Data Exchange (ETDEWEB)

Nikanjam, Mina; Blakely, Eleanor A.; Bjornstad, Kathleen A.; Shu,Xiao; Budinger, Thomas F.; Forte, Trudy M.

2006-06-14

This paper discribes a synthetic low density lipoprotein(LDL) made by complexing a 29 amino acid that consists of a lipid bindingdomain and the LDL receptor binding domain with a lipid microemulsion.The nano-LDL particles were intermdiate in size between LDL and HDL andbound to LDL receptors on GBM brain tumor cells. Synthetic nano-LDLuptake by GBM cells was LDL receptor specific and dependent on cellreceptor number. It is suggested that these synthetic particles can serveas a delivery vehicle for hydophobic anti-tumor drugs by targeting theLDL receptor.

The Dual Role of Lipids of the Lipoproteins in Trumenba, a Self-Adjuvanting Vaccine Against Meningococcal Meningitis B Disease.

Science.gov (United States)

Luo, Yin; Friese, Olga V; Runnels, Herbert A; Khandke, Lakshmi; Zlotnick, Gary; Aulabaugh, Ann; Gore, Thomas; Vidunas, Eugene; Raso, Stephen W; Novikova, Elena; Byrne, Emilia; Schlittler, Michael; Stano, Donald; Dufield, Robert L; Kumar, Sandeep; Anderson, Annaliesa S; Jansen, Kathrin U; Rouse, Jason C

2016-11-01

Trumenba (bivalent rLP2086) is a vaccine licensed for the prevention of meningococcal meningitis disease caused by Neisseria meningitidis serogroup B (NmB) in individuals 10-25 years of age in the USA. The vaccine is composed of two factor H binding protein (fHbp) variants that were recombinantly expressed in Escherichia coli as native lipoproteins: rLP2086-A05 and rLP2086-B01. The vaccine was shown to induce potent bactericidal antibodies against a broad range of NmB isolates expressing fHbp that were different in sequence from the fHbp vaccine antigens. Here, we describe the characterization of the vaccine antigens including the elucidation of their structure which is characterized by two distinct motifs, the polypeptide domain and the N-terminal lipid moiety. In the vaccine formulation, the lipoproteins self-associate to form micelles driven by the hydrophobicity of the lipids and limited by the size of the folded polypeptides. The micelles help to increase the structural stability of the lipoproteins in the absence of bacterial cell walls. Analysis of the lipoproteins in Toll-like receptor (TLR) activation assays revealed their TLR2 agonist activity. This activity was lost with removal of the O-linked fatty acids, similar to removal of all lipids, demonstrating that this moiety plays an adjuvant role in immune activation. The thorough understanding of the structure and function of each moiety of the lipoproteins, as well as their relationship, lays the foundation for identifying critical parameters to guide vaccine development and manufacture.

The G protein Gi1 exhibits basal coupling but not preassembly with G protein-coupled receptors.

Science.gov (United States)

Bondar, Alexey; Lazar, Josef

2017-06-09

The G i/o protein family transduces signals from a diverse group of G protein-coupled receptors (GPCRs). The observed specificity of G i/o -GPCR coupling and the high rate of G i/o signal transduction have been hypothesized to be enabled by the existence of stable associates between G i/o proteins and their cognate GPCRs in the inactive state (G i/o -GPCR preassembly). To test this hypothesis, we applied the recently developed technique of two-photon polarization microscopy (2PPM) to Gα i1 subunits labeled with fluorescent proteins and four GPCRs: the α 2A -adrenergic receptor, GABA B , cannabinoid receptor type 1 (CB 1 R), and dopamine receptor type 2. Our experiments with non-dissociating mutants of fluorescently labeled Gα i1 subunits (exhibiting impaired dissociation from activated GPCRs) showed that 2PPM is capable of detecting GPCR-G protein interactions. 2PPM experiments with non-mutated fluorescently labeled Gα i1 subunits and α 2A -adrenergic receptor, GABA B , or dopamine receptor type 2 receptors did not reveal any interaction between the G i1 protein and the non-stimulated GPCRs. In contrast, non-stimulated CB 1 R exhibited an interaction with the G i1 protein. Further experiments revealed that this interaction is caused solely by CB 1 R basal activity; no preassembly between CB 1 R and the G i1 protein could be observed. Our results demonstrate that four diverse GPCRs do not preassemble with non-active G i1 However, we also show that basal GPCR activity allows interactions between non-stimulated GPCRs and G i1 (basal coupling). These findings suggest that G i1 interacts only with active GPCRs and that the well known high speed of GPCR signal transduction does not require preassembly between G proteins and GPCRs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Defective Lipoprotein Sorting Induces lolA Expression through the Rcs Stress Response Phosphorelay System

OpenAIRE

Tao, Kazuyuki; Narita, Shin-ichiro; Tokuda, Hajime

2012-01-01

The Escherichia coli LolA protein is a lipoprotein-specific chaperone that carries lipoproteins from the inner to the outer membrane. A dominant negative LolA mutant, LolA(I93C/F140C), in which both 93Ile and 140Phe are replaced by Cys, binds tightly to the lipoprotein-dedicated ABC transporter LolCDE complex on the inner membrane and therefore inhibits the detachment of outer membrane-specific lipoproteins from the inner membrane. We found that the expression of this mutant strongly induced ...

Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein

International Nuclear Information System (INIS)

Williamson, K.; Dickey, B.F.; Pyun, H.Y.; Navarro, J.

1988-01-01

The authors describe the solubilization, resolution, and reconstitution of the formylmethionylleucylphenylalanine (fMet-Leu-Phe) receptor and guanine nucleotide regulatory proteins (G-proteins). The receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Guanine nucleotides decreased the number of high-affinity binding sites and accelerated the rate of dissociation of the receptor-ligand complex, suggesting that the solubilized receptor remained coupled to endogenous G-proteins. The solubilized receptor was resolved from endogenous G-proteins by fractionation on a wheat germ agglutinin (WGA)-Sepharose 4B column. High-affinity [ 3 H]fMet-Leu-Phe binding to the WGA-purified receptor was diminished and exhibited reduced guanine nucleotide sensitivity. High-affinity [ 3 H]fMET-Leu-Phe binding and guanine nucleotide sensitivity were reconstituted upon the addition of purified brain G-proteins. Similar results were obtained when the receptor was reconstituted with brain G-proteins into phospholipid vesicles by gel filtration chromatography. In addition, they also demonstrated fMET-Leu-Phe-dependent GTP hydrolysis in the reconstituted vesicles. The results of this work indicate that coupling of the fMet-Leu-Phe receptor to G-proteins converts the receptor to a high-affinity binding state and that agonist produces activation of G-proteins. The resolution and functional reconstitution of this receptor should provide an important step toward the elucidation of the molecular mechanism of the fMet-Leu-Phe transduction system in neutrophils

Elevated plasma low-density lipoprotein and high-density lipoprotein cholesterol levels in amenorrheic athletes: effects of endogenous hormone status and nutrient intake.

Science.gov (United States)

Friday, K E; Drinkwater, B L; Bruemmer, B; Chesnut, C; Chait, A

1993-12-01

To determine the interactive effects of hormones, exercise, and diet on plasma lipids and lipoproteins, serum estrogen and progesterone levels, nutrient intake, and plasma lipid, lipoprotein, and apolipoprotein concentrations were measured in 24 hypoestrogenic amenorrheic and 44 eumenorrheic female athletes. When compared to eumenorrheic athletes, amenorrheic athletes had higher levels of plasma cholesterol (5.47 +/- 0.17 vs. 4.84 +/- 0.12 mmol/L, P = 0.003), triglyceride (0.75 +/- 0.06 vs. 0.61 +/- 0.03 mmol/L, P = 0.046), low-density lipoprotein (LDL; 3.16 +/- 0.15 vs. 2.81 +/- 0.09 mmol/L, P = 0.037), high-density lipoprotein (HDL; 1.95 +/- 0.07 vs. 1.73 +/- 0.05 mmol/L, P = 0.007), and HDL2 (0.84 +/- 0.06 vs. 0.68 +/- 0.04 mmol/L, P = 0.02) cholesterol. Plasma LDL/HDL cholesterol ratios, very low-density lipoprotein and HDL3 cholesterol, and apolipoprotein A-I and A-II levels were similar in the two groups. Amenorrheic athletes consumed less fat than eumenorrheic subjects (52 +/- 5 vs. 75 +/- 3 g/day, P = 0.02), but similar amounts of calories, cholesterol, protein, carbohydrate, and ethanol. HDL cholesterol levels in amenorrheic subjects correlated positively with the percent of dietary calories from fat (r = 0.42, n = 23, P = 0.045) but negatively with the percent from protein (r = -0.49, n = 23, P = 0.017). Thus, exercise-induced amenorrhea may adversely affect cardiovascular risk by increasing plasma LDL and total cholesterol. However, cardioprotective elevations in plasma HDL and HDL2 cholesterol may neutralize the risk of cardiovascular disease in amenorrheic athletes.

Characterization of the β-barrel assembly machine accessory lipoproteins from Borrelia burgdorferi.

Science.gov (United States)

Dunn, Joshua P; Kenedy, Melisha R; Iqbal, Henna; Akins, Darrin R

2015-03-24

Like all diderm bacteria studied to date, Borrelia burgdorferi possesses a β-barrel assembly machine (BAM) complex. The bacterial BAM complexes characterized thus far consist of an essential integral outer membrane protein designated BamA and one or more accessory proteins. The accessory proteins are typically lipid-modified proteins anchored to the inner leaflet of the outer membrane through their lipid moieties. We previously identified and characterized the B. burgdorferi BamA protein in detail and more recently identified two lipoproteins encoded by open reading frames bb0324 and bb0028 that associate with the borrelial BamA protein. The role(s) of the BAM accessory lipoproteins in B. burgdorferi is currently unknown. Structural modeling of B. burgdorferi BB0028 revealed a distinct β-propeller fold similar to the known structure for the E. coli BAM accessory lipoprotein BamB. Additionally, the structural model for BB0324 was highly similar to the known structure of BamD, which is consistent with the prior finding that BB0324 contains tetratricopeptide repeat regions similar to other BamD orthologs. Consistent with BB0028 and BB0324 being BAM accessory lipoproteins, mutants lacking expression of each protein were found to exhibit altered membrane permeability and enhanced sensitivity to various antimicrobials. Additionally, BB0028 mutants also exhibited significantly impaired in vitro growth. Finally, immunoprecipitation experiments revealed that BB0028 and BB0324 each interact specifically and independently with BamA to form the BAM complex in B. burgdorferi. Combined structural studies, functional assays, and co-immunoprecipitation experiments confirmed that BB0028 and BB0324 are the respective BamB and BamD orthologs in B. burgdorferi, and are important in membrane integrity and/or outer membrane protein localization. The borrelial BamB and BamD proteins both interact specifically and independently with BamA to form a tripartite BAM complex in B

Synthesis of Lipidated Proteins.

Science.gov (United States)

Mejuch, Tom; Waldmann, Herbert

2016-08-17

Protein lipidation is one of the major post-translational modifications (PTM) of proteins. The attachment of the lipid moiety frequently determines the localization and the function of the lipoproteins. Lipidated proteins participate in many essential biological processes in eukaryotic cells, including vesicular trafficking, signal transduction, and regulation of the immune response. Malfunction of these cellular processes usually leads to various diseases such as cancer. Understanding the mechanism of cellular signaling and identifying the protein-protein and protein-lipid interactions in which the lipoproteins are involved is a crucial task. To achieve these goals, fully functional lipidated proteins are required. However, access to lipoproteins by means of standard expression is often rather limited. Therefore, semisynthetic methods, involving the synthesis of lipidated peptides and their subsequent chemoselective ligation to yield full-length lipoproteins, were developed. In this Review we summarize the commonly used methods for lipoprotein synthesis and the development of the corresponding chemoselective ligation techniques. Several key studies involving full-length semisynthetic lipidated Ras, Rheb, and LC3 proteins are presented.

Characteristics of 2,4,5,2',4',5'-hexachlorobiphenyl distribution among lipoproteins in vitro

International Nuclear Information System (INIS)

Vomachka, M.S.; Vodicnik, M.J.; Lech, J.J.

1983-01-01

The uptake, distribution, and transfer of 2,4,5,2',4',5'-hexachlorobiphenyl (6-CB) were examined in vitro with human and rat whole blood, plasma, and lipoprotein fractions. 6-CB distribution between plasma and erythrocytes as well as among lipoproteins was determined following sedimentation of erythrocytes and ultracentrifugal fractionation of plasma. In both rat and human whole blood, 70 to 75% of 6-CB partitioned into plasma and 25 to 30% into erythrocytes. The uptake of 6-CB into plasma was extremely rapid and the rate of uptake was found to be dependent upon temperature. The distribution of 6-CB among lipoproteins was relatively homogeneous with 20 to 30% being distributed in very low-density lipoproteins (VLDL, d . 0.95-1.006 g/ml), 15 to 20% in low-density lipoproteins (LDL, d . 1.006-1.063 g/ml), and 15 to 25% in high-density lipoproteins (HDL, d . 1.063-1.21 g/ml). Over 25% of 6-CB was found in the remaining bottom fraction. In addition, each isolated fraction when incubated alone with 6-CB was shown capable of uptake. The relative proportion of 6-CB among the lipoproteins was independent of the level taken up by plasma. 6-CB was also found to transfer among lipoproteins. This exchange of 6-CB proved to be dependent upon the concentrations of both protein and triacylglycerol in the incubations. Two proteins in the bottom fraction (Bf), albumin and a steroid binding globulin, were capable of competing with the lipoproteins for 6-CB uptake

Semiotic Selection of Mutated or Misfolded Receptor Proteins

DEFF Research Database (Denmark)

Giorgi, Franco; Bruni, Luis Emilio; Maggio, Roberto

2013-01-01

contention that the plasma membrane acts as the locus where several contextual cues may be integrated. As such it allows the semiotic selection of those receptor configurations that provide cells with the minimum essential requirements for agency. The occurrence of protein misfolding makes it impossible...... focused on the significance and semiotic nature of the interplay between membrane receptors and the epigenetic control of gene expression, as mediated by the control of mismatched repairing and protein folding mechanisms....

Effect of I125 on oxidation behavior of lipoprotein subpopulations

International Nuclear Information System (INIS)

Majtenyi, S.

2002-07-01

Lipoproteins play a central role in lipid metabolism. They serve as a transport vehicle for cholesterol and triglycerides keeping them in plasma in solution. Lipoproteins are characterized by the content of specific apoproteins and differences in the hydrated density ranges. Moreover, they are distinguished by electrophoretic mobility and other characteristics as high and low-density lipoproteins, respectively lipoprotein (a). More specifically, HDL is classified into HDL 2 and HDL 3 . In atherogenesis, lipoproteins are considered to play a key-role. Oxidatively modified LDL is selectively taken up via scavenger receptors of the macrophage-monocyte system. These cells are transformed into foam cells promoting atherogenesis in vessels in the subendothelial space. Oxidized HDL essentially appears to loose its protective effects on LDL and its beneficial function in reverse cholesterol transport. Thus, it turns proatherogenic. The effects various species of free radicals exert on lipoproteins are the reason for this oxidative modification. Thyroid function also influences lipoproteins in a complex manner. Based on their hydrated density ranges, lipoprotein subpopulations were fractionated and isolated via isopycnic density gradient ultracentrifugation. After investigation of the general oxidation behavior, initiated by addition of CuSO 4 to the isolated samples of HDL 3 , HDL 2 , LDL and Lp(a), the influence of different activities of radioiodine-125 on the kinetics of the formation of conjugated dienes was assessed. This was achieved by coincubation of plasma with I 125 . The spectrophotometrical measurement of the concentration of conjugated dienes in the course of CuSO 4 -induced lipid peroxidation leads to measurable changes in absorption at 234 nm. These changes in absorption over time result in a characteristically shaped curve graphically plotted. The shape of these curves mirrors different indicators of lipid peroxidation. Therefrom lag time, maximal

Liver protein profiles in insulin receptor-knockout mice reveal novel molecules involved in the diabetes pathophysiology.

Science.gov (United States)

Capuani, Barbara; Della-Morte, David; Donadel, Giulia; Caratelli, Sara; Bova, Luca; Pastore, Donatella; De Canio, Michele; D'Aguanno, Simona; Coppola, Andrea; Pacifici, Francesca; Arriga, Roberto; Bellia, Alfonso; Ferrelli, Francesca; Tesauro, Manfredi; Federici, Massimo; Neri, Anna; Bernardini, Sergio; Sbraccia, Paolo; Di Daniele, Nicola; Sconocchia, Giuseppe; Orlandi, Augusto; Urbani, Andrea; Lauro, Davide

2015-05-01

Liver has a principal role in glucose regulation and lipids homeostasis. It is under a complex control by substrates such as hormones, nutrients, and neuronal impulses. Insulin promotes glycogen synthesis, lipogenesis, and lipoprotein synthesis and inhibits gluconeogenesis, glycogenolysis, and VLDL secretion by modifying the expression and enzymatic activity of specific molecules. To understand the pathophysiological mechanisms leading to metabolic liver disease, we analyzed liver protein patterns expressed in a mouse model of diabetes by proteomic approaches. We used insulin receptor-knockout (IR(-/-)) and heterozygous (IR(+/-)) mice as a murine model of liver metabolic dysfunction associated with diabetic ketoacidosis and insulin resistance. We evaluated liver fatty acid levels by microscopic examination and protein expression profiles by orthogonal experimental strategies using protein 2-DE MALDI-TOF/TOF and peptic nLC-MS/MS shotgun profiling. Identified proteins were then loaded into Ingenuity Pathways Analysis to find possible molecular networks. Twenty-eight proteins identified by 2-DE analysis and 24 identified by nLC-MS/MS shotgun were differentially expressed among the three genotypes. Bioinformatic analysis revealed a central role of high-mobility group box 1/2 and huntigtin never reported before in association with metabolic and related liver disease. A different modulation of these proteins in both blood and hepatic tissue further suggests their role in these processes. These results provide new insight into pathophysiology of insulin resistance and hepatic steatosis and could be useful in identifying novel biomarkers to predict risk for diabetes and its complications. Copyright © 2015 the American Physiological Society.

A molecular pharmacologist's guide to G protein-coupled receptor crystallography

NARCIS (Netherlands)

Piscitelli, Chayne L.; Kean, James; de Graaf, C.; Deupi, Xavier

2015-01-01

G protein-coupled receptor (GPCR) structural biology has progressed dramatically in the last decade. There are now over 120 GPCR crystal structures deposited in the Protein Data Bank of 32 different receptors from families scattered across the phylogenetic tree, including class B, C, and Frizzled

Dopamine D2L receptor-interacting proteins regulate dopaminergic signaling

Directory of Open Access Journals (Sweden)

Norifumi Shioda

2017-10-01

Full Text Available Dopamine receptor family proteins include seven transmembrane and trimeric GTP-binding protein-coupled receptors (GPCRs. Among them, the dopamine D2 receptor (D2R is most extensively studied. All clinically used antipsychotic drugs serve as D2R antagonists in the mesolimbic dopamine system, and their ability to block D2R signaling is positively correlated with antipsychotic efficiency. Human genetic studies also show a significant association of DRD2 polymorphisms with disorders including schizophrenia and Parkinson's disease. D2R exists as two alternatively spliced isoforms, the long isoform (D2LR and the short isoform (D2SR, which differ in a 29-amino acid (AA insert in the third cytoplasmic loop. Importantly, previous reports demonstrate functional diversity between the two isoforms in humans. In this review, we focus on binding proteins that specifically interact with the D2LR 29AA insert. We discuss how D2R activities are mediated not only by heterotrimeric G proteins but by D2LR-interacting proteins, which in part regulate diverse D2R activities. Keywords: Dopamine D2L receptor, Antipsychotic drugs, DRD2 polymorphisms, Alternatively spliced isoforms, D2LR-interacting proteins

A library of 7TM receptor C-terminal tails - Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP)

DEFF Research Database (Denmark)

Heydorn, A.; Sondergaard, B.P.; Ersbøll, Bjarne Kjær

2004-01-01

Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor...... only a single receptor tail, i.e. the beta(2)-adrenergic receptor, whereas N-ethylmaleimide-sensitive factor bound 11 of the tail-fusion proteins. Of the two proteins proposed to target receptors for lysosomal degradation, sorting nexin 1 (SNX1) bound 10 and the C-terminal domain of G protein...... the expected nanomolar affinities for interaction with SNX1. Truncations of the NK1 receptor revealed that an extended binding epitope is responsible for the interaction with both SNX1 and G protein-coupled receptor-associated sorting protein as well as with N-ethylmaleimide-sensitive factor. It is concluded...

A library of 7TM receptor C-terminal tails. Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP)

DEFF Research Database (Denmark)

Heydorn, Arne; Søndergaard, Birgitte P; Ersbøll, Bjarne

2004-01-01

Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor...... only a single receptor tail, i.e. the beta(2)-adrenergic receptor, whereas N-ethylmaleimide-sensitive factor bound 11 of the tail-fusion proteins. Of the two proteins proposed to target receptors for lysosomal degradation, sorting nexin 1 (SNX1) bound 10 and the C-terminal domain of G protein...... the expected nanomolar affinities for interaction with SNX1. Truncations of the NK(1) receptor revealed that an extended binding epitope is responsible for the interaction with both SNX1 and G protein-coupled receptor-associated sorting protein as well as with N-ethylmaleimide-sensitive factor. It is concluded...

The lactate receptor, G-protein-coupled receptor 81/hydroxycarboxylic acid receptor 1

DEFF Research Database (Denmark)

Morland, Cecilie; Lauritzen, Knut Huso; Puchades, Maja

2015-01-01

We have proposed that lactate is a “volume transmitter” in the brain and underpinned this by showing that the lactate receptor, G-protein-coupled receptor 81 (GPR81, also known as HCA1 or HCAR1), which promotes lipid storage in adipocytes, is also active in the mammalian brain. This includes......, energy metabolism, and energy substrate availability, including a glucose- and glycogen-saving response. HCAR1 may contribute to optimizing the cAMP concentration. For instance, in the prefrontal cortex, excessively high cAMP levels are implicated in impaired cognition in old age, fatigue, stress...

Examination of the relation between periodontal health status and cardiovascular risk factors: serum total and high density lipoprotein cholesterol, C-reactive protein, and plasma fibrinogen.

Science.gov (United States)

Wu, T; Trevisan, M; Genco, R J; Falkner, K L; Dorn, J P; Sempos, C T

2000-02-01

Using data from the Third National Health and Nutrition Examination Survey (1988-1994), the authors examined the relation between periodontal health and cardiovascular risk factors: serum total and high density lipoprotein cholesterol, C-reactive protein, and plasma fibrinogen. A total of 10,146 participants were included in the analyses of cholesterol and C-reactive protein and 4,461 in the analyses of fibrinogen. Periodontal health indicators included the gingival bleeding index, calculus index, and periodontal disease status (defined by pocket depth and attachment loss). While cholesterol and fibrinogen were analyzed as continuous variables, C-reactive protein was dichotomized into two levels. The results show a significant relation between indicators of poor periodontal status and increased C-reactive protein and fibrinogen. The association between periodontal status and total cholesterol level is much weaker. No consistent association between periodontal status and high density lipoprotein cholesterol was detectable. Similar patterns of association were observed for participants aged 17-54 years and those 55 years and older. In conclusion, this study suggests that total cholesterol, C-reactive protein, and fibrinogen are possible intermediate factors that may link periodontal disease to elevated cardiovascular risk.

Association of dopamine D(3) receptors with actin-binding protein 280 (ABP-280).

Science.gov (United States)

Li, Ming; Li, Chuanyu; Weingarten, Paul; Bunzow, James R; Grandy, David K; Zhou, Qun Yong

2002-03-01

Proteins that bind to G protein-coupled receptors have been identified as regulators of receptor localization and signaling. In our previous studies, a cytoskeletal protein, actin-binding protein 280 (ABP-280), was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. In this study, we demonstrate that ABP-280 also interacts with dopamine D(3) receptors, but not with D(4) receptors. Similar to the dopamine D(2) receptor, the D(3)/ABP-280 association is of signaling importance. In human melanoma M2 cells lacking ABP-280, D(3) receptors were unable to inhibit forskolin-stimulated cyclic AMP (cAMP) production significantly. D(4) receptors, however, exhibited a similar degree of inhibition of forskolin-stimulated cAMP production in ABP-280-deficient M2 cells and ABP-280-replent M2 subclones (A7 cells). Further experiments revealed that the D(3)/ABP-280 interaction was critically dependent upon a 36 amino acid carboxyl domain of the D(3) receptor third loop, which is conserved in the D(2) receptor but not in the D(4) receptor. Our results demonstrate a subtype-specific regulation of dopamine D(2)-family receptor signaling by the cytoskeletal protein ABP-280.

Characterization of G-protein coupled receptor kinase interaction with the neurokinin-1 receptor using bioluminescence resonance energy transfer

DEFF Research Database (Denmark)

Jorgensen, Rasmus; Holliday, Nicholas D; Hansen, Jakob L

2007-01-01

To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase-inactive muta......To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase...

G-Protein Coupled Receptors: Surface Display and Biosensor Technology

Science.gov (United States)

McMurchie, Edward; Leifert, Wayne

Signal transduction by G-protein coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to the activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. With growing interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high-throughput screening of drugs and biosensors, greater attention will focus on assay development to allow for miniaturization, ultrahigh-throughput and, eventually, microarray/biochip assay formats that will require nanotechnology-based approaches. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR/G-protein platforms, which should be able to be adapted to such applications as microarrays and biosensors. This chapter focuses on cell-free GPCR assay nanotechnologies and describes some molecular biological approaches for the construction of more sophisticated, surface-immobilized, homogeneous, functional GPCR sensors. The latter points should greatly extend the range of applications to which technologies based on GPCRs could be applied.

Lipoproteins in Drosophila melanogaster—Assembly, Function, and Influence on Tissue Lipid Composition

Science.gov (United States)

Palm, Wilhelm; Sampaio, Julio L.; Brankatschk, Marko; Carvalho, Maria; Mahmoud, Ali; Shevchenko, Andrej; Eaton, Suzanne

2012-01-01

Interorgan lipid transport occurs via lipoproteins, and altered lipoprotein levels correlate with metabolic disease. However, precisely how lipoproteins affect tissue lipid composition has not been comprehensively analyzed. Here, we identify the major lipoproteins of Drosophila melanogaster and use genetics and mass spectrometry to study their assembly, interorgan trafficking, and influence on tissue lipids. The apoB-family lipoprotein Lipophorin (Lpp) is the major hemolymph lipid carrier. It is produced as a phospholipid-rich particle by the fat body, and its secretion requires Microsomal Triglyceride Transfer Protein (MTP). Lpp acquires sterols and most diacylglycerol (DAG) at the gut via Lipid Transfer Particle (LTP), another fat body-derived apoB-family lipoprotein. The gut, like the fat body, is a lipogenic organ, incorporating both de novo–synthesized and dietary fatty acids into DAG for export. We identify distinct requirements for LTP and Lpp-dependent lipid mobilization in contributing to the neutral and polar lipid composition of the brain and wing imaginal disc. These studies define major routes of interorgan lipid transport in Drosophila and uncover surprising tissue-specific differences in lipoprotein lipid utilization. PMID:22844248

Genetically elevated apolipoprotein A-I, high-density lipoprotein cholesterol levels, and risk of ischemic heart disease

DEFF Research Database (Denmark)

Lundegaard, Christiane; Tybjærg-Hansen, Anne; Grande, Peer

2010-01-01

Epidemiologically, levels of high-density lipoprotein (HDL) cholesterol and its major protein constituent, apolipoprotein A-I (apoA-I), are inversely related to risk of ischemic heart disease (IHD).......Epidemiologically, levels of high-density lipoprotein (HDL) cholesterol and its major protein constituent, apolipoprotein A-I (apoA-I), are inversely related to risk of ischemic heart disease (IHD)....

Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor

DEFF Research Database (Denmark)

Chen, Y; Grall, D; Salcini, A E

1996-01-01

The serine protease thrombin activates G protein signaling systems that lead to Ras activation and, in certain cells, proliferation. Whereas the steps leading to Ras activation by G protein-coupled receptors are not well defined, the mechanisms of Ras activation by receptor tyrosine kinases have......-insensitive proteins appear to mediate this effect, since (i) pertussis toxin pre-treatment of cells does not blunt the action of thrombin and (ii) Shc phosphorylation on tyrosine can be stimulated by the muscarinic m1 receptor. Shc phosphorylation does not appear to involve protein kinase C, since the addition of 4...

Oxidized low-density lipoproteins upregulate proline oxidase to initiate ROS-dependent autophagy

OpenAIRE

Zabirnyk, Olga; Liu, Wei; Khalil, Shadi; Sharma, Anit; Phang, James M.

2009-01-01

Epidemiological studies showed that high levels of oxidized low-density lipoproteins (oxLDLs) are associated with increased cancer risk. We examined the direct effect of physiologic concentrations oxLDL on cancer cells. OxLDLs were cytotoxic and activate both apoptosis and autophagy. OxLDLs have ligands for peroxisome proliferator-activated receptor gamma and upregulated proline oxidase (POX) through this nuclear receptor. We identified 7-ketocholesterol (7KC) as a main component responsible ...

Central nervous system neuropeptide Y signaling via the Y1 receptor partially dissociates feeding behavior from lipoprotein metabolism in lean rats.

Science.gov (United States)

Rojas, Jennifer M; Stafford, John M; Saadat, Sanaz; Printz, Richard L; Beck-Sickinger, Annette G; Niswender, Kevin D

2012-12-15

Elevated plasma triglyceride (TG) levels contribute to an atherogenic dyslipidemia that is associated with obesity, diabetes, and metabolic syndrome. Numerous models of obesity are characterized by increased central nervous system (CNS) neuropeptide Y (NPY) tone that contributes to excess food intake and obesity. Previously, we demonstrated that intracerebroventricular (icv) administration of NPY in lean fasted rats also elevates hepatic production of very low-density lipoprotein (VLDL)-TG. Thus, we hypothesize that elevated CNS NPY action contributes to not only the pathogenesis of obesity but also dyslipidemia. Here, we sought to determine whether the effects of NPY on feeding and/or obesity are dissociable from effects on hepatic VLDL-TG secretion. Pair-fed, icv NPY-treated, chow-fed Long-Evans rats develop hypertriglyceridemia in the absence of increased food intake and body fat accumulation compared with vehicle-treated controls. We then modulated CNS NPY signaling by icv injection of selective NPY receptor agonists and found that Y1, Y2, Y4, and Y5 receptor agonists all induced hyperphagia in lean, ad libitum chow-fed Long-Evans rats, with the Y2 receptor agonist having the most pronounced effect. Next, we found that at equipotent doses for food intake NPY Y1 receptor agonist had the most robust effect on VLDL-TG secretion, a Y2 receptor agonist had a modest effect, and no effect was observed for Y4 and Y5 receptor agonists. These findings, using selective agonists, suggest the possibility that the effect of CNS NPY signaling on hepatic VLDL-TG secretion may be relatively dissociable from effects on feeding behavior via the Y1 receptor.

Effect of apolipoprotein M on high density lipoprotein metabolism and atherosclerosis in low density lipoprotein receptor knock-out mice

DEFF Research Database (Denmark)

Christoffersen, Christina; Jauhiainen, Matti; Moser, Markus

2008-01-01

To investigate the role of apoM in high density lipoprotein (HDL) metabolism and atherogenesis, we generated human apoM transgenic (apoM-Tg) and apoM-deficient (apoM(-/-)) mice. Plasma apoM was predominantly associated with 10-12-nm alpha-migrating HDL particles. Human apoM overexpression (11-fol...

Lipoprotein Apheresis for Lipoprotein(a)-Associated Cardiovascular Disease

DEFF Research Database (Denmark)

Roeseler, Eberhard; Julius, Ulrich; Heigl, Franz

2016-01-01

OBJECTIVE: Lipoprotein(a)-hyperlipoproteinemia (Lp(a)-HLP) along with progressive cardiovascular disease has been approved as indication for regular lipoprotein apheresis (LA) in Germany since 2008. We aimed to study the long-term preventive effect of LA and to assess hypothetical clinical correl...

Mycobacterium tuberculosis lipoproteins in virulence and immunity - fighting with a double-edged sword.

Science.gov (United States)

Becker, Katja; Sander, Peter

2016-11-01

Bacterial lipoproteins are secreted membrane-anchored proteins characterized by a lipobox motif. This lipobox motif directs post-translational modifications at the conserved cysteine through the consecutive action of three enzymes: Lgt, LspA and Lnt, which results in di- or triacylated forms. Lipoproteins are abundant in all bacteria including Mycobacterium tuberculosis and often involved in virulence and immunoregulatory processes. On the one hand, disruption of the biosynthesis pathway of lipoproteins leads to attenuation of M. tuberculosis in vivo, and mycobacteria deficient for certain lipoproteins have been assessed as attenuated live vaccine candidates. On the other hand, several mycobacterial lipoproteins form immunodominant antigens which promote an immune response. Some of these have been explored in DNA or subunit vaccination approaches against tuberculosis. The immune recognition of specific lipoproteins, however, might also benefit long-term survival of M. tuberculosis through immune modulation, while others induce protective responses. Exploiting lipoproteins as vaccines is thus a complex matter which requires deliberative investigation. The dual role of lipoproteins in the immunity to and pathogenicity of mycobacteria is discussed here. © 2016 Federation of European Biochemical Societies.

TAM receptors, Gas6, and protein S: roles in inflammation and hemostasis.

Science.gov (United States)

van der Meer, Jonathan H M; van der Poll, Tom; van 't Veer, Cornelis

2014-04-17

TAM receptors (Tyro3, Axl, and Mer) belong to a family of receptor tyrosine kinases that have important effects on hemostasis and inflammation. Also, they affect cell proliferation, survival, adhesion, and migration. TAM receptors can be activated by the vitamin K-dependent proteins Gas6 and protein S. Protein S is more commonly known as an important cofactor for protein C as well as a direct inhibitor of multiple coagulation factors. To our knowledge, the functions of Gas6 are limited to TAM receptor activation. When activated, the TAM receptors have effects on primary hemostasis and coagulation and display an anti-inflammatory or a proinflammatory effect, depending on cell type. To comprehend the effects that the TAM receptors and their ligands have on hemostasis and inflammation, we compare studies that report the different phenotypes displayed by mice with deficiencies in the genes of this receptor family and its ligands (protein S(+/-), Gas6(-/-), TAM(-/-), and variations of these). In this manner, we aim to display which features are attributable to the different ligands. Because of the effects TAM receptors have on hemostasis, inflammation, and cancer growth, their modulation could make interesting therapeutic targets in thromboembolic disease, atherosclerosis, sepsis, autoimmune disease, and cancer.

Effects of APOA5 S19W polymorphism on growth, insulin sensitivity and lipoproteins in normoweight neonates

Science.gov (United States)

Apolipoprotein (Apo) A5 is a protein involved in the activation of lipoprotein lipase (LPL) and the metabolism of triglyceride (TG)-rich lipoproteins. LPL plays a major role in the metabolism of TG-rich lipoproteins, and placental LPL activity is known to correlate positively with foetal fat deposit...

Effects of soy bean on serum paraoxonase 1 activity and lipoproteins in hyperlipidemic postmenopausal women.

Science.gov (United States)

Shidfar, Farzad; Ehramphosh, Elham; Heydari, Iraj; Haghighi, Ladan; Hosseini, Sharieh; Shidfar, Shahrzad

2009-05-01

Because of an unfavorable serum lipoprotein profile, postmenopausal women are at risk of cardiovascular disease. Soy protein may help protect against these risk factors, although its effect on paraoxonase 1 (PON1) is not clear. The aim of the present study was to determine the effects of soy protein on serum concentration of lipoproteins and PON1 activity in hypercholesterolemic postmenopausal women. In a double-blind randomized clinical trial with a parallel design, 52 hypercholesterolemic postmenopausal women were randomly assigned to 50 g/day soy protein containing 164 mg isoflavones or placebo, for 10 weeks. Serum lipoproteins and PON1 activity were measured at baseline and at the 10th week. There was significant increase in PON1 activity (P=0.029) and a significant decrease in low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), LDL-C/high-density lipoprotein cholesterol (HDL-C), triacylglycerol/HDL-C and TC/HDL-C in the soy group compared with the placebo group (P=0.001, P=0.008, P=0.012, P=0.04 and P=0.029, respectively) at the end of the study. Similarly, PON1 activity was significantly increased (P=0.015) and LDL-C, TC, LDL-C/HDL-C, triacylglycerol/HDL-C and TC/HDL-C were significantly decreased (P=0.001, P=0.002, P=0.001, P=0.016 and P=0.001) at the end of the study compared with the beginning value in soy group. Soy protein reduces the cardiovascular disease risk in postmenopausal women because of both modest reductions in serum lipoproteins and an increase in PON1 activity.

G protein-coupled receptors in Anopheles gambiae.

Science.gov (United States)

Hill, Catherine A; Fox, A Nicole; Pitts, R Jason; Kent, Lauren B; Tan, Perciliz L; Chrystal, Mathew A; Cravchik, Anibal; Collins, Frank H; Robertson, Hugh M; Zwiebel, Laurence J

2002-10-04

We used bioinformatic approaches to identify a total of 276 G protein-coupled receptors (GPCRs) from the Anopheles gambiae genome. These include GPCRs that are likely to play roles in pathways affecting almost every aspect of the mosquito's life cycle. Seventy-nine candidate odorant receptors were characterized for tissue expression and, along with 76 putative gustatory receptors, for their molecular evolution relative to Drosophila melanogaster. Examples of lineage-specific gene expansions were observed as well as a single instance of unusually high sequence conservation.

Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2

OpenAIRE

Li, Qiong; Kumar, Ashok; Gui, Jian-Fang; Yu, Fu-Shin X.

2007-01-01

Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-κB, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-κB activation in HUCL cells after lipoprotein lipa...

Challenging the roles of CD44 and lipolysis stimulated lipoprotein receptor in conveying Clostridium perfringens iota toxin cytotoxicity in breast cancer.

Science.gov (United States)

Fagan-Solis, Katerina D; Reaves, Denise K; Rangel, M Cristina; Popoff, Michel R; Stiles, Bradley G; Fleming, Jodie M

2014-07-02

Translational exploration of bacterial toxins has come to the forefront of research given their potential as a chemotherapeutic tool. Studies in select tissues have demonstrated that Clostridium perfringens iota toxin binds to CD44 and lipolysis stimulated lipoprotein receptor (LSR) cell-surface proteins. We recently demonstrated that LSR expression correlates with estrogen receptor positive breast cancers and that LSR signaling directs aggressive, tumor-initiating cell behaviors. Herein, we identify the mechanisms of iota toxin cytotoxicity in a tissue-specific, breast cancer model with the ultimate goal of laying the foundation for using iota toxin as a targeted breast cancer therapy. In vitro model systems were used to determine the cytotoxic effect of iota toxin on breast cancer intrinsic subtypes. The use of overexpression and knockdown technologies confirmed the roles of LSR and CD44 in regulating iota toxin endocytosis and induction of cell death. Lastly, cytotoxicity assays were used to demonstrate the effect of iota toxin on a validated set of tamoxifen resistant breast cancer cell lines. Treatment of 14 breast cancer cell lines revealed that LSR+/CD44- lines were highly sensitive, LSR+/CD44+ lines were slightly sensitive, and LSR-/CD44+ lines were resistant to iota cytotoxicity. Reduction in LSR expression resulted in a significant decrease in toxin sensitivity; however, overexpression of CD44 conveyed toxin resistance. CD44 overexpression was correlated with decreased toxin-stimulated lysosome formation and decreased cytosolic levels of iota toxin. These findings indicated that expression of CD44 drives iota toxin resistance through inhibition of endocytosis in breast cancer cells, a role not previously defined for CD44. Moreover, tamoxifen-resistant breast cancer cells exhibited robust expression of LSR and were highly sensitive to iota-induced cytotoxicity. Collectively, these data are the first to show that iota toxin has the potential to be an

Structural basis of G protein-coupled receptor-Gi protein interaction: formation of the cannabinoid CB2 receptor-Gi protein complex.

Science.gov (United States)

Mnpotra, Jagjeet S; Qiao, Zhuanhong; Cai, Jian; Lynch, Diane L; Grossfield, Alan; Leioatts, Nicholas; Hurst, Dow P; Pitman, Michael C; Song, Zhao-Hui; Reggio, Patricia H

2014-07-18

In this study, we applied a comprehensive G protein-coupled receptor-Gαi protein chemical cross-linking strategy to map the cannabinoid receptor subtype 2 (CB2)-Gαi interface and then used molecular dynamics simulations to explore the dynamics of complex formation. Three cross-link sites were identified using LC-MS/MS and electrospray ionization-MS/MS as follows: 1) a sulfhydryl cross-link between C3.53(134) in TMH3 and the Gαi C-terminal i-3 residue Cys-351; 2) a lysine cross-link between K6.35(245) in TMH6 and the Gαi C-terminal i-5 residue, Lys-349; and 3) a lysine cross-link between K5.64(215) in TMH5 and the Gαi α4β6 loop residue, Lys-317. To investigate the dynamics and nature of the conformational changes involved in CB2·Gi complex formation, we carried out microsecond-time scale molecular dynamics simulations of the CB2 R*·Gαi1β1γ2 complex embedded in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer, using cross-linking information as validation. Our results show that although molecular dynamics simulations started with the G protein orientation in the β2-AR*·Gαsβ1γ2 complex crystal structure, the Gαi1β1γ2 protein reoriented itself within 300 ns. Two major changes occurred as follows. 1) The Gαi1 α5 helix tilt changed due to the outward movement of TMH5 in CB2 R*. 2) A 25° clockwise rotation of Gαi1β1γ2 underneath CB2 R* occurred, with rotation ceasing when Pro-139 (IC-2 loop) anchors in a hydrophobic pocket on Gαi1 (Val-34, Leu-194, Phe-196, Phe-336, Thr-340, Ile-343, and Ile-344). In this complex, all three experimentally identified cross-links can occur. These findings should be relevant for other class A G protein-coupled receptors that couple to Gi proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Studies on the metabolism and possible mechanisms of atherogenesis of lipoprotein (a)

International Nuclear Information System (INIS)

Krempler, F.

1984-01-01

The mechanisms of atherogenesis are under intensive clinical and experimental investigation. It is commonly accepted that lipoproteins play a major role in atherogenesis. The results of several clinical studies suggest that lipoprotein(a) [Lp(a)] represents an independent risk factor for atherosclerosis. In order to obtain information on the physiological and pathological role of LP(a), studies were undertaken to investigate the metabolism, removal sites, and possible atherogenic mechanism of Lp(a). It was found that Lp(a) is not metabolic product of other apoprotein B containing lipoproteins, but appears to be synthesized as a separate lipoprotein. The turnover parameters of Lp(a) resemble those of LDL. Binding studies of Lp(a) with cultured human fibroblasts demonstrated that Lp(a) is bound by the B-E receptor. After binding, Lp(a) is internalized and inhibits cellular cholesterol synthesis. In the presence of dextran sulfate or antibodies to the specific Lp(a) apoprotein or apoprotein B, Lp(a) is avidly taken up by macrophages. A similar mechanism might be responsible for the atherogenic effect of Lp(a). (Author)

Model of mouth-to-mouth transfer of bacterial lipoproteins through inner membrane LolC, periplasmic LolA, and outer membrane LolB.

Science.gov (United States)

Okuda, Suguru; Tokuda, Hajime

2009-04-07

Outer membrane-specific lipoproteins in Escherichia coli are released from the inner membrane by an ATP-binding cassette transporter, the LolCDE complex, which causes the formation of a soluble complex with a periplasmic molecular chaperone, LolA. LolA then transports lipoproteins to the outer membrane where an outer membrane receptor, LolB, incorporates lipoproteins into the outer membrane. The molecular mechanisms underlying the Lol-dependent lipoprotein sorting have been clarified in detail. However, it remained unclear how Lol factors interact with each other to conduct very efficient lipoprotein transfer in the periplasm where ATP is not available. To address this issue, a photo-reactive phenylalanine analogue, p-benzoyl-phenylalanine, was introduced at various positions of LolA and LolB, of which the overall structures are very similar and comprise an incomplete beta-barrel with a hydrophobic cavity inside. Cells expressing LolA or LolB derivatives containing the above analogue were irradiated with UV for in vivo photo-cross-linking. These analyses revealed a hot area in the same region of LolA and LolB, through which LolA and LolB interact with each other. This area is located at the entrance of the hydrophobic cavity. Moreover, this area in LolA is involved in the interaction with a membrane subunit, LolC, whereas no cross-linking occurs between LolA and the other membrane subunit, LolE, or ATP-binding subunit LolD, despite the structural similarity between LolC and LolE. The hydrophobic cavities of LolA and LolB were both found to bind lipoproteins inside. These results indicate that the transfer of lipoproteins through Lol proteins occurs in a mouth-to-mouth manner.

Hepatitis C Virus, Cholesterol and Lipoproteins — Impact for the Viral Life Cycle and Pathogenesis of Liver Disease

Science.gov (United States)

Felmlee, Daniel J.; Hafirassou, Mohamed Lamine; Lefevre, Mathieu; Baumert, Thomas F.; Schuster, Catherine

2013-01-01

Hepatitis C virus (HCV) is a leading cause of chronic liver disease, including chronic hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Hepatitis C infection associates with lipid and lipoprotein metabolism disorders such as hepatic steatosis, hypobetalipoproteinemia, and hypocholesterolemia. Furthermore, virus production is dependent on hepatic very-low-density lipoprotein (VLDL) assembly, and circulating virions are physically associated with lipoproteins in complexes termed lipoviral particles. Evidence has indicated several functional roles for the formation of these complexes, including co-opting of lipoprotein receptors for attachment and entry, concealing epitopes to facilitate immune escape, and hijacking host factors for HCV maturation and secretion. Here, we review the evidence surrounding pathogenesis of the hepatitis C infection regarding lipoprotein engagement, cholesterol and triglyceride regulation, and the molecular mechanisms underlying these effects. PMID:23698400

TAM receptors, Gas6, and protein S: roles in inflammation and hemostasis

NARCIS (Netherlands)

van der Meer, Jonathan H. M.; van der Poll, Tom; van 't Veer, Cornelis

2014-01-01

TAM receptors (Tyro3, Axl, andMer) belong to a family of receptor tyrosine kinases that have important effects on hemostasis and inflammation. Also, they affect cell proliferation, survival, adhesion, and migration. TAM receptors can be activated by the vitamin K-dependent proteins Gas6 and protein

The lipid- and lipoprotein- [LDL-Lp(a)] apheresis techniques. Updating.

Science.gov (United States)

Stefanutti, C; Morozzi, C; Perrone, G; Di Giacomo, S; Vivenzio, A; D'Alessandri, G

2012-01-01

Therapeutic plasmapheresis allows the extracorporeal removal of plasmatic lipoproteins (Lipid-apheresis) (LA). It can be non selective (non specific), semi - selective or selective low density lipoprotein-lipoprotein(a) (specific [LDL- Lp(a)] apheresis) (Lipoprotein apheresis, LDLa). The LDL removal rate is a perfect parameter to assess the system efficiency. Plasma-Exchange (PEX) cannot be considered either specific nor, selective. In PEX the whole blood is separated into plasma and its corpuscular components usually through centrifugation or rather filtration. The corpuscular components mixed with albumin solution plus saline (NaCl 0.9%) solution at 20%-25%, are then reinfused to the patient, to substitute the plasma formerly removed. PEX eliminates atherogenic lipoproteins, but also other essential plasma proteins, such as albumin, immunoglobulins, and hemocoagulatory mediators. Cascade filtration (CF) is a method based on plasma separation and removal of plasma proteins through double filtration. During the CF two hollow-fiber filters with pores of different diameter are used to eliminate the plasma components of different weight and molecular diameter. A CF system uses a first polypropylene filter with 0.55 µm diameter pores and a second one of diacetate of cellulose with 0.02 µm pores. The first filter separates the whole blood, and the plasma is then perfused through a second filter which allows the recovery of molecules with a diameter lower than 0.02 µm, and the removal of molecules larger in diameter as apoB100-containing lipoproteins. Since both albumin and immunoglobulins are not removed, or to a negligible extent, plasma-expanders, substitution fluids, and in particular albumin, as occurs in PEX are not needed. CF however, is characterized by lower selectivity since removes also high density lipoprotein (HDL) particles which have an antiatherogenic activity. In the 80's, a variation of Lipid-apheresis has been developed which allows the LDL

Expression of G(alpha)(s) proteins and TSH receptor signalling in hyperfunctioning thyroid nodules with TSH receptor mutations.

Science.gov (United States)

Holzapfel, Hans-Peter; Bergner, Beate; Wonerow, Peter; Paschke, Ralf

2002-07-01

Constitutively activating mutations of the thyrotrophin receptor (TSHR) are the main molecular cause of hyperfunctioning thyroid nodules (HTNs). The G protein coupling is an important and critical step in the TSHR signalling which mainly includes G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins. We investigated the in vitro consequences of overexpressing G(alpha) proteins on signalling of the wild-type (WT) or mutated TSHR. Moreover, we investigated whether changes in G(alpha) protein expression are pathophysiologically relevant in HTNs or cold thyroid nodules (CTNs). Wild-type TSH receptor and mutated TSH receptors were coexpressed with G(alpha)(s), G(alpha)(i) or G(alpha)(q)/11, and cAMP and inositol phosphate (IP) production was measured after stimulation with TSH. The expression of G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins was examined by Western blotting in 28 HTNs and 14 CTNs. Coexpression of G(alpha)(s) with the WT TSH receptor in COS 7 cells significantly increased the basal and TSH-stimulated cAMP accumulation while coexpression of the G(alpha)(q) or G(alpha)11 protein significantly increased the production of cAMP and inositol triphosphate (IP(3)). The coexpression of the TSH receptor mutants (I486F, DEL613-621), known to couple constitutively to G(alpha)(s) and G(alpha)(q) with G(alpha)(s) and G(alpha)(q)/11, significantly increased the basal and stimulated cAMP and IP(3) accumulation. Coexpression of the TSH receptor mutant V556F with G(alpha)(s) only increased the basal and stimulated cAMP production while its coexpression with G(alpha)(q)/11 increased the basal and stimulated IP(3) signalling. The expression of G(alpha)(s) protein subunits determined by Western blotting was significantly decreased in 14 HTNs with a constitutively activating TSH receptor mutation in comparison with the corresponding surrounding tissue, while in 14 HTNs without TSH receptor or G(alpha)(s) protein mutation and in 14 CTNs the expression of G

The Role of Cgrp-Receptor Component Protein (Rcp in Cgrp-Mediated Signal Transduction

Directory of Open Access Journals (Sweden)

M. A. Prado

2001-01-01

Full Text Available The calcitonin gene-related peptide (CGRP-receptor component protein (RCP is a 17-kDa intracellular peripheral membrane protein required for signal transduction at CGRP receptors. To determine the role of RCP in CGRP-mediated signal transduction, RCP was depleted from NIH3T3 cells using antisense strategy. Loss of RCP protein correlated with loss of cAMP production by CGRP in the antisense cells. In contrast, loss of RCP had no effect on CGRP-mediated binding; therefore RCP is not acting as a chaperone for the CGRP receptor. Instead, RCP is a novel signal transduction molecule that couples the CGRP receptor to the cellular signal transduction machinery. RCP thus represents a prototype for a new class of signal transduction proteins that are required for regulation of G protein-coupled receptors.

Contribution of lipoproteins and lipoprotein processing to endocarditis virulence in Streptococcus sanguinis.

Science.gov (United States)

Das, Sankar; Kanamoto, Taisei; Ge, Xiuchun; Xu, Ping; Unoki, Takeshi; Munro, Cindy L; Kitten, Todd

2009-07-01

Streptococcus sanguinis is an important cause of infective endocarditis. Previous studies have identified lipoproteins as virulence determinants in other streptococcal species. Using a bioinformatic approach, we identified 52 putative lipoprotein genes in S. sanguinis strain SK36 as well as genes encoding the lipoprotein-processing enzymes prolipoprotein diacylglyceryl transferase (lgt) and signal peptidase II (lspA). We employed a directed signature-tagged mutagenesis approach to systematically disrupt these genes and screen each mutant for the loss of virulence in an animal model of endocarditis. All mutants were viable. In competitive index assays, mutation of a putative phosphate transporter reduced in vivo competitiveness by 14-fold but also reduced in vitro viability by more than 20-fold. Mutations in lgt, lspA, or an uncharacterized lipoprotein gene reduced competitiveness by two- to threefold in the animal model and in broth culture. Mutation of ssaB, encoding a putative metal transporter, produced a similar effect in culture but reduced in vivo competiveness by >1,000-fold. [(3)H]palmitate labeling and Western blot analysis confirmed that the lgt mutant failed to acylate lipoproteins, that the lspA mutant had a general defect in lipoprotein cleavage, and that SsaB was processed differently in both mutants. These results indicate that the loss of a single lipoprotein, SsaB, dramatically reduces endocarditis virulence, whereas the loss of most other lipoproteins or of normal lipoprotein processing has no more than a minor effect on virulence.

Non-conventional Frizzled ligands and Wnt receptors.

Science.gov (United States)

Hendrickx, Marijke; Leyns, Luc

2008-05-01

The Wnt family of secreted signaling factors plays numerous roles in embryonic development and in stem cell biology. In the adult, Wnt signaling is involved in tissue homeostasis and mutations that lead to the overexpression of Wnt can be linked to cancer. Wnt signaling is transduced intracellularly by the Frizzled (Fzd) family of receptors. In the canonical pathway, accumulation of beta-catenin and the subsequent formation of a complex with T cell factors (TCF) or lymphoid enhancing factors (Lef) lead to target gene activation. The identification of Ryk as an alternative Wnt receptor and the discovery of the novel Fzd ligands Norrie disease protein (NDP) and R-Spondin, changed the traditional view of Wnts binding to Fzd receptors. Mouse R-Spondin cooperates with Wnt signaling and Low density lipoprotein (LDL) receptor related protein (LRP) to activate beta-catenin dependent gene expression and is involved in processes such as limb and placental development in the mouse. NDP is the product of the Norrie disease gene and controls vascular development in the retina, inner ear and in the female reproductive system during pregnancy. In this review a functional overview of the interactions of the different Wnt and non-Wnt ligands with the Fzd receptors is given as well as a survey of Wnts binding to Ryk and we discuss the biological significance of these interactions.

Stimulation of LDL receptor activity in Hep-G2 cells by a serum factor(s)

International Nuclear Information System (INIS)

Ellsworth, J.L.; Brown, C.; Cooper, A.D.

1988-01-01

The regulation of low-density lipoprotein (LDL) receptor activity in the human hepatoma cell line Hep-G2 by serum components was examined. Incubation of dense monolayers of Hep-G2 cells with fresh medium containing 10% fetal calf serum (FM) produced a time-dependent increase in LDL receptor activity. Uptake and degradation of 125I-LDL was stimulated two- to four-fold, as compared with that of Hep-G2 cells cultured in the same media in which they had been grown to confluence (CM); the maximal 125I-LDL uptake plus degradation increased from 0.2 microgram/mg cell protein/4 h to 0.8 microgram/mg cell protein/4 h. In addition, a two-fold increase in cell surface binding of 125I-LDL to Hep-G2 cells was observed when binding was measured at 4 degrees C. There was no change in the apparent Kd. The stimulation of LDL receptor activity was suppressed in a concentration-dependent manner by the addition of cholesterol, as LDL, to the cell medium. In contrast to the stimulation of LDL receptor activity, FM did not affect the uptake or degradation of 125I-asialoorosomucoid. Addition of FM increased the protein content per dish, and DNA synthesis was stimulated approximately five-fold, as measured by [3H]thymidine incorporation into DNA; however, the cell number did not change. Cellular cholesterol biosynthesis was also stimulated by FM; [14C]acetate incorporation into unesterified and esterified cholesterol was increased approximately five-fold. Incubation of Hep-G2 cells with high-density lipoproteins (200 micrograms protein/ml) or albumin (8.0 mg/ml) in the absence of the serum factor did not significantly increase the total processed 125I-LDL. Stimulation of LDL receptor activity was dependent on a heat-stable, nondialyzable serum component that eluted in the inclusion volume of a Sephadex G-75 column

Expressions of the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase genes are stimulated by recombinant platelet-derived growth factor isomers

International Nuclear Information System (INIS)

Roth, M.; Emmons, L.R.; Perruchoud, A.; Block, L.H.

1991-01-01

The plausible role that platelet-derived growth factor (PDGF) has in the localized pathophysiological changes that occur in the arterial wall during development of atherosclerotic lesions led the authors to investigate the influence of recombinant (r)PDGF isomers -AA, -AB, and -BB on the expression of low density lipoprotein receptor (LDL-R) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG0CoA) reductase [(S)-mevalonate:NAD + oxidoreductase (CoA-acylating), EC 1.1.1.88] genes. In addition, they clarified the role of protein kinase C (PKC) in expression of the two genes in human skin fibroblasts and vascular smooth muscle cells. The various rPDGF isoforms are distinct in their ability to activate transcription of both genes: (i) both rPDGF-AA and -BB stimulate transcription of the LDL-R gene; in contrast, rPDGF-BB but not -AA, activates transcription of the HMG-CoA reductase gene; (ii) all recombinant isoforms of PDGF activate transcription of the c-fos gene; (iii) while rPDGF-dependent transcription of the lDL-R gene occurs independently of PKC, transcription of the HMG-CoA reductase gene appears to involve the action of that enzyme

Role of protein dynamics in transmembrane receptor signalling

DEFF Research Database (Denmark)

Wang, Yong; Bugge, Katrine Østergaard; Kragelund, Birthe Brandt

2018-01-01

Cells are dependent on transmembrane receptors to communicate and transform chemical and physical signals into intracellular responses. Because receptors transport 'information', conformational changes and protein dynamics play a key mechanistic role. We here review examples where experiment...... to function. Because the receptors function in a heterogeneous environment and need to be able to switch between distinct functional states, they may be particularly sensitive to small perturbations that complicate studies linking dynamics to function....

Computational studies of G protein-coupled receptor complexes : Structure and dynamics

NARCIS (Netherlands)

Sensoy, Ozge; Almeida, Jose G; Shabbir, Javeria; de Sousa Moreira, Irina; Morra, Giulia

2017-01-01

G protein-coupled receptors (GPCRs) are ubiquitously expressed transmembrane proteins associated with a wide range of diseases such as Alzheimer's, Parkinson, schizophrenia, and also implicated in in several abnormal heart conditions. As such, this family of receptors is regarded as excellent drug

Unfavorable apoAI-containing lipoproteins profile in Tunisian obese ...

African Journals Online (AJOL)

hope&shola

1Ecole Supérieure des Sciences et Techniques De la Santé, Sfax, Tunisia. 2Laboratoire de Génie Enzymatique ... lipoproteins metabolism. Control and obese women ... cholesteryl ester transfer protein; CHD, coronary heart disease. studied.

Presynaptic G Protein-Coupled Receptors: Gatekeepers of Addiction?

Directory of Open Access Journals (Sweden)

Kari A Johnson

2016-11-01

Full Text Available Drug abuse and addiction cause widespread social and public health problems, and the neurobiology underlying drug actions and drug use and abuse is an area of intensive research. Drugs of abuse alter synaptic transmission, and these actions contribute to acute intoxication as well as the chronic effects of abused substances. Transmission at most mammalian synapses involves neurotransmitter activation of two receptor subtypes, ligand-gated ion channels that mediate fast synaptic responses, and G protein-coupled receptors (GPCRs that have slower neuromodulatory actions. The GPCRs represent a large proportion of neurotransmitter receptors involved in almost all facets of nervous system function. In addition, these receptors are targets for many pharmacotherapeutic agents. Drugs of abuse directly or indirectly affect neuromodulation mediated by GPCRs, with important consequences for intoxication, drug taking and responses to prolonged drug exposure, withdrawal and addiction. Among the GPCRs are several subtypes involved in presynaptic inhibition, most of which are coupled to the Gi/o class of G protein. There is increasing evidence that these presynaptic Gi/o-coupled GPCRs have important roles in the actions of drugs of abuse, as well as behaviors related to these drugs. This topic will be reviewed, with particular emphasis on receptors for three neurotransmitters, dopamine (D1- and D2-like receptors, endocannabinoids (CB1 receptors and glutamate (group II metabotropic glutamate (mGlu receptors. The focus is on recent evidence from laboratory animal models (and some evidence in humans implicating these receptors in the acute and chronic effects of numerous abused drugs, as well as in the control of drug seeking and taking. The ability of drugs targeting these receptors to modify drug seeking behavior has raised the possibility of using compounds targeting these receptors for addiction pharmacotherapy. This topic is also discussed, with emphasis on

G Protein-Coupled Receptor Signaling in Stem Cells and Cancer.

Science.gov (United States)

Lynch, Jennifer R; Wang, Jenny Yingzi

2016-05-11

G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.

G Protein-Coupled Receptor Signaling in Stem Cells and Cancer

Directory of Open Access Journals (Sweden)

Jennifer R. Lynch

2016-05-01

Full Text Available G protein-coupled receptors (GPCRs are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84 and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.

Hypervariable region 1 deletion and required adaptive envelope mutations confer decreased dependency on scavenger receptor class B type I and low-density lipoprotein receptor for hepatitis C virus

DEFF Research Database (Denmark)

Prentoe, Jannick; Serre, Stéphanie B N; Ramirez, Santseharay

2014-01-01

-deleted viruses. Apolipoprotein E (ApoE)-specific HCV neutralization was similar for H77, J6, and S52 viruses with and without HVR1. In conclusion, HVR1 and HVR1-related adaptive envelope mutations appeared to be involved in LDLr and SR-BI dependency, respectively. Also, LDLr served Apo....../S733F), S52(ΔHVR1/A369V), and S52(A369V), but not for J6(ΔHVR1). Low-density lipoprotein receptor (LDLr) dependency was decreased for HVR1-deleted viruses, but not for H77(N476D/S733F) and S52(A369V). Soluble LDLr neutralization revealed strong inhibition of parental HCV but limited effect against HVR1...

Delivery of circulating lipoproteins to specific neurons in the Drosophila brain regulates systemic insulin signaling.

Science.gov (United States)

Brankatschk, Marko; Dunst, Sebastian; Nemetschke, Linda; Eaton, Suzanne

2014-10-02

The Insulin signaling pathway couples growth, development and lifespan to nutritional conditions. Here, we demonstrate a function for the Drosophila lipoprotein LTP in conveying information about dietary lipid composition to the brain to regulate Insulin signaling. When yeast lipids are present in the diet, free calcium levels rise in Blood Brain Barrier glial cells. This induces transport of LTP across the Blood Brain Barrier by two LDL receptor-related proteins: LRP1 and Megalin. LTP accumulates on specific neurons that connect to cells that produce Insulin-like peptides, and induces their release into the circulation. This increases systemic Insulin signaling and the rate of larval development on yeast-containing food compared with a plant-based food of similar nutritional content.

Effects of High Fat Feeding and Diabetes on Regression of Atherosclerosis Induced by Low-Density Lipoprotein Receptor Gene Therapy in LDL Receptor-Deficient Mice.

Directory of Open Access Journals (Sweden)

Florian Willecke

Full Text Available We tested whether a high fat diet (HFD containing the inflammatory dietary fatty acid palmitate or insulin deficient diabetes altered the remodeling of atherosclerotic plaques in LDL receptor knockout (Ldlr-/- mice. Cholesterol reduction was achieved by using a helper-dependent adenovirus (HDAd carrying the gene for the low-density lipoprotein receptor (Ldlr; HDAd-LDLR. After injection of the HDAd-LDLR, mice consuming either HFD, which led to insulin resistance but not hyperglycemia, or low fat diet (LFD, showed regression compared to baseline. However there was no difference between the two groups in terms of atherosclerotic lesion size, or CD68+ cell and lipid content. Because of the lack of effects of these two diets, we then tested whether viral-mediated cholesterol reduction would lead to defective regression in mice with greater hyperglycemia. In both normoglycemic and streptozotocin (STZ-treated hyperglycemic mice, HDAd-LDLR significantly reduced plasma cholesterol levels, decreased atherosclerotic lesion size, reduced macrophage area and lipid content, and increased collagen content of plaque in the aortic sinus. However, reductions in anti-inflammatory and ER stress-related genes were less pronounced in STZ-diabetic mice compared to non-diabetic mice. In conclusion, HDAd-mediated Ldlr gene therapy is an effective and simple method to induce atherosclerosis regression in Ldlr-/- mice in different metabolic states.

Interrelationships between postprandial lipoprotein B:CIII particle changes and high-density lipoprotein subpopulation profiles in mixed hyperlipoproteinemia.

Science.gov (United States)

Saïdi, Y; Sich, D; Camproux, A; Egloff, M; Federspiel, M C; Gautier, V; Raisonnier, A; Turpin, G; Beucler, I

1999-01-01

We studied the relationships postprandially between triglyceride-rich lipoprotein (TRL) and high-density lipoprotein (HDL) in 11 mixed hyperlipoproteinemia (MHL) and 11 hypercholesterolemia (HCL) patients. The high and prolonged postprandial triglyceridemia response observed in MHL but not HCL patients was essentially dependent on very-low-density lipoprotein (VLDL) changes. This abnormal response was related to decreased lipoprotein lipase (LPL) activity (-48.7%, P<.01) in MHL compared with HCL subjects. Cholesteryl ester transfer protein (CETP) activity was postprandially enhanced only in MHL patients, and this elevation persisted in the late period (+19% at 12 hours, P<.05), sustaining the delayed enrichment of VLDL with cholesteryl ester (CE). The late postprandial period in MHL patients was also characterized by high levels of apolipoprotein B (apoB)-containing lipoproteins with apoCIII ([LpB:CIII] +36% at 12 hours, P<.01) and decreased levels of apoCIII contained in HDL ([LpCIII-HDL] -34% at 12 hours, P<.01), reflecting probably a defective return of apoCIII from TRL toward HDL. In MHL compared with HCL patients, decreased HDL2 levels were related to both HDL2b and HDL2a subpopulations (-57% and -49%, respectively, P<.01 for both) and decreased apoA-I levels (-53%, P<.01) were equally linked to decreased HDL2 with apoA-I only (LpA-I) and HDL2 with both apoA-I and apoA-II ([LpA-I:A-II] -55% and -52%, respectively, P<.01 for both). The significant inverse correlations between the postprandial magnitude of LpB:CIII and HDL2-LpA-I and HDL2b levels in MHL patients underline the close TRL-HDL interrelationships. Our findings indicate that TRL and HDL abnormalities evidenced at fasting were postprandially amplified, tightly interrelated, and persistent during the late fed period in mixed hyperlipidemia. Thus, these fasting abnormalities are likely postprandially originated and may constitute proatherogenic lipoprotein disorders additional to the HCL in MHL patients.

Background diet and fat type alters plasma lipoprotein response but not aortic cholesterol accumulation in F1B Golden Syrian hamsters.

Science.gov (United States)

Dillard, Alice; Matthan, Nirupa R; Spartano, Nicole L; Butkowski, Ann E; Lichtenstein, Alice H

2013-12-01

Dietary modification alters plasma lipoprotein profiles and atherosclerotic lesion progression in humans and some animal models. Variability in response to diet induced atherosclerosis has been reported in hamsters. Assessed was the interaction between background diet composition and dietary fat type on aortic cholesterol accumulation, lipoprotein profiles, hepatic lipids and selected genes. F1B Golden Syrian hamsters (20/group) were fed (12 weeks) semi-purified or non-purified diets containing either 10 % (w/w) coconut oil or safflower oil and 0.15 % (w/w) cholesterol. The non-purified diets relative to semi-purified diets resulted in significantly higher TC (72 % [percent difference] and 38 %, coconut oil and safflower oil, respectively) and nHDL-C (84 and 61 %, coconut oil and safflower oil, respectively), and lower HDL-C (-47 and -45 %, coconut oil and safflower oil, respectively) concentrations. Plasma triacylglycerol concentrations in the hamsters fed the non-purified coconut oil-supplemented diets were three- to fourfold higher than non-purified safflower oil-supplemented, and both semi-purified diets. With the exception of HDL-C, a significant effect of fat type was observed in TC, nHDL-C and triacylglycerol (all P < 0.05) concentrations. Regardless of diet induced differences in lipoprotein profiles, there was no significant effect on aortic cholesterol accumulation. There was an inverse relationship between plasma nHDL-C and triacylglycerol, and hepatic cholesteryl ester content (P < 0.001). Diet induced differences in hepatic gene transcription (LDL receptor, apoB-100, microsomal transfer protein) were not reflected in protein concentrations. Although hamsters fed non-purified and/or saturated fatty acid-supplemented diets had more atherogenic lipoprotein profiles compared to hamsters fed semi-purified and/or polyunsaturated fatty acid-supplemented diets these differences were not reflected in aortic cholesterol accumulation.

Receptor-interacting protein (RIP) kinase family

OpenAIRE

Zhang, Duanwu; Lin, Juan; Han, Jiahuai

2010-01-01

Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, incl...

Receptor activity modifying proteins (RAMPs) interact with the VPAC1 receptor: evidence for differential RAMP modulation of multiple signalling pathways

International Nuclear Information System (INIS)

Christopoulos, G.; Morfis, M.; Sexton, P.M.; Christopoulos, A.; Laburthe, M.; Couvineau, A.

2001-01-01

Full text: Receptor activity modifying proteins (RAMP) constitute a family of three accessory proteins that affect the expression and/or phenotype of the calcitonin receptor (CTR) or CTR-like receptor (CRLR). In this study we screened a range of class II G protein-coupled receptors (PTH1, PTH2, GHRH, VPAC1, VPAC2 receptors) for possible RAMP interactions by measurement of receptor-induced translocation of c-myc tagged RAMP1 or HA tagged RAMP3. Of these, only the VPAC1 receptor caused significant translocation of c-myc-RAMP1 or HA-RAMP3 to the cell surface. Co-transfection of VPAC1 and RAMPs did not alter 125 I-VIP binding and specificity. VPAC1 receptor function was subsequently analyzed through parallel determinations of cAMP accumulation and phosphoinositide (PI) hydrolysis in the presence and absence of each of the three RAMPs. In contrast to CTR-RAMP interaction, where there was an increase in cAMP Pharmacologisand a decrease in PI hydrolysis, VPAC1-RAMP interaction was characterized by a specific increase in agonist-mediated PI hydrolysis when co-transfected with RAMP2. This change was due to an enhancement of Emax with no change in EC 50 value for VIP. No significant change in cAMP accumulation was observed. This is the first demonstration of an interaction of RAMPs with a G protein-coupled receptor outside the CTR family and may suggest a more generalized role for RAMPs in modulating G protein-coupled receptor signaling. Copyright (2001) Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists

PDZ domain-mediated interactions of G protein-coupled receptors with postsynaptic density protein 95

DEFF Research Database (Denmark)

Møller, Thor C; Wirth, Volker F; Roberts, Nina Ingerslev

2013-01-01

G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome. Their signaling is regulated by scaffold proteins containing PDZ domains, but although these interactions are important for GPCR function, they are still poorly understood. We here present...

[Plasma lipoproteins as drug carriers. Effect of phospholipid formulations].

Science.gov (United States)

Torkhovskaia, T I; Ipatova, O M; Medvedeva, N V; Ivanov, V S; Ivanova, L I

2010-01-01

The extensive development of nanotechnologies in the last two decades has brought about new understanding of plasma lipoproteins (LP) as natural drug nanocarriers that escape interaction with immune and reticuloendothelial systems. Drugs bound to LP (especially LDL) can more actively penetrate into cells of many cancer and inflammation tissues with enhanced expression or/and dysregulation of B,E receptors or possibly scavenger SR-BI receptors. Relevant studies are focused on the development of new dosage forms by conjugating lipophilic drugs either with isolated plasma LP or with their model formulations, such as nanoemulsions, mimetics, lipid nanospheres, etc. Some authors include in these particles serum or recombinant apoproteins, peptides, and modified polymer products. As shown recently, protein-free lipid nanoemulsions in plasma take up free apoA and apoE. Complexes with various LP also form after direct administration of lypophilic drugs into blood especially those enclosed in phospholipid formulations, e.g. liposomes. Results of evaluation of some lipophilic dugs (mainly cytostatics, amphotericin B, cyclosporine A, etc.) are discussed. Original data are presented on the influence of phospholipid formulations on the distribution of doxorubicin and indomethacin between LP classes after in vitro incubation in plasma. On the whole, the review illustrates the importance of research on LP and phospholi pid forms as drug nanocarriers to be used to enhance effect of therapy.

Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2)

DEFF Research Database (Denmark)

Theilade, Juliane; Lerche Hansen, Jakob; Haunsø, Stig

2002-01-01

G protein-coupled receptor kinase 2 (GRK2) phosphorylates G protein-coupled receptors resulting in uncoupling from G proteins. Receptors modulate GRK2 expression, however the mechanistic basis for this effect is largely unknown. Here we report a novel mechanism by which receptors use...

Cholesteryl ester transfer activity in plasma measured by using solid-phase-bound high-density lipoprotein

International Nuclear Information System (INIS)

Sparks, D.L.; Frohlich, J.; Cullis, P.; Pritchard, P.H.

1987-01-01

We studied the ability of lipid-transfer factors in plasma to promote transfer, to endogenous lipoproteins, of [ 3 H]cholesteryl ester from high-density lipoprotein (HDL) covalently bound to Sepharose 4B beads. After incubation for 2 h at 37 degrees C, 12 to 14% of the [ 3 H]cholesteryl ester had been transferred to the lipoproteins of the plasma, in the proportions 57% to HDL and 43% to low- and very-low-density lipoproteins. This process was a function of the amount of plasma present and was stimulated by addition of partly purified lipid-transfer protein. Transfer also depended on the concentration of donor HDL but was independent of the amount of acceptor lipoprotein. This simple evaluation of cholesteryl ester transfer does not require removal of lipoproteins from the plasma before incubation

Synaptic proteins and receptors defects in autism spectrum disorders

Directory of Open Access Journals (Sweden)

Jianling eChen

2014-09-01

Full Text Available Recent studies have found that hundreds of genetic variants, including common and rare variants, rare and de novo mutations, and common polymorphisms have contributed to the occurrence of autism spectrum disorders (ASDs. The mutations in a number of genes such as neurexin, neuroligin, postsynaptic density protein 95 (PSD-95, SH3 and multiple ankyrin repeat domains 3 (SHANK3, synapsin, gephyrin, cadherin (CDH and protocadherin (PCDH, thousand-and-one-amino acid 2 kinase (TAOK2, and contactin (CNTN, have been shown to play important roles in the development and function of synapses. In addition, synaptic receptors, such as gamma-aminobutyric acid (GABA receptors and glutamate receptors, have also been associated with ASDs. This review will primarily focus on the defects of synaptic proteins and receptors associated with ASDs and their roles in the pathogenesis of ASDs via synaptic pathways.

Kaempferol stimulates gene expression of low-density lipoprotein receptor through activation of Sp1 in cultured hepatocytes

Science.gov (United States)

Ochiai, Ayasa; Miyata, Shingo; Iwase, Masamori; Shimizu, Makoto; Inoue, Jun; Sato, Ryuichiro

2016-01-01

A high level of plasma low-density lipoprotein (LDL) cholesterol is considered a risk factor for atherosclerosis. Because the hepatic LDL receptor (LDLR) is essential for clearing plasma LDL cholesterol, activation of LDLR is a promising therapeutic target for patients with atherosclerotic disease. Here we demonstrated how the flavonoid kaempferol stimulated the gene expression and activity of LDLR in HepG2 cells. The kaempferol-mediated stimulation of LDLR gene expression was completely inhibited by knockdown of Sp1 gene expression. Treatment of HepG2 cells with kaempferol stimulated the recruitment of Sp1 to the promoter region of the LDLR gene, as well as the phosphorylation of Sp1 on Thr-453 and Thr-739. Moreover, these kaempferol-mediated processes were inhibited in the presence of U0126, an ERK pathway inhibitor. These results suggest that kaempferol may increase the activity of Sp1 through stimulation of Sp1 phosphorylation by ERK1/2 and subsequent induction of LDLR expression and activity. PMID:27109240

Direct protein-protein interaction between PLCγ1 and the bradykinin B2 receptor-Importance of growth conditions

International Nuclear Information System (INIS)

Duchene, Johan; Chauhan, Sharmila D.; Lopez, Frederic; Pecher, Christiane; Esteve, Jean-Pierre; Girolami, Jean-Pierre; Bascands, Jean-Loup; Schanstra, Joost P.

2005-01-01

Recently, we have described a novel protein-protein interaction between the G-protein coupled bradykinin B2 receptor and tyrosine phosphatase SHP-2 via an immunoreceptor tyrosine-based inhibition motif (ITIM) sequence located in the C-terminal part of the B2 receptor and the Src homology (SH2) domains of SHP-2. Here we show that phospholipase C (PLC)γ1, another SH2 domain containing protein, can also interact with this ITIM sequence. Using surface plasmon resonance analysis, we observed that PLCγ1 interacted with a peptide containing the phosphorylated form of the bradykinin B2 receptor ITIM sequence. In CHO cells expressing the wild-type B2 receptor, bradykinin-induced transient recruitment and activation of PLCγ1. Interestingly, this interaction was only observed in quiescent and not in proliferating cells. Mutation of the key ITIM residue abolished this interaction with and activation of PLCγ1. Finally we also identified bradykinin-induced PLCγ1 recruitment and activation in primary culture renal mesangial cells

Interfacial Tension and Surface Pressure of High Density Lipoprotein, Low Density Lipoprotein, and Related Lipid Droplets

DEFF Research Database (Denmark)

Ollila, O. H. S.; Lamberg, A.; Lehtivaara, M.

2012-01-01

) are essentially lipid droplets surrounded by specific proteins, their main function being to transport cholesterol. Interfacial tension and surface pressure of these particles are of great interest because they are related to the shape and the stability of the droplets and to protein adsorption at the interface....... Here we use coarse-grained molecular-dynamics simulations to consider a number of related issues by calculating the interfacial tension in protein-free lipid droplets, and in HDL and LDL particles mimicking physiological conditions. First, our results suggest that the curvature dependence......Lipid droplets play a central role in energy storage and metabolism on a cellular scale. Their core is comprised of hydrophobic lipids covered by a surface region consisting of amphiphilic lipids and proteins. For example, high and low density lipoproteins (HDL and LDL, respectively...

Receptor-like proteins involved in plant disease resistance

NARCIS (Netherlands)

Kruijt, M.; Kock, de M.J.D.; Wit, de P.J.G.M.

2005-01-01

Race-specific resistance in plants against microbial pathogens is governed by several distinct classes of resistance (R) genes. This review focuses on the class that consists of the plasma membrane-bound leucine-rich repeat proteins known as receptor-like proteins (RLPs). The first isolated

G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na+ channel interaction

International Nuclear Information System (INIS)

Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

1988-01-01

The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na + channels is a coupled event mediated by guanine nucleotide binding protein(s) [G-protein(s)]. These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of [ 3 H] acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of [ 3 H]batrachotoxin to Na + channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced 22 Na + uptake in the presence and absence of tetrodotoxin, which blocks Na + channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na + channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na + channel-is such that at resting potential the muscarinic receptor induces opening of Na + channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues

G protein-coupled receptors: the inside story.

Science.gov (United States)

Jalink, Kees; Moolenaar, Wouter H

2010-01-01

Recent findings necessitate revision of the traditional view of G protein-coupled receptor (GPCR) signaling and expand the diversity of mechanisms by which receptor signaling influences cell behavior in general. GPCRs elicit signals at the plasma membrane and are then rapidly removed from the cell surface by endocytosis. Internalization of GPCRs has long been thought to serve as a mechanism to terminate the production of second messengers such as cAMP. However, recent studies show that internalized GPCRs can continue to either stimulate or inhibit cAMP production in a sustained manner. They do so by remaining associated with their cognate G protein subunit and adenylyl cyclase at endosomal compartments. Once internalized, the GPCRs produce cellular responses distinct from those elicited at the cell surface.

Recent Progress in Understanding Subtype Specific Regulation of NMDA Receptors by G Protein Coupled Receptors (GPCRs

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Kai Yang

2014-02-01

Full Text Available G Protein Coupled Receptors (GPCRs are the largest family of receptors whose ligands constitute nearly a third of prescription drugs in the market. They are widely involved in diverse physiological functions including learning and memory. NMDA receptors (NMDARs, which belong to the ionotropic glutamate receptor family, are likewise ubiquitously expressed in the central nervous system (CNS and play a pivotal role in learning and memory. Despite its critical contribution to physiological and pathophysiological processes, few pharmacological interventions aimed directly at regulating NMDAR function have been developed to date. However, it is well established that NMDAR function is precisely regulated by cellular signalling cascades recruited downstream of G protein coupled receptor (GPCR stimulation. Accordingly, the downstream regulation of NMDARs likely represents an important determinant of outcome following treatment with neuropsychiatric agents that target selected GPCRs. Importantly, the functional consequence of such regulation on NMDAR function varies, based not only on the identity of the GPCR, but also on the cell type in which relevant receptors are expressed. Indeed, the mechanisms responsible for regulating NMDARs by GPCRs involve numerous intracellular signalling molecules and regulatory proteins that vary from one cell type to another. In the present article, we highlight recent findings from studies that have uncovered novel mechanisms by which selected GPCRs regulate NMDAR function and consequently NMDAR-dependent plasticity.

Cholesterol transfer from normal and atherogenic low density lipoproteins to Mycoplasma membranes

International Nuclear Information System (INIS)

Mitschelen, J.J.; St Clair, R.W.; Hester, S.H.

1981-01-01

The purpose of this study was to determine whether the free cholesterol of hypercholesterolemic low density lipoprotein from cholesterol-fed nonhuman primates has a greater potential for surface transfer to cell membranes than does the free cholesterol of normal low density lipoprotein. The low density lipoproteins were isolated from normal and hypercholesterolemic rhesus and cynomolgus monkeys, incubated with membranes from Acholeplasma laidlawii, a mycoplasma species devoid of cholesterol in its membranes, and the mass transfer of free cholesterol determined by measuring membrane cholesterol content. Since these membranes neither synthesize nor esterify cholesterol, nor degrade the protein or cholesterol ester moieties of low density lipoprotein, they are an ideal model with which to study differences in the cholesterol transfer potential of low density lipoprotein independent of the uptake of the intact low density lipoprotein particle. These studies indicate that, even though there are marked differences in the cholesterol composition of normal and hypercholesterolemic low density lipoproteins, this does not result in a greater chemical potential for surface transfer of free cholesterol. Consequently, if a difference in the surface transfer of free cholesterol is responsible for the enhanced ability of hypercholesterolemic low density lipoprotein to promote cellular cholesterol accumulation and, perhaps, also atherosclerosis, it must be the result of differences in the interaction to the hypercholesterolemic low density lipoprotein with the more complicated mammalian cell membranes, rather than differences in the chemical potential for cholesterol transfer

Contribution of Lipoproteins and Lipoprotein Processing to Endocarditis Virulence in Streptococcus sanguinis▿ §

Science.gov (United States)

Das, Sankar; Kanamoto, Taisei; Ge, Xiuchun; Xu, Ping; Unoki, Takeshi; Munro, Cindy L.; Kitten, Todd

2009-01-01

Streptococcus sanguinis is an important cause of infective endocarditis. Previous studies have identified lipoproteins as virulence determinants in other streptococcal species. Using a bioinformatic approach, we identified 52 putative lipoprotein genes in S. sanguinis strain SK36 as well as genes encoding the lipoprotein-processing enzymes prolipoprotein diacylglyceryl transferase (lgt) and signal peptidase II (lspA). We employed a directed signature-tagged mutagenesis approach to systematically disrupt these genes and screen each mutant for the loss of virulence in an animal model of endocarditis. All mutants were viable. In competitive index assays, mutation of a putative phosphate transporter reduced in vivo competitiveness by 14-fold but also reduced in vitro viability by more than 20-fold. Mutations in lgt, lspA, or an uncharacterized lipoprotein gene reduced competitiveness by two- to threefold in the animal model and in broth culture. Mutation of ssaB, encoding a putative metal transporter, produced a similar effect in culture but reduced in vivo competiveness by >1,000-fold. [3H]palmitate labeling and Western blot analysis confirmed that the lgt mutant failed to acylate lipoproteins, that the lspA mutant had a general defect in lipoprotein cleavage, and that SsaB was processed differently in both mutants. These results indicate that the loss of a single lipoprotein, SsaB, dramatically reduces endocarditis virulence, whereas the loss of most other lipoproteins or of normal lipoprotein processing has no more than a minor effect on virulence. PMID:19395487

Down-regulation of lipoprotein lipase increases glucose uptake in L6 muscle cells

Energy Technology Data Exchange (ETDEWEB)

Lopez, Veronica; Saraff, Kumuda [Department of Chemistry and Biochemistry, California State University Northridge, Northridge, CA 91330-8262 (United States); Medh, Jheem D., E-mail: jheem.medh@csun.edu [Department of Chemistry and Biochemistry, California State University Northridge, Northridge, CA 91330-8262 (United States)

2009-11-06

Thiazolidinediones (TZDs) are synthetic hypoglycemic agents used to treat type 2 diabetes. TZDs target the peroxisome proliferator activated receptor-gamma (PPAR-{gamma}) and improve systemic insulin sensitivity. The contributions of specific tissues to TZD action, or the downstream effects of PPAR-{gamma} activation, are not very clear. We have used a rat skeletal muscle cell line (L6 cells) to demonstrate that TZDs directly target PPAR-{gamma} in muscle cells. TZD treatment resulted in a significant repression of lipoprotein lipase (LPL) expression in L6 cells. This repression correlated with an increase in glucose uptake. Down-regulation of LPL message and protein levels using siRNA resulted in a similar increase in insulin-dependent glucose uptake. Thus, LPL down-regulation improved insulin sensitivity independent of TZDs. This finding provides a novel method for the management of insulin resistance.

Endoplasmic Reticulum Stress Caused by Lipoprotein Accumulation Suppresses Immunity against Bacterial Pathogens and Contributes to Immunosenescence

Directory of Open Access Journals (Sweden)

Jogender Singh

2017-05-01

Full Text Available The unfolded protein response (UPR is a stress response pathway that is activated upon increased unfolded and/or misfolded proteins in the endoplasmic reticulum (ER, and enhanced ER stress response prolongs life span and improves immunity. However, the mechanism by which ER stress affects immunity remains poorly understood. Using the nematode Caenorhabditis elegans, we show that mutations in the lipoproteins vitellogenins, which are homologs of human apolipoprotein B-100, resulted in upregulation of the UPR. Lipoprotein accumulation in the intestine adversely affects the immune response and the life span of the organism, suggesting that it could be a contributing factor to immunosenescence. We show that lipoprotein accumulation inhibited the expression of several immune genes encoding proteins secreted by the intestinal cells in an IRE-1-independent manner. Our studies provide a mechanistic explanation for adverse effects caused by protein aggregation and ER stress on immunity and highlight the role of an IRE-1-independent pathway in the suppression of the expression of genes encoding secreted proteins.

Applications of molecular replacement to G protein-coupled receptors

International Nuclear Information System (INIS)

Kruse, Andrew C.; Manglik, Aashish; Kobilka, Brian K.; Weis, William I.

2013-01-01

The use of molecular replacement in solving the structures of G protein-coupled receptors is discussed, with specific examples being described in detail. G protein-coupled receptors (GPCRs) are a large class of integral membrane proteins involved in regulating virtually every aspect of human physiology. Despite their profound importance in human health and disease, structural information regarding GPCRs has been extremely limited until recently. With the advent of a variety of new biochemical and crystallographic techniques, the structural biology of GPCRs has advanced rapidly, offering key molecular insights into GPCR activation and signal transduction. To date, almost all GPCR structures have been solved using molecular-replacement techniques. Here, the unique aspects of molecular replacement as applied to individual GPCRs and to signaling complexes of these important proteins are discussed

Elevated Lipoprotein(A Impairs Platelet Radiolabeling Yield

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Susanne Granegger

2015-02-01

Full Text Available Objectives: Platelet radiolabeling in clinical routine usually results in labeling efficiencies (LE above 80%. A variety of risk factors and clinical conditions are known to impair platelet labeling yield, among them elevated triglycerides and low-density lipoproteins. The potential influence of lipoprotein(a (Lp(a, an atherogenic lipoprotein particle containing a kringle subunit, which is widely found in the proteins of fibrinolysis pathway, has never been studied. Normal Lp(a levels range below 30 mg/ dl. The exact prevalence of elevated Lp(a is unknown, most likely ranging below 10%. Even more rare is an isolated elevation despite an otherwise normal lipoprotein profile. Methods: We examined the role of isolated elevated Lp(a (> 50 mg/dl, ranging up to 440 mg/dl compared to patients with normal lipid profile. Platelets were radiolabeled with in-111-oxine at 37 °C for 5 minutes using ISORBE-consensus methodology. Results: The findings indicate that already at levels below 100 mg/dl Lp(a decreases LE. LE assessment after cross-incubation of hyper-Lp(a platelets with normal Lp(a plasma and vice versa reveals that platelets rather than the plasmatic environment are responsible for the deterioration of labeling yield. This behavior already has been reported for elevated low-density lipoproteins. Apparently, the quantitative influence of LDL and Lp(a/mg is comparable. Plotting the sum of LDL and Lp(a versus LE reveals a clear significant negative correlation. Conclusion: As extremely elevated Lp(a, particularly above 150 mg/dl, may significantly impair labeling results. We therefore recommend to include extremely elevated Lp(a into the list of parameters, which should be known before performing radiolabeling of human platelets.

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

Science.gov (United States)

Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

1993-01-01

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

Biased and g protein-independent signaling of chemokine receptors

DEFF Research Database (Denmark)

Steen, Anne; Larsen, Olav; Thiele, Stefanie

2014-01-01

), different receptors (with the same ligand), or different tissues or cells (for the same ligand-receptor pair). Most often biased signaling is differentiated into G protein-dependent and β-arrestin-dependent signaling. Yet, it may also cover signaling differences within these groups. Moreover, it may...

Serum amyloid A is found on ApoB-containing lipoproteins in obese humans with diabetes.

Science.gov (United States)

Jahangiri, Anisa; Wilson, Patricia G; Hou, Tianfei; Brown, Aparna; King, Victoria L; Tannock, Lisa R

2013-05-01

In murine models of obesity/diabetes, there is an increase in plasma serum amyloid A (SAA) levels along with redistribution of SAA from high-density lipoprotein (HDL) to apolipoprotein B (apoB)-containing lipoprotein particles, namely, low-density lipoprotein and very low-density lipoprotein. The goal of this study was to determine if obesity is associated with similar SAA lipoprotein redistribution in humans. Three groups of obese individuals were recruited from a weight loss clinic: healthy obese (n = 14), metabolic syndrome (MetS) obese (n = 8), and obese with type 2 diabetes (n = 6). Plasma was separated into lipoprotein fractions by fast protein liquid chromatography, and SAA was measured in lipid fractions using enzyme-linked immunosorbent assay and Western blotting. Only the obese diabetic group had SAA detectable in apoB-containing lipoproteins, and SAA reverted back to HDL with active weight loss. In human subjects, SAA is found in apoB-containing lipoprotein particles only in obese subjects with type 2 diabetes, but not in healthy obese or obese subjects with MetS. Copyright © 2012 The Obesity Society.

[Roles of G protein-coupled estrogen receptor in the male reproductive system].

Science.gov (United States)

Chen, Kai-hong; Zhang, Xian; Jiang, Xue-wu

2016-02-01

The G protein-coupled estrogen receptor (GPER), also known as G protein-coupled receptor 30 (GPR30), was identified in the recent years as a functional membrane receptor different from the classical nuclear estrogen receptors. This receptor is widely expressed in the cortex, cerebellum, hippocampus, heart, lung, liver, skeletal muscle, and the urogenital system. It is responsible for the mediation of nongenomic effects associated with estrogen and its derivatives, participating in the physiological activities of the body. The present study reviews the molecular structure, subcellular localization, signaling pathways, distribution, and function of GPER in the male reproductive system.

Isolation and characterization of rhamnose-binding lectins from eggs of steelhead trout (Oncorhynchus mykiss) homologous to low density lipoprotein receptor superfamily.

Science.gov (United States)

Tateno, H; Saneyoshi, A; Ogawa, T; Muramoto, K; Kamiya, H; Saneyoshi, M

1998-07-24

methodology to investigate the interactions between STL2 and major egg yolk proteins from steelhead trout, lipovitellin, and beta'-component, which are known as vitellogenin digests. Interestingly, STL2 showed distinct interactions with both egg yolk proteins. The estimated values for the affinity constant (Ka) of STL2 to lipovitellin and beta' component were 3.44 x 10(6) and 4.99 x 10(6), respectively. These results suggest that the fish egg lectins belong to a new family of animal lectin structurally related to the low density lipoprotein receptor super- family.

Effect of phospholipase A treatment of low density lipoproteins on the dextran sulfate--lipoprotein interaction.

Science.gov (United States)

Nishida, T

1968-09-01

The effect of phospholipase A on the interaction of low density lipoproteins of the S(f) 0-10 class with dextran sulfate was studied in phosphate buffer of pH 7.4, ionic strength 0.1, by chemical, spectrophotometric, and centrifugal methods. When low density lipoproteins that had been treated with phospholipase A were substituted for untreated lipoproteins, the amount of insoluble dextran sulfate-lipoprotein complex formed was greatly reduced. Hydrolysis of over 20% of the lecithin and phosphatidyl ethanolamine constituents of the lipoproteins prevented the formation of insoluble complex. However, even the lipoproteins in which almost all the phosphoglycerides were hydrolyzed produced soluble complex, which was converted to insoluble complex upon addition of magnesium sulfate. It is apparent that the lipoproteins altered extensively by treatment with phospholipase A retain many characteristic properties of native low density lipoproteins. Fatty acids, but not lysolecithin, released by the action of phospholipase A interfered with the formation of insoluble complex; this interference was due to association of the fatty acids with the lipoproteins. With increases in the concentration of the associated fatty acids, the amounts of magnesium ion required for the conversion of soluble complex to insoluble complex increased progressively. Charge interaction is evidently of paramount importance in the formation of sulfated polysaccharide-lipoprotein complexes.

Correlation of structural stability with functional remodeling of high-density lipoproteins: the importance of being disordered.

Science.gov (United States)

Guha, Madhumita; Gao, Xuan; Jayaraman, Shobini; Gursky, Olga

2008-11-04

High-density lipoproteins (HDLs) are protein-lipid assemblies that remove excess cell cholesterol and prevent atherosclerosis. HDLs are stabilized by kinetic barriers that decelerate protein dissociation and lipoprotein fusion. We propose that similar barriers modulate metabolic remodeling of plasma HDLs; hence, changes in particle composition that destabilize HDLs and accelerate their denaturation may accelerate their metabolic remodeling. To test this notion, we correlate existing reports on HDL-mediated cell cholesterol efflux and esterification, which are obligatory early steps in cholesterol removal, with our kinetic studies of HDL stability. The results support our hypothesis and show that factors accelerating cholesterol efflux and esterification in model discoidal lipoproteins (including reduced protein size, reduced fatty acyl chain length, and/or increased level of cis unsaturation) destabilize lipoproteins and accelerate their fusion and apolipoprotein dissociation. Oxidation studies of plasma spherical HDLs show a similar trend: mild oxidation by Cu(2+) or OCl(-) accelerates cell cholesterol efflux, protein dissociation, and HDL fusion, while extensive oxidation inhibits these reactions. Consequently, moderate destabilization may be beneficial for HDL functions by facilitating insertion of cholesterol and lipophilic enzymes, promoting dissociation of lipid-poor apolipoproteins, which are primary acceptors of cell cholesterol, and thereby accelerating HDL metabolism. Therefore, HDL stability must be delicately balanced to maintain the structural integrity of the lipoprotein assembly and ensure structural specificity necessary for interactions of HDL with its metabolic partners, while facilitating rapid HDL remodeling and turnover at key junctures of cholesterol transport. The inverse correlation between HDL stability and remodeling illustrates the functional importance of structural disorder in macromolecular assemblies stabilized by kinetic barriers.

Drosophila lipophorin receptors mediate the uptake of neutral lipids in oocytes and imaginal disc cells by an endocytosis-independent mechanism.

Directory of Open Access Journals (Sweden)

Esmeralda Parra-Peralbo

2011-02-01

Full Text Available Lipids are constantly shuttled through the body to redistribute energy and metabolites between sites of absorption, storage, and catabolism in a complex homeostatic equilibrium. In Drosophila, lipids are transported through the hemolymph in the form of lipoprotein particles, known as lipophorins. The mechanisms by which cells interact with circulating lipophorins and acquire their lipidic cargo are poorly understood. We have found that lipophorin receptor 1 and 2 (lpr1 and lpr2, two partially redundant genes belonging to the Low Density Lipoprotein Receptor (LDLR family, are essential for the efficient uptake and accumulation of neutral lipids by oocytes and cells of the imaginal discs. Females lacking the lpr2 gene lay eggs with low lipid content and have reduced fertility, revealing a central role for lpr2 in mediating Drosophila vitellogenesis. lpr1 and lpr2 are transcribed into multiple isoforms. Interestingly, only a subset of these isoforms containing a particular LDLR type A module mediate neutral lipid uptake. Expression of these isoforms induces the extracellular stabilization of lipophorins. Furthermore, our data indicate that endocytosis of the lipophorin receptors is not required to mediate the uptake of neutral lipids. These findings suggest a model where lipophorin receptors promote the extracellular lipolysis of lipophorins. This model is reminiscent of the lipolytic processing of triglyceride-rich lipoproteins that occurs at the mammalian capillary endothelium, suggesting an ancient role for LDLR-like proteins in this process.

Effects of medium-chain fatty acids and oleic acid on blood lipids, lipoproteins, glucose, insulin, and lipid transfer protein activities

DEFF Research Database (Denmark)

Tholstrup, T.; Ehnholm, C.; Jauhiainen, M.

2004-01-01

Background: Dietary medium-chain fatty acids (MCFAs) are of nutritional interest because they are more easily absorbed from dietary medium-chain triacylglycerols (MCTs) than are long-chain fatty acids from, for example, vegetable oils. It has generally been claimed that MCFAs do not increase plasma...... cholesterol, although this claim is poorly documented. Objective: We compared the effects of a diet rich in either MCFAs or oleic acid on fasting blood lipids, lipoproteins, glucose, insulin, and lipid transfer protein activities in healthy men. Design: In a study with a double-blind, randomized, crossover...... plasma total triacylglycerol (P = 0.0361), and higher plasma glucose (P = 0.033). Plasma HDL-cholesterol and insulin concentrations and activities of cholesterol ester transfer protein and phospholipid transfer protein did not differ significantly between the diets. Conclusions: Compared with fat high...

Muscarinic supersensitivity and impaired receptor desensitization in G protein-coupled receptor kinase 5-deficient mice.

Science.gov (United States)

Gainetdinov, R R; Bohn, L M; Walker, J K; Laporte, S A; Macrae, A D; Caron, M G; Lefkowitz, R J; Premont, R T

1999-12-01

G protein-coupled receptor kinase 5 (GRK5) is a member of a family of enzymes that phosphorylate activated G protein-coupled receptors (GPCR). To address the physiological importance of GRK5-mediated regulation of GPCRs, mice bearing targeted deletion of the GRK5 gene (GRK5-KO) were generated. GRK5-KO mice exhibited mild spontaneous hypothermia as well as pronounced behavioral supersensitivity upon challenge with the nonselective muscarinic agonist oxotremorine. Classical cholinergic responses such as hypothermia, hypoactivity, tremor, and salivation were enhanced in GRK5-KO animals. The antinociceptive effect of oxotremorine was also potentiated and prolonged. Muscarinic receptors in brains from GRK5-KO mice resisted oxotremorine-induced desensitization, as assessed by oxotremorine-stimulated [5S]GTPgammaS binding. These data demonstrate that elimination of GRK5 results in cholinergic supersensitivity and impaired muscarinic receptor desensitization and suggest that a deficit of GPCR desensitization may be an underlying cause of behavioral supersensitivity.

Purification and properties of a mitochondrial lipoprotein inhibitor of sterol synthesis

International Nuclear Information System (INIS)

Madhosingh, C.; Migicovsky, B.B.; Starratt, A.N.

1976-01-01

A lipoprotein inhibitor of hydroxymethylglutaryl CoA reductase (EC 1.1.1.34) and of cholesterol synthesis by rat liver homogenates, was isolated from the mitochondria of starved rats' livers. The isolated lipoprotein complex contained a low molecular weight protein and fatty acids. The fatty acids identified were arachidonic, linoleic, oleic, stearic and palmitic. The saturated fatty acids and oleic acid did not inhibit. Inhibition of the enzyme was to a large extent related to the degree of fatty acid unsaturation. (auth.)

High-density lipoproteins: a novel therapeutic target for cardiovascular disease

Directory of Open Access Journals (Sweden)

TS Mohamed Saleem

2011-01-01

Full Text Available TS Mohamed Saleem1, PV Sandhya Rani1, K Gauthaman21Department of Pharmacology, Annamacharya College of Pharmacy, New Boyanapalli, Andhrapradesh, India; 2Department of Drug Technology, Faculty of Medical Technology, Derna, LibyaAbstract: Cardiovascular disease has a high rate of mortality in both Western and developing countries. Atherosclerosis and generation of reactive oxygen species through oxidative stress is the major risk factor for cardiovascular disease. Atherothrombosis with low levels of high-density lipoprotein (HDL and high levels of low-density lipoprotein is a major risk factor for atherosclerosis-induced cardiovascular disease. Lipid-lowering drugs like statins, niacin, fibrates, and some newer agents, ie, the apolipoprotein A-I mimetics and the cholesteryl ester transfer protein inhibitors, not only increase HDL levels but are also effective in reducing key atherogenic lipid components, including triglyceride-rich lipoproteins. The aim of this review is to discuss the accumulating evidence suggesting that HDL possesses a diverse range of biological actions, and that increasing HDL levels by drug treatment may be beneficial in the prevention of cardiovascular disease.Keywords: cardiovascular disease, lipoproteins, statins, apolipoprotein, atherosclerosis

Regulation of G protein-coupled receptor signalling: focus on the cardiovascular system and regulator of G protein signalling proteins

NARCIS (Netherlands)

Hendriks-Balk, Mariëlle C.; Peters, Stephan L. M.; Michel, Martin C.; Alewijnse, Astrid E.

2008-01-01

G protein-coupled receptors (GPCRs) are involved in many biological processes. Therefore, GPCR function is tightly controlled both at receptor level and at the level of signalling components. Well-known mechanisms by which GPCR function can be regulated comprise desensitization/resensitization

Modeling structure of G protein-coupled receptors in huan genome

KAUST Repository

Zhang, Yang

2016-01-01

G protein-coupled receptors (or GPCRs) are integral transmembrane proteins responsible to various cellular signal transductions. Human GPCR proteins are encoded by 5% of human genes but account for the targets of 40% of the FDA approved drugs. Due

The AMPA receptor-associated protein Shisa7 regulates hippocampal synaptic function and contextual memory

NARCIS (Netherlands)

Schmitz, Leanne J M; Klaassen, Remco V; Ruiperez-Alonso, Marta; Zamri, Azra Elia; Stroeder, Jasper; Rao-Ruiz, Priyanka; Lodder, Johannes C; van der Loo, Rolinka J; Mansvelder, Huib D; Smit, August B; Spijker, Sabine; Verhage, Matthijs

2017-01-01

Glutamatergic synapses rely on AMPA receptors (AMPARs) for fast synaptic transmission and plasticity. AMPAR auxiliary proteins regulate receptor trafficking, and modulate receptor mobility and its biophysical properties. The AMPAR auxiliary protein Shisa7 (CKAMP59) has been shown to interact with

G protein-coupled receptor kinase 2 negatively regulates chemokine signaling at a level downstream from G protein subunits

NARCIS (Netherlands)

Jimenez-Sainz, MC; Murga, C; Kavelaars, A; Jurado-Pueyo, M; Krakstad, BF; Heijnen, CJ; Mayor, F; Aragay, AM

The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes ligand-activated G protein-coupled-receptors. Here, evidence is shown for a novel role of GRK2 in regulating chemokine-mediated signals. The presence of increased levels of GRK2 in human embryonic kidney (HEK) 293 cells

Liver lipase and high-density lipoprotein. Lipoprotein changes after incubation of human serum with rat liver lipase.

Science.gov (United States)

Groot, P H; Scheek, L M; Jansen, H

1983-05-16

Human sera were incubated with rat liver lipase after inactivation of lecithin:cholesterol acyltransferase, and the changes in serum lipoprotein composition were measured. In the presence of liver lipase serum triacylglycerol and phosphatidylcholine were hydrolyzed. The main changes in the concentrations of these lipids were found in the high-density lipoprotein fraction. Subfractionation of high-density lipoprotein by rate-zonal ultracentrifugation showed a prominent decrease in all constituents of high-density lipoprotein2, a smaller decrease in the 'light' high-density lipoprotein3 and an increase in the 'heavy' high-density lipoprotein3. These data support a concept in which liver lipase is involved in high-density lipoprotein2 phospholipid and triacylglycerol catabolism and suggest that as a result of this action high-density lipoprotein2 is converted into high-density lipoprotein3.

PDZ Protein Regulation of G Protein-Coupled Receptor Trafficking and Signaling Pathways.

Science.gov (United States)

Dunn, Henry A; Ferguson, Stephen S G

2015-10-01

G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membrane-associated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na(+)/H(+) exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domain-containing kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Gα-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and

Improving lipoprotein profiles by liver-directed gene transfer of low ...

Indian Academy of Sciences (India)

cesses, where a myriad of proteins necessary for metabolism and immune .... containing 5% nonfat milk for several hours, the membrane .... high-density lipoprotein cholesterol; mice were fed with a high fat diet (21% fat, 0.15% choles- terol).

Delayed Toxicity Associated with Soluble Anthrax Toxin Receptor Decoy-Ig Fusion Protein Treatment

Science.gov (United States)

Cote, Christopher; Welkos, Susan; Manchester, Marianne; Young, John A. T.

2012-01-01

Soluble receptor decoy inhibitors, including receptor-immunogloubulin (Ig) fusion proteins, have shown promise as candidate anthrax toxin therapeutics. These agents act by binding to the receptor-interaction site on the protective antigen (PA) toxin subunit, thereby blocking toxin binding to cell surface receptors. Here we have made the surprising observation that co-administration of receptor decoy-Ig fusion proteins significantly delayed, but did not protect, rats challenged with anthrax lethal toxin. The delayed toxicity was associated with the in vivo assembly of a long-lived complex comprised of anthrax lethal toxin and the receptor decoy-Ig inhibitor. Intoxication in this system presumably results from the slow dissociation of the toxin complex from the inhibitor following their prolonged circulation. We conclude that while receptor decoy-Ig proteins represent promising candidates for the early treatment of B. anthracis infection, they may not be suitable for therapeutic use at later stages when fatal levels of toxin have already accumulated in the bloodstream. PMID:22511955

The endothelial protein C receptor and activated protein C play a limited role in host defense during experimental tuberculosis

NARCIS (Netherlands)

Kager, Liesbeth M.; Roelofs, Joris J. T. H.; de Vos, Alex F.; Wieland, Catharina W.; Schouten, Marcel; Meijers, Joost C. M.; Isermann, Berend; van't Veer, Cornelis; Esmon, Charles T.; van der Poll, Tom

2013-01-01

The protein C (PC) system is an important regulator of both coagulation and inflammation. Activated PC (APC), together with its receptor the endothelial protein C receptor (EPCR), has anticoagulant and anti-inflammatory properties. During tuberculosis (TB), a devastating chronic pulmonary disease

Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors

DEFF Research Database (Denmark)

Tian, He; Naganathan, Saranga; Kazmi, Manija A

2014-01-01

Novel methods are required for site-specific, quantitative fluorescent labeling of G-protein-coupled receptors (GPCRs) and other difficult-to-express membrane proteins. Ideally, fluorescent probes should perturb the native structure and function as little as possible. We evaluated bioorthogonal...

Central nervous system regulation of intestinal lipid and lipoprotein metabolism.

Science.gov (United States)

Farr, Sarah; Taher, Jennifer; Adeli, Khosrow

2016-02-01

In response to nutrient availability, the small intestine and brain closely communicate to modulate energy homeostasis and metabolism. The gut-brain axis involves complex nutrient sensing mechanisms and an integration of neuronal and hormonal signaling. This review summarizes recent evidence implicating the gut-brain axis in regulating lipoprotein metabolism, with potential implications for the dyslipidemia of insulin resistant states. The intestine and brain possess distinct mechanisms for sensing lipid availability, which triggers subsequent regulation of feeding, glucose homeostasis, and adipose tissue metabolism. More recently, central receptors, neuropeptides, and gut hormones that communicate with the brain have been shown to modulate hepatic and intestinal lipoprotein metabolism via parasympathetic and sympathetic signaling. Gut-derived glucagon-like peptides appear to be particularly important in modulating the intestinal secretion of chylomicron particles via a novel brain-gut axis. Dysregulation of these pathways may contribute to postprandial diabetic dyslipidemia. Emerging evidence implicates the central and enteric nervous systems in controlling many aspects of lipid and lipoprotein metabolism. Bidirectional communication between the gut and brain involving neuronal pathways and gut peptides is critical for regulating feeding and metabolism, and forms a neuroendocrine circuit to modulate dietary fat absorption and intestinal production of atherogenic chylomicron particles.

Recruitment of SHP-1 protein tyrosine phosphatase and signalling by a chimeric T-cell receptor-killer inhibitory receptor

DEFF Research Database (Denmark)

Christensen, M D; Geisler, C

2000-01-01

Receptors expressing the immunoreceptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic tail play an important role in the negative regulation of natural killer and B-cell activation. A subpopulation of T cells expresses the ITIM containing killer cell inhibitory receptor (KIR), which...... recognize MHC class I molecules. Following coligation of KIR with an activating receptor, the tyrosine in the ITIM is phosphorylated and the cytoplasmic protein tyrosine phosphatase SHP-1 is recruited to the ITIM via its SH2 domains. It is still not clear how SHP-1 affects T-cell receptor (TCR) signalling...... regarding total protein tyrosine phosphorylation, TCR down-regulation, mobilization of intracellular free calcium, or induction of the activation markers CD69 and CD25....

The levels of plasma low density lipoprotein are independent of cholesterol ester transfer protein in fish-oil fed F1B hamsters

Directory of Open Access Journals (Sweden)

Davis Phillip J

2005-03-01

Full Text Available Abstract Background Cholesterol ester transfer protein (CETP plays a major role in regulating the levels of LDL- and HDL-cholesterol. We previously observed a fish-oil-induced elevation of low-density lipoprotein (LDL-and very-low-density lipoprotein (VLDL-cholesterol concentrations and a decrease in high-density lipoprotein (HDL-cholesterol concentration in F1B hamsters. The molecular mechanism/s by which fish oil induces hyperlipidaemic effect was investigated in this study. We examined whether the effects of dietary fish oil on plasma lipoprotein concentrations are due to fish-oil-induced alterations in plasma CETP activity. MIX diet, a diet supplemented with a mixture of lard and safflower oil, was used as the control diet. Results We found that fish oil feeding in hamsters reduced CETP mass as well as CETP activity. Increasing the dietary fat level of fish-oil from 5% to 20% (w/w led to a further decrease in CETP mass. Supplementation with dietary cholesterol increased both CETP mass and CETP activity in fish-oil and MIX-diet fed hamsters. However, there was no correlation between CETP mass as well as CETP activity and LDL-cholesterol concentrations. Conclusion These findings suggest that cholesterol ester transfer between HDL and LDL is not likely to play a major role in determining fish-oil-induced changes in LDL- and HDL-cholesterol concentrations in F1B hamsters. A possible role of reduced clearance of LDL-particles as well as dietary fat level and dietary cholesterol dependent changes in LDL-lipid composition have been discussed.

Regulation of AMPA Receptor Trafficking by Protein Ubiquitination

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Jocelyn Widagdo

2017-10-01

Full Text Available The molecular mechanisms underlying plastic changes in the strength and connectivity of excitatory synapses have been studied extensively for the past few decades and remain the most attractive cellular models of learning and memory. One of the major mechanisms that regulate synaptic plasticity is the dynamic adjustment of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA-type glutamate receptor content on the neuronal plasma membrane. The expression of surface AMPA receptors (AMPARs is controlled by the delicate balance between the biosynthesis, dendritic transport, exocytosis, endocytosis, recycling and degradation of the receptors. These processes are dynamically regulated by AMPAR interacting proteins as well as by various post-translational modifications that occur on their cytoplasmic domains. In the last few years, protein ubiquitination has emerged as a major regulator of AMPAR intracellular trafficking. Dysregulation of AMPAR ubiquitination has also been implicated in the pathophysiology of Alzheimer’s disease. Here we review recent advances in the field and provide insights into the role of protein ubiquitination in regulating AMPAR membrane trafficking and function. We also discuss how aberrant ubiquitination of AMPARs contributes to the pathogenesis of various neurological disorders, including Alzheimer’s disease, chronic stress and epilepsy.

Comparative reactivity of the myeloperoxidase-derived oxidants HOCl and HOSCN with low-density lipoprotein (LDL)

DEFF Research Database (Denmark)

Ismael, Fahd O; Proudfoot, Julie M; Brown, Bronwyn E

2015-01-01

Atherosclerosis is characterised by the accumulation of lipids within macrophages in the artery wall. Low-density lipoprotein (LDL) is the source of this lipid, owing to the uptake of oxidised LDL by scavenger receptors. Myeloperoxidase (MPO) released by leukocytes during inflammation produces ox...

Comparison of human plasma low- and high-density lipoproteins as substrates for lecithin: cholesterol acyltransferase.

Science.gov (United States)

Barter, P J; Hopkins, G J; Gorjatschko, L

1984-01-17

A recent observation that lecithin: cholesterol acyltransferase (EC 2.3.1.43) interacts with both low-density lipoproteins (LDL) and high-density lipoproteins (HDL) in human plasma is in apparent conflict with an earlier finding that the purified enzyme, while highly reactive with isolated HDL, was only minimally reactive with LDL. There is evidence, however, that lecithin: cholesterol acyltransferase may exist physiologically as a component of a complex with other proteins and that studies with the isolated enzyme may therefore provide misleading results. Consequently, interactions of the enzyme with isolated human lipoproteins have been re-examined in incubations containing lecithin: cholesterol acyltransferase as a component of human lipoprotein-free plasma in which a physiologically active complex of the enzyme with other proteins may have been preserved. In this system there was a ready esterification of the free cholesterol associated with both LDL and HDL-subfraction 3 (HDL3) in reactions that obeyed typical enzyme-saturation kinetics. For a given preparation of lipoprotein-free plasma the Vmax values with LDL and with HDL3 were virtually identical. The apparent Km for free cholesterol associated with HDL3 was 5.6 X 10(-5) M, while for that associated with LDL it was 4.1 X 10(-4) M. This implied that, in terms of free cholesterol concentration, the affinity of HDL3 for lecithin: cholesterol acyltransferase was about 7-times greater than that of LDL. When expressed in terms of lipoprotein particle concentration, however, it was apparent that the affinity of LDL for the enzyme was considerably greater than that of HDL3. When the lipoprotein fractions were equated in terms of lipoprotein surface area, the apparent affinities of the two fractions for the enzyme were found to be comparable.

Vitamin D receptor and estrogen receptor gene polymorphisms in postmenopausal Danish women

DEFF Research Database (Denmark)

Bagger, Y Z; Hassager, C; Heegaard, Anne-Marie

2000-01-01

To investigate the polymorphisms of the vitamin D receptor (VDR) and estrogen receptor (ER) genes in relation to biochemical markers of bone turnover (serum osteocalcin and urinary collagen type I degradation products (CrossLaps), and to study ER genotypes in relation to serum lipoproteins, blood...... pressure, or changes in these parameters after 2 years of hormone replacement therapy (HRT) in 499 Danish postmenopausal women....

Identification of the Oxidized Low-Density Lipoprotein Scavenger Receptor CD36 in Plasma

DEFF Research Database (Denmark)

Handberg, Aase; Levin, Klaus; Højlund, Kurt

2006-01-01

BACKGROUND: Macrophage CD36 scavenges oxidized low-density lipoprotein, leading to foam cell formation, and appears to be a key proatherogenic molecule. Increased expression of CD36 has been attributed to hyperglycemia and to defective macrophage insulin signaling in insulin resistance. Premature...

A monoclonal antibody for G protein-coupled receptor crystallography

DEFF Research Database (Denmark)

Day, Peter W; Rasmussen, Søren Gøgsig Faarup; Parnot, Charles

2007-01-01

G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural inf...

The structure and function of G-protein-coupled receptors

DEFF Research Database (Denmark)

Rosenbaum, Daniel M; Rasmussen, Søren Gøgsig Faarup; Kobilka, Brian K

2009-01-01

G-protein-coupled receptors (GPCRs) mediate most of our physiological responses to hormones, neurotransmitters and environmental stimulants, and so have great potential as therapeutic targets for a broad spectrum of diseases. They are also fascinating molecules from the perspective of membrane-protein...

G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins from endosomes.

Science.gov (United States)

Tsvetanova, Nikoleta G; Irannejad, Roshanak; von Zastrow, Mark

2015-03-13

Some G protein-coupled receptors (GPCRs), in addition to activating heterotrimeric G proteins in the plasma membrane, appear to elicit a "second wave" of G protein activation after ligand-induced internalization. We briefly summarize evidence supporting this view and then discuss what is presently known about the functional significance of GPCR-G protein activation in endosomes. Endosomal activation can shape the cellular response temporally by prolonging its overall duration, and may shape the response spatially by moving the location of intracellular second messenger production relative to effectors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Single-Particle Tracking of Human Lipoproteins.

Science.gov (United States)

de Messieres, Michel; Ng, Abby; Duarte, Cornelio J; Remaley, Alan T; Lee, Jennifer C

2016-01-05

Lipoproteins, such as high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very-low density lipoprotein (VLDL), play a critical role in heart disease. Lipoproteins vary in size and shape as well as in their apolipoprotein content. Here, we developed a new experimental framework to study freely diffusing lipoproteins from human blood, allowing analysis of even the smallest HDL with a radius of 5 nm. In an easily constructed confinement chamber, individual HDL, LDL, and VLDL particles labeled with three distinct fluorophores were simultaneously tracked by wide-field fluorescence microscopy and their sizes were determined by their motion. This technique enables studies of individual lipoproteins in solution and allows characterization of the heterogeneous properties of lipoproteins which affect their biological function but are difficult to discern in bulk studies.

Estrogen receptor accessory proteins augment receptor-DNA interaction and DNA bending.

Science.gov (United States)

Landel, C C; Potthoff, S J; Nardulli, A M; Kushner, P J; Greene, G L

1997-01-01

Increasing evidence suggests that accessory proteins play an important role in the ability of the estrogen receptor (ER) and other nuclear hormone receptors to modulate transcription when bound to cis-acting hormone response elements in target genes. We have previously shown that four proteins, hsp70, protein disulfide isomerase (PDI) and two unknown proteins (p48 and p45), copurify with ER that has been isolated by site-specific DNA chromatography (BERE) and influence the interaction of ER with DNA in vitro. To better define the nature of these effects, we used filter binding and electrophoretic mobility shift assays to study the ability of these proteins to alter the kinetics of ER-DNA interaction and to influence the ability of ER to bend DNA when bound to an estrogen response element (ERE). The results of both assays indicate that ERE-purified ER, with its four associated proteins (hsp70, PDI, p48, p45), has a greater ability to bind to the vitellogenin A2 ERE than ER purified by estradiol-Sepharose chromatography in the absence (ESeph) or presence (EATP) of ATP, in which p48, p45 (ESeph) and hsp70 (EATP) are removed. Surprisingly, the rates of association and dissociation of ER and ERE were essentially the same for all three mixtures, suggesting that one or more ER-associated proteins, especially p45 and p48, may be required for ER to attain maximum DNA binding activity. In addition, circular permutation and phasing analyses demonstrated that the same ER-associated proteins produced higher order ER-DNA complexes that significantly increased the magnitude of DNA distortion, but did not alter the direction of the ER-induced bend of ERE-containing DNA fragments, which was toward the major groove of the DNA helix. These results suggest that p45 and/or p48 and possibly hsp70, play an important role both in the specific DNA binding and bending activities of ER and thus contribute to the overall stimulation of transcription in target genes that contain cis

Two novel mutations in exon 3 and 4 of low density lipoprotein (LDL) receptor gene in patients with heterozygous familial hypercholesterolemia

International Nuclear Information System (INIS)

Khan, S.P.

2011-01-01

Objective: To determine the common mutation of low density lipoprotein receptor in hypercholesterolemia patients requiring screening for heterozygous familial hypercholesterolemia (HeFH) in Karachi. Study Design: Case-series. Place and Duration of Study: Dr. Ziauddin Hospital Laboratory and Dr. Rubina Ghani's Pathological and Molecular Laboratories, Karachi, for the PCR bench work from June 2008 to October 2009. Methodology: All the patients selected for this study were from Dr. Ziauddin Hospital and National Institute of Cardiovascular Diseases. All the patients having high total cholesterol and LDL-cholesterol were included in this study with premature coronary artery diseases or a family history of hypercholesterolemia. Exclusion criteria included Diabetes mellitus, hypertension, renal disease, hypothyroidism and steroid therapy. After lipid profile with overnight fasting, DNA was extracted from whole blood collected in EDTA (ethylenediamine tetra acetic acid) tube and multiplex PCR (polymerase chain reaction) using forward and reverse primers of exons 3, 4, 9 and 14 of base pairs 162, 431, 550 and 496 respectively. Results: Out of total of 120 hypercholesterolemia cases, 42 patients were classical cases of HeFH (heterozygous familial hypercholesterolemia) with xanthomas, xanthelasmas and LDL-C > 160 mg/dl. The total cholesterol (260 +- 57 mg/dL) and LDL-C (192 +- 39 mg/dL ) of cases was significantly high as compared to, controls having total cholesterol (184 9 +- 27 mg/dL) and LDL-C (105 +- 22 mg/dL), p > 0.001. Two novel point mutations were noted in exon 3 and exon 4. The other 78 cases were probable with raised LDL-C (low density lipoprotein cholesterol) and family history of premature coronary heart diseases. Conclusion: The frequency of HeFH was 35% classical and 65% probable cases out of total 120 hypercholesterolemia patients from two tertiary care hospitals in Karachi. The point mutation on exon 3 and exon 4 of LDLR gene was the most common. PCR is

Lipoprotein glomerulopathy treated with LDL-apheresis (Heparin-induced Extracorporeal Lipoprotein Precipitation system: a case report

Directory of Open Access Journals (Sweden)

Rivasi Paolo

2009-12-01

Full Text Available Abstract Introduction Lipoprotein glomerulopathy is a glomerulonephritis which was described for the first time by Saito in 1989 and is currently acknowledged as a separate nosological entity. It is histologically characterized by a marked dilatation of the glomerular capillaries and the presence of lipoprotein thrombi in the glomerular lumens. The dyslipidemic profile is similar to that of type III dyslipoproteinemia with Apolipoprotein E values that are often high; proteinuria and renal dysfunction are present. Proteinuria often does not respond to steroid and cytostatic treatments. The phenotypic expression of lipoprotein glomerulopathy is most probably correlated to a genetic alteration of the lipoprotein metabolism (mutation of the Apolipoprotein E coding gene. In literature, lipoprotein glomerulopathies have mainly been reported in Japanese and Chinese subjects, except for three cases in the Caucasian race, reported in France and the USA. Case presentation We describe the case of a 60-year-old female, Caucasian patient suffering from lipoprotein glomerulopathy, carrier of a new mutation on the Apolipoprotein E gene (Apolipoprotein EMODENA, and treated successfully with low density lipoprotein-apheresis with the Heparin induced extracorporeal lipoprotein precipitation system. After a first phase of therapeutic protocol with statins, the patient was admitted for nephrotic syndrome, renal failure and hypertension. Since conventional treatment alone was not able to control dyslipidemia, aphaeretic treatment with heparin-induced Extracorporeal Lipoprotein Precipitation - apheresis (HELP-apheresis was started to maintain angiotensin converting enzyme inhibitor therapy for the treatment of hypertension. Treatment with HELP-apheresis led to a complete remission of the proteinuria in a very short time (four months, as well as control of hypercholesterolemia and renal function recovery. Conclusion According to this case of lipoprotein glomerulopathy

Protein-induced satiation and the calcium-sensing receptor

OpenAIRE

Ojha,Utkarsh

2018-01-01

Utkarsh Ojha Faculty of Medicine, Imperial College School of Medicine, Imperial College London, London, UK Abstract: Obesity is a major global health issue. High-protein diets have been shown to be associated with weight loss and satiety. The precise mechanism by which protein-rich diets promote weight loss remains unclear. Evidence suggests amino acids, formed as a consequence of protein digestion, are sensed by specific receptors on L-cells in the gastrointestinal (GI) tract. These L-cells ...

Comparative microscopic and biochemical study of the uptake of fluorescent and 125I-labeled lipoproteins by skin fibroblasts, smooth muscle cells, and peritoneal macrophages in culture

International Nuclear Information System (INIS)

Reynolds, G.D.; St Clair, R.W.

1985-01-01

Uptake of low density lipoprotein (LDL) and of acetyl LDL was compared in skin fibroblasts, smooth muscle cells, and peritoneal macrophages with the use of lipoproteins labeled with either 125 I or the fluorescent probe 3,3'-dioctadecylindocarbocyanine (DiI). The uptake of DiI-labeled lipoproteins was assessed by quantitative spectrofluorometry and by fluorescence microscopy. The DiI was quantitatively retained by the cells, while the 125 I-LDL was degraded and 125 I-labeled degradation products were excreted from the cells. In smooth muscle cells and fibroblasts the uptake of LDL was virtually the same whether measured with the use of the DiI or 125 I-label. The labeling of acetyl LDL with DiI enhanced its uptake in peritoneal macrophages by an average of 18%. With the DiI label, lipoprotein uptake could be determined after as little as 10 minutes of incubation at 37 C. The pattern of uptake of the DiI-labeled lipoproteins was consistent with binding to specific receptors, because no DiI could be detected in mutant cells without LDL receptors, and uptake was competitively inhibited by addition of excess unlabeled lipoprotein. When the DiI-labeled lipoproteins were removed from the medium, there was a 5-15% loss of DiI from all cell types studied over the first 24 hours

Scotopic vision in the monkey is modulated by the G protein-coupled receptor 55

DEFF Research Database (Denmark)

Bouskila, Joseph; Harrar, Vanessa; Javadi, Pasha

2016-01-01

The endogenous cannabinoid system plays important roles in the retina of mice and monkeys via their classic CB1 and CB2 receptors. We have previously reported that the G protein-coupled receptor 55 (GPR55), a putative cannabinoid receptor, is exclusively expressed in rod photoreceptors in the mon......The endogenous cannabinoid system plays important roles in the retina of mice and monkeys via their classic CB1 and CB2 receptors. We have previously reported that the G protein-coupled receptor 55 (GPR55), a putative cannabinoid receptor, is exclusively expressed in rod photoreceptors...

DMPD: G-protein-coupled receptor expression, function, and signaling in macrophages. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 17456803 G-protein-coupled receptor expression, function, and signaling in macropha...2007 Apr 24. (.png) (.svg) (.html) (.csml) Show G-protein-coupled receptor expression, function, and signali...ng in macrophages. PubmedID 17456803 Title G-protein-coupled receptor expression, function

Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor.

Science.gov (United States)

Roskoski, Robert

2005-11-11

Signaling by stem cell factor and Kit, its receptor, plays important roles in gametogenesis, hematopoiesis, mast cell development and function, and melanogenesis. Moreover, human and mouse embryonic stem cells express Kit transcripts. Stem cell factor exists as both a soluble and a membrane-bound glycoprotein while Kit is a receptor protein-tyrosine kinase. The complete absence of stem cell factor or Kit is lethal. Deficiencies of either produce defects in red and white blood cell production, hypopigmentation, and sterility. Gain-of-function mutations of Kit are associated with several human neoplasms including acute myelogenous leukemia, gastrointestinal stromal tumors, and mastocytomas. Kit consists of an extracellular domain, a transmembrane segment, a juxtamembrane segment, and a protein kinase domain that contains an insert of about 80 amino acid residues. Binding of stem cell factor to Kit results in receptor dimerization and activation of protein kinase activity. The activated receptor becomes autophosphorylated at tyrosine residues that serve as docking sites for signal transduction molecules containing SH2 domains. The adaptor protein APS, Src family kinases, and Shp2 tyrosyl phosphatase bind to phosphotyrosine 568. Shp1 tyrosyl phosphatase and the adaptor protein Shc bind to phosphotyrosine 570. C-terminal Src kinase homologous kinase and the adaptor Shc bind to both phosphotyrosines 568 and 570. These residues occur in the juxtamembrane segment of Kit. Three residues in the kinase insert domain are phosphorylated and attract the adaptor protein Grb2 (Tyr703), phosphatidylinositol 3-kinase (Tyr721), and phospholipase Cgamma (Tyr730). Phosphotyrosine 900 in the distal kinase domain binds phosphatidylinositol 3-kinase which in turn binds the adaptor protein Crk. Phosphotyrosine 936, also in the distal kinase domain, binds the adaptor proteins APS, Grb2, and Grb7. Kit has the potential to participate in multiple signal transduction pathways as a result of

Guanine nucleotide-binding protein regulation of melatonin receptors in lizard brain

International Nuclear Information System (INIS)

Rivkees, S.A.; Carlson, L.L.; Reppert, S.M.

1989-01-01

Melatonin receptors were identified and characterized in crude membrane preparations from lizard brain by using 125 I-labeled melatonin ( 125 I-Mel), a potent melatonin agonist. 125 I-Mel binding sites were saturable; Scatchard analysis revealed high-affinity and lower affinity binding sites, with apparent K d of 2.3 ± 1.0 x 10 -11 M and 2.06 ± 0.43 x 10 -10 M, respectively. Binding was reversible and inhibited by melatonin and closely related analogs but not by serotonin or norepinephrine. Treatment of crude membranes with the nonhydrolyzable GTP analog guanosine 5'-[γ-thio]triphosphate (GTP[γS]), significantly reduced the number of high-affinity receptors and increased the dissociation rate of 125 I-Mel from its receptor. Furthermore, GTP[γS] treatment of ligand-receptor complexes solubilized by Triton X-100 also led to a rapid dissociation of 125 I-Mel from solubilized ligand-receptor complexes. Gel filtration chromatography of solubilized ligand-receptor complexes revealed two major peaks of radioactivity corresponding to M r > 400,000 and M r ca. 110,000. This elution profile was markedly altered by pretreatment with GTP[γS] before solubilization; only the M r 110,000 peak was present in GTP[γS]-pretreated membranes. The results strongly suggest that 125 I-mel binding sites in lizard brain are melatonin receptors, with agonist-promoted guanine nucleotide-binding protein (G protein) coupling and that the apparent molecular size of receptors uncoupled from G proteins is about 110,000

Analyzing the molecular mechanism of lipoprotein localization in Brucella

CSIR Research Space (South Africa)

Goolab, S

2015-10-01

Full Text Available doi: 10.3389/fmicb.2015.01189 Edited by: Lee Mark Wetzler, Boston University School of Medicine, USA Reviewed by: Moriya Tsuji, Aaron Diamond AIDS Research Center, USA Thomas A. Ficht, Texas A&M University, USA *Correspondence: Shivani Goolab...-associated lipoproteins; POTRA, polypeptide-transport-associated; PRR, pattern-recognition receptors; RGD, Arg- Gly- Asp; Sec, system, general secretory; Tat system, twin-arginine translocation; T1SS, type I secretion system; T2SS, type II secretion system; T3SS, type III...

Low-density lipoprotein receptor genetic polymorphism in chronic hepatitis C virus Egyptian patients affects treatment response.

Science.gov (United States)

Naga, Mazen; Amin, Mona; Algendy, Dina; Elbadry, Ahmed; Fawzi, May; Foda, Ayman; Esmat, Serag; Sabry, Dina; Rashed, Laila; Gabal, Samia; Kamal, Manal

2015-10-21

To correlate a genetic polymorphism of the low-density lipoprotein (LDL) receptor with antiviral responses in Egyptian chronic hepatitis C virus (HCV) patients. Our study included 657 HCV-infected patients with genotype 4 who received interferon-based combination therapy. Patients were divided into two groups based on their response to therapy: 356 were responders, and 301 were non-responders. Patients were compared to 160 healthy controls. All patients and controls underwent a thorough physical examination, measurement of body mass index (BMI) and the following laboratory tests: serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, albumin, total bilirubin, direct bilirubin, prothrombin time, prothrombin concentration, INR, complete blood count, serum creatinine, fasting blood sugar, HCV antibody, and hepatitis B surface antigen. All HCV patients were further subjected to the following laboratory tests: HCV-RNA using quantitative polymerase chain reaction (PCR), antinuclear antibodies, thyroid-stimulating hormone, an LDL receptor (LDLR) genotype study of LDLR exon8c.1171G>A and exon10c.1413G>A using real-time PCR-based assays, abdominal ultrasonography, ultrasonographic-guided liver biopsy, and histopathological examination of liver biopsies. Correlations of LDL receptor polymorphisms with HAI, METAVIR score, presence of steatosis, and BMI were performed in all cases. There were no statistically significant differences in response rates between the different types of interferon used or LDLR exon10c.1413G>A. However, there was a significant difference in the frequency of the LDL receptor exon8c.1171G>A genotype between cases (AA: 25.9%, GA: 22.2%, GG: 51.9%) and controls (AA: 3.8%, GA: 53.1% and GG: 43.1%) (P A polymorphism between responders (AA: 3.6%, GA: 15.2%, GG: 81.2%) and non-responders (AA: 52.2%, GA: 30.6%, GG: 17.2%) (P A predominated in cases and controls over the A allele, and a statistically significant association with

Evolution of a G protein-coupled receptor response by mutations in regulatory network interactions

DEFF Research Database (Denmark)

Di Roberto, Raphaël B; Chang, Belinda; Trusina, Ala

2016-01-01

All cellular functions depend on the concerted action of multiple proteins organized in complex networks. To understand how selection acts on protein networks, we used the yeast mating receptor Ste2, a pheromone-activated G protein-coupled receptor, as a model system. In Saccharomyces cerevisiae......, Ste2 is a hub in a network of interactions controlling both signal transduction and signal suppression. Through laboratory evolution, we obtained 21 mutant receptors sensitive to the pheromone of a related yeast species and investigated the molecular mechanisms behind this newfound sensitivity. While...... demonstrate that a new receptor-ligand pair can evolve through network-altering mutations independently of receptor-ligand binding, and suggest a potential role for such mutations in disease....

Utility of soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) in the postmortem diagnosis of ischemic heart disease.

Science.gov (United States)

Takasu, Shojiro; Matsumoto, Sari; Kanto, Yuko; Iwadate, Kimiharu

2018-04-01

Ischemic heart disease (IHD) is a major cause of death in developed countries. Postmortem IHD diagnosis using biochemical markers is difficult because of the postmortem changes. In the present study, we investigated the utility of soluble lectin-like low-density lipoprotein receptor-1 (sLOX-1) in body fluids obtained from forensic autopsy cases. We measured pericardial fluid, urine, and serum sLOX-1 levels; these samples were obtained from medicolegal autopsy cases (n = 149, postmortem interval fluid and urine of patients with acute IHD had higher sLOX-1 levels (p fluid and urine samples obtained postmortem are useful markers of acute IHD. Copyright © 2018 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Combined analysis of six lipoprotein lipase genetic variants on triglycerides, high-density lipoprotein, and ischemic heart disease

DEFF Research Database (Denmark)

Wittrup, Hans H; Andersen, Rolf V; Tybjaerg-Hansen, Anne

2006-01-01

Genetic variants in lipoprotein lipase may affect triglycerides, high-density lipoprotein (HDL), and risk of ischemic heart disease (IHD).......Genetic variants in lipoprotein lipase may affect triglycerides, high-density lipoprotein (HDL), and risk of ischemic heart disease (IHD)....

p75 Neurotrophin Receptor Signaling Activates Sterol Regulatory Element-binding Protein-2 in Hepatocyte Cells via p38 Mitogen-activated Protein Kinase and Caspase-3.

Science.gov (United States)

Pham, Dan Duc; Do, Hai Thi; Bruelle, Céline; Kukkonen, Jyrki P; Eriksson, Ove; Mogollón, Isabel; Korhonen, Laura T; Arumäe, Urmas; Lindholm, Dan

2016-05-13

Nerve growth factor (NGF) influences the survival and differentiation of a specific population of neurons during development, but its role in non-neuronal cells has been less studied. We observed here that NGF and its pro-form, pro-NGF, are elevated in fatty livers from leptin-deficient mice compared with controls, concomitant with an increase in low density lipoprotein receptors (LDLRs). Stimulation of mouse primary hepatocytes with NGF or pro-NGF increased LDLR expression through the p75 neurotrophin receptor (p75NTR). Studies using Huh7 human hepatocyte cells showed that the neurotrophins activate the sterol regulatory element-binding protein-2 (SREBP2) that regulates genes involved in lipid metabolism. The mechanisms for this were related to stimulation of p38 mitogen-activated protein kinase (p38 MAPK) and activation of caspase-3 and SREBP2 cleavage following NGF and pro-NGF stimulations. Cell fractionation experiments showed that caspase-3 activity was increased particularly in the membrane fraction that harbors SREBP2 and caspase-2. Experiments showed further that caspase-2 interacts with pro-caspase-3 and that p38 MAPK reduced this interaction and caused caspase-3 activation. Because of the increased caspase-3 activity, the cells did not undergo cell death following p75NTR stimulation, possibly due to concomitant activation of nuclear factor-κB (NF-κB) pathway by the neurotrophins. These results identify a novel signaling pathway triggered by ligand-activated p75NTR that via p38 MAPK and caspase-3 mediate the activation of SREBP2. This pathway may regulate LDLRs and lipid uptake particularly after injury or during tissue inflammation accompanied by an increased production of growth factors, including NGF and pro-NGF. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor

DEFF Research Database (Denmark)

Cherezov, Vadim; Rosenbaum, Daniel M; Hanson, Michael A

2007-01-01

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to t...

Structural–Functional Features of the Thyrotropin Receptor: A Class A G-Protein-Coupled Receptor at Work

Directory of Open Access Journals (Sweden)

Gerd Krause

2017-04-01

Full Text Available The thyroid-stimulating hormone receptor (TSHR is a member of the glycoprotein hormone receptors, a sub-group of class A G-protein-coupled receptors (GPCRs. TSHR and its endogenous ligand thyrotropin (TSH are of essential importance for growth and function of the thyroid gland and proper function of the TSH/TSHR system is pivotal for production and release of thyroid hormones. This receptor is also important with respect to pathophysiology, such as autoimmune (including ophthalmopathy or non-autoimmune thyroid dysfunctions and cancer development. Pharmacological interventions directly targeting the TSHR should provide benefits to disease treatment compared to currently available therapies of dysfunctions associated with the TSHR or the thyroid gland. Upon TSHR activation, the molecular events conveying conformational changes from the extra- to the intracellular side of the cell across the membrane comprise reception, conversion, and amplification of the signal. These steps are highly dependent on structural features of this receptor and its intermolecular interaction partners, e.g., TSH, antibodies, small molecules, G-proteins, or arrestin. For better understanding of signal transduction, pathogenic mechanisms such as autoantibody action and mutational modifications or for developing new pharmacological strategies, it is essential to combine available structural data with functional information to generate homology models of the entire receptor. Although so far these insights are fragmental, in the past few decades essential contributions have been made to investigate in-depth the involved determinants, such as by structure determination via X-ray crystallography. This review summarizes available knowledge (as of December 2016 concerning the TSHR protein structure, associated functional aspects, and based on these insights we suggest several receptor complex models. Moreover, distinct TSHR properties will be highlighted in comparison to other

Structural-Functional Features of the Thyrotropin Receptor: A Class A G-Protein-Coupled Receptor at Work.