Background: Familial hypercholesterolemia type IIA (FH) is due to mutations in the low-densitylipoproteinreceptor (LDLR) resulting in elevated levels of low-densitylipoprotein cholesterol (LDL-c) in plasma and in premature cardiovascular diseases. As hepatocytes are the only cells capable of metabolizing cholesterol, they are therefore the target cells for cell/ gene therapy approaches in the treatment of lipid metabolism disorders. Furthermore, the LDLR has been reported to be involved in hepatitis C virus (HCV) entry into hepatocytes; however, its role in the virus infection cycle is still disputed. Methods: We generated induced pluripotent stem cells (iPSCs) from a homozygous LDLR-null FH-patient (FH-iPSCs). We constructed a correction cassette bearing LDLR cDNA under the control of human hepatic apolipoprotein A2 promoter that targets the adeno-associated virus integration site AAVS1. We differentiated both FH-iPSCs and corrected FH-iPSCs (corr-FH-iPSCs) into hepatocytes to study statin-mediated regulation of genes involved in cholesterol metabolism. Upon HCV particle inoculation, viral replication and production were quantified in these cells.
described ( 12 –15 ). Several receptor candidates have been identified, including CD81 ( 16 ), scavenger receptor-BI ( 17 ), DC-SIGN and/or L-SIGN ( 18 ), asialoglycoprotein receptor ( 19 ) and low-densitylipoproteinreceptor (LDLR) ( 20 –22 ).
LDLR is a membrane glycoprotein that controls the primary pathway through which cholesterol enters the cells. The N-terminal ligand binding domain spans over residues 1 292 arranged in 7 repeats. Each repeat contains 6 S-S linked cysteines ( – 23 24 , ). The physiological ligand LDL contains a single copy of apolipoprotein B-100 and carries 65 70 of plasma cholesterol. The receptor also binds – % apolipoprotein E-containing lipoproteins such as very low-density lipoproteins ( -VLDL). LDLR has been suspected to play a role in HCV β infection on the basis of the well documented interaction between HCV and lipoproteins ( 25 –27 ). Ultracentrifugation of infected plasma on density gradients revealed two compartments in which HCV is abundant. The first one (1.25 g/ml) contains poorly infectious particles linked to immunoglobulins. The second (1.06 g/ml) contains infectious lipoviroparticles that are rich in triglycerides, and apolipoprotein B and E. Further observations supporting a role for LDLR in HCV infection came from analysis of the effects of lovastatin ( 22 ), anti-LDL antibodies ( , 8 20 21 28 , , ), LDLR genetic deficiency ( 21 25 28 , , ) and LDLR ectopic expression ( 21 ).
Tissue inhibitor of metalloproteinases-1 (TIMP-1) regulates the extracellular matrix turnover by inhibiting the proteolytic activity of matrix metalloproteinases (MMPs). TIMP-1 also displays MMP-independent activities that influence the behavior of various cell types including neuronal plasticity, but the underlying molecular mechanisms remain mostly unknown. The trans-membrane receptorlow-densitylipoproteinreceptor-related protein-1 (LRP-1) consists of a large extracellular chain with distinct ligand-binding domains that interact with numerous ligands including TIMP-2 and TIMP-3 and a short transmembrane chain with intracellular motifs that allow endocytosis and confer signaling properties to LRP-1. We addressed TIMP-1 interaction with recombinant ligand-binding domains of LRP-1 expressed by CHO cells for endocytosis study, or linked onto sensor chips for surface plasmon resonance analysis. Primary cortical neurons bound and internalized endogenous TIMP-1 through a mechanism mediated by LRP-1. This resulted in inhibition of neurite outgrowth and increased growth cone volume. Using a mutated inactive TIMP-1 variant we showed that TIMP-1 effect on neurone morphology was independent of its MMP inhibitory activity. We conclude that TIMP-1 is a new ligand of LRP-1 and we highlight a new example of its MMP-independent, cytokine-like functions.
1. Rashid S, Tavori H, Brown PE, Linton MF, He J, Giunzioni I, Fazio S. Proprotein convertase subtilisin kexin type 9 promotes intesti- nal overproduction of triglyceride-rich apolipoprotein B lipoproteins through both low-densitylipoproteinreceptor-dependent and -inde- pendent mechanisms. Circulation. 2014;130:431–441. doi: 10.1161/ CIRCULATIONAHA.113.006720.
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important factor in plasma cholesterol regulation through modu- lation of lowdensitylipoproteinreceptor (LDLR) levels. Naturally occurring mutations can lead to hyper- or hypocholesterolemia in human. Recently, we reported that PCSK9 was also able to modu- late CD81 in Huh7 cells. In the present study, several gain-of-func- tion and loss-of-function mutants as well as engineered mutants of PCSK9 were compared for their ability to modulate the cell surface expression of LDLR and CD81. Although PCSK9 gain-of-function D374Y enhanced the degradation both receptors, D374H and D129N seemed to only reduce LDLR levels. In contrast, mutations in the C-terminal hinge-cysteine-histidine-rich domain segment primarily affected the PCSK9-induced CD81 degradation. Fur- thermore, when C-terminally fused to an ACE2 transmembrane anchor, the secretory N-terminal catalytic or hinge-cysteine-histi- dine-rich domain domains of PCSK9 were able to reduce CD81 and LDLR levels. These data confirm that PCSK9 reduces CD81 levels via an intracellular pathway as reported for LDLR. Using immuno- cytochemistry, a proximity ligation assay, and co-immunoprecipi- tation, we found that the cell surface level of PCSK9 was enhanced upon overexpression of CD81 and that both PCSK9 and LDLR interact with this tetraspanin protein. Interestingly, using CHO-A7 cells lacking LDLR expression, we revealed that LDLR was not required for the degradation of CD81 by PCSK9, but its presence strengthened the PCSK9 effect.
ABSTRACT Low-densitylipoproteinreceptor-related protein-1 (LRP-1) is a plasma membrane scavenger and signaling receptor, composed of a large ligand-binding subunit (515-kDa ␣-chain) linked to a shorter trans- membrane subunit (85-kDa ␤-chain). LRP-1 cell-surface level and function are controlled by proteolytic shed- ding of its ectodomain. Here, we identified ectodomain sheddases in human HT1080 cells and demonstrated regulation of the cleavage by cholesterol by comparing the classical fibroblastoid type with a spontaneous epithelioid variant, enriched ⬃2-fold in cholesterol. Two membrane-associated metalloproteinases were in- volved in LRP-1 shedding: a disintegrin and metallopro- teinase-12 (ADAM-12) and membrane-type 1 matrix metalloproteinase (MT1-MMP). Although both variants expressed similar levels of LRP-1, ADAM-12, MT1- MMP, and specific tissue inhibitor of metalloprotei- nases-2 (TIMP-2), LRP-1 shedding from epithelioid cells was ⬃4-fold lower than from fibroblastoid cells. Release of the ectodomain was triggered by cholesterol depletion in epithelioid cells and impaired by choles- terol overload in fibroblastoid cells. Modulation of LRP-1 shedding on clearance was reflected by accumu- lation of gelatinases (MMP-2 and MMP-9) in the me- dium. We conclude that cholesterol exerts an important control on LRP-1 levels and function at the plasma membrane by modulating shedding of its ectodomain, and therefore represents a novel regulator of extracel- lular proteolytic activities.—Selvais, C., D’Auria, L., Tyteca, D., Perrot, G, Lemoine, P., Troeberg, L., Dedieu, S., Noe¨l, A., Nagase, H., Henriet, P., Courtoy, P. J., Marbaix, E., Emonard, H. Cell cholesterol mod- ulates metalloproteinase-dependent shedding of low- densitylipoproteinreceptor-related protein-1 (LRP-1) and clearance function. FASEB J. 25, 000 – 000 (2011). www.fasebj.org
Familial hypercholesterolemia (FH) is an inherited metabolic disease associated with high levels of circulating low-densitylipoprotein cholesterol (LDL-C) and premature cardiovascular disease (CVD)(1).
Heterozygous FH (HeFH) is a common genetic disorder resulting from an autosomal dominant or co- dominant inheritance pattern with an estimated prevalence of 1 in 250 subjects in most countries. However, the prevalence is much higher in regions or localized populations with founder effects (2, 3). Homozygous FH (HoFH) is characterized by a much lower prevalence, around 1 case in 160000 to 300000 subjects (4). In rare instances, HoFH is transmitted as a recessive trait (5). Here we focus on the clinical phenotypes of FH, the genetic variants at the origin of the phenotype as well as, on the most recent experimental approaches used to investigate molecular defects affecting the LDL receptor pathway in FH. The perturbations of lipoprotein metabolism beyond LDL as well as the mode of action and efficacy of the currently approved hypocholesterolemic agents used to treat FH patients are also reviewed.
capturing machinery like other classical maturation-inducing signals. FITC-dextran has been
used to measure mannose receptor-mediated endocytosis, fluorescent beads for
macropinocytosis and LY as nonspecific fluid phase marker. Figure 3A shows that although
oxLDL does not induce a fully mature DC phenotype when added early on monocytes, it still
Modelling subsoil bulk density increase of Australian low activity clay soils
Bruand A. 1 , Gilkes R.J. 2
Institut des Sciences de la Terre d'Orléans (ISTO) UMR6113 CNRS/Université d'Orléans 1A, rue de la Férollerie 45071 Orléans, France.
Figure 1: Infrared spectra of a LLDPE film (80 microns)
at different times of photooxidation in the MHE device (90 W.m -2 and 60°C)
The changes in the spectrum indicate that ketones (1718 cm -1 ) are formed during the initial steps, and carboxylic acids (1713 cm -1 ), esters (1735 cm -1 ) and lactones (1780 cm -1 ) are formed during secondary processes. One can also notice the formation of isolated double bonds (1640 cm -1 ). In the range of hydroxyl frequencies, not shown here, a broad band with a maximum at 3420 cm -1 and a sharp absorption band at 3550 cm -1 appeared in the spectrum. These bands were attributed to the formation of monomeric hydroperoxides (3550 cm -1 ) and hydrogen-bonded alcohols and hydroperoxides (3420 cm -1 ). The intensities of both bands remained very low, confirming that the stationary concentrations of hydroperoxides were rather small under the conditions of photooxidation . At present, the mechanism of photooxidation accounting for the main routes of degradation of LLDPE is fairly well understood. LLDPE is a non-absorbing polymer in the sense that it does not contain chromophoric functions in its chemical structure, which can absorb in the range of solar light. The basic law of photochemistry should be recalled: light absorption is necessary to provoke photochemical reactions. In the case of non-absorbing polymers, the absorption of light is attributed to unidentified chromophores, which can vary from one LLDPE to another. Extensive work has been devoted to the nature of chromophoric species [33, 34, 35] and it is well known that oxidized species formed during processing, such as hydroperoxides or ketones, are potential
Schmidt number increases. The effect of a wake on the charge
density distribution is shown in Fig. 6.2. Figure 6.2 shows that
for the flow with wake (Re = 40), the same phenomenon as in the
case of cylinder in cross-flow (Fig. 5.1) occurs in the rear stagnation région. In both cases this concentration of charge
G-protein-coupled receptors (GPCRs) are dynamically regulated by various mechanisms, which tune their response to external stimuli. Modulation of their plasma membrane density, via trafficking between sub-cellular compartments, constitutes an important process in this context. Substantial information has been accumulated on cellular pathways, which remove GPCRs from the cell surface for subsequent degradation or recycling. In comparison, much less is known about mechanisms controlling trafficking of neo-synthesised GPCRs from intracellular compartments to the cell surface. Although GPCR export to the plasma membrane is commonly considered to mostly implicate the default, unregulated secretory pathway, an increasing number of observations indicate that trafficking to the plasma membrane from the endoplasmic reticulum may be tightly regulated and involve specific protein partners. Moreover, a new paradigm is emerging in some cellular contexts, in which stocks of functional receptors retained within intracellular compartments may be rapidly mobilized to the plasma membrane to maintain sustained physiological responsiveness.
The chemical equilibrium constants of the α-particles measured in the three different experimental data sets are displayed in the bottom panel of Fig. 2. The constants are seen to increasingly deviate from the ideal gas calcula- tion, as the density increases, reflecting the increased in- medium suppression. The results of the different systems almost perfectly overlap, confirming the expectation that chemical constants are isospin-independent. The same is true for the chemical constants of all the other clusters (not shown).
The grain size statistics were calculated using the Folk-Word method with the Gradistat software [Blott and Pye, 2001]. The sediments were classi ﬁed according to Friedman and Sanders [1978. Sediment porosity and density were calculated. The concentrations of both chlorophyll a (Chl a) and pheopigments (Pheo) were summed to a total and referred to as the chloroplast pigment equivalent (CPE). Chlorophyll a was expressed both in μg g 1 and percent of chlorophyll in the CPE. The chlorophyll a to POC (%) ratio was calculated. The differences in the sediment characteristics (Chl a, Pheo, Chl a, CPE concentrations, Chl a/POC, POC, carbon to nitrogen ratio (POC/TN), δ 13 C, δ 15 N, mean grain size, and ﬁne sand content) between the bottom types (Bottom-vegetated bottom versus bare sands) and among locations (Loc) were tested using a two-way univariate PERMANOVA model (with two ﬁxed factors: Bottom and Loc) based on a simi- larity matrix created with the Euclidean distances among the samples (untransformed data). When signi ﬁ- cant effects were found with the main test, post hoc pairwise tests were conducted. Carbon enhancement at vegetated bottom for three locations was expressed as the ratio of POC recorded at vegetated to unve- getated bottom. Spearman correlation tests were performed to check whether the organic matter and sediment descriptors (Chl a, Pheo, Chl a, CPE concentrations, Chl a/POC, POC, carbon to nitrogen ratio (POC/TN), δ 13 C, δ 15 N, mean grain size, and ﬁne sand content) were correlated to the characteristics of the seagrass meadows (seagrass shoot density, aboveground seagrass biomass, total seagrass biomass, and total macrophyte biomass).
Subject headings: planetary systems: individual: WASP-17b – stars: individual: WASP-17
The first measurement of the radius and density of an extrasolar planet was made when HD 209458b was seen to transit its parent star (Charbonneau et al. 2000, Henry et al. 2000). The large radius (1.32 R Jup ) of HD 209458b, confirmed by later observations (e.g., Knut- son et al. 2007), could not be explained by standard models of planet evolution (Guillot & Showman 2002). Since the discovery of HD 209458b, other bloated plan- ets have been found, including TrES-4 (Mandushev et al. 2007), WASP-12b (Hebb et al. 2008), WASP-4b (Wil- son et al. 2008; Gillon et al. 2009a, Winn et al. 2009a, Southworth et al. 2009), WASP-6b (Gillon et al. 2009b), XO-3b (Johns-Krull et al. 2008; Winn et al. 2008) and HAT-P-1b (Bakos et al. 2007; Winn et al. 2007; Johnson et al. 2008). Of those, TrES-4 is the most bloated, with a density 15 per cent that of Jupiter, and a radius larger by a factor 1.78 (Sozzetti et al. 2009).