Low-density lipoprotein (LDL) receptor (LDLR) and LDLR-related protein (LRP) are members of the LDLR family of endocytic receptors. LRP recognizes a wide spectrum of structurally and functionally unrelated ligands, including coagulation factor VIII (FVIII). In contrast, the ligand specificity of LDLR is restricted to apolipoproteins E and B-100. Ligand binding to the LDLR family is inhibited by receptor-associated protein (RAP). We have previously reported that, apart from LRP, other RAP-sensitive mechanisms contribute to the regulation of FVIII in vivo. In the present study, we showed that the extracellular ligand-binding domain of LDLR interacts with FVIII in vitro and that binding was inhibited by RAP. The physiologic relevance of the FVIII-LDLR interaction was addressed using mouse models of LDLR or hepatic LRP deficiency. In the absence of hepatic LRP, LDLR played a dominant role in the regulation and clearance of FVIII in vivo. IntroductionLow-density lipoprotein (LDL) receptor (LDLR) plays a pivotal role in the metabolism of large cholesterol-rich lipoproteins from the circulation in a process known as receptor-mediated endocytosis. 1 This process is dependent on the high-affinity interaction between LDLR and its protein ligands, apolipoprotein E (apoE) and apoB-100, both of which are present at the surfaces of lipoprotein particles in plasma. 1,2 The importance of LDLR is illustrated by the fact that genetic defects within the LDLR gene are the underlying cause of familial hypercholesterolemia. 3 These patients display elevated plasma LDL cholesterol concentrations and increased risk for atherosclerosis and coronary artery disease.LDLR belongs to the LDLR family of cell-surface endocytic receptors that also includes apoE-receptor-2, very low-density lipoprotein (VLDL) receptor, megalin, and LDLR-related protein (LRP). 2,4 The extracellular ligand-binding domains of these receptors are composed of 1 to 4 homolog clusters of complement-type repeats. The chaperone receptor-associated protein (RAP) blocks all ligand binding to these clusters. 5 Disruption of the LRP gene in LDLR knockout mice accumulates apoE-rich VLDL/LDL-sized lipoproteins in plasma, indicating that LDLR works in concert with LRP in lipoprotein metabolism in vivo. 6 Unlike LDLR, however, LRP also recognizes a broad spectrum of lipoprotein-unrelated ligands. 4 In the past few years, LRP has been established to interact with coagulation factor VIII (FVIII) and to mediate its cellular uptake into the lysosomal degradation pathway. 7,8 Within the blood coagulation cascade, the cofactor activity of FVIII is indispensable for appropriate hemostasis. 9 Deficiency or dysfunction of FVIII is associated with the bleeding disorder hemophilia A, whereas elevated plasma FVIII markedly increases the risk for arterial and venous thrombosis. [9][10][11] The cofactor circulates in plasma in complex with its carrier protein von Willebrand factor (VWF). 12 Using conditional hepatic LRP-deficient mice, we recently demonstrated that LRP contributes to the rem...

A common dose-limiting side effect of treatment with the retinoid X receptor agonist bexarotene is dyslipidemia. We evaluated the effects of bexarotene on plasma lipid metabolism in patients with metastatic differentiated thyroid carcinoma and investigated the underlying mechanism(s) in apolipoprotein (APO) E*3-Leiden mice without (E3L) and with human cholesteryl ester transfer protein (CETP; E3L.CETP). To this end, 10 patients with metastatic differentiated thyroid carcinoma were treated with bexarotene (300 mg/d) for 6 wk. Bexarotene increased plasma triglyceride (TG; +150%), primarily associated with very low-density lipoprotein (VLDL), and raised plasma total cholesterol (+50%). However, whereas bexarotene increased VLDL-cholesterol (C) and low-density lipoprotein (LDL)-C (+63%), it decreased high-density lipoprotein (HDL)-C (-30%) and tended to decrease apoAI (-18%) concomitant with an increase in endogenous CETP activity (+44%). To evaluate the cause of the bexarotene-induced hypertriglyceridemia and the role of CETP in the bexarotene-induced shift in cholesterol distribution, E3L and E3L.CETP mice were treated with bexarotene through dietary supplementation [0.03% (wt/wt)]. Bexarotene increased VLDL-associated TG in both E3L (+47%) and E3L.CETP (+29%) mice by increasing VLDL-TG production (+68%). Bexarotene did not affect the total cholesterol levels or distribution in E3L mice but increased VLDL-C (+11%) and decreased HDL-C (-56%) as well as apoAI (-31%) in E3L.CETP mice, concomitant with increased endogenous CETP activity (+41%). This increased CETP activity by bexarotene-treatment is likely due to the increase in VLDL-TG, a CETP substrate that drives CETP activity. In conclusion, bexarotene causes combined dyslipidemia as reflected by increased TG, VLDL-C, and LDL-C and decreased HDL-C, which is the result of an increased VLDL-TG production that causes an increase of the endogenous CETP activity.

The electrogenic Na(+)-HCO(3)(-) cotransporter NBCe1 is important for the regulation of intracellular pH (pH(i)) and for epithelial HCO(3)(-) transport in many tissues, including kidney, pancreas, and brain. In the present study, we investigate glycosylation sites in NBCe1. Treatment of rat kidney membrane extracts with peptide N-glycosidase F (PNGase F) shifted the apparent molecular weight (MW) of NBCe1 from 130 to 116, the MW predicted from the deduced amino acid sequence. Treatment with endoglycosidase F(2) or H or O-glycosidase did not affect the MW of NBCe1. Lectin-binding studies, together with the enzyme data, suggest that the N-linked carbohydrates are of tri- or tetra-antennary type. To localize glycosylation sites, we individually mutated the seven consensus N-glycosylation sites by replacing asparagine (N) with glutamine (Q) and assessing mutant transporters in Xenopus laevis oocytes. Immunoblotting of oocyte membrane extracts treated with PNGase F indicates that NBCe1 is normally glycosylated at N597 and N617 (both on the third extracellular loop). However, N592 (on the same loop) is glycosylated when the other two sites are mutated. The triple mutant (N592Q/N597Q/N617Q) is completely unglycosylated but, based on microelectrode measurements of membrane potential and pH(i) in oocytes, preserves the Na(+) and HCO(3)(-) dependence and electrogenicity of wild-type NBCe1.

The variable susceptibility to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment observed in various types of leukemia cells is related to the difference in the expression levels of death receptors, DR4 and DR5, on the cell surfaces. Quantifying the DR4/DR5 expression status on leukemia cell surfaces is of vital importance to the development of diagnostic tools to guide death receptor-based leukemia treatment. Taking the full advantages of novel nanobiotechnology, we have developed a robust electrochemical cytosensing approach toward ultrasensitive detection of leukemia cells with detection limit as low as ~40 cells and quantitative evaluation of DR4/DR5 expression on leukemia cell surfaces. The optimization of electron transfer and cell capture processes at specifically tailored nanobiointerfaces and the incorporation of multiple functions into rationally designed nanoprobes provide unique opportunities of integrating high specificity and signal amplification on one electrochemical cytosensor. The high sensitivity and selectivity of this electrochemical cytosensing approach also allows us to evaluate the dynamic alteration of DR4/DR5 expression on the surfaces of living cells in response to drug treatments. Using the TRAIL-resistant HL-60 cells and TRAIL-sensitive Jurkat cells as model cells, we have further verified that the TRAIL susceptibility of various types of leukemia cells is directly correlated to the surface expression levels of DR4/DR5. This versatile electrochemical cytosensing platform is believed to be of great clinical value for the early diagnosis of human leukemia and the evaluation of therapeutic effects on leukemia patients after radiation therapy or drug treatment.

LPL activity plays an important role in preceding the VLDL remnant clearance via the three major apolipoprotein E (apoE)-recognizing receptors: the LDL receptor (LDLr), LDL receptor-related protein (LRP), and VLDL receptor (VLDLr). The aim of this study was to determine whether LPL activity is also important for VLDL remnant clearance irrespective of these receptors and to determine the mechanisms involved in the hepatic remnant uptake. Administration of an adenovirus expressing LPL (AdLPL) into lrp 2 ldlr 2/2 vldlr 2/2 mice reduced both VLDL-triglyceride (TG) and VLDL-total cholesterol (TC) levels. Conversely, inhibition of LPL by AdAPOC1 increased plasma VLDL-TG and VLDL-TC levels. Metabolic studies with radiolabeled VLDL-like emulsion particles showed that the clearance and hepatic association of their remnants positively correlated with LPL activity. This hepatic association was independent of the bridging function of LPL and HL, since heparin did not reduce the liver association. In vitro studies demonstrated that VLDL-like emulsion particles avidly bound to the cell surface of primary hepatocytes from lrp 2 ldlr 2/2 vldlr 2/2 mice, followed by slow internalization, and involved heparin-releaseable cell surface proteins as well as scavenger receptor class B type I (SR-BI). Collectively, we conclude that hepatic VLDL remnant uptake in the absence of the three classical apoE-recognizing receptors is regulated by LPL activity and involves heparan sulfate proteoglycans and SR-BI.-Hu, L., C. C. van LPL is the key enzyme responsible for the hydrolysis of triglycerides (TGs) in TG-rich lipoproteins such as chylomicrons and VLDL (1, 2). During lipolysis, the lipoproteins are reduced in size and enriched with apolipoprotein E (apoE). Subsequently, their core remnants are taken up mainly by the liver via apoE-recognizing receptors [i.e., the LDL receptor (LDLr) and the LDLr-related protein (LRP)] (2). Therefore, mice deficient for the LDLr and hepatic LRP show marked accumulation of TG-rich lipoprotein remnants (3). Although core remnants may be directly internalized via the LDLr, the binding and internalization via the LRP is thought to involve previous binding of core remnants to heparan sulfate proteoglycans (HSPGs) in the space of Disse via heparin binding proteins such as apoE (4, 5).The third major apoE-recognizing receptor, the VLDL receptor (VLDLr), is expressed abundantly in tissues active in fatty acid metabolism [i.e., heart, skeletal muscle,

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Chikungunya virus (CHIKV) has been detected sporadically since the 1950s and includes three distinct co-circulating genotypes. In late 2013, the Asian genotype of CHIKV was responsible for the Caribbean outbreak (CO) that rapidly became an epidemic throughout the Americas. There is a limited understanding of the molecular evolution of CHIKV in the Americas during this epidemic. We sequenced 185 complete CHIKV genomes collected mainly from Nicaragua in Central America and Florida in the United States during the 2014–2015 Caribbean/Americas epidemic. Our comprehensive phylogenetic analyses estimated the epidemic history of the Asian genotype and the recent Caribbean outbreak (CO) clade, revealed considerable genetic diversity within the CO clade, and described different epidemiological dynamics of CHIKV in the Americas. Specifically, we identified multiple introductions in both Nicaragua and Florida, with rapid local spread of viruses in Nicaragua but limited autochthonous transmission in Florida in the US. Our phylogenetic analysis also showed phylogeographic clustering of the CO clade. In addition, we identified the significant amino acid substitutions that were observed across the entire Asian genotype during its evolution and examined amino acid changes that were specific to the CO clade. Deep sequencing analysis identified specific minor variants present in clinical specimens below-consensus levels. Finally, we investigated the association between viral phylogeny and geographic/clinical metadata in Nicaragua. To date, this study represents the largest single collection of CHIKV complete genomes during the Caribbean/Americas epidemic and significantly expands our understanding of the emergence and evolution of CHIKV CO clade in the Americas.

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