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1.
Biochemistry ; 49(45): 9866-73, 2010 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-20879789

RESUMO

Serum opacity factor (SOF), a virulence determinant of Streptococcus pyogenes, converts plasma high-density lipoproteins (HDL) to three distinct species: lipid-free apolipoprotein (apo) A-I, neo HDL, a small discoidal HDL-like particle, and a large cholesteryl ester-rich microemulsion (CERM) that contains the cholesterol esters (CE) of up to ∼400000 HDL particles and apo E as its major protein. Similar SOF reaction products are obtained with HDL, total plasma lipoproteins, and whole plasma. We hypothesized that hepatic uptake of CERM-CE via multiple apo E-dependent receptors would be faster than that of HDL-CE. We tested our hypothesis using human hepatoma cells and lipoprotein receptor-specific Chinese hamster ovary (CHO) cells. The uptake of [(3)H]CE by HepG2 and Huh7 cells from HDL after SOF treatment, which transfers >90% of HDL-CE to CERM, was 2.4 and 4.5 times faster, respectively, than from control HDL. CERM-[(3)H]CE uptake was inhibited by LDL and HDL, suggestive of uptake by both the LDL receptor (LDL-R) and scavenger receptor class B type I (SR-BI). Studies in CHO cells specifically expressing LDL-R and SR-BI confirmed CERM-[(3)H]CE uptake by both receptors. RAP and heparin inhibit CERM-[(3)H]CE but not HDL-[(3)H]CE uptake, thereby implicating LRP-1 and cell surface proteoglycans in this process. These data demonstrate that SOF treatment of HDL increases the rate of CE uptake via multiple hepatic apo E receptors. In so doing, SOF might increase the level of hepatic disposal of plasma cholesterol in a way that is therapeutically useful.


Assuntos
HDL-Colesterol/metabolismo , Hepatócitos/metabolismo , Peptídeo Hidrolases/farmacologia , Streptococcus pyogenes/metabolismo , Animais , Compostos de Boro/metabolismo , Células CHO/metabolismo , Técnicas de Cultura de Células , Ésteres do Colesterol/metabolismo , HDL-Colesterol/efeitos dos fármacos , Cricetinae , Cricetulus , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Cinética , Fígado/metabolismo , Microscopia Confocal , Streptococcus pyogenes/patogenicidade , Virulência
2.
Biochim Biophys Acta ; 1791(12): 1125-32, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19635584

RESUMO

Whereas hepatocytes secrete the major human plasma high density lipoproteins (HDL)-protein, apo A-I, as lipid-free and lipidated species, the biogenic itineraries of apo A-II and apo E are unknown. Human plasma and HepG2 cell-derived apo A-II and apo E occur as monomers, homodimers and heterodimers. Dimerization of apo A-II, which is more lipophilic than apo A-I, is catalyzed by lipid surfaces. Thus, we hypothesized that lipidation of intracellular and secreted apo A-II exceeds that of apo A-I, and once lipidated, apo A-II dimerizes. Fractionation of HepG2 cell lysate and media by size exclusion chromatography showed that intracellular apo A-II and apo E are fully lipidated and occur on nascent HDL and VLDL respectively, while only 45% of intracellular apo A-I is lipidated. Secreted apo A-II and apo E occur on small HDL and on LDL and large HDL respectively. HDL particles containing both apo A-II and apo A-I form only after secretion from both HepG2 and Huh7 hepatoma cells. Apo A-II dimerizes intracellularly while intracellular apo E is monomeric but after secretion associates with HDL and subsequently dimerizes. Thus, HDL apolipoproteins A-I, A-II and E have distinct intracellular and post-secretory pathways of hepatic lipidation and dimerization in the process of HDL formation. These early forms of HDL are expected to follow different apolipoprotein-specific pathways through plasma remodeling and reverse cholesterol transport.


Assuntos
Apolipoproteína A-II/metabolismo , Apolipoproteína A-I/metabolismo , Apolipoproteínas E/metabolismo , Carcinoma Hepatocelular/metabolismo , Espaço Intracelular/metabolismo , Lipoproteínas HDL/metabolismo , Neoplasias Hepáticas/metabolismo , Cromatografia em Gel , Células Hep G2 , Humanos , Modelos Biológicos , Ligação Proteica , Multimerização Proteica , Sefarose/análogos & derivados , Fatores de Tempo , Ultracentrifugação
3.
Lipids ; 45(12): 1117-26, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20972840

RESUMO

Serum opacity factor (SOF) is a streptococcal protein that disrupts the structure of human high density lipoproteins (HDL) releasing lipid-free apo A-I while forming a large cholesteryl ester-rich particle and a small neo HDL. Given its low cholesterol and high phospholipid contents, we tested the hypotheses that neo HDL is a better substrate for cholesterol esterification via lecithin:cholesterol acyltransferase (LCAT), better than HDL as an acceptor of THP-1 macrophage cholesterol efflux, and improves reduction of oxidized LDL-induced production of inflammatory markers. We observed that both cholesterol efflux and esterification were improved by recombinant (r)SOF treatment of whole plasma and that the underlying cause of the improved cholesterol esterification in plasma and macrophage cholesterol efflux to rSOF-treated plasma was due to the rSOF-mediated conversion of HDL to neo HDL. Moreover, the reduction of secretion of TNF-α and IL-6 by THP-1 cells by neo HDL was twice that of HDL. Studies in BHK cells overexpressing cholesterol transporters showed that efflux to neo HDL occurred primarily via ABCA1 not ABCG1. Thus, rSOF improves two steps in reverse cholesterol transport with a concomitant reduction in the release of macrophage markers of inflammation. We conclude that rSOF catalyzes a novel reaction that might be developed as a new therapy that prevents or reverses atherosclerosis via improved reverse cholesterol transport.


Assuntos
Anti-Inflamatórios/metabolismo , HDL-Colesterol/metabolismo , Colesterol/metabolismo , Peptídeo Hidrolases/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Células Cultivadas , Cricetinae , Esterificação , Humanos , Inflamação/metabolismo , Macrófagos/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo
4.
Biochemistry ; 46(25): 7449-59, 2007 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-17530866

RESUMO

The distribution of apolipoprotein (apo) A-I between human high-density lipoproteins (HDL) and water is an important component of reverse cholesterol transport and the atheroprotective effects of HDL. Chaotropic perturbation (CP) with guanidinium chloride (Gdm-Cl) reveals HDL instability by inducing the unfolding and transfer of apo A-I but not apo A-II into the aqueous phase while forming larger apo A-I deficient HDL-like particles and small amounts of cholesteryl ester-rich microemulsions (CERMs). Our kinetic and hydrodynamic studies of the CP of HDL species separated according to size and density show that (1) CP mediated an increase in HDL size, which involves quasi-fusion of surface and core lipids, and release of lipid-free apo A-I (these processes correlate linearly), (2) >94% of the HDL lipids remain with an apo A-I deficient particle, (3) apo A-II remains associated with a very stable HDL-like particle even at high levels of Gdm-Cl, and (4) apo A-I unfolding and transfer from HDL to water vary among HDL subfractions with the larger and more buoyant species exhibiting greater stability. Our data indicate that apo A-I's on small HDL (HDL-S) are highly dynamic and, relative to apo A-I on the larger more mature HDL, partition more readily into the aqueous phase, where they initiate the formation of new HDL species. Our data suggest that the greater instability of HDL-S generates free apo A-I and an apo A-I deficient HDL-S that readily fuses with the more stable HDL-L. Thus, the presence of HDL-L drives the CP remodeling of HDL to an equilibrium with even larger HDL-L and more lipid-free apo A-I than with either HDL-L or HDL-S alone. Moreover, according to dilution studies of HDL in 3 M Gdm-Cl, CP of HDL fits a model of apo A-I partitioning between HDL phospholipids and water that is controlled by the principal of opposing forces. These findings suggest that the size and relative amount of HDL lipid determine the HDL stability and the fraction of apo A-I that partitions into the aqueous phase where it is destined for interaction with ABCA1 transporters, thereby initiating reverse cholesterol transport or, alternatively, renal clearance.


Assuntos
Apolipoproteína A-I/sangue , Lipoproteínas HDL/sangue , Apolipoproteína A-I/metabolismo , Apolipoproteína A-II/sangue , Cromatografia em Gel , Dicroísmo Circular , Relação Dose-Resposta a Droga , Guanidina/farmacologia , Humanos , Cinética , Lipoproteínas HDL/isolamento & purificação , Modelos Biológicos , Peso Molecular , Água/química
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