RESUMO
Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18 interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor complex. Twelve of these 28 interactions are supported by prior reports, and we have directly validated novel interactions with SEC22A, TMCO4, and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites, interactors included groups of microtubule/membrane-remodeling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP is a Δ8-Δ7 sterol isomerase, and ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We found that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Furthermore, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated or in which ORP2 expression is disrupted. Our data demonstrate that guanine nucleotide exchange factor-dependent Rab interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.
Assuntos
Biotinilação , Esteróis , Proteínas rab de Ligação ao GTP , Humanos , Colesterol/biossíntese , Colesterol/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab3 de Ligação ao GTP/metabolismo , Esteróis/biossíntese , Esteróis/metabolismo , Células Cultivadas , Técnicas de Silenciamento de Genes , Transporte Proteico/genéticaRESUMO
Apolipoprotein (apo) E functions in lipoprotein metabolism as a low density lipoprotein receptor ligand. ApoE is comprised of two structural domains, a 22 kDa N-terminal (NT) domain that adopts a helix bundle conformation and a 10 kDa C-terminal domain with strong lipid binding affinity. The NT domain is capable of transforming aqueous phospholipid dispersions into discoidal reconstituted high density lipoprotein (rHDL) particles. Given the utility of apoE-NT as a structural component of rHDL, expression studies were conducted. A plasmid construct encoding a pelB leader sequence fused to the N-terminus of human apoE4 (residues 1-183) was transformed into Escherichia coli. Upon expression, the fusion protein is directed to the periplasmic space where leader peptidase cleaves the pelB sequence, generating mature apoE4-NT. In shaker flask expression cultures, apoE4-NT escapes the bacteria and accumulates in the medium. In a bioreactor setting, however, apoE4-NT was found to combine with gas and liquid components in the culture medium to generate large quantities of foam. When this foam was collected in an external vessel and collapsed into a liquid foamate, analysis revealed that apoE4-NT was the sole major protein present. The product protein was further isolated by heparin affinity chromatography (60-80 mg/liter bacterial culture), shown to be active in rHDL formulation, and documented to serve as an acceptor of effluxed cellular cholesterol. Thus, foam fractionation provides a streamlined process to produce recombinant apoE4-NT for biotechnology applications.
Assuntos
Apolipoproteína E4 , Apolipoproteínas E , Humanos , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/química , Apolipoproteínas E/metabolismo , Proteínas de Transporte , Proteínas Recombinantes/químicaRESUMO
Apolipoprotein (apo) A-I is the major protein component of high-density lipoprotein (HDL) and plays key roles in the Reverse Cholesterol Transport pathway. In the past decade, reconstituted HDL (rHDL) has been employed as a therapeutic agent for treatment of atherosclerosis. The ability of rHDL to promote cholesterol efflux from peripheral cells has been documented to reduce the size of atherosclerotic plaque lesions. However, development of apoA-I rHDL-based therapeutics for human use requires a cost effective process to generate an apoA-I product that meets "Good Manufacturing Practice" standards. Methods available for production and isolation of unmodified recombinant human apoA-I at scale are cumbersome, laborious and complex. To overcome this obstacle, a streamlined two-step procedure has been devised for isolation of recombinant untagged human apoA-I from E. coli that takes advantage of its ability to re-fold to a native conformation following denaturation. Heat treatment of a sonicated E. coli supernatant fraction induced precipitation of a large proportion of host cell proteins (HCP), yielding apoA-I as the major soluble protein. Reversed-phase HPLC of this material permitted recovery of apoA-I largely free of HCP and endotoxin. Purified apoA-I possessed α-helix secondary structure, formed rHDL upon incubation with phospholipid and efficiently promoted cholesterol efflux from cholesterol loaded J774 macrophages.
Assuntos
Apolipoproteína A-I , Escherichia coli/metabolismo , Redobramento de Proteína , Apolipoproteína A-I/biossíntese , Apolipoproteína A-I/química , Apolipoproteína A-I/isolamento & purificação , Apolipoproteína A-I/farmacocinética , Transporte Biológico Ativo/efeitos dos fármacos , Linhagem Celular , Colesterol/metabolismo , Escherichia coli/química , Escherichia coli/genética , Humanos , Macrófagos/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologiaRESUMO
OBJECTIVE: The hypothesis that cholesterol that enters the cell within low-density lipoprotein (LDL) particles rapidly equilibrates with the regulatory pool of intracellular cholesterol and maintains cholesterol homeostasis by reducing cholesterol and LDL receptor synthesis was validated in the fibroblast but not in the hepatocyte. Accordingly, the present studies were designed to compare the effects of cholesterol that enters the hepatocyte within an LDL particle with those of cholesterol that enters via other lipoprotein particles. APPROACH AND RESULTS: We measured cholesterol synthesis and esterification in hamster hepatocytes treated with LDL and other lipoprotein particles, including chylomicron remnants and VLDL. Endogenous cholesterol synthesis was not significantly reduced by uptake of LDL, but cholesterol esterification (280%) and acyl CoA:cholesterol acyltransferase 2 expression (870%) were increased. In contrast, cholesterol synthesis was significantly reduced (70% decrease) with other lipoprotein particles. Furthermore, more cholesterol that entered the hepatocyte within LDL particles was secreted within VLDL particles (480%) compared with cholesterol from other sources. CONCLUSIONS: Much of the cholesterol that enters the hepatocyte within LDL particles is shunted through the cell and resecreted within VLDL particles without reaching equilibrium with the regulatory pool.
Assuntos
LDL-Colesterol/metabolismo , VLDL-Colesterol/metabolismo , Hepatócitos/metabolismo , Homeostase/fisiologia , Metabolismo dos Lipídeos/fisiologia , Animais , Ésteres do Colesterol/biossíntese , Ésteres do Colesterol/metabolismo , HDL-Colesterol/biossíntese , HDL-Colesterol/metabolismo , LDL-Colesterol/biossíntese , VLDL-Colesterol/biossíntese , Quilomícrons/metabolismo , Cricetinae , Fibroblastos/metabolismo , Homeostase/genética , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Metabolismo dos Lipídeos/genética , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMO
Apolipoprotein A-I (apoA-I), the primary protein component of plasma high-density lipoproteins (HDL), is comprised of two structural regions, an N-terminal amphipathic α-helix bundle domain (residues 1-184) and a hydrophobic C-terminal domain (residues 185-243). When a recombinant fusion protein construct [bacterial pelB leader sequence - human apoA-I (1-243)] was expressed in Escherichia coli shaker flask cultures, apoA-I was recovered in the cell lysate. By contrast, when the C-terminal domain was deleted from the construct, large amounts of the truncated protein, apoA-I (1-184), were recovered in the culture medium. Consequently, following pelB leader sequence cleavage in the E. coli periplasmic space, apoA-I (1-184) was secreted from the bacteria. When the pelB-apoA-I (1-184) fusion construct was expressed in a 5 L bioreactor, substantial foam production (~30 L) occurred. Upon foam collection and collapse into a liquid foamate, SDS-PAGE revealed that apoA-I (1-184) was the sole major protein present. Incubation of apoA-I (1-184) with phospholipid vesicles yielded reconstituted HDL (rHDL) particles that were similar in size and cholesterol efflux capacity to those generated with full-length apoA-I. Mass spectrometry analysis confirmed that pelB leader sequence cleavage occurred and that foam fractionation did not result in unwanted protein modifications. The facile nature and scalability of bioreactor-based apolipoprotein foam fractionation provide a novel means to generate a versatile rHDL scaffold protein.
RESUMO
BACKGROUND: Epidemiological evidence links the proprotein convertase subtilisin/kexin 7 (PCSK7) to triglyceride (TG) metabolism. We associated the known PCSK7 gain-of-function non-coding SNP rs236918 with higher levels of plasma apolipoprotein B (apoB) and the loss-of-function coding variant p.Pro777Leu (SNP rs201598301) with lower apoB and TG. Herein, we aimed to unravel the in vivo role of liver PCSK7. METHODS: We biochemically defined the functional role of PCSK7 in lipid metabolism using hepatic cell lines and Pcsk7-/- mice. Our findings were validated following subcutaneous administration of hepatocyte-targeted N-acetylgalactosamine (GalNAc)-antisense oligonucleotides (ASOs) against Pcsk7. RESULTS: Independent of its proteolytic activity, membrane-bound PCSK7 binds apoB100 in the endoplasmic reticulum and enhances its secretion. Mechanistically, the loss of PCSK7/Pcsk7 leads to apoB100 degradation, triggering an unfolded protein response, autophagy, and ß-oxidation, eventually reducing lipid accumulation in hepatocytes. Non-alcoholic fatty liver disease (NAFLD) was induced by a 12-week high fat/fructose/cholesterol diet in wild type (WT) and Pcsk7-/- mice that were then allowed to recover on a 4-week control diet. Pcsk7-/- mice recovered more effectively than WT mice from all NAFLD-related liver phenotypes. Finally, subcutaneous administration of GalNAc-ASOs targeting hepatic Pcsk7 to WT mice validated the above results. CONCLUSIONS: Our data reveal hepatic PCSK7 as one of the major regulators of apoB, and its absence reduces apoB secretion from hepatocytes favoring its ubiquitination and degradation by the proteasome. This results in a cascade of events, eventually reducing hepatic lipid accumulation, thus supporting the notion of silencing PCSK7 mRNA in hepatocytes for targeting NAFLD.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Camundongos , Animais , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Subtilisina/metabolismo , Triglicerídeos/metabolismo , Fígado/metabolismo , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Pró-Proteína Convertases/metabolismo , Apolipoproteína B-100/genética , Apolipoproteína B-100/metabolismoRESUMO
Transforming growth factor ß (TGF-ß) is a key regulatory molecule with pleiotropic effects on cell growth, migration, and invasion. As a result, impairment of proper TGF-ß signaling is central to tumorigenesis and metastasis. The TGF-ß receptor V (TGFBRV or LRP1) has been shown to be responsible for TGF-ß-mediated cell growth inhibition in Chinese hamster ovary (CHO) cells. The LRP1 adapter protein GULP mediates internalization of the various LRP1-specific ligands, and we hypothesize that GULP acts as a novel regulator of TGF-ß signaling in ovarian cells. CHO cells that overexpress exogenous GULP (FL) demonstrate enhancement in growth inhibition, migration, and invasion from TGF-ß treatment, whereas cells that lack GULP (AS) show impairment of growth inhibition and decreased migration and invasion. The enhanced TGF-ß response in FL cells was confirmed by a prolonged TGF-ß-induced SMAD3 phosphorylation, whereas a shortening of the phosphorylation event is observed in AS cells. Mechanistically, the presence of GULP retains the TGF-ß in a signaling-competent early endosome for enhanced signaling. To address this mechanism in a physiological setting, TGF-ß insensitive ovarian adenocarcinoma cells (HEY) have a very low GULP expression level, similar to the observation made in a wide selection of human ovarian adenocarcinomas. Transfection of GULP into the HEY cells restored the TGF-ß responsiveness, as measured by SMAD3 phosphorylation and impairment of cell growth. Because GULP expression positively regulates TGF-ß signaling leading to growth inhibition, this may represent an attractive target to achieve TGF-ß responsiveness in ovarian cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Adenocarcinoma/metabolismo , Movimento Celular , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Células CHO , Cricetinae , Cricetulus , Endossomos/genética , Endossomos/metabolismo , Endossomos/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/patologia , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/genéticaRESUMO
Mechanisms to increase plasma high-density lipoprotein (HDL) or to promote egress of cholesterol from cholesterol-loaded cells (e.g., foam cells from atherosclerotic lesions) remain an important target to regress heart disease. Reconstituted HDL (rHDL) serves as a valuable vehicle to promote cellular cholesterol efflux in vitro and in vivo. rHDL were prepared with wild type apolipoprotein (apo) A-I and the rare variant, apoA-I Milano (M), and each apolipoprotein was reconstituted with phosphatidylcholine (PC) or sphingomyelin (SM). The four distinct rHDL generated were incubated with CHO cells, J774 macrophages, and BHK cells in cellular cholesterol efflux assays. In each cell type, apoA-I(M) SM-rHDL promoted the greatest cholesterol efflux. In BHK cells, the cholesterol efflux capacities of all four distinct rHDL were greatly enhanced by increased expression of ABCG1. Efflux to PC-containing rHDL was stimulated by transfection of a nonfunctional ABCA1 mutant (W590S), suggesting that binding to ABCA1 represents a competing interaction. This interpretation was confirmed by binding experiments. The data show that cholesterol efflux activity is dependent upon the apoA-I protein employed, as well as the phospholipid constituent of the rHDL. Future studies designed to optimize the efflux capacity of therapeutic rHDL may improve the value of this emerging intervention strategy.
Assuntos
Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Células CHO , Células Cultivadas , Cricetinae , TransfecçãoRESUMO
We have identified a novel mutation in apoA-I (serine 36 to alanine; S36A) in a human subject with severe hypoalphalipoproteinemia. The mutation is located in the N-terminal region of the protein, which has been implicated in several functions, including lipid binding and lecithin:cholesterol acyltransferase (LCAT) activity. In the present study, the S36A protein was produced recombinantly and characterized both structurally and functionally. While the helical content of the mutant protein was lower compared with wild-type (WT) apoA-I, it retained its helical character. The protein stability, measured as the resistance to guanidine-induced denaturation, decreased significantly. Interestingly, native gel electrophoresis, cross-linking, and sedimentation equilibrium analysis showed that the S36A mutant was primarily present as a monomer, notably different from the WT protein, which showed considerable oligomeric forms. Although the ability of S36A apoA-I to solubilize phosphatidylcholine vesicles and bind to lipoprotein surfaces was not altered, a significantly impaired LCAT activation compared with the WT protein was observed. These results implicate a region around S36 in apoA-I self-association, independent of the intact C terminus. Furthermore, the region around S36 in the N-terminus of human apoA-I is necessary for LCAT activation.
Assuntos
Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Mutação , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Fosfatidilcolina-Esterol O-Aciltransferase/química , Fosfatidilcolinas/metabolismo , Relação Estrutura-AtividadeRESUMO
RATIONALE: Expression of the vasoactive peptide Urotensin II (UII) is elevated in a number of cardiovascular diseases. OBJECTIVE: Here, we sought to determine the effect of UII receptor (UT) gene deletion in a mouse model of atherosclerosis. METHODS AND RESULTS: UT knockout (KO) mice were crossed with ApoE KO mice to generate UT/ApoE double knockout (DKO) mice. Mice were placed on a high-fat Western-type diet for 12 weeks. We evaluated the degree of atherosclerosis and hepatic steatosis by histology. In addition, serum glucose, insulin, and lipids were determined. DKO mice exhibited significantly increased atherosclerosis compared to ApoE KO mice (P<0.05). This was associated with a significant increase in serum insulin and lipids (P<0.001) but a decrease in hepatic steatosis (P<0.001). UT gene deletion led to a significant increase in systolic pressure and pulse pressure. RT-PCR and immunoblot analyses showed significant reductions in hepatic scavenger receptors, nuclear receptors, and acyl-CoA:cholesterol acyltransferase (ACAT1) expression in DKO mice. UII induced a significant increase in intracellular cholesteryl ester formation in primary mouse hepatocytes, which was blocked by the MEK inhibitor, PD98059. Hepatocytes of UTKO mice showed a significant reduction in lipoprotein uptake compared to wild-type mice. CONCLUSIONS: We propose that UT gene deletion in an ApoE-deficient background promotes downregulation of ACAT1, which in turn attenuates hepatic lipoprotein receptor-mediated uptake and lipid transporter expression. As the liver is the main organ for uptake of lipoprotein-derived lipids, DKO leads to an increase in hyperlipidemia, with a concomitant decrease in hepatic steatosis, and consequently increased atherosclerotic lesion formation. Furthermore, the hypertension associated with UT gene deletion is likely to contribute to the increased atherosclerotic burden.
Assuntos
Aorta/metabolismo , Apolipoproteínas E/deficiência , Aterosclerose/metabolismo , Hiperlipidemias/metabolismo , Fígado/metabolismo , Receptores Acoplados a Proteínas G/deficiência , Urotensinas/metabolismo , Acetil-CoA C-Acetiltransferase/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/patologia , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Glicemia/metabolismo , Pressão Sanguínea , Células Cultivadas , Ésteres do Colesterol/metabolismo , Gorduras na Dieta/administração & dosagem , Modelos Animais de Doenças , Fígado Gorduroso/metabolismo , Fígado Gorduroso/prevenção & controle , Genótipo , Hiperlipidemias/genética , Hiperlipidemias/patologia , Hiperlipidemias/fisiopatologia , Insulina/sangue , Lipídeos/sangue , Fígado/efeitos dos fármacos , Fígado/patologia , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Depuradores/metabolismo , Fatores de TempoRESUMO
Objective: Probucol is a cholesterol-lowering agent whose ability to prevent atherosclerosis is currently under study. Herein, we investigate the putative mechanism of probucol by observation of changes in cellular cholesterol efflux and lipid droplet morphology in macrophages. Results: The inhibitory activity of probucol was assessed in non-foam or foam cell macrophages expressing ABCA1 generated by treatment with fetal calf serum (FCS) alone or in combination with acetylated LDL, respectively. Probucol inhibited cholesterol efflux to apolipoprotein A-I (apoA-I) by 31.5±0.1% in THP-1 non-foam cells and by 18.5±0.2% in foam cells. In probucol-treated non-foam THP-1 cells, nascent high density lipoprotein (nHDL) particles with a diameter < 7 nm were generated, while in probucol-treated THP-1 foam cells nHDL particles of > 7 nm in diameter containing cholesterol were produced. Foam cells also displayed a significant accumulation of free cholesterol at the plasma membrane, as measured by percent cholestenone formed. Intracellularly, there was a significant decrease in lipid droplet number and an increase in size in probucol-treated THP-1 foam cells when compared to non-treated cells. Conclusions: We report for the first time that probucol is unable to completely inhibit cholesterol efflux in foam cells to the same extent as in non-foam cells. Indeed, functional nHDL is released from foam cells in the presence of probucol. This difference in inhibitory effect could potentially be explained by changes in the plasma membrane pool as well as intracellular cholesterol storage independently of ABCA1.
RESUMO
The objectives of this analysis are to re-examine the foundational studies of the in vivo metabolism of plasma LDL (low-density lipoprotein) particles in humans and, based on them, to reconstruct our understanding of the governance of the concentration of plasma LDL and the maintenance of cholesterol homoeostasis in the hepatocyte. We believe that regulation of cholesterol homoeostasis within the hepatocyte is demonstrably more complex than envisioned by the LDL receptor paradigm, the conventional model to explain the regulation of plasma LDL and the fluxes of cholesterol into the liver, a model which was generated in the fibroblast but has never been fully validated in the hepatocyte. We suggest that the LDL receptor paradigm should be reconfigured as the apoB (apolipoprotein B) paradigm, which states that the rate at which LDL particles are produced is at least an important determinant of their concentration in plasma as the rate at which they are cleared from plasma and that secretion of cholesterol within VLDL (very-low-density lipoprotein) particles is an important mechanism of maintaining cholesterol homoeostasis within the hepatocyte. These two paradigms are not mutually exclusive. The LDL receptor paradigm, however, includes only one critical aspect of the regulation of plasma LDL, namely the rate at which LDL particles are cleared through the LDL receptor pathway, but ignores another - the rate at which LDL particles are added to the plasma compartment. The apoB paradigm includes both and points to a different model of how the hepatocyte achieves cholesterol homoeostasis in a complex metabolic environment.
Assuntos
Apolipoproteínas B/sangue , Lipoproteínas LDL/sangue , Modelos Biológicos , Colesterol/metabolismo , Hepatócitos/metabolismo , Homeostase/fisiologia , Humanos , Receptores de LDL/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Apolipoproteins are the protein components of lipoproteins that have the innate ability to inter convert between a lipid-free and a lipid-bound form in a facile manner, a remarkable property conferred by the helix bundle motif. Composed of a series of four or five amphipathic alpha-helices that fold to form a helix bundle, this motif allows the en face orientation of the hydrophobic faces of the alpha-helices in the protein interior in the lipid-free state. A conformational switch then permits helix-helix interactions to be substituted by helix-lipid interactions upon lipid binding interaction. This review compares the apolipoprotein high-resolution structures and the factors that trigger this switch in insect apolipophorin III and the mammalian apolipoproteins, apolipoprotein E and apolipoprotein A-I, pointing out the commonalities and key differences in the mode of lipid interaction. Further insights into the lipid-bound conformation of apolipoproteins are required to fully understand their functional role under physiological conditions.
Assuntos
Apolipoproteínas/química , Lipídeos/química , Dobramento de Proteína , Estrutura Secundária de Proteína , Animais , Apolipoproteínas/metabolismo , Humanos , Metabolismo dos Lipídeos , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
Engulfment of apoptotic cells by phagocytes is important throughout development and adult life. When phagocytes engulf apoptotic cells, they increase their cellular contents including cholesterol and phospholipids, but how the phagocytes respond to this increased load is poorly understood. Here, we identify one type of a phagocyte response, wherein the recognition of apoptotic cells triggers enhanced cholesterol efflux (to apolipoprotein A-I) from macrophages. Phosphatidylserine (PS) exposed on apoptotic cells was necessary and sufficient to stimulate the efflux response. A major mechanism for this enhanced efflux by macrophages was the upregulation of the mRNA and protein for ABCA1, a membrane transporter independently linked to cholesterol efflux as well as engulfment of apoptotic cells. This increase in phagocyte ABCA1 levels required the function of nuclear receptor LXRalpha/beta, a known regulator of cholesterol homeostasis in humans and mice. Taken together, these data reveal a "homeostatic program" initiated in phagocytes that include a proximal membrane signaling event initiated by PS recognition, a downstream signaling event acting through nuclear receptors, and an effector arm involving upregulation of ABCA1, in turn promoting reverse cholesterol transport from the phagocytes. These data also have implications for macrophage handling of contents derived from apoptotic versus necrotic cells in atherosclerotic lesions.
Assuntos
Apoptose/fisiologia , Homeostase/fisiologia , Fagócitos/metabolismo , Fagocitose/fisiologia , Fosfatidilserinas/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Western Blotting , Colesterol/metabolismo , Humanos , Células Jurkat , Macrófagos , Camundongos , Reação em Cadeia da Polimerase , Transdução de Sinais/fisiologiaRESUMO
OBJECTIVE: Reduced plasma concentrations of high-density lipoprotein-cholesterol (HDL-C) are a significant risk factor for cardiovascular disease. Mechanisms that regulate HDL-C concentrations represent an important area of investigation. METHODS AND RESULTS: Comparative transcriptome analyses of monocyte-derived macrophages (MDM) from a large population of low HDL-C subjects and age- and sex-matched controls revealed a cluster of inflammatory genes highly expressed in low HDL-C subjects. The expression levels of peroxisome proliferator activated receptor (PPAR) gamma and several antioxidant metallothionein genes were decreased in MDM from all low HDL-C groups compared with controls, as was the expression of other genes regulated by PPARgamma, including CD36, adipocyte fatty acid binding protein (FABP4), and adipophilin (ADFP). In contrast, PPARdelta expression was increased in MDM from low HDL-C groups. Quantitative RT-PCR corroborated all major findings from the microarray analysis in two separate patient cohorts. Expression of several inflammatory cytokine genes including interleukin 1beta, interleukin 8, and tumor necrosis factor alpha were highly increased in low HDL-C subjects. CONCLUSIONS: The activated proinflammatory state of monocytes and MDM in low HDL-C subjects constitutes a novel parameter of risk associated with HDL deficiency, related to altered expression of metallothionein genes and the reciprocal regulation of PPARgamma and PPARdelta.
Assuntos
HDL-Colesterol/deficiência , Expressão Gênica , Hipolipoproteinemias/sangue , Macrófagos/metabolismo , PPAR delta/genética , PPAR gama/genética , RNA/genética , Aterosclerose/sangue , Aterosclerose/etiologia , Biomarcadores/sangue , HDL-Colesterol/sangue , Proteínas de Ligação a Ácido Graxo/biossíntese , Proteínas de Ligação a Ácido Graxo/genética , Genótipo , Humanos , Hipolipoproteinemias/complicações , Hipolipoproteinemias/genética , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Interleucina-8/biossíntese , Interleucina-8/genética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Análise em Microsséries , Mutação , PPAR delta/biossíntese , PPAR gama/biossíntese , Perilipina-2 , Fenótipo , Reação em Cadeia da Polimerase , Fatores de Risco , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
OBJECTIVE: We have used a multitiered approach to identify genetic and cellular contributors to high-density lipoprotein (HDL) deficiency in 124 human subjects. METHODS AND RESULTS: We resequenced 4 candidate genes for HDL regulation and identified several functional nonsynonymous mutations including 2 in apolipoprotein A-I (APOA1), 4 in lecithin:cholesterol acyltransferase (LCAT), 1 in phospholipid transfer protein (PLTP), and 7 in the ATP-binding cassette transporter ABCA1, leaving 88% (110/124) of HDL deficient subjects without a genetic diagnosis. Cholesterol efflux assays performed using cholesterol-loaded monocyte-derived macrophages from the 124 low HDL subjects and 48 control subjects revealed that 33% (41/124) of low HDL subjects had low efflux, despite the fact that the majority of these subjects (34/41) were not carriers of dysfunctional ABCA1 alleles. In contrast, only 2% of control subjects presented with low efflux (1/48). In 3 families without ABCA1 mutations, efflux defects were found to cosegregate with low HDL. CONCLUSIONS: Efflux defects are frequent in low HDL syndromes, but the majority of HDL deficient subjects with cellular cholesterol efflux defects do not harbor ABCA1 mutations, suggesting that novel pathways contribute to this phenotype.
Assuntos
Apolipoproteína A-I/genética , HDL-Colesterol/sangue , Hipercolesterolemia/genética , Mutação , RNA/genética , Apolipoproteína A-I/metabolismo , Biomarcadores/sangue , Western Blotting , LDL-Colesterol/sangue , Eletroforese em Gel de Ágar , Predisposição Genética para Doença , Humanos , Hipercolesterolemia/sangue , Pessoa de Meia-Idade , Fenótipo , Proteínas de Transferência de Fosfolipídeos/sangue , Proteínas de Transferência de Fosfolipídeos/genética , Esterol O-Aciltransferase/sangue , Esterol O-Aciltransferase/genética , SíndromeRESUMO
The N-terminal domain of human apolipoprotein E (apoE-NT) harbors residues critical for interaction with members of the low-density lipoprotein receptor (LDLR) family. Whereas lipid free apoE-NT adopts a stable four-helix bundle conformation, a lipid binding induced conformational adaptation is required for manifestation of LDLR binding ability. To investigate the structural basis for this conformational change, the short helix connecting helix 1 and 2 in the four-helix bundle was replaced by the sequence NPNG, introducing a beta-turn. Recombinant helix-to-turn (HT) variant apoE3-NT was produced in Escherichia coli, isolated and characterized. Stability studies revealed a denaturation transition midpoint of 1.9 m guanidine hydrochloride for HT apoE3-NT vs. 2.5 M for wild-type apoE3-NT. Wild-type and HT apoE3-NT form dimers in solution via an intermolecular disulfide bond. Native PAGE showed that reconstituted high-density lipoprotein prepared with HT apoE3-NT have a diameter in the range of 9 nm and possess binding activity for the LDLR on cultured human skin fibroblasts. In phospholipid vesicle solubilization assays, HT apoE3-NT was more effective than wild-type apoE3-NT at inducing a time dependent decrease in dimyristoylphosphatidylglycerol vesicle light scattering intensity. In lipoprotein binding assays, HT apoE3-NT protected human low-density lipoprotein from phospholipase C induced aggregation to a greater extent that wild-type apoE3-NT. The results indicate that a mutation at one end of the apoE3-NT four-helix bundle markedly enhances the lipid binding activity of this protein. In the context of lipoprotein associated full-length apoE, increased lipid binding affinity of the N-terminal domain may alter the balance between receptor-active and -inactive conformational states.
Assuntos
Apolipoproteínas E/química , Apolipoproteínas E/metabolismo , Lipídeos/química , Lipídeos/fisiologia , Apolipoproteína E3 , Apolipoproteínas E/isolamento & purificação , Escherichia coli/metabolismo , Corantes Fluorescentes/química , Regulação da Expressão Gênica , Humanos , Fosfatidilgliceróis/química , Ligação Proteica/fisiologia , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína/fisiologia , Receptores de LDL/química , Receptores de LDL/fisiologia , Solubilidade , Fatores de TempoRESUMO
Macrophages clear millions of apoptotic cells daily and, during this process, take up large quantities of cholesterol. The membrane transporter ABCA1 is a key player in cholesterol efflux from macrophages and has been shown via human genetic studies to provide protection against cardiovascular disease. How the apoptotic cell clearance process is linked to macrophage ABCA1 expression is not known. Here, we identified a plasma membrane-initiated signaling pathway that drives a rapid upregulation of ABCA1 mRNA and protein. This pathway involves the phagocytic receptor brain-specific angiogenesis inhibitor 1 (BAI1), which recognizes phosphatidylserine on apoptotic cells, and the intracellular signaling intermediates engulfment cell motility 1 (ELMO1) and Rac1, as ABCA1 induction was attenuated in primary macrophages from mice lacking these molecules. Moreover, this apoptotic cell-initiated pathway functioned independently of the liver X receptor (LXR) sterol-sensing machinery that is known to regulate ABCA1 expression and cholesterol efflux. When placed on a high-fat diet, mice lacking BAI1 had increased numbers of apoptotic cells in their aortic roots, which correlated with altered lipid profiles. In contrast, macrophages from engineered mice with transgenic BAI1 overexpression showed greater ABCA1 induction in response to apoptotic cells compared with those from control animals. Collectively, these data identify a membrane-initiated pathway that is triggered by apoptotic cells to enhance ABCA1 within engulfing phagocytes and with functional consequences in vivo.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/biossíntese , Apoptose/fisiologia , Transportador 1 de Cassete de Ligação de ATP/deficiência , Transportador 1 de Cassete de Ligação de ATP/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/metabolismo , Animais , Membrana Celular/metabolismo , Colesterol/metabolismo , Feminino , Humanos , Células Jurkat , Metabolismo dos Lipídeos , Receptores X do Fígado , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neuropeptídeos/deficiência , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Receptores Nucleares Órfãos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Regulação para Cima , Proteínas rac1 de Ligação ao GTP/deficiência , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
OBJECTIVES: Peroxisome proliferator-activated receptor γ coactivator 1 (PPARGCA1, PGC-1) transcriptional coactivators control gene programs important for nutrient metabolism. Islets of type 2 diabetic subjects have reduced PGC-1α expression and this is associated with decreased insulin secretion, yet little is known about why this occurs or what role it plays in the development of diabetes. Our goal was to delineate the role and importance of PGC-1 proteins to ß-cell function and energy homeostasis. METHODS: We investigated how nutrient signals regulate coactivator expression in islets and the metabolic consequences of reduced PGC-1α and PGC-1ß in primary and cultured ß-cells. Mice with inducible ß-cell specific double knockout of Pgc-1α/Pgc-1ß (ßPgc-1 KO) were created to determine the physiological impact of reduced Pgc1 expression on glucose homeostasis. RESULTS: Pgc-1α and Pgc-1ß expression was increased in primary mouse and human islets by acute glucose and palmitate exposure. Surprisingly, PGC-1 proteins were dispensable for the maintenance of mitochondrial mass, gene expression, and oxygen consumption in response to glucose in adult ß-cells. However, islets and mice with an inducible, ß-cell-specific PGC-1 knockout had decreased insulin secretion due in large part to loss of the potentiating effect of fatty acids. Consistent with an essential role for PGC-1 in lipid metabolism, ß-cells with reduced PGC-1s accumulated acyl-glycerols and PGC-1s controlled expression of key enzymes in lipolysis and the glycerolipid/free fatty acid cycle. CONCLUSIONS: These data highlight the importance of PGC-1s in coupling ß-cell lipid metabolism to promote efficient insulin secretion.