RESUMEN
High-density lipoprotein (HDL) is an anti-atherosclerotic lipoprotein. Thanks to the activity of apolipoprotein ApoA1, the principal protein component of HDL, this last is responsible for converting cholesterol into ester form and transporting excessive cholesterol to the liver ("reverse cholesterol transport" RCT). When HDL undergoes oxidation, it becomes dysfunctional and proatherogenic. ApoA1 is a target of oxidation, and its alteration affects RCT and contributes to atherosclerosis development. Until now, the mechanism of HDL oxidation is not fully understood and only hydroxyl radicals seem to induce direct oxidation of protein and lipidic components of lipoproteins. Here we demonstrate that superoxide radical, widely produced in early atherosclerosis, directly oxidizes HDL, and as a consequence, ApoA1 undergoes structural alterations impairing its anti-atherosclerotic functions. Our results highlight in an in vitro system the potential mechanism by which O2·- triggers atherosclerotic pathogenesis in vivo. Our study gets the basis for therapeutic approaches focused on the management of superoxide generation in early atherosclerosis onset.
Asunto(s)
Aterosclerosis , Lipoproteínas HDL , Humanos , Superóxidos , Colesterol/metabolismo , Transporte Biológico , Apolipoproteína A-I/metabolismo , HDL-ColesterolRESUMEN
Syndromes associated with LCAT deficiency, a rare autosomal recessive condition, include fish-eye disease (FED) and familial LCAT deficiency (FLD). FLD is more severe and characterized by early and progressive chronic kidney disease (CKD). No treatment is currently available for FLD, but novel therapeutics are under development. Furthermore, although biomarkers of LCAT deficiency have been identified, their suitability to monitor disease progression and therapeutic efficacy is unclear, as little data exist on the rate of progression of renal disease. Here, we systematically review observational studies of FLD, FED, and heterozygous subjects, which summarize available evidence on the natural history and biomarkers of LCAT deficiency, in order to guide the development of novel therapeutics. We identified 146 FLD and 53 FED patients from 219 publications, showing that both syndromes are characterized by early corneal opacity and markedly reduced HDL-C levels. Proteinuria/hematuria were the first signs of renal impairment in FLD, followed by rapid decline of renal function. Furthermore, LCAT activity toward endogenous substrates and the percentage of circulating esterified cholesterol (EC%) were the best discriminators between these two syndromes. In FLD, higher levels of total, non-HDL, and unesterified cholesterol were associated with severe CKD. We reveal a nonlinear association between LCAT activity and EC% levels, in which subnormal levels of LCAT activity were associated with normal EC%. This review provides the first step toward the identification of disease biomarkers to be used in clinical trials and suggests that restoring LCAT activity to subnormal levels may be sufficient to prevent renal disease progression.
Asunto(s)
Deficiencia de la Lecitina Colesterol Aciltransferasa , Humanos , Biomarcadores , Heterocigoto , Deficiencia de la Lecitina Colesterol Aciltransferasa/complicaciones , Deficiencia de la Lecitina Colesterol Aciltransferasa/genética , Mutación , Fosfatidilcolina-Esterol O-Aciltransferasa/genéticaRESUMEN
Because of its critical role in HDL formation, significant efforts have been devoted to studying apolipoprotein A-I (APOA1) structural transitions in response to lipid binding. To assess the requirements for the conformational freedom of its termini during HDL particle formation, we generated three dimeric APOA1 molecules with their termini covalently joined in different combinations. The dimeric (d)-APOA1C-N mutant coupled the C-terminus of one APOA1 molecule to the N-terminus of a second with a short alanine linker, whereas the d-APOA1C-C and d-APOA1N-N mutants coupled the C-termini and the N-termini of two APOA1 molecules, respectively, using introduced cysteine residues to form disulfide linkages. We then tested the ability of these constructs to generate reconstituted HDL by detergent-assisted and spontaneous phospholipid microsolubilization methods. Using cholate dialysis, we demonstrate WT and all APOA1 mutants generated reconstituted HDL particles of similar sizes, morphologies, compositions, and abilities to activate lecithin:cholesterol acyltransferase. Unlike WT, however, the mutants were incapable of spontaneously solubilizing short chain phospholipids into discoidal particles. We found lipid-free d-APOA1C-N and d-APOA1N-N retained most of WT APOA1's ability to promote cholesterol efflux via the ATP binding cassette transporter A1, whereas d-APOA1C-C exhibited impaired cholesterol efflux. Our data support the double belt model for a lipid-bound APOA1 structure in nascent HDL particles and refute other postulated arrangements like the "double super helix." Furthermore, we conclude the conformational freedom of both the N- and C-termini of APOA1 is important in spontaneous microsolubilization of bulk phospholipid but is not critical for ABCA1-mediated cholesterol efflux.
Asunto(s)
Apolipoproteína A-I , Colesterol , Transportador 1 de Casete de Unión a ATP/metabolismo , Apolipoproteína A-I/metabolismo , Transporte Biológico , Colesterol/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Fosfolípidos/metabolismoRESUMEN
Production of hydroxy fatty acids (HFAs) in transgenic crops represents a promising strategy to meet our demands for specialized plant oils with industrial applications. The expression of Ricinus communis (castor) OLEATE 12-HYDROXYLASE (RcFAH12) in Arabidopsis has resulted in only limited accumulation of HFAs in seeds, which probably results from inefficient transfer of HFAs from their site of synthesis (phosphatidylcholine; PC) to triacylglycerol (TAG), especially at the sn-1/3 positions of TAG. Phospholipase As (PLAs) may be directly involved in the liberation of HFAs from PC, but the functions of their over-expression in HFA accumulation and distribution at TAG in transgenic plants have not been well studied. In this work, the functions of lecithin:cholesterol acyltransferase-like PLAs (LCAT-PLAs) in HFA biosynthesis were characterized. The LCAT-PLAs were shown to exhibit homology to LCAT and mammalian lysosomal PLA2 , and to contain a conserved and functional Ser/His/Asp catalytic triad. In vitro assays revealed that LCAT-PLAs from the HFA-accumulating plant species Physaria fendleri (PfLCAT-PLA) and castor (RcLCAT-PLA) could cleave acyl chains at both the sn-1 and sn-2 positions of PC, and displayed substrate selectivity towards sn-2-ricinoleoyl-PC over sn-2-oleoyl-PC. Furthermore, co-expression of RcFAH12 with PfLCAT-PLA or RcLCAT-PLA, but not Arabidopsis AtLCAT-PLA, resulted in increased occupation of HFA at the sn-1/3 positions of TAG as well as small but insignificant increases in HFA levels in Arabidopsis seeds compared with RcFAH12 expression alone. Therefore, PfLCAT-PLA and RcLCAT-PLA may contribute to HFA turnover on PC, and represent potential candidates for engineering the production of unusual fatty acids in crops.
Asunto(s)
Brassicaceae/enzimología , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Fosfatidilcolinas/metabolismo , Proteínas de Plantas/metabolismo , Ricinus/enzimología , Arabidopsis/metabolismo , Brassicaceae/genética , Ácidos Grasos/metabolismo , Lisofosfolípidos , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Proteínas de Plantas/genética , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Estructura Terciaria de Proteína , Ricinus/genética , Semillas/metabolismo , Especificidad por SustratoRESUMEN
OBJECTIVE: Enhancement of LCAT (lecithin:cholesterol acyltransferase) activity has possibility to be beneficial for atherosclerosis. To evaluate this concept, we characterized our novel, orally administered, small-molecule LCAT activator DS-8190a, which was created from high-throughput screening and subsequent derivatization. We also focused on its mechanism of LCAT activation and the therapeutic activity with improvement of HDL (high-density lipoprotein) functionality. Approach and Results: DS-8190a activated human and cynomolgus monkey but not mouse LCAT enzymes in vitro. DS-8190a was orally administered to cynomolgus monkeys and dose dependently increased LCAT activity (2.0-fold in 3 mg/kg group on day 7), resulting in HDL cholesterol elevation without drastic changes of non-HDL cholesterol. Atheroprotective effects were then evaluated using Ldl-r KO×hLcat Tg mice fed a Western diet for 8 weeks. DS-8190a treatment achieved significant reduction of atherosclerotic lesion area (48.3% reduction in 10 mg/kg treatment group). Furthermore, we conducted reverse cholesterol transport study using Ldl-r KO×hLcat Tg mice intraperitoneally injected with J774A.1 cells loaded with [3H]-cholesterol and confirmed significant increases of [3H] count in plasma (1.4-fold) and feces (1.4-fold on day 2 and 1.5-fold on day3) in the DS-8190a-treated group. With regard to the molecular mechanism involved, direct binding of DS-8190a to human LCAT protein was confirmed by 2 different approaches: affinity purification by DS-8190a-immobilized beads and thermal shift assay. In addition, the candidate binding site of DS-8190a in human LCAT protein was identified by photoaffinity labeling. CONCLUSIONS: This study demonstrates the potential of DS-8190a as a novel therapeutic for atherosclerosis. In addition, this compound proves that a small-molecule direct LCAT activator can achieve HDL-C elevation in monkey and reduction of atherosclerotic lesion area with enhanced HDL function in rodent.
Asunto(s)
Aterosclerosis/prevención & control , Activadores de Enzimas/farmacología , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Placa Aterosclerótica , Animales , Aterosclerosis/enzimología , Aterosclerosis/genética , Aterosclerosis/patología , Línea Celular , HDL-Colesterol/sangre , Modelos Animales de Enfermedad , Activación Enzimática , Humanos , Macaca fascicularis , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Receptores de LDL/deficiencia , Receptores de LDL/genética , Especificidad de la Especie , Regulación hacia ArribaRESUMEN
The metabolic syndrome (MetS) is a cluster of cardiovascular risk factors characterised by central obesity, atherogenic dyslipidaemia, and changes in the circulating lipidome; the underlying mechanisms that lead to this lipid remodelling have only been partially elucidated. This study used an integrated "omics" approach (untargeted whole serum lipidomics, targeted proteomics, and lipoprotein lipidomics) to study lipoprotein remodelling and HDL composition in subjects with central obesity diagnosed with MetS (vs. controls). Compared with healthy subjects, MetS patients showed higher free fatty acids, diglycerides, phosphatidylcholines, and triglycerides, particularly those enriched in products of de novo lipogenesis. On the other hand, the "lysophosphatidylcholines to phosphatidylcholines" and "cholesteryl ester to free cholesterol" ratios were reduced, pointing to a lower activity of lecithin cholesterol acyltransferase (LCAT) in MetS; LCAT activity (directly measured and predicted by lipidomic ratios) was positively correlated with high-density lipoprotein cholesterol (HDL-C) and negatively correlated with body mass index (BMI) and insulin resistance. Moreover, many phosphatidylcholines and sphingomyelins were significantly lower in the HDL of MetS patients and strongly correlated with BMI and clinical metabolic parameters. These results suggest that MetS is associated with an impairment of phospholipid metabolism in HDL, partially led by LCAT, and associated with obesity and underlying insulin resistance. This study proposes a candidate strategy to use integrated "omics" approaches to gain mechanistic insights into lipoprotein remodelling, thus deepening the knowledge regarding the molecular basis of the association between MetS and atherosclerosis.
Asunto(s)
Resistencia a la Insulina , Síndrome Metabólico , Colesterol/metabolismo , HDL-Colesterol , Humanos , Lipidómica , Lipoproteínas , Síndrome Metabólico/complicaciones , Síndrome Metabólico/diagnóstico , Obesidad/complicaciones , Obesidad Abdominal/complicaciones , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , FosfatidilcolinasRESUMEN
Triacylglycerols have important physiological roles in photosynthetic organisms, and are widely used as food, feed and industrial materials in our daily life. Phospholipid:diacylglycerol acyltransferase (PDAT) is the pivotal enzyme catalyzing the acyl-CoA-independent biosynthesis of triacylglycerols, which is unique in plants, algae and fungi, but not in animals, and has essential functions in plant and algal growth, development and stress responses. Currently, this enzyme has yet to be examined in an evolutionary context at the level of the green lineage. Some fundamental questions remain unanswered, such as how PDATs evolved in photosynthetic organisms and whether the evolution of terrestrial plant PDATs from a lineage of charophyte green algae diverges in enzyme function. As such, we used molecular evolutionary analysis and biochemical assays to address these questions. Our results indicated that PDAT underwent divergent evolution in the green lineage: PDATs exist in a wide range of plants and algae, but not in cyanobacteria. Although PDATs exhibit the conservation of several features, phylogenetic and selection-pressure analyses revealed that overall they evolved to be highly divergent, driven by different selection constraints. Positive selection, as one major driving force, may have resulted in enzymes with a higher functional importance in land plants than green algae. Further structural and mutagenesis analyses demonstrated that some amino acid sites under positive selection are critically important to PDAT structure and function, and may be central in lecithin:cholesterol acyltransferase family enzymes in general.
Asunto(s)
Aciltransferasas/genética , Proteínas Algáceas/genética , Evolución Biológica , Proteínas de Plantas/genética , Plantas/enzimología , Aciltransferasas/química , Aciltransferasas/metabolismo , Proteínas Algáceas/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Plantas/genética , Estructura Terciaria de Proteína , Alineación de Secuencia , Triglicéridos/metabolismoRESUMEN
BACKGROUND AND AIMS: Relative adrenal insufficiency (RAI) in patients with cirrhosis is associated with increased mortality. Although the pathogenesis of RAI remains unclear, disordered cholesterol metabolism may contribute. METHODS: We performed a prospective cohort study of 96 non-critically ill subjects with decompensated cirrhosis at a tertiary care centre. Subjects were administered 250 µcg cosyntropin, with RAI defined as an increase in total cortisol <9 µg/dL. High-density lipoprotein (HDL) levels and serum cholesterol esterification percentage (%CE), a validated surrogate marker of lecithin-cholesterol acyltransferase (LCAT) activity, were measured to assess the relationship between disordered cholesterol metabolism and the presence of RAI. Subjects were followed until death, liver transplantation or a maximum of 6 months. RESULTS: Subjects with RAI had decreased levels of HDL (18 vs 29 mg/dL, P < .01) and %CE (64% vs 66%, P = .03). Correlation was seen between HDL and %CE (r = 0.7, R2 = 0.49; P < .01) and each integer decrease in %CE predicted an approximately 2% increase in the probability of RAI. Transplant-free survival was reduced in subjects with RAI at both 6 months (43% vs 71%, P = .01) and 90 days (54% vs 81%, P < .01). CONCLUSIONS: Disruption in cholesterol metabolism contributes to the development of RAI in cirrhosis, as decreased LCAT activity leads to reduced HDL trafficking to the adrenal gland.
Asunto(s)
Insuficiencia Suprarrenal , Colesterol , Humanos , Metabolismo de los Lípidos , Cirrosis Hepática , Estudios ProspectivosRESUMEN
OBJECTIVE: LCAT (lecithin cholesterol acyltransferase) deficiency results in severe low HDL (high-density lipoprotein). Although whether LCAT is pro- or antiatherosclerosis was in debate in mouse studies, our previous study clearly shows that LCAT deficiency (LCAT-/-) in hamster accelerates atherosclerotic development on high-fat diet. However, unlike in hypercholesterolemia and hypertriglyceridemia, whether LCAT deficiency could lead to spontaneous atherosclerosis has not been studied yet in animal models. We, therefore, sought to investigate the atherosclerosis in LCAT-/- hamsters on standard laboratory diet and explore the potential underlying mechanisms. Approach and Results: Young (<8 months) and aged (>16 months) male and female wild-type and LCAT-/- hamsters on standard laboratory diet were used. Compared with age- and sex-matched wild-type hamsters, LCAT-/- hamsters showed a complete loss of plasma HDL and an increase in triglyceride by 2- to 8-fold at different stages of age. In aged LCAT-/- hamsters, the lesion areas at the aortic roots were ≈40×104 µm3 in males and 18×104 µm3 in females, respectively, which were consistent with the en face plaques observed in male (1.2%) and (1.5%) female groups, respectively. The results of plasma malondialdehyde measurement showed that malondialdehyde concentrations were markedly elevated to 54.4 µmol/L in males and 30 µmol/L in females, which are significantly associated with the atherosclerotic lesions. CONCLUSIONS: Our study demonstrates the development of spontaneous atherosclerotic lesions in aged male and female LCAT-/- hamsters with higher plasma oxidative lipid levels independent of plasma total cholesterol levels, further confirming the antiatherosclerotic role of LCAT.
Asunto(s)
Aorta/metabolismo , Enfermedades de la Aorta/etiología , Aterosclerosis/etiología , Deficiencia de la Lecitina Colesterol Aciltransferasa/complicaciones , Estrés Oxidativo , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Placa Aterosclerótica , Animales , Animales Modificados Genéticamente , Aorta/patología , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Biomarcadores/sangre , Modelos Animales de Enfermedad , Femenino , Técnicas de Inactivación de Genes , Deficiencia de la Lecitina Colesterol Aciltransferasa/enzimología , Deficiencia de la Lecitina Colesterol Aciltransferasa/genética , Lípidos/sangre , Masculino , Malondialdehído/sangre , Mesocricetus/genética , Fosfatidilcolina-Esterol O-Aciltransferasa/genéticaRESUMEN
Familial LCAT deficiency (FLD) is a rare genetic disorder of HDL metabolism, caused by loss-of-function mutations in the LCAT gene and characterized by a variety of symptoms including corneal opacities and kidney failure. Renal disease represents the leading cause of morbidity and mortality in FLD cases. However, the prognosis is not known and the rate of deterioration of kidney function is variable and unpredictable from patient to patient. In this article, we present data from a follow-up of the large Italian cohort of FLD patients, who have been followed for an average of 12 years. We show that renal failure occurs at the median age of 46 years, with a median time to a second recurrence of 10 years. Additionally, we identify high plasma unesterified cholesterol level as a predicting factor for rapid deterioration of kidney function. In conclusion, this study highlights the severe consequences of FLD, underlines the need of correct early diagnosis and referral of patients to specialized centers, and highlights the urgency for effective treatments to prevent or slow renal disease in patients with LCAT deficiency.
Asunto(s)
Deficiencia de la Lecitina Colesterol Aciltransferasa/complicaciones , Insuficiencia Renal Crónica/complicaciones , Colesterol/metabolismo , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Humanos , Italia , Masculino , Persona de Mediana EdadRESUMEN
The acyltransferase LCAT mediates FA esterification of plasma cholesterol. In vitro studies have shown that LCAT also FA-esterifies several oxysterols, but in vivo evidence is lacking. Here, we measured both free and FA-esterified forms of sterols in 206 healthy volunteers and 8 individuals with genetic LCAT deficiency, including familial LCAT deficiency (FLD) and fish-eye disease (FED). In the healthy volunteers, the mean values of the ester-to-total molar ratios of the following sterols varied: 4ß-hydroxycholesterol (4ßHC), 0.38; 5,6α-epoxycholesterol (5,6αEC), 0.46; 5,6ß-epoxycholesterol (5,6ßEC), 0.51; cholesterol, 0.70; cholestane-3ß,5α,6ß-triol (CT), 0.70; 7-ketocholesterol (7KC), 0.75; 24S-hydroxycholesterol (24SHC), 0.80; 25-hydroxycholesterol (25HC), 0.81; 27-hydroxycholesterol (27HC), 0.86; and 7α-hydroxycholesterol (7αHC), 0.89. In the individuals with LCAT deficiency, the plasma levels of the FA-esterified forms of cholesterol, 5,6αEC, 5,6ßEC, CT, 7αHC, 7KC, 24SHC, 25HC, and 27HC, were significantly lower than those in the healthy volunteers. The individuals with FLD had significantly lower FA-esterified forms of 7αHC, 24SHC, and 27HC than those with FED. It is of note that, even in the three FLD individuals with negligible plasma cholesteryl ester, substantial amounts of the FA-esterified forms of 4ßHC, 5,6αEC, 7αHC, 7KC, and 27HC were present. We conclude that LCAT has a major role in the FA esterification of many plasma oxysterols but contributes little to the FA esterification of 4ßHC. Substantial FA esterification of 4ßHC, 5,6αEC, 7αHC, 7KC, and 27HC is independent of LCAT.
Asunto(s)
Hidroxicolesteroles/sangre , Hidroxicolesteroles/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Adulto , Estudios de Casos y Controles , Esterificación , Femenino , Humanos , Masculino , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Adulto JovenRESUMEN
BACKGROUND: The functionality of high-density lipoproteins (HDL) is a better cardiovascular risk predictor than HDL concentrations. One of the key elements of HDL functionality is its apolipoprotein composition. Lecithin-cholesterol acyl transferase (LCAT) and cholesterol-ester transfer protein (CETP) are enzymes involved in HDL-mediated reverse cholesterol transport. This study assessed the concentration and activity of LCAT and CETP in HDL subspecies defined by their content of apolipoproteins E (apoE) and C-III (apoC-III) in humans. METHODS: Eighteen adults (ten women and eight men, mean age 55.6, BMI 26.9 Kg/m2, HbA1c 5.4%) were studied. HDL from each participant were isolated and divided into four subspecies containing respectively: No apoE and no apoC-III (E-C-), apoE but not apoC-III (E + C-), apoC-III but no apoE (E-C+) and both apoE and apoC-III (E + C+). The concentration and enzymatic activity of LCAT and CETP were measured within each HDL subspecies using immunoenzymatic and fluorometric methods. Additionally, the size distribution of HDL in each apolipoprotein-defined fraction was determined using non-denaturing electrophoresis and anti-apoA-I western blotting. RESULTS: HDL without apoE or apoC-III was the predominant HDL subtype. The size distribution of HDL was very similar in all the four apolipoprotein-defined subtypes. LCAT was most abundant in E-C- HDL (3.58 mg/mL, 59.6% of plasma LCAT mass), while HDL with apoE or apoC-III had much less LCAT (19.8, 12.2 and 8.37% of plasma LCAT respectively for E + C-, E-C+ and E + C+). LCAT mass was lower in E + C- HDL relative to E-C- HDL, but LCAT activity was similar in both fractions, signaling a greater activity-to-mass ratio associated with the presence of apoE. Both CETP mass and CETP activity showed only slight variations across HDL subspecies. There was an inverse correlation between plasma LCAT activity and concentrations of both E-C+ pre-beta HDL (r = - 0.55, P = 0.017) and E-C- alpha 1 HDL (r = - 0.49, P = 0.041). Conversely, there was a direct correlation between plasma CETP activity and concentrations of E-C+ alpha 1 HDL (r = 0.52, P = 0.025). CONCLUSIONS: The presence of apoE in small HDL is correlated with increased LCAT activity and esterification of plasma cholesterol. These results favor an interpretation that LCAT and apoE interact to enhance anti-atherogenic pathways of HDL.
Asunto(s)
Apolipoproteína C-III/análisis , Apolipoproteínas E/análisis , Proteínas de Transferencia de Ésteres de Colesterol/análisis , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/análisis , Adulto , Anciano , Proteínas de Transferencia de Ésteres de Colesterol/sangre , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Ésteres del Colesterol/metabolismo , Femenino , Humanos , Lipoproteínas HDL/química , Lipoproteínas HDL/clasificación , Masculino , Persona de Mediana Edad , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismoRESUMEN
Familial LCAT deficiency (FLD) patients accumulate lipoprotein-X (LP-X), an abnormal nephrotoxic lipoprotein enriched in free cholesterol (FC). The low neutral lipid content of LP-X limits the ability to detect it after separation by lipoprotein electrophoresis and staining with Sudan Black or other neutral lipid stains. A sensitive and accurate method for quantitating LP-X would be useful to examine the relationship between plasma LP-X and renal disease progression in FLD patients and could also serve as a biomarker for monitoring recombinant human LCAT (rhLCAT) therapy. Plasma lipoproteins were separated by agarose gel electrophoresis and cathodal migrating bands corresponding to LP-X were quantified after staining with filipin, which fluoresces with FC, but not with neutral lipids. rhLCAT was incubated with FLD plasma and lipoproteins and LP-X changes were analyzed by agarose gel electrophoresis. Filipin detects synthetic LP-X quantitatively (linearity 20-200 mg/dl FC; coefficient of variation <20%) and sensitively (lower limit of quantitation <1 mg/ml FC), enabling LP-X detection in FLD, cholestatic, and even fish-eye disease patients. rhLCAT incubation with FLD plasma ex vivo reduced LP-X dose dependently, generated HDL, and decreased lipoprotein FC content. Filipin staining after agarose gel electrophoresis sensitively detects LP-X in human plasma and accurately quantifies LP-X reduction after rhLCAT incubation ex vivo.
Asunto(s)
Filipina/química , Deficiencia de la Lecitina Colesterol Aciltransferasa/tratamiento farmacológico , Lipoproteína X/sangre , Lipoproteínas/sangre , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre , Biomarcadores/sangre , Geles/química , Humanos , Deficiencia de la Lecitina Colesterol Aciltransferasa/sangre , Deficiencia de la Lecitina Colesterol Aciltransferasa/enzimología , Lipoproteína X/síntesis química , Lipoproteína X/química , Proteínas Recombinantes/sangreRESUMEN
Human apolipoprotein C1 (APOC1) is a 57 amino acid long polypeptide that, through its potent inhibition of cholesteryl ester transferase protein, helps regulate the transfer of lipids between lipid particles. We have now determined the structure of APOC1 in four crystal forms by X-ray diffraction. A molecule of APOC1 is a single, slightly bent, α-helix having 13-14 turns and a length of about 80 Å. APOC1 exists as a dimer, but the dimers are not the same in the four crystals. In two monoclinic crystals, two helices closely engage one another in an antiparallel fashion. The interactions between monomers are almost entirely hydrophobic with sparse electrostatic complements. In the third monoclinic crystal, the two monomers spread at one end of the dimer, like a scissor opening, and, by translation along the crystallographic a axis, form a continuous, contiguous sheet through the crystal. In the orthorhombic crystals, two molecules of APOC1 are related by a noncrystallographic 2-fold axis to create an arc of about 120 Å length. This symmetrical dimer utilizes interactions not present in dimers of the monoclinic crystals. Versatility of APOC1 monomer association shown by these crystals is suggestive of physiological function.
Asunto(s)
Apolipoproteína C-I/química , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación Proteica , Electricidad EstáticaRESUMEN
INTRODUCTION: Lecithin cholesterol acyltransferase (LCAT) deficiency is an autosomal recessive disorder occurred by different mutations in the LCAT gene that cause two extremely rare syndromes including familial LCAT deficiency (FLD) and fish-eye disease (FED). Unlike FED in FLD renal failure is the most important defect due to deposition of abnormal lipoproteins in the renal stroma. In this study, FLD patients from the North of Iran were investigated for mutations in the LCAT gene. MATERIALS AND METHODS: Eight patients with corneal opacification and renal defect were analyzed for mutations in the LCAT gene by PCR sequencing. RESULTS: Sequencing analysis revealed a missense pathogenic variation c.301 G>A in exon 2 of LCAT gene in all patients changing the amino acid aspartate to asparagine at the conserved position of amino acid 101 of LCAT protein. CONCLUSION: In this study, a very rare variation was reported for the first time in this part of the world. Investigation of a larger number of LCAT patients in different parts of Iran can provide availability of mutations panel that is useful for phenotype prediction and also prenatal diagnosis programming in families with a previous history of the disease.
RESUMEN
RATIONALE & OBJECTIVE: Lecithin-cholesterol acyltransferase (LCAT) catalyzes the maturation of high-density lipoprotein. Homozygosity for loss-of-function mutations causes familial LCAT deficiency (FLD), characterized by corneal opacities, anemia, and renal involvement. This study sought to characterize kidney biopsy findings and clinical outcomes in a family with FLD. STUDY DESIGN: Prospective observational study. SETTING & PARTICIPANTS: 2 (related) index patients with clinically apparent FLD were initially identified. 110 of 122 family members who consented to genetic analysis were also studied. PREDICTORS: Demographic and laboratory parameters (including lipid profiles and LCAT activity) and full sequence analysis of the LCAT gene. Kidney histologic examination was performed with samples from 6 participants. OUTCOMES: Cardiovascular and renal events during a median follow-up of 12 years. Estimation of annual rate of decline in glomerular filtration rate. ANALYTICAL APPROACH: Analysis of variance, linear regression analysis, and Fine-Gray competing-risk survival analysis. RESULTS: 9 homozygous, 57 heterozygous, and 44 unaffected family members were identified. In all affected individuals, full sequence analysis of the LCAT gene revealed a mutation (c.820C>T) predicted to cause a proline to serine substitution at amino acid 274 (P274S). Homozygosity caused a complete loss of LCAT activity. Kidney biopsy findings demonstrated lipid deposition causing glomerular basement membrane thickening, mesangial expansion, and "foam-cell" infiltration of kidney tissue. Tubular atrophy, glomerular sclerosis, and complement fixation were associated with worse kidney outcomes. Estimated glomerular filtration rate deteriorated among homozygous family members at an average annual rate of 3.56 mL/min/1.73 m2. The incidence of cardiovascular and renal complications was higher among homozygous family members compared with heterozygous and unaffected members. Mild thrombocytopenia was a common finding among homozygous participants. LIMITATIONS: The presence of cardiovascular disease was mainly based on medical history. CONCLUSIONS: The P274S LCAT mutation was found to cause FLD with renal involvement. Tubular atrophy, glomerular sclerosis, and complement fixation were associated with a worse renal prognosis.
Asunto(s)
Enfermedades Renales/diagnóstico , Enfermedades Renales/genética , Deficiencia de la Lecitina Colesterol Aciltransferasa/diagnóstico , Deficiencia de la Lecitina Colesterol Aciltransferasa/genética , Mutación/genética , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
AIMS: To review the formation, catabolism, and the possible atherogenic properties of Lp-X. DATA SYNTHESIS: The conversion of cholesterol to bile acids is regulated by several mechanisms including cholesterol 7 alpha hydroxylase, fibroblast growth factor 19, and farnesoid X receptors. During cholestasis these mechanisms are altered and there is an accumulation of bile acids and cholesterol in plasma. The hypercholesterolemia observed in cholestasis is due to the presence of an anomalous lipoprotein called lipoprotein-X (Lp-X). Lp-X is a lipoprotein rich in phospholipid and free cholesterol present in plasma of patients with cholestasis and, with some variations, in patients with lecithin-cholesterol-acyl-transferase deficiency (LCAT), and after lipid infusion. Lp-X is formed from a bile lipoprotein moving to the blood vessels where it incorporates small quantities of triglycerides, apo-C and esterified cholesterol and becomes a "mature" Lp-X. The activity of the phosphatidilcholine canalicular transporter Mdr2 P-glycoprotein (homologous to the human ABCB4) is essential for LpX appearance, since its suppression abolishes Lp-X formation. However, the concentration of Lp-X in plasma is determined also by the degree of the cholestasis, the residual liver function, and the LCAT deficiency. The Lp-X catabolism seems to be mediated by the reticuloendothelial system and possibly the kidney. CONCLUSIONS: Lp-X might be considered a defense mechanism against the toxic effect of free cholesterol in cholestasis. The frequency of cardiovascular events in patients affected by primary biliary cholangitis, in whom the Lp-X is present in high concentration, are not increased. Further studies could now clarify the remaining open questions on the role of Lp-X in the dyslipidemia of cholestasis.
Asunto(s)
Colestasis/sangre , Hipercolesterolemia/sangre , Lipoproteína X/sangre , Hígado/metabolismo , Animales , Transporte Biológico , Colestasis/epidemiología , Colestasis/historia , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Hipercolesterolemia/epidemiología , Hipercolesterolemia/historia , Deficiencia de la Lecitina Colesterol Aciltransferasa/sangre , Deficiencia de la Lecitina Colesterol Aciltransferasa/epidemiología , Lipoproteína X/historia , Pronóstico , Factores de RiesgoRESUMEN
BACKGROUND: Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme that esterifies cholesterol in high- and low-density lipoproteins (HDL and LDL). Mutations in LCAT gene causes familial LCAT deficiency, which is characterized by very low plasma HDL-cholesterol levels (Hypoalphalipoproteinemia), corneal opacity and anemia, among other lipid-related traits. Our aim is to evaluate clinical/biochemical features of a Chilean family with a proband showing clinical signs of familial LCAT deficiency, as well as to identify and assess the functional effects of LCAT mutations. METHODS: An adult female proband with hypoalphalipoproteinemia, corneal opacity and mild anemia, as well as her first-degree relatives, were recruited for clinical, biochemical, genetic, in-silico and in-vitro LCAT analysis. Sequencing of exons and intron-exon boundaries was performed to identify mutations. Site-directed mutagenesis was carried out to generate plasmids containing cDNA with wild type or mutant sequences. Such expression vectors were transfected to HEK-239 T cells to asses the effect of LCAT variants in expression, synthesis, secretion and enzyme activity. In-silico prediction analysis and molecular modeling was also used to evaluate the effect of LCAT variants. RESULTS: LCAT sequencing identified rare p.V333 M and p.M404 V missense mutations in compound heterozygous state in the proband, as well the common synonymous p.L363 L variant. LCAT protein was detected in proband's plasma, but with undetectable enzyme activity compared to control relatives. HEK-293 T transfected cells with vector expression plasmids containing either p.M404 V or p.V333 M cDNA showed detectable LCAT protein expression both in supernatants and lysates from cultured cells, but with much lower enzyme activity compared to cells transfected with the wild-type sequence. Bioinformatic analyses also supported a causal role of such rare variations in LCAT lack of function. Additionally, the proband carried the minor allele of the synonymous p.L363 L variant. However, this variant is unlikely to affect the clinical phenotype of the proband given its relatively high frequency in the Chilean population (4%) and its small putative effect on plasma HDL-cholesterol levels. CONCLUSION: Genetic, biochemical, in vitro and in silico analyses indicate that the rare mutations p.M404 V and p.V333 M in LCAT gene lead to suppression of LCAT enzyme activity and cause clinical features of familial LCAT deficiency.
Asunto(s)
Hipoalfalipoproteinemias/genética , Deficiencia de la Lecitina Colesterol Aciltransferasa/genética , Lípidos/sangre , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Adulto , Anciano , Chile/epidemiología , Colesterol/sangre , HDL-Colesterol/sangre , Opacidad de la Córnea/genética , Opacidad de la Córnea/patología , Exones/genética , Femenino , Células HEK293 , Humanos , Hipoalfalipoproteinemias/sangre , Hipoalfalipoproteinemias/epidemiología , Hipoalfalipoproteinemias/patología , Deficiencia de la Lecitina Colesterol Aciltransferasa/sangre , Deficiencia de la Lecitina Colesterol Aciltransferasa/epidemiología , Deficiencia de la Lecitina Colesterol Aciltransferasa/patología , Lipoproteínas HDL/sangre , Simulación de Dinámica Molecular , Mutación Missense/genética , Linaje , Fosfatidilcolina-Esterol O-Aciltransferasa/química , Relación Estructura-ActividadRESUMEN
Glomerulopathy associated with lecithin-cholesterol-acyltransferase deficiency (LCAT) is a rare automosal recessive disease. Acquired LCAT deficiency due to inhibitory autoantibodies against LCAT are also described. This disease is induced by systemic deposits related to a lipid metabolism disorder and lead to multi-organ involvement including renal involvement. Lipid profile usually shows variable cholesterol levels but very low HDL levels. Here we describe the case of a 33-year-old man presenting a nephrotic syndrome associated with moderate renal insufficiency for which the pathological analysis allowed to guide towards the diagnosis of LCAT deficiency. Laboratory and genetic data confirmed this diagnosis. Familial history and lipid profile abnormalities are important in the identification of this disease.
Asunto(s)
Glomérulos Renales , Deficiencia de la Lecitina Colesterol Aciltransferasa/complicaciones , Síndrome Nefrótico/etiología , Insuficiencia Renal/etiología , Adulto , Humanos , MasculinoRESUMEN
We assessed secondary and genetic causes of severe HDL deficiency in 258,252 subjects, of whom 370 men (0.33%) and 144 women (0.099%) had HDL cholesterol levels <20 mg/dl. We excluded 206 subjects (40.1%) with significant elevations of triglycerides, C-reactive protein, glycosylated hemoglobin, myeloperoxidase, or liver enzymes and men receiving testosterone. We sequenced 23 lipid-related genes in 201 (65.3%) of 308 eligible subjects. Mutations (23 novel) and selected variants were found at the following gene loci: 1) ABCA1 (26.9%): 2 homozygotes, 7 compound or double heterozygotes, 30 heterozygotes, and 2 homozygotes and 13 heterozygotes with variants rs9282541/p.R230C or rs111292742/c.-279C>G; 2) LCAT (12.4%): 1 homozygote, 3 compound heterozygotes, 13 heterozygotes, and 8 heterozygotes with variant rs4986970/p.S232T; 3) APOA1 (5.0%): 1 homozygote and 9 heterozygotes; and 4) LPL (4.5%): 1 heterozygote and 8 heterozygotes with variant rs268/p.N318S. In addition, 4.5% had other mutations, and 46.8% had no mutations. Atherosclerotic cardiovascular disease (ASCVD) prevalence rates in the ABCA1, LCAT, APOA1, LPL, and mutation-negative groups were 37.0%, 4.0%, 40.0%, 11.1%, and 6.4%, respectively. Severe HDL deficiency is uncommon, with 40.1% having secondary causes and 48.8% of the subjects sequenced having ABCA1, LCAT, APOA1, or LPL mutations or variants, with the highest ASCVD prevalence rates being observed in the ABCA1 and APOA1 groups.