RESUMEN

Genome sequencing from wastewater has emerged as an accurate and cost-effective tool for identifying SARS-CoV-2 variants. However, existing methods for analyzing wastewater sequencing data are not designed to detect novel variants that have not been characterized in humans. Here, we present an unsupervised learning approach that clusters co-varying and time-evolving mutation patterns leading to the identification of SARS-CoV-2 variants. To build our model, we sequenced 3,659 wastewater samples collected over a span of more than two years from urban and rural locations in Southern Nevada. We then developed a multivariate independent component analysis (ICA)-based pipeline to transform mutation frequencies into independent sources with co-varying and time-evolving patterns and compared variant predictions to >5,000 SARS-CoV-2 clinical genomes isolated from Nevadans. Using the source patterns as data-driven reference "barcodes", we demonstrated the model's accuracy by successfully detecting the Delta variant in late 2021, Omicron variants in 2022, and emerging recombinant XBB variants in 2023. Our approach revealed the spatial and temporal dynamics of variants in both urban and rural regions; achieved earlier detection of most variants compared to other computational tools; and uncovered unique co-varying mutation patterns not associated with any known variant. The multivariate nature of our pipeline boosts statistical power and can support accurate and early detection of SARS-CoV-2 variants. This feature offers a unique opportunity for novel variant and pathogen detection, even in the absence of clinical testing.

RESUMEN

Real-time surveillance of infectious diseases at schools or in communities is often hampered by delays in reporting due to resource limitations and infrastructure issues. By incorporating quantitative PCR and genome sequencing, wastewater surveillance has been an effective complement to public health surveillance at the community and building-scale for pathogens such as poliovirus, SARS-CoV-2, and even the monkeypox virus. In this study, we asked whether wastewater surveillance programs at elementary schools could be leveraged to detect RNA from influenza viruses shed in wastewater. We monitored for influenza A and B viral RNA in wastewater from six elementary schools from January to May 2022. Quantitative PCR led to the identification of influenza A viral RNA at three schools, which coincided with the lifting of COVID-19 restrictions and a surge in influenza A infections in Las Vegas, Nevada, USA. We performed genome sequencing of wastewater RNA, leading to the identification of a 2021-2022 vaccine-resistant influenza A (H3N2) 3C.2a1b.2a.2 subclade. We next tested wastewater samples from a treatment plant that serviced the elementary schools, but we were unable to detect the presence of influenza A/B RNA. Together, our results demonstrate the utility of near-source wastewater surveillance for the detection of local influenza transmission in schools, which has the potential to be investigated further with paired school-level influenza incidence data.

Asunto(s)

COVID-19 , Vacunas contra la Influenza , Gripe Humana , Humanos , Gripe Humana/genética , Aguas Residuales , Subtipo H3N2 del Virus de la Influenza A/genética , Nevada/epidemiología , COVID-19/epidemiología , SARS-CoV-2/genética , Monitoreo Epidemiológico Basado en Aguas Residuales , Vacunas contra la Influenza/genética , ARN Viral , Instituciones Académicas

RESUMEN

The identification of novel SARS-CoV-2 variants can predict new patterns of COVID-19 community transmission and lead to the deployment of public health resources. However, increased access to at-home antigen tests and reduced free PCR tests have recently led to data gaps for the surveillance of evolving SARS-CoV-2 variants. To overcome such limitations, we asked whether wastewater surveillance could be leveraged to detect rare variants circulating in a community before local detection in human cases. Here, we performed whole genome sequencing (WGS) of SARS-CoV-2 from a wastewater treatment plant serving Las Vegas, Nevada in April 2022. Using metrics that exceeded 100× depth at a coverage of >90 % of the viral genome, we identified a variant profile similar to the XL recombinant lineage containing 26 mutations found in BA.1 and BA.2 and three private mutations. Prompted by the discovery of this rare lineage in wastewater, we analyzed clinical COVID-19 sequencing data from Southern Nevada and identified two cases infected with the XL lineage. Taken together, our data highlight how wastewater genome sequencing data can be used to discover rare SARS-CoV-2 lineages in a community and complement local public health surveillance.

Asunto(s)

COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Aguas Residuales , Monitoreo Epidemiológico Basado en Aguas Residuales

RESUMEN

To identify residues and segments in the central region of apolipoprotein A-I (apoA-I) that are important for the protein structure and stability, we studied the effects of four double charge ablations, D102A/D103A, E110A/E111A, R116V/K118A, and R160V/H162A, and two deletion mutations, Delta(61-78) and Delta(121-142), on the conformation and stability of apoA-I in the lipid-free state and in reconstituted discoidal phospholipid-cholesterol-apoA-I particles (rHDL). The findings suggest that D102/D103 and E110/E111 located in helix 4 and segment(s) between residues 61 and 78 are involved in maintenance of the conformation and stability of apoA-I in both the lipid-free state and in rHDL. R116/K118 located in helix 4 are essential for the conformation and stabilization of apoA-I in rHDL but not vital for the lipid-free state of the protein. The R160V/H162A substitutions in helix 6 lead to a less compact tertiary structure of lipid-free apoA-I without notable effects on the lipid-free or lipid-bound secondary conformation, suggesting involvement of R160/H162 in important interhelical interactions. The results on the Delta(121-142) mutant, together with our earlier findings, suggest disordered structure of a major segment between residues 121 and 143, likely including residues 131-143, in lipid-free apoA-I. Our findings provide the first experimental evidence for stabilization of rHDL by specific electrostatic interhelical interactions, in agreement with the double belt model. The effects of alterations in the conformation and stability of the apoA-I mutants on in vitro and in vivo functions of apoA-I and lipid homeostasis are discussed.

Asunto(s)

Apolipoproteína A-I/química , Eliminación de Gen , Lipoproteínas HDL/farmacología , Naftalenosulfonatos de Anilina/química , Animales , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Guanidina/metabolismo , Guanidina/farmacología , Humanos , Lípidos/química , Lípidos/genética , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Modelos Biológicos , Mutación/efectos de los fármacos , Desnaturalización Proteica , Estructura Secundaria de Proteína/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Soluciones/metabolismo , Soluciones/farmacología , Espectrometría de Fluorescencia , Electricidad Estática , Temperatura , Triptófano/química , Células Tumorales Cultivadas

RESUMEN

We have analyzed the effect of charged to neutral amino acid substitutions around the kinks flanking helices 4 and 6 of apoA-I and of the deletion of helix 6 on the in vivo activity of LCAT and the biogenesis of HDL. The LCAT activation capacity of apoA-I in vitro was nearly abolished by the helix 6 point (helix 6P-apoA-I[R160V/H162A]) and deletion {helix 6Delta-apoA-I[Delta(144-165)]} mutants, but was reduced to 50% in the helix 4 point mutant (helix 4P-apoA-I[D102A/D103A]). Following adenovirus-mediated gene transfer in apoA-I deficient mice, the level of plasma HDL cholesterol was greatly reduced in helix 6P and helix 6Delta mutants. Electron microscopy and two-dimensional gel electrophoresis showed that the helix 6P mutant formed predominantly high levels of apoA-I containing discoidal particles and had an increased prebeta1-HDL/alpha-HDL ratio. The helix 6Delta mutant formed few spherical particles and had an increased prebeta1-HDL/alpha-HDL ratio. Mice infected with adenovirus expressing the helix 4P mutant or wild-type apoA-I had normal HDL cholesterol and formed spherical alpha-HDL particles. Coinfection of mice with adenoviruses expressing human LCAT and the helix 6P mutant dramatically increased plasma HDL and apoA-I levels and converted the discoidal into spherical HDL, indicating that the LCAT activity was rate-limiting for the biogenesis of HDL. The LCAT treatment caused only a small increase in HDL cholesterol and apoA-I levels and in alpha-HDL particle numbers in the helix 6Delta mutant. The findings indicate a critical contribution of residue 160 of apoA-I to the in vivo activity of LCAT and the subsequent maturation of HDL and explain the low HDL levels in heterozygous subjects carrying this mutation.

Asunto(s)

Apolipoproteína A-I/genética , Fosfatidilcolinas/deficiencia , Mutación Puntual , Esterol O-Aciltransferasa/deficiencia , Animales , Apolipoproteína A-I/metabolismo , Secuencia de Bases , Lipoproteínas de Alta Densidad Pre-beta , Humanos , Lipoproteínas HDL/metabolismo , Ratones , Imitación Molecular , Fenotipo , Fosfatidilcolinas/genética , Fosfatidilcolinas/metabolismo , Esterol O-Aciltransferasa/genética , Esterol O-Aciltransferasa/metabolismo

RESUMEN

The objective of this study was to determine the effect of two amino-terminal apolipoprotein A-I (apoA-I) deletions on high-density lipoprotein (HDL) biosynthesis and lipid homeostasis. Adenovirus-mediated gene transfer showed that the apoA-I[Delta(89-99)] deletion mutant caused hypercholesterolemia, characterized by increased plasma cholesterol and phospholipids, that were distributed in the very low density/intermediate density/low-density lipoprotein (VLDL/IDL/LDL) region, and normal triglycerides. The capacity of the mutant protein to promote ATP-binding cassette transporter A1- (ABCA1-) mediated cholesterol efflux and to activate lecithin:cholesterol acyltranserase (LCAT) was approximately 70-80% of the wild-type (WT) control. The phospholipid transfer protein (PLTP) activity of plasma containing the apoA-I[Delta(89-99)] mutant was decreased to 32% of the WT control. Similar analysis showed that the apoA-I[Delta(62-78)] deletion mutant in apoA-I-deficient mice caused combined hyperlipidemia characterized by increased triglycerides, cholesterol, and phospholipids in the VLDL/IDL region. There was enrichment of the VLDL/IDL with mutant apoA-I that resulted in reduction of in vitro lipolysis. The capacity of this mutant to promote ABCA1-mediated cholesterol efflux was normal, and the capacity to activate LCAT in vitro was reduced by 53%. The WT apoA-I and the apoA-I[Delta(62-78)] mutant formed spherical HDL particles, whereas the apoA-I[Delta(89-99)] mutant formed discoidal HDL particles. We conclude that alterations in apoA-I not only may have adverse effects on HDL biosynthesis but also may promote dyslipidemia due to interference of the apoA-I mutants on the overall cholesterol and triglycerides homeostasis.

Asunto(s)

Adenoviridae/genética , Apolipoproteína A-I/deficiencia , Apolipoproteína A-I/genética , Técnicas de Transferencia de Gen , Hipercolesterolemia/genética , Eliminación de Secuencia , Animales , Apolipoproteína A-I/biosíntesis , Apolipoproteína A-I/sangre , Línea Celular , Cromatografía Líquida de Alta Presión , Humanos , Hidrólisis , Hipercolesterolemia/sangre , Lipoproteínas/antagonistas & inhibidores , Lipoproteínas/sangre , Lipoproteínas HDL/sangre , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/ultraestructura , Lipoproteínas IDL , Lipoproteínas VLDL/antagonistas & inhibidores , Lipoproteínas VLDL/sangre , Hígado/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/genética , Proteínas de Transferencia de Fosfolípidos/antagonistas & inhibidores , Proteínas de Transferencia de Fosfolípidos/sangre , Estructura Secundaria de Proteína/genética , ARN Mensajero/biosíntesis , Triglicéridos/antagonistas & inhibidores , Triglicéridos/sangre

RESUMEN

Hypertriglyceridemia is a common pathological condition in humans of mostly unknown etiology. Here we report induction of dyslipidemia characterized by severe hypertriglyceridemia as a result of point mutations in human apolipoprotein A-I (apoA-I). Adenovirus-mediated gene transfer in apoA-I-deficient (apoA-I(-)(/)(-)) mice showed that mice expressing an apoA-I[E110A/E111A] mutant had comparable hepatic mRNA levels with WT controls but greatly increased plasma triglyceride and elevated plasma cholesterol levels. In addition, they had decreased apoE and apoCII levels and increased apoB48 levels in very low-density lipoprotein (VLDL)/intermediate-density lipoprotein (IDL). Fast protein liquid chromatography (FPLC) analysis of plasma showed that most of cholesterol and approximately 15% of the mutant apoA-I were distributed in the VLDL and IDL regions and all the triglycerides in the VLDL region. Hypertriglyceridemia was corrected by coinfection of mice with recombinant adenoviruses expressing the mutant apoA-I and human lipoprotein lipase. Physicochemical studies indicated that the apoA-I mutation decreased the alpha-helical content, the stability, and the unfolding cooperativity of both lipid-free and lipid-bound apoA-I. In vitro functional analyses showed that reconstituted HDL (rHDL) particles containing the mutant apoA-I had 53% of scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux capacity and 37% capacity to activate lecithin:cholesterol acyltransferase (LCAT) as compared to the WT control. The mutant lipid-free apoA-I had normal capacity to promote ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux. The findings indicate that subtle structural alterations in apoA-I may alter the stability and functions of apoA-I and high-density lipoprotein (HDL) and may cause hypertriglyceridemia.

Asunto(s)

Alanina/genética , Apolipoproteína A-I/química , Apolipoproteína A-I/fisiología , Ácido Glutámico/genética , Hipertrigliceridemia/etiología , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenoviridae/genética , Sustitución de Aminoácidos , Animales , Apolipoproteína C-II , Apolipoproteínas C/sangre , Apolipoproteínas E/sangre , Colesterol/sangre , Cromatografía Liquida , Dicroismo Circular , Humanos , Técnicas In Vitro , Lipoproteínas/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas IDL , Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Triglicéridos/sangre

RESUMEN

We have generated and studied the pattern of expression of transgenic mouse lines carrying the human apoA-I and apoCIII gene cluster mutated at different sites. In two lines, we have either mutated the hormone-response element (HRE) of element G of the apoCIII enhancer or the C/EBP binding site of the proximal apoA-I promoter. In a third line, we have mutated the two HREs of the apoA-I promoter and the HRE of the apoCIII enhancer. Mutations in the HRE of element G reduced the hepatic and intestinal expressions of the reporter chloramphenicol acetyltransferase (CAT) gene (which substituted the apoCIII gene) to 4 and 13% of the wild-type (WT) control, whereas the hepatic and intestinal expressions of the apoA-I gene were reduced to 92 and 25% of the WT control, respectively. A mutation in the C/EBP site increased the hepatic and intestinal expressions of the apoA-I gene approximately 1.25- and 1.6-fold, respectively, and did not affect the expression of the CAT gene. The mutation in the three HNF-4 binding sites of the apoA-I promoter/apoCIII enhancer nearly abolished the expression of apoA-I and the reporter CAT gene in all tissues. These findings establish the importance of the HREs for the hepatic and intestinal expressions of the apoA-I and apoCIII genes and suggest that C/EBP does not play a central role in the expression of the apoA-I gene.

Asunto(s)

Apolipoproteína A-I/genética , Apolipoproteínas C/genética , Elementos de Facilitación Genéticos , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Regiones Promotoras Genéticas , Elementos de Respuesta , Animales , Apolipoproteína C-III , Secuencia de Bases , Sitios de Unión , Línea Celular , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Familia de Multigenes , Mutación , Transcripción Genética , Activación Transcripcional

RESUMEN

PURPOSE OF THE REVIEW: This review clarifies the functions of key proteins of the chylomicron and the HDL pathways. RECENT FINDINGS: Adenovirus-mediated gene transfer of several apolipoprotein (apo)E forms in mice showed that the amino-terminal 1-185 domain of apoE can direct receptor-mediated lipoprotein clearance in vivo. Clearance is mediated mainly by the LDL receptor. The carboxyl-terminal 261-299 domain of apoE induces hypertriglyceridemia, because of increased VLDL secretion, diminished lipolysis and inefficient VLDL clearance. Truncated apoE forms, including apoE2-202, have a dominant effect in remnant clearance and may have future therapeutic applications for the correction of remnant removal disorders. Permanent expression of apoE and apoA-I following adenoviral gene transfer protected mice from atherosclerosis. Functional assays, protein cross-linking, and adenovirus-mediated gene transfer of apoA-I mutants in apoA-I deficient mice showed that residues 220-231, as well as the central helices of apoA-I, participate in ATP-binding cassette transporter A1-mediated lipid efflux and HDL biogenesis. Following apoA-I gene transfer, an amino-terminal deletion mutant formed spherical alpha-HDL, a double amino- and carboxyl-terminal deletion mutant formed discoidal HDL, and a carboxyl-terminal deletion mutant formed only pre-beta-HDL. The findings support a model of cholesterol efflux that requires direct physical interactions between apoA-I and ATP-binding cassette transporter A1, and can explain Tangier disease and other HDL deficiencies. SUMMARY: New insights are provided into the role of apoE in cholesterol and triglyceride homeostasis, and of apoA-I in the biogenesis of HDL. Clearance of the lipoprotein remnants and increase in HDL synthesis are obvious targets for therapeutic interventions.

Asunto(s)

Adenoviridae/genética , Apolipoproteína A-I/genética , Apolipoproteínas E/genética , Quilomicrones/metabolismo , Lipoproteínas HDL/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Apolipoproteína A-I/metabolismo , Apolipoproteínas E/metabolismo , Arteriosclerosis/genética , Arteriosclerosis/metabolismo , Vectores Genéticos , Humanos , Hiperlipoproteinemia Tipo III/genética , Hiperlipoproteinemia Tipo III/metabolismo , Ratones , Modelos Animales , Transducción de Señal , Enfermedad de Tangier/genética , Enfermedad de Tangier/metabolismo

RESUMEN

A pharmacogenomic approach to therapy requires systematic knowledge of the regulatory regions of the genes, as well as basic understanding of transcriptional regulatory mechanisms of genes. Using the apolipoprotein (apo) A-I/CIII gene cluster as a model system, we have identified by in vitro and in vivo studies the regulatory elements and the factors which control its transcription. Studies in transgenic mice established that the hepatocyte nuclear factor (HNF-4) binding site of the apoCIII enhancer, which controls transcription of both genes, is required for the intestinal expression of apoA-I and apoCIII genes, and enhances synergistically their hepatic transcription in vivo. The three Sp1 sites of the enhancer are also required for the intestinal expression of apoA-land apoCIII genes in vivo, and for the enhancement of the hepatic transcription. The regulation of the apoE/apoCI/apoCIV/apoCII cluster is also cited. It is expected that identification of the regulatory regions of genes will be soon accelerated by the sequencing of several mammalian genomes. The functional analyses of the regulatory domains of genes involved in lipid homeostasis, combined with cross-species sequence comparisons in the near future, may identify natural regulatory gene polymorphisms in the general population that will permit rational pharmacogenomic approaches for treatment of dyslipidemias.

Asunto(s)

Apolipoproteína A-I/genética , Apolipoproteínas C/genética , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Mutación , Animales , Apolipoproteína C-III , Apolipoproteínas C/metabolismo , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Genes Reguladores , Factor Nuclear 4 del Hepatocito , Humanos , Técnicas In Vitro , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Farmacogenética , Fosfoproteínas/metabolismo , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/metabolismo

RESUMEN

We have mapped the domains of lipid-free apoA-I that promote cAMP-dependent and cAMP-independent cholesterol and phospholipid efflux. The cAMP-dependent lipid efflux in J774 mouse macrophages was decreased by approximately 80-92% by apoA-I[delta(185-243)], only by 15% by apoA-I[delta(1-41)] or apoA-I[delta(1-59)], and was restored to 75-80% of the wild-type apoA-I control value by double deletion mutants apoA-I[delta(1-41)delta(185-243)] and apoA-I[delta(1-59)delta(185-243)]. Similar results were obtained in HEK293 cells transfected with an ATP-binding cassette transporter A1 (ABCA1) expression plasmid. The double deletion mutant of apoA-I had reduced thermal and chemical stability compared with wild-type apoA-I. Sequential carboxyl-terminal deletions showed that cAMP-dependent cholesterol efflux was diminished in all the mutants tested, except the apoA-I[delta(232-243)] which had normal cholesterol efflux. In cAMP-untreated or in mock-transfected cells, cholesterol efflux was not affected by the amino-terminal deletions, but decreased by 30-40% and 50-65% by the carboxyl-terminal and double deletions, respectively. After adenovirus-mediated gene transfer in apoA-I-deficient mice, wild-type apoA-I and apoA-I[delta(1-41)] formed spherical high density lipoprotein (HDL) particles, whereas apoA-I[delta(1-41)delta(185-243)] formed discoidal HDL. The findings suggest that although the central helices of apoA-I alone can promote ABCA1-mediated lipid efflux, residues 220-231 are necessary to allow functional interactions between the full-length apoA-I and ABCA1 that are required for lipid efflux and HDL biogenesis.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/metabolismo , Apolipoproteína A-I/química , Apolipoproteína A-I/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/química , Adenosina Trifosfato/metabolismo , Adenoviridae/genética , Animales , Baculoviridae/metabolismo , Línea Celular , Células Cultivadas , Centrifugación por Gradiente de Densidad , Colesterol/metabolismo , Dicroismo Circular , AMP Cíclico/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Eliminación de Gen , Guanidina/farmacología , Humanos , Cinética , Ligandos , Metabolismo de los Lípidos , Hígado/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Mutación , Fosfolípidos/metabolismo , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica , Isoformas de Proteínas , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Temperatura , Factores de Tiempo , Transfección , Triglicéridos/metabolismo

RESUMEN

Apolipoprotein E (apoE) isoforms are key determinants of susceptibility to late-onset Alzheimer's disease (AD). The epsilon 4 and epsilon 2 alleles have been associated with increased and decreased risk for AD, respectively. We have generated and characterized transgenic mice in which the human apoE2 gene is expressed under the control of the platelet-derived growth factor B-chain (PDGF-B) promoter, or the transferrin (TF) promoter. S1 nuclease analysis and immunoblotting showed that the PDGF-B apoE2 mice express apoE2 exclusively in the brain whereas the TF apoE2 mice express apoE2 in the liver and in the brain. In the TF apoE2 mouse line, apoE2 is also detected in the plasma. The PDGF-B apoE2 and the TF apoE2 transgenic mice were bred back to apoE(-)(/)(-) background. Immunohistochemical analysis showed that the PDGF apoE2 x apoE(-)(/)(-) and the TF apoE2 x apoE(-)(/)(-) mice express human apoE2 within the neocortex in hippocampal neurons and glial cells, respectively. ApoE(-)(/)(-) mice have been shown to develop age-dependent loss of synaptophysin. Immunoblotting of mouse brain extracts and immunohistochemical analysis of brain sections showed that apoE expression in both apoE2 x apoE(-)(/)(-) transgenic lines was associated with significant recovery of brain synaptophysin levels as compared to the levels of apoE(-)(/)(-) littermates of the same age. These apoE2-expressing mice, when bred back on amyloid precursor protein (APP) transgenic mice or other mouse lines featuring alterations in lipoprotein metabolism, may provide new mouse models for elucidating the role of apoE2 in lipid homeostasis in the brain and in the pathogenesis of AD.

Asunto(s)

Apolipoproteínas E/genética , Neuroglía/metabolismo , Neuronas/metabolismo , Animales , Apolipoproteína E2 , Secuencia de Bases , Western Blotting , Encéfalo/metabolismo , Cartilla de ADN , Humanos , Inmunohistoquímica , Ratones , Ratones Transgénicos , Factor de Crecimiento Derivado de Plaquetas/genética , Regiones Promotoras Genéticas , Sinaptofisina/metabolismo

RESUMEN

We have studied the effects of mutations in apoA-I on reconstituted high density lipoprotein (HDL) particle (rHDL(apoA-I)) binding to and cholesterol efflux from wild-type (WT) and mutant forms of the HDL receptor SR-BI expressed by ldlA-7 cells. Mutations in helix 4 or helix 6 of the apoA-I reduced efflux by 79 and 51%, respectively, without substantially altering receptor binding (apparent K(d) values of 1.1-4.4 microg of protein/ml). SR-BI with an M158R mutation bound poorly to rHDL with WT and helix 4 mutant apoA-I; the helix 6 mutant restored tight binding to SR-BI(M158R) (K(d) values of 48, 60, and 7 microg of protein/ml, respectively). SR-BI(M158R)-mediated cholesterol efflux rates, normalized for binding, were high for all three rHDLs (71-111% of control). In contrast, absolute (12-19%) and binding-corrected (24-47%) efflux rates for all three rHDLs mediated by SR-BI with Q402R/Q418R mutations were very low. We propose that formation of a productive complex between apoA-I in rHDL and SR-BI, in which the lipoprotein and the receptor must either be precisely aligned or have the capacity to undergo appropriate conformational changes, is required for efficient SR-BI-mediated cholesterol efflux. Some mutations in apoA-I and/or SR-BI can result in high affinity, but non-productive, binding that does not permit efficient cholesterol efflux.

Asunto(s)

Apolipoproteína A-I/química , Antígenos CD36/química , Antígenos CD36/genética , Colesterol/metabolismo , Metabolismo de los Lípidos , Proteínas de la Membrana , Mutación , Receptores Inmunológicos , Receptores de Lipoproteína , Animales , Apolipoproteína A-I/metabolismo , Transporte Biológico , Células CHO , Cricetinae , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Cinética , Lipoproteínas HDL/metabolismo , Modelos Biológicos , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica , Receptores Depuradores , Receptores Depuradores de Clase B , Factores de Tiempo

RESUMEN

The high density lipoprotein receptor, scavenger receptor class B type I (SR-BI), recognizes lipid-bound apolipoprotein A-I (apoA-I) and other apolipoproteins. Here, we have used large scale cultures of apoE-expressing cells to purify apoE and prepare apoE containing reconstituted discoidal 1-palmitoyl-2-oleoyl-l-phosphatidylcholine (POPC)-apoE particles. These particles have been used to examine their binding to wild-type and mutant forms of SR-BI expressed in transfected ldlA-7 cells. Specific binding to SR-BI was determined by subtracting from the total binding, nonspecific values measured using either control untransfected ldlA-7 cells or by inhibiting SR-BI-mediated binding with a high titer antireceptor-blocking antibody. POPC-apoE particles generated using apoE2, apoE3, apoE4, or the carboxyl-terminally truncated forms apoE165, apoE202, apoE229, and apoE259 all bound tightly to wild-type SR-BI with similar affinities (K(d) = 35-45 microg/ml). Binding was nearly abolished in a cell line expressing the ldlA (Q402R/Q418R) double mutant form of SR-BI that is unable to bind native high density lipoprotein but binds low density lipoprotein normally. The findings establish that apoE is a ligand for SR-BI and that the receptor binding domain is located in the amino-terminal 1-165-region of the protein. SR-BI-apoE interactions may contribute to cholesterol homeostasis in tissues and cells expressing SR-BI that are accessible to apoE-containing lipoproteins.

Asunto(s)

Apolipoproteínas E/química , Apolipoproteínas E/metabolismo , Antígenos CD36/química , Antígenos CD36/metabolismo , Proteínas de la Membrana , Fosfolípidos/química , Receptores Inmunológicos , Receptores de Lipoproteína , Animales , Secuencia de Bases , Unión Competitiva , Encéfalo/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Cinética , Ligandos , Neoplasias Mamarias Animales/metabolismo , Ratones , Microscopía Electrónica , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Receptores Depuradores , Receptores Depuradores de Clase B , Transfección , Células Tumorales Cultivadas