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The increasing diffusion of antimicrobial resistance (AMR) across more and more bacterial species emphasizes the urgency of identifying innovative treatment strategies to counter its diffusion. Pathogen infection prevention is among the most effective strategies to prevent the spread of both disease and AMR. Since their discovery, vaccines have been the strongest prophylactic weapon against infectious diseases, with a multitude of different antigen types and formulative strategies developed over more than a century to protect populations from different pathogens. In this review, we review the main characteristics of vaccine formulations in use and under development against AMR pathogens, focusing on the importance of administering multiple antigens where possible, and the challenges associated with their development and production. The most relevant antigen classes and adjuvant systems are described, highlighting their mechanisms of action and presenting examples of their use in clinical trials against AMR. We also present an overview of the analytical and formulative strategies for multivalent vaccines, in which we discuss the complexities associated with mixing multiple components in a single formulation. This review emphasizes the importance of combining existing knowledge with advanced technologies within a Quality by Design development framework to efficiently develop vaccines against AMR pathogens.
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Paramagnetic restraints have been used in biomolecular NMR for the last three decades to elucidate and refine biomolecular structures, but also to characterize protein-ligand interactions. A common technique to generate such restraints in proteins, which do not naturally contain a (paramagnetic) metal, consists in the attachment to the protein of a lanthanide-binding-tag (LBT). In order to design such LBTs, it is important to consider the efficiency and stability of the conjugation, the geometry of the complex (conformational exchanges and coordination) and the chemical inertness of the ligand. Here we describe a photo-catalyzed thiol-ene reaction for the cysteine-selective paramagnetic tagging of proteins. As a model, we designed an LBT with a vinyl-pyridine moiety which was used to attach our tag to the protein GB1 in fast and irreversible fashion. Our tag T1 yields magnetic susceptibility tensors of significant size with different lanthanides and has been characterized using NMR and relaxometry measurements.
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Proteínas/química , Compuestos de Sulfhidrilo/química , Catálisis , Cisteína/química , Elementos de la Serie de los Lantanoides/química , Ligandos , Espectroscopía de Resonancia Magnética/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Procesos Fotoquímicos , Piridinas/químicaRESUMEN
Indole alkaloids are important natural compounds with interesting bio-activities that can be found in various species belonging to the Amaryllidaceae, Apocynaceae, or Strychnaceae families. Although these compounds have different connections, substituents, and functionalities, their main core can be produced via the formation of a common functionalized tetracyclic subunit, which is rapidly obtained by an oxidative de-aromatization process mediated by a hypervalent iodine reagent from an inexpensive phenol containing a lactate moiety as the chiral auxiliary. A subsequent stereoselective aza-Michael addition and an intramolecular Heck-type reaction lead to the formation of a common key intermediate. This approach provides a solid foundation for the further elaborations of several natural products. The asymmetric syntheses of (-)-strychnopivotine and the polycyclic main cores of (-)-strychnosplendine, (+)-isosplendine, and (+)-malagashanol, three other indole alkaloids, are reported.
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Apocynaceae , Strychnos , Ciclización , Humanos , Alcaloides Indólicos , FenolRESUMEN
Raman microscopy can reveal a compound-specific vibrational "fingerprint" from micrograms of material with no sample preparation. We expect this increasingly available instrumentation to routinely assist synthetic chemists in structure determination; however, interpreting the information-dense spectra can be challenging for unreported compounds. Appropriate theoretical calculations using the highly efficient r2SCAN-3c method can accurately predict peak positions but are less precise in matching peak heights. To limit incorrect biases while comparing experimental and theoretical spectra, we introduce a user-friendly software that gives a match score to assist with structure determination. The capabilities and limitations of this approach are demonstrated for several proof-of-concept examples including the characterization of intermediates in the total synthesis of deoxyaspidodispermine.
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BACKGROUND: The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of its enzymatic activity the degradation of the low-density lipoprotein (LDL) receptor. PCSK9 gain of function in humans leads to autosomal dominant hypercholesterolemia, whereas the absence of functional PCSK9 results in ≈7-fold lower levels of LDL cholesterol. This suggests that lowering PCSK9 may protect against atherosclerosis. METHODS AND RESULTS: We investigated the role of PCSK9 in atherosclerosis in C57BL/6 wild-type (WT), apolipoprotein E-deficient, and LDL receptor-deficient mouse models. Circulating cholesterol levels, fast protein liquid chromatography profiles, aortic cholesteryl esters (CE), and plaque sizes were determined. Intima-media thicknesses were measured by ultrasound biomicroscopy. First, mice expressing null (knockout [KO]), normal (WT), or high (transgenic [Tg]) levels of PCSK9 were fed a 12-month Western diet. KO mice accumulated 4-fold less aortic CE than WT mice, whereas Tg mice exhibited high CE and severe aortic lesions. Next we generated apolipoprotein E-deficient mice, known to spontaneously develop lesions, that expressed null (KO/e), normal (WT/e), or high (Tg/e) levels of PCSK9. After a 6-month regular diet, KO/e mice showed a 39% reduction compared with WT/e mice in aortic CE accumulation, whereas Tg/e mice showed a 137% increase. Finally, LDL receptor-deficient mice expressing no (KO/L), normal (WT/L), or high (Tg/L) levels of PCSK9 were fed a Western diet for 3 months. KO/L and Tg/L mice exhibited levels of plasma cholesterol and CE accumulation similar to those of WT/L mice, suggesting that PCSK9 modulates atherosclerosis mainly via the LDL receptor. CONCLUSIONS: Altogether, our results show a direct relationship between PCSK9 and atherosclerosis. PCSK9 overexpression is proatherogenic, whereas its absence is protective.
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Aterosclerosis/etiología , Serina Endopeptidasas/fisiología , Factores de Edad , Animales , Apolipoproteínas E/deficiencia , Aterosclerosis/prevención & control , LDL-Colesterol/sangre , Dieta , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Proproteína Convertasa 9 , Proproteína Convertasas , Receptores de LDL/fisiología , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/genética , Factores SexualesRESUMEN
Hypervalent iodine reagents are among the most fascinating reagents described in the last 30 years since they allow a plethora of different transformations and are environmentally friendly compounds that avoid the use of toxic heavy metals in most cases. Hence, their versatility has been widely used in multi-step syntheses for the formation of complex structures. In particular, the use of iodanes can easily generate complexity from simple substrates, leading to polyfunctionalized systems that allow the rapid formation of natural products or related intricate architectures. In this review, we describe a plethora of recent routes and strategies to produce complex natural products based on several key steps mediated by hypervalent iodines that help in building the desired molecule framework, highlighting the numerous advantages of these key reactants yet not hiding their possible limitations.
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With the deepening of our understanding of adaptive immunity at the cellular and molecular level, targeting antigens directly to immune cells has proven to be a successful strategy to develop innovative and potent vaccines. Indeed, it offers the potential to increase vaccine potency and/or modulate immune response quality while reducing off-target effects. With mRNA-vaccines establishing themselves as a versatile technology for future applications, in the last years several approaches have been explored to target nanoparticles-enabled mRNA-delivery systems to immune cells, with a focus on dendritic cells. Dendritic cells (DCs) are the most potent antigen presenting cells and key mediators of B- and T-cell immunity, and therefore considered as an ideal target for cell-specific antigen delivery. Indeed, improved potency of DC-targeted vaccines has been proved in vitro and in vivo. This review discusses the potential specific targets for immune system-directed mRNA delivery, as well as the different targeting ligand classes and delivery systems used for this purpose.
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Células Dendríticas , Vacunas , Inmunidad Adaptativa , Linfocitos T , AntígenosRESUMEN
AIMS: The adenylate cyclase type 9 (ADCY9) gene appears to determine atherosclerotic outcomes in patients treated with dalcetrapib. In mice, we recently demonstrated that Adcy9 inactivation potentiates endothelial function and inhibits atherogenesis. The objective of this study was to characterize the contribution of ADCY9 to the regulation of endothelial signalling pathways involved in atherosclerosis. METHODS AND RESULTS: We show that ADCY9 is expressed in the endothelium of mouse aorta and femoral arteries. We demonstrate that ADCY9 inactivation in cultured endothelial cells paradoxically increases cAMP accumulation in response to the adenylate cyclase activators forskolin and vasoactive intestinal peptide (VIP). Reciprocally, ADCY9 overexpression decreases cAMP production. Using mouse femoral artery arteriography, we show that Adcy9 inactivation potentiates VIP-induced endothelial-dependent vasodilation. Moreover, Adcy9 inactivation reduces mouse atheroma endothelial permeability in different vascular beds. ADCY9 overexpression reduces forskolin-induced phosphorylation of Ser157-vasodilator-stimulated phosphoprotein (VASP) and worsens thrombin-induced fall of RAP1 activity, both leading to increased endothelial permeability. ADCY9 inactivation in thrombin-stimulated human coronary artery endothelial cells results in cAMP accumulation, increases p-Ser157-VASP, and inhibits endothelial permeability. MLC2 phosphorylation and actin stress fibre increases in response to thrombin were reduced by ADCY9 inactivation, suggesting actin cytoskeleton regulation. Finally, using the Miles assay, we demonstrate that Adcy9 regulates thrombin-induced endothelial permeability in vivo in normal and atherosclerotic animals. CONCLUSION: Adcy9 is expressed in endothelial cells and regulates local cAMP and endothelial functions including permeability relevant to atherogenesis.
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Adenilil Ciclasas , Aterosclerosis , Animales , Humanos , Ratones , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Aterosclerosis/genética , Aterosclerosis/prevención & control , Aterosclerosis/metabolismo , Colforsina/farmacología , Colforsina/metabolismo , Células Endoteliales/metabolismo , Endotelio/metabolismo , Trombina/metabolismo , AMP Cíclico/metabolismoRESUMEN
Macrophage foam cell is the predominant cell type in atherosclerotic lesions. Removal of excess cholesterol from macrophages thus offers effective protection against atherosclerosis. Here we report that a protein kinase A (PKA)-anchoring inhibitor, st-Ht31, induces robust cholesterol/phospholipid efflux, and ATP-binding cassette transporter A1 (ABCA1) greatly facilitates this process. Remarkably, we found that st-Ht31 completely reverses foam cell formation, and this process is ABCA1-dependent. The reversal is also accompanied by the restoration of well modulated inflammatory response to LPS. There is no detectable toxicity associated with st-Ht31, even when cells export up to 20% cellular cholesterol per hour. Using FRET-based PKA biosensors in live cells, we provide evidence that st-Ht31 drives cholesterol efflux by elevating PKA activity specifically in the cytoplasm. Furthermore, ABCA1 facilitates st-Ht31 uptake. This allows st-Ht31 to effectively remove cholesterol from ABCA1-expressing cells. We speculate that de-anchoring of PKA offers a novel therapeutic strategy to remove excess cholesterol from lipid-laden lesion macrophages.
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Transportadoras de Casetes de Unión a ATP/fisiología , Colesterol/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Células Espumosas/efectos de los fármacos , Proteínas/farmacología , Transportador 1 de Casete de Unión a ATP , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Células Espumosas/citología , Macrófagos/citología , RatonesRESUMEN
Langerin is a mammalian C-type lectin expressed on Langerhans cells in the skin. As an innate immune cell receptor, Langerin is involved in coordinating innate and adaptive immune responses against various incoming threats. We have previously reported a series of thiazolopyrimidines as murine Langerin ligands. Prompted by the observation that its human homologue exhibits different binding specificities for these small molecules, we report here our investigations to define their exact binding site. By using structural comparison and molecular dynamics simulations, we showed that the nonconserved short loops have a high degree of conformational flexibility between the human and murine homologues. Sequence analysis and mutational studies indicated that a pair of residues are essential for the recognition of the thiazolopyrimidines. Taking solvent paramagnetic relaxation enhancement NMR studies together with a series of peptides occupying the same site, we could define the cleft between the short and long loops as the allosteric binding site for these aromatic heterocycles.
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Lectinas Tipo C , Lectinas de Unión a Manosa , Humanos , Ratones , Animales , Lectinas Tipo C/metabolismo , Lectinas de Unión a Manosa/metabolismo , Sitio Alostérico , Ligandos , Antígenos CD/metabolismo , Sitios de Unión , Solventes , Mamíferos/metabolismoRESUMEN
Carbohydrate-protein interactions are key for cell-cell and host-pathogen recognition and thus, emerged as viable therapeutic targets. However, their hydrophilic nature poses major limitations to the conventional development of drug-like inhibitors. To address this shortcoming, four fragment libraries were screened to identify metal-binding pharmacophores (MBPs) as novel scaffolds for inhibition of Ca2+-dependent carbohydrate-protein interactions. Here, we show the effect of MBPs on the clinically relevant lectins DC-SIGN, Langerin, LecA and LecB. Detailed structural and biochemical investigations revealed the specificity of MBPs for different Ca2+-dependent lectins. Exploring the structure-activity relationships of several fragments uncovered the functional groups in the MBPs suitable for modification to further improve lectin binding and selectivity. Selected inhibitors bound efficiently to DC-SIGN-expressing cells. Altogether, the discovery of MBPs as a promising class of Ca2+-dependent lectin inhibitors creates a foundation for fragment-based ligand design for future drug discovery campaigns.
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DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) is a pattern recognition receptor expressed on immune cells and involved in the recognition of carbohydrate signatures present on various pathogens, including HIV, Ebola, and SARS-CoV-2. Therefore, developing inhibitors blocking the carbohydrate-binding site of DC-SIGN could generate a valuable tool to investigate the role of this receptor in several infectious diseases. Herein, we performed a fragment-based ligand design using 4-quinolone as a scaffold. We synthesized a library of 61 compounds, performed a screening against DC-SIGN using an STD reporter assay, and validated these data using protein-based 1H-15N HSQC NMR. Based on the structure-activity relationship data, we demonstrate that ethoxycarbonyl or dimethylaminocarbonyl in position 2 or 3 is favorable for the DC-SIGN binding activity, especially in combination with fluorine, ethoxycarbonyl, or dimethylaminocarbonyl in position 7 or 8. Furthermore, we demonstrate that these quinolones can allosterically modulate the carbohydrate binding site, which offers an alternative approach toward this challenging protein target.
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Two orthogonal, metal free click reactions, enabled to glycosylate ubiquitin and its mutant A28C forming two protein scaffolds with high affinity for BambL, a lectin from the human pathogen Burkholderia ambifaria. A new fucoside analogue, with high affinity with BambL, firstly synthetized and co-crystallized with the protein target, provided the insights for sugar determinants grafting onto ubiquitin. Three ubiquitin-based glycosides were thus assembled. Fuc-Ub, presented several copies of the fucoside analogue, with proper geometry for multivalent effect; Rha-A28C, displayed one thio-rhamnose, known for its ability to tuning the immunological response; finally, Fuc-Rha-A28C, included both multiple fucoside analogs and the rhamnose residue. Fuc-Ub and Fuc-Rha-A28C ligands proved high affinity for BambL and unprecedented immune modulatory properties towards macrophages activation.
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Although cholesterol is synthesized in the endoplasmic reticulum (ER), compared with other cellular membranes, ER membrane has low cholesterol (3-6%). Most of the molecular machinery that regulates cellular cholesterol homeostasis also resides in the ER. Little is known about how cholesterol itself affects the ER membrane. Here, we demonstrate that acute cholesterol depletion in ER membranes impairs ER-to-Golgi transport of secretory membrane proteins. Cholesterol depletion is achieved by a brief inhibition of cholesterol synthesis with statins in cells grown in cholesterol-depleted medium. We provide evidence that secretory membrane proteins vesicular stomatitis virus glycoprotein and scavenger receptor A failed to be efficiently transported from the ER upon cholesterol depletion. Fluorescence photobleaching recovery experiments indicated that cholesterol depletion by statins leads to a severe loss of lateral mobility on the ER membrane of these transmembrane proteins, but not loss of mobility of proteins in the ER lumen. This impaired lateral mobility is correlated with impaired ER-to-Golgi transport. These results provide evidence for the first time that cholesterol is required in the ER membrane to maintain mobility of membrane proteins and thus protein secretion.
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Colesterol/deficiencia , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Retículo Endoplásmico/efectos de los fármacos , Recuperación de Fluorescencia tras Fotoblanqueo , Aparato de Golgi/ultraestructura , Hidroximetilglutaril-CoA Reductasas/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Glicoproteínas de Membrana/metabolismo , Ácido Mevalónico/farmacología , Transporte de Proteínas , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/metabolismo , Proteínas del Envoltorio Viral/metabolismoRESUMEN
BACKGROUND: Given the link between cholesterol and activation of inflammation via interleukin 1ß (IL-1ß), we tested the effects of IL-1ß inhibition on atherosclerotic calcification in mice. Patients with familial hypercholesterolemia develop extensive aortic calcification and calcific aortic stenosis. Although statins delay this process, low-density lipoprotein (LDL) cholesterol lowering alone is not enough to avert it. Data suggest that vascular inflammation initiated by hypercholesterolemia is followed by unchecked mineralization at sites of atherosclerotic plaques. The LDL-receptor (LDLR)-deficient (Ldlr(-/-)) and LDLR-attenuated Pcsk9(Tg) mice are available animal models for pharmacological testing. METHODS: A mouse monoclonal antibody (mAb) against IL-1ß or placebo was administered subcutaneously in Ldlr(-/-) and Pcsk9(Tg) models fed a Western diet. Drug level, anthropometric, lipid, and glucose profiles were determined. Expressions of proprotein convertase subtilisin/kexin type 9 (PCSK9), serum amyloid A1, and cytokine were measured by enzyme-linked immunosorbent assay. Aortic calcification was determined by microcomputerized tomography (micro-CT) and X-ray densitometry, and aortic flow velocity was assessed by ultrasound. RESULTS: Circulating levels of IL-1ß in Ldlr(-/-) mice were significantly greater (2-fold) than observed in Pcsk9(Tg) mice. Placebo- and mAb-treated mice did not differ in their growth, lipid, glucose profiles, and other cytokines. Calcifications were significantly diminished in mAb-treatment Ldlr(-/-) mice (a reduction of â¼ 75% by X-ray and â¼ 90% by micro-CT) and reduced insignificantly in mAb-treatment Pcsk9(Tg) mice, whereas aortic flow velocity was unchanged in both models. CONCLUSIONS: Herein, we demonstrate that aortic calcifications can be inhibited by an IL-1ß mAb in LDLR-deficient mice. These results have a translational component to prevent vascular calcification in human and represent new evidence to rationalize targeting inflammation in cardiovascular disease.
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Antiinflamatorios/farmacología , Anticuerpos Monoclonales/farmacología , Aorta/efectos de los fármacos , Enfermedades de la Aorta/prevención & control , Hiperlipoproteinemia Tipo II/tratamiento farmacológico , Interleucina-1beta/antagonistas & inhibidores , Calcificación Vascular/prevención & control , Animales , Aorta/diagnóstico por imagen , Aorta/metabolismo , Aorta/fisiopatología , Enfermedades de la Aorta/sangre , Enfermedades de la Aorta/diagnóstico , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/fisiopatología , Aortografía/métodos , Biomarcadores/sangre , Velocidad del Flujo Sanguíneo , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Hiperlipoproteinemia Tipo II/sangre , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo II/inmunología , Interleucina-1beta/sangre , Interleucina-1beta/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Proproteína Convertasas/genética , Proproteína Convertasas/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Flujo Sanguíneo Regional , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Ultrasonografía , Calcificación Vascular/diagnóstico , Calcificación Vascular/genética , Calcificación Vascular/inmunología , Calcificación Vascular/metabolismo , Calcificación Vascular/fisiopatología , Microtomografía por Rayos XRESUMEN
BACKGROUND: Of the cells that compose the atherosclerotic plaque, vascular endothelial cells are the most resistant to cholesterol accumulation. Cholesterol efflux pathways may play an important role in endothelial cholesterol homeostasis. METHODS AND RESULTS: We examined the global genetic response of endothelial cells to cholesterol and in particular the contribution of the cholesterol efflux proteins ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), and scavenger receptor B-I (SR-BI) to endothelial cell cholesterol efflux. The ABCG1 gene is induced in endothelial cells by cholesterol, whereas ABCA1 is not. Using specific chemical inhibitors of ABC transporters and SR-BI, we have shown that neither ABC transporters nor SR-BI is required for apolipoprotein A-1-mediated endothelial cholesterol efflux. CONCLUSIONS: Endothelial cells may use nontraditional pathways for cholesterol efflux.
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Colesterol/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/fisiología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Transportador 1 de Casete de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Aorta , Apolipoproteína A-I/farmacología , Antígenos CD36 , Células COS , Bovinos , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Chlorocebus aethiops , Colesterol/farmacología , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Gliburida/farmacología , Humanos , Hidroxicolesteroles/farmacología , Lipoproteínas HDL/farmacología , Lipoproteínas HDL3 , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Receptores Depuradores , Proteínas Recombinantes de Fusión/fisiología , Albúmina Sérica Bovina/farmacología , Enfermedad de Tangier/genética , Enfermedad de Tangier/patología , Transfección , Venas UmbilicalesRESUMEN
BACKGROUND: Impairment of acid sphingomyelinase (SMase) results in accumulation of sphingomyelin (SM) and cholesterol in late endosomes, the hallmarks of a lysosomal storage disease. OBJECTIVE: We describe cellular lipid metabolism in fibroblasts from two patients with novel compound heterozygote mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene manifesting as Niemann-Pick disease type B (NPB) and demonstrate mechanisms to overcome the storage defect. METHODS: Using biochemical assays and confocal microscopy, we provide evidence that accumulated lysosomal SM and cholesterol can be released by different treatments. RESULTS: Defective SMase activity in these fibroblasts results in a 2.5-fold increased cellular mass of SM and cholesterol, increased de novo endogenous cholesterol synthesis, and decreased cholesterol esterification, demonstrating impaired intracellular cholesterol homeostasis. Depletion of exogenous addition of cholesterol for 24 hours or addition of the cholesterol acceptor apolipoprotein A-I are sufficient to restore normal homeostatic responses. In an effort to correct the lysosomal storage phenotype of NPB, we infected the fibroblasts with a lentivirus expressing the phosphotyrosine binding domain of the adapter protein GULP (PTB-GULP). We have previously shown that expression of PTB-GULP in Chinese hamster ovary cells promotes intracellular cholesterol trafficking and ABCA1-mediated cholesterol efflux. We find that expression of PTB-GULP in NPB fibroblasts results in increased ABCA1 expression, increased cellular cholesterol efflux and lysosomal cholesterol redistribution, independent of the impaired SMase and cholesterol presence. CONCLUSION: We provide extensive functional characterization of a novel compound heterozygote mutation and provide a novel functional mechanism to overcome lysosomal storage disease defects.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Colesterol/metabolismo , Fibroblastos/metabolismo , Fosfotirosina/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Esterificación , Femenino , Fibroblastos/citología , Heterocigoto , Humanos , Lisosomas/metabolismo , Masculino , Microscopía Fluorescente , Persona de Mediana Edad , Mutación , Enfermedad de Niemann-Pick Tipo B/metabolismo , Enfermedad de Niemann-Pick Tipo B/patología , Fosfotirosina/química , Unión Proteica , Esfingomielina Fosfodiesterasa/genética , Esfingomielinas/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , TransfecciónRESUMEN
Proprotein convertase subtilisin/kexin-9 (PCSK9) enhances the degradation of hepatic low-density lipoprotein receptor (LDLR). Deletion of PCSK9, and loss-of-function mutants in humans result in lower levels of circulating LDL-cholesterol and a strong protection against coronary heart disease. Accordingly, the quest for PCSK9 inhibitors has major clinical implications. We have previously identified annexin A2 (AnxA2) as an endogenous binding partner and functional inhibitor of PCSK9. Herein, we studied the relevance of AnxA2 in PCSK9 inhibition and lipid metabolism in vivo. Plasma analyses of AnxA2(-/-) mice revealed: i) a â¼1.4-fold increase in LDL-cholesterol without significant changes in VLDLs or HDLs, and ii) a â¼2-fold increase in circulating PCSK9 levels. Western blotting and immunohistochemistry of AnxA2(-/-) tissues revealed that the LDLR was decreased by â¼50% in extrahepatic tissues, such as adrenals and colon. We also show that AnxA2-derived synthetic peptides block the PCSK9≡LDLR interaction in vitro, and adenoviral overexpression of AnxA2 in mouse liver increases LDLR protein levels in vivo. These results suggest that AnxA2 acts as an endogenous regulator of LDLR degradation, mostly in extrahepatic tissues. Finally, we identified an AnxA2 coding polymorphism, V98L, that correlates with lower circulating levels of PCSK9 thereby extending our results on the physiological role of AnxA2 in humans.
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Anexina A2/metabolismo , Hígado/metabolismo , Proproteína Convertasas/metabolismo , Proteolisis , Receptores de LDL/metabolismo , Serina Endopeptidasas/metabolismo , Adenoviridae/genética , Secuencia de Aminoácidos , Animales , Anexina A2/química , Anexina A2/deficiencia , Anexina A2/genética , Línea Celular , LDL-Colesterol/sangre , Cricetinae , Exones/genética , Espacio Extracelular/metabolismo , Humanos , Hígado/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Proproteína Convertasa 9 , Proproteína Convertasas/sangre , Estructura Terciaria de Proteína , Serina Endopeptidasas/sangreRESUMEN
OBJECTIVE: Patients with familial hypercholesterolemia (FH) due mutations in the low-density lipoprotein receptor (LDLR) suffer premature aortic calcification, an effect that is age- and gene dosage-dependent and cholesterol level independent later in life. To better understand this process, we examined a murine model. METHODS: We compared chow fed Ldlr(-/-) mice to controls at 6, 12 and 18 months and on a Western diet (WD) at 6 months. Additionally, we compared controls to Ldlr(-/-) mice and transgenic mice Tg(Pcsk9) overexpressing PCSK9, which promotes LDLR degradation. Aortas were perfused-fixed, embedded in paraffin, and sections were stained with alizarin red. Micro-computerized tomography (micro-CT) was used to quantify vascular calcification. RESULTS: Ldlr(-/-) mice develop calcification in the ascending, transverse aorta and neck vessels with a distribution similar to that of human. Calcification was most prominent in 18-month-old Ldlr(-/-) mice fed a chow diet and in 6-month-old Ldlr(-/-) mice fed a WD. Interestingly, Tg(Pcsk9) mice fed a WD develop aortic calcifications as well. Histology confirmed that the calcification were predominantly sub-intimal. Marked expression of LRP5 and WNT was observed in the Ldlr(-/-) and Tg(Pcsk9) models, but not in age-matched controls. CONCLUSIONS: The two mouse models develop aortic calcification in an age- and diet-dependent manner. Abnormal regulation of the LRP5/Wnt pathway may play a role in the calcification process. Further analysis of these aortic calcification models using this micro-CT imaging technique may provide a better understanding of the link between FH and arterial calcification.
Asunto(s)
Enfermedades de la Aorta/diagnóstico por imagen , Enfermedades de la Aorta/metabolismo , Aortografía/métodos , Receptores de LDL/deficiencia , Calcificación Vascular/diagnóstico por imagen , Calcificación Vascular/metabolismo , Microtomografía por Rayos X , Factores de Edad , Envejecimiento , Animales , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Biomarcadores/sangre , Colesterol/sangre , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Adhesión en Parafina , Proproteína Convertasa 9 , Proproteína Convertasas , Receptores de LDL/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Índice de Severidad de la Enfermedad , Coloración y Etiquetado , Calcificación Vascular/genética , Calcificación Vascular/patología , Proteínas Wnt/metabolismoRESUMEN
ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol efflux to lipid-poor apolipoprotein A-I (apoA-I) and generates HDL. Here, we demonstrate that ABCA1 also directly mediates the production of apoA-I free microparticles. In baby hamster kidney (BHK) cells and RAW macrophages, ABCA1 expression led to lipid efflux in the absence of apoA-I and released large microparticles devoid of apoB and apoE. We provide evidence that these microparticles are an integral component of the classical cholesterol efflux pathway when apoA-I is present and accounted for approximately 30% of the total cholesterol released to the medium. Furthermore, microparticle release required similar ABCA1 activities as was required for HDL production. For instance, a nucleotide binding domain mutation in ABCA1 (A937V) that impaired HDL generation also abolished microparticle release. Similarly, inhibition of protein kinase A (PKA) prevented the release of both types of particles. Interestingly, physical modulation of membrane dynamics affected HDL and microparticle production, rigidifying the plasma membrane with wheat germ agglutinin inhibited HDL and microparticle release, whereas increasing the fluidity promoted the production of these particles. Given the established role of ABCA1 in expending nonraft or more fluid-like membrane domains, our results suggest that both HDL and microparticle release is favored by a more fluid plasma membrane. We speculate that ABCA1 enhances the dynamic movement of the plasma membrane, which is required for apoA-I lipidation and microparticle formation.