RESUMEN
Cytotoxic T lymphocytes (CTLs) recognize peptides presented by HLA class I molecules on the cell surface. The C terminus of these CTL epitopes is considered to be produced by the proteasome. Here we demonstrate that the cytosolic endopeptidases nardilysin and thimet oligopeptidase (TOP) complemented proteasome activity. Nardilysin and TOP were required, either together or alone, for the generation of a tumor-specific CTL epitope from PRAME, an immunodominant CTL epitope from Epstein-Barr virus protein EBNA3C, and a clinically important epitope from the melanoma protein MART-1. TOP functioned as C-terminal trimming peptidase in antigen processing, and nardilysin contributed to both the C-terminal and N-terminal generation of CTL epitopes. By broadening the antigenic peptide repertoire, nardilysin and TOP strengthen the immune defense against intracellular pathogens and cancer.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Epítopos de Linfocito T/metabolismo , Metaloendopeptidasas/metabolismo , Linfocitos T Citotóxicos/metabolismo , Presentación de Antígeno/genética , Antígenos de Neoplasias/química , Antígenos de Neoplasias/inmunología , Citotoxicidad Inmunológica/genética , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Antígeno HLA-A3/metabolismo , Humanos , Células K562 , Metaloendopeptidasas/genética , Metaloendopeptidasas/inmunología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , ARN Interferente Pequeño/genética , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/patología , Transgenes/genéticaRESUMEN
Elevated plasma lipoprotein(a) (Lp(a)) is an independent, causal risk factor for atherosclerotic cardiovascular disease and calcific aortic valve stenosis. Lp(a) is formed in or on hepatocytes from successive noncovalent and covalent interactions between apo(a) and apoB, although the subcellular location of these interactions and the nature of the apoB-containing particle involved remain unclear. Sortilin, encoded by the SORT1 gene, modulates apoB secretion and LDL clearance. We used a HepG2 cell model to study the secretion kinetics of apo(a) and apoB. Overexpression of sortilin increased apo(a) secretion, while siRNA-mediated knockdown of sortilin expression correspondingly decreased apo(a) secretion. Sortilin binds LDL but not apo(a) or Lp(a), indicating that its effect on apo(a) secretion is likely indirect. Indeed, the effect was dependent on the ability of apo(a) to interact noncovalently with apoB. Overexpression of sortilin enhanced internalization of Lp(a), but not apo(a), by HepG2 cells, although neither sortilin knockdown in these cells or Sort1 deficiency in mice impacted Lp(a) uptake. We found several missense mutations in SORT1 in patients with extremely high Lp(a) levels; sortilin containing some of these mutations was more effective at promoting apo(a) secretion than WT sortilin, though no differences were found with respect to Lp(a) internalization. Our observations suggest that sortilin could play a role in determining plasma Lp(a) levels and corroborate in vivo human kinetic studies which imply that secretion of apo(a) and apoB are coupled, likely within the hepatocyte.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular , Apolipoproteínas B , Hiperlipidemias , Lipoproteína(a) , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Apolipoproteínas A/metabolismo , Apolipoproteínas B/metabolismo , Apoproteína(a) , Células Hep G2 , Humanos , Cinética , Lipoproteína(a)/metabolismo , RatonesRESUMEN
Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes lysosomal degradation of the LDL receptor and is a key regulator of cholesterol metabolism. After the liver, the small intestine is the second organ that highly expresses PCSK9. However, the small intestine's ability to secrete PCSK9 remains a matter of debate. While liver-specific PCSK9-deficient mice present no PCSK9 in systemic blood, human intestinal Caco-2 cells can actively secrete PCSK9. This raises the possibility for active intestinal secretion via the portal blood. Here, we aimed to determine whether enterocytes can secrete PCSK9 using in vitro, ex vivo, and in vivo approaches. We first observed that PCSK9 secretion from Caco-2 cells was biphasic and dependent on Caco-2 maturation status. Transcriptional analysis suggested that this transient reduction in PCSK9 secretion might be due to loss of SREBP2-mediated transcription of PCSK9. Consistently, PCSK9 secretion was not detected ex vivo in human or mouse intestinal biopsies mounted in Ussing chambers. Finally, direct comparison of systemic versus portal blood PCSK9 concentrations in WT or liver-specific PCSK9-deficient mice confirmed the inability of the small intestine to secrete PCSK9 into the portal compartment. Altogether, our data demonstrate that mature enterocytes do not secrete PCSK9 and reinforce the central role of the liver in the regulation of the concentration of circulating PCSK9 and consequently of cellular LDL receptors.
Asunto(s)
Proproteína Convertasa 9/metabolismo , Animales , Células CACO-2 , Diferenciación Celular , Células Cultivadas , Humanos , Intestino Delgado/citología , Intestino Delgado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proproteína Convertasa 9/sangre , Proproteína Convertasa 9/deficienciaRESUMEN
OBJECTIVE: Increased postprandial lipemia (PPL) is an independent risk factor for atherosclerotic cardiovascular diseases. PCSK9 (Proprotein convertase subtilisin kexin type 9) is an endogenous inhibitor of the LDLR (low-density lipoprotein receptor) pathway. We previously showed that PCSK9 inhibition in mice reduces PPL. However, the relative contribution of intracellular intestinal PCSK9 or liver-derived circulating PCSK9 to this effect is still unclear. Approach and Results: To address this issue, we generated the first intestine-specific Pcsk9-deficient (i-Pcsk9-/-) mouse model. PPL was measured in i-Pcsk9-/- as well as in wild-type and streptozotocin-induced diabetic mice following treatment with a PCSK9 monoclonal antibody (alirocumab). Blocking the circulating form of PCSK9 with alirocumab significantly reduced PPL, while overexpressing human PCSK9 in the liver of full Pcsk9-/- mice had the opposite effect. Alirocumab regulated PPL in a LDLR-dependent manner as this effect was abolished in Ldlr-/- mice. In contrast, i-Pcsk9-/- mice did not exhibit alterations in plasma lipid parameters nor in PPL. Finally, PPL was highly exacerbated by streptozotocin-induced diabetes mellitus in Pcsk9+/+ but not in Pcsk9-/- mice, an effect that was mimicked by the use of alirocumab in streptozotocin-treated Pcsk9+/+ mice. CONCLUSIONS: Taken together, our data demonstrate that PPL is significantly altered by full but not intestinal PCSK9 deficiency. Treatment with a PCSK9 monoclonal antibody mimics the effect of PCSK9 deficiency on PPL suggesting that circulating PCSK9 rather than intestinal PCSK9 is a critical regulator of PPL. These data validate the clinical relevance of PCSK9 inhibitors to reduce PPL, especially in patients with type 2 diabetes mellitus.
Asunto(s)
Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Tipo 2/sangre , Hiperlipidemias/sangre , Intestinos/enzimología , Lípidos/sangre , Proproteína Convertasa 9/sangre , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/enzimología , Hiperlipidemias/enzimología , Hiperlipidemias/genética , Hiperlipidemias/prevención & control , Hipolipemiantes/farmacología , Intestinos/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibidores de PCSK9 , Periodo Posprandial , Proproteína Convertasa 9/deficiencia , Proproteína Convertasa 9/genética , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMEN
RATIONALE: Monocyte infiltration into the subintimal space and its intracellular lipid accumulation are the most prominent features of atherosclerosis. To understand the pathophysiology of atherosclerotic disease, we need to understand the characteristics of lipid-laden foamy macrophages in the subintimal space during atherosclerosis. OBJECTIVE: We sought to examine the transcriptomic profiles of foamy and nonfoamy macrophages isolated from atherosclerotic intima. METHODS AND RESULTS: Single-cell RNA sequencing analysis of CD45+ leukocytes from murine atherosclerotic aorta revealed that there are macrophage subpopulations with distinct differentially expressed genes involved in various functional pathways. To specifically characterize the intimal foamy macrophages of plaque, we developed a lipid staining-based flow cytometric method for analyzing the lipid-laden foam cells of atherosclerotic aortas. We used the fluorescent lipid probe BODIPY493/503 and assessed side-scattered light as an indication of cellular granularity. BODIPYhiSSChi foamy macrophages were found residing in intima and expressing CD11c. Foamy macrophage accumulation determined by flow cytometry was positively correlated with the severity of atherosclerosis. Bulk RNA sequencing analysis showed that compared with nonfoamy macrophages, foamy macrophages expressed few inflammatory genes but many lipid-processing genes. Intimal nonfoamy macrophages formed the major population expressing IL (interleukin)-1ß and many other inflammatory transcripts in atherosclerotic aorta. CONCLUSIONS: RNA sequencing analysis of intimal macrophages from atherosclerotic aorta revealed that lipid-loaded plaque macrophages are not likely the plaque macrophages that drive lesional inflammation.
Asunto(s)
Macrófagos/metabolismo , Placa Aterosclerótica/metabolismo , Transcriptoma , Animales , Aorta/metabolismo , Aorta/patología , Células Cultivadas , Humanos , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Placa Aterosclerótica/patologíaRESUMEN
The secretory proprotein convertase (PC) family comprises nine members, as follows: PC1/3, PC2, furin, PC4, PC5/6, paired basic amino acid cleaving enzyme 4, PC7, subtilisin kexin isozyme 1/site 1 protease (SKI-1/S1P), and PC subtilisin/kexin type 9 (PCSK9). The first seven PCs cleave their substrates at single/paired basic residues and exhibit specific and often essential functions during development and/or in adulthood. The essential SKI-1/S1P cleaves membrane-bound transcription factors at nonbasic residues. In contrast, PCSK9 cleaves itself once, and the secreted inactive protease drags the low-density lipoprotein receptors (LDLR) and very LDLR (VLDLR) to endosomal/lysosomal degradation. Inhibitory PCSK9 monoclonal antibodies are now prescribed to treat hypercholesterolemia. This review focuses on the implication of PCs in cardiovascular functions and diseases, with a major emphasis on PCSK9. We present a phylogeny of the PCs and the analysis of PCSK9 haplotypes in modern and archaic human species. The absence of PCSK9 in mice led to the discovery of a sex- and tissue-specific subcellular distribution of the LDLR and VLDLR. PCSK9 inhibition may have other applications because it reduces inflammation and sepsis in a LDLR-dependent manner. Our present understanding of the cellular mechanism(s) that enables PCSK9 to induce the degradation of receptors is reviewed, as well as the consequences of its key natural mutations. The PCSK9 ongoing clinical trials are reviewed. Finally, how the other PCs may impact cardiovascular disease and the metabolic syndrome, and become relevant targets, is discussed.
Asunto(s)
Colesterol/sangre , Hipercolesterolemia/enzimología , Proproteína Convertasa 9/metabolismo , Animales , Anticolesterolemiantes/uso terapéutico , Biomarcadores/sangre , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/enzimología , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/prevención & control , Femenino , Haplotipos , Humanos , Hipercolesterolemia/sangre , Hipercolesterolemia/complicaciones , Hipercolesterolemia/tratamiento farmacológico , Lipoproteína(a)/sangre , Masculino , Mutación , Inhibidores de PCSK9 , Filogenia , Proproteína Convertasa 9/genética , Proteolisis , Receptores de LDL/metabolismo , Inhibidores de Serina Proteinasa/uso terapéuticoRESUMEN
Accumulating evidence implicates endoplasmic reticulum (ER) stress as a mediator of impaired lipid metabolism, thereby contributing to fatty liver disease and atherosclerosis. Previous studies demonstrated that ER stress can activate the sterol regulatory element-binding protein-2 (SREBP2), an ER-localized transcription factor that directly up-regulates sterol regulatory genes, including PCSK9 Given that PCSK9 contributes to atherosclerosis by targeting low density lipoprotein (LDL) receptor (LDLR) degradation, this study investigates a novel mechanism by which ER stress plays a role in lipid metabolism by examining its ability to modulate PCSK9 expression. Herein, we demonstrate the existence of two independent effects of ER stress on PCSK9 expression and secretion. In cultured HuH7 and HepG2 cells, agents or conditions that cause ER Ca2+ depletion, including thapsigargin, induced SREBP2-dependent up-regulation of PCSK9 expression. In contrast, a significant reduction in the secreted form of PCSK9 protein was observed in the media from both thapsigargin- and tunicamycin (TM)-treated HuH7 cells, mouse primary hepatocytes, and in the plasma of TM-treated C57BL/6 mice. Furthermore, TM significantly increased hepatic LDLR expression and reduced plasma LDL concentrations in mice. Based on these findings, we propose a model in which ER Ca2+ depletion promotes the activation of SREBP2 and subsequent transcription of PCSK9. However, conditions that cause ER stress regardless of their ability to dysregulate ER Ca2+ inhibit PCSK9 secretion, thereby reducing PCSK9-mediated LDLR degradation and promoting LDLR-dependent hepatic cholesterol uptake. Taken together, our studies provide evidence that the retention of PCSK9 in the ER may serve as a potential strategy for lowering LDL cholesterol levels.
Asunto(s)
Calcio/metabolismo , Estrés del Retículo Endoplásmico , Regulación Enzimológica de la Expresión Génica , Hepatocitos/metabolismo , Metabolismo de los Lípidos , Proproteína Convertasa 9/biosíntesis , Animales , Células Hep G2 , Humanos , Masculino , Ratones , Proproteína Convertasa 9/genética , Proteolisis , Receptores de LDL/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismoRESUMEN
Protein C, a secretory vitamin K-dependent anticoagulant serine protease, inactivates factors Va/VIIIa. It is exclusively synthesized in liver hepatocytes as an inactive zymogen (proprotein C). In humans, thrombin cleavage of the propeptide at PR221↓ results in activated protein C (APC; residues 222-461). However, the propeptide is also cleaved by a furin-like proprotein convertase(s) (PCs) at KKRSHLKR199↓ (underlined basic residues critical for the recognition by PCs), but the order of cleavage is unknown. Herein, we present evidence that at the surface of COS-1 cells, mouse proprotein C is first cleaved by the convertases furin, PC5/6A, and PACE4. In mice, this cleavage occurs at the equivalent site, KKRKILKR198↓, and requires the presence of Arg198 at P1 and a combination of two other basic residues at either P2 (Lys197), P6 (Arg193), or P8 (Lys191) positions. Notably, the thrombin-resistant R221A mutant is still cleaved by these PCs, revealing that convertase cleavage can precede thrombin activation. This conclusion was supported by the fact that the APC-specific activity in the medium of COS-1 cells is exclusively dependent on prior cleavage by the convertases, because both R198A and R221A lack protein C activity. Primary cultures of hepatocytes derived from wild-type or hepatocyte-specific furin, PC5/6, or complete PACE4 knock-out mice suggested that the cleavage of overexpressed proprotein C is predominantly performed by furin intracellularly and by all three proprotein convertases at the cell surface. Indeed, plasma analyses of single-proprotein convertase-knock-out mice showed that loss of the convertase furin or PC5/6 in hepatocytes results in a â¼30% decrease in APC levels, with no significant contribution from PACE4. We conclude that prior convertase cleavage of protein C in hepatocytes is critical for its thrombin activation.
Asunto(s)
Hepatocitos/enzimología , Hígado/enzimología , Proproteína Convertasa 5/metabolismo , Proteína C/metabolismo , Sustitución de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Activación Enzimática/fisiología , Células Hep G2 , Humanos , Ratones , Ratones Noqueados , Mutación Missense , Proproteína Convertasa 5/genética , Proproteína Convertasas/genética , Proproteína Convertasas/metabolismo , Proteína C/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Trombina/genética , Trombina/metabolismoRESUMEN
BACKGROUND & AIMS: Inefficient fatty acid oxidation in mitochondria and increased oxidative damage are features of non-alcoholic fatty liver disease (NAFLD). In rodent models and patients with NAFLD, hepatic expression of peroxisome proliferator-activated receptor-γ (PPARG) coactivator 1α (PPARGC1A or PGC1A) is inversely correlated with liver fat and disease severity. A common polymorphism in this gene (rs8192678, encoding Gly482Ser) has been associated with NAFLD. We investigated whether reduced expression of PGC1A contributes to development of NAFLD using mouse models, primary hepatocytes, and human cell lines. METHODS: HepG2 cells were transfected with variants of PPARGC1A and protein and messenger RNA levels were measured. Mice with liver-specific hemizygous or homozygous disruption of Ppargc1a (Ppargc1af/+Alb-cre+/0 and Ppargc1af/f Alb-cre+/0 mice, respectively) were fed regular chow (control) or a high-fat diet supplemented with 30% d-fructose in drinking water (obesogenic diet) for 25-33 weeks. Liver tissues were analyzed by histology and by immunoblotting. Primary hepatocytes were analyzed for insulin signaling, reactive oxygen species, and estrogen response. Luciferase reporter expression was measured in transfected H2.35 cells expressing an estrogen receptor reporter gene, estrogen receptor 1, and/or PGC1A/B. RESULTS: The serine 482 variant of the human PGC1A protein had a shorter half-life than the glycine 482 variant when expressed in HepG2 cells. Liver tissues from mice with liver-specific hemizygous disruption of Ppargc1a placed on an obesogenic diet expressed increased markers of inflammation and fibrosis and decreased levels of antioxidant enzymes compared with the Ppargc1a+/+ on the same diet. Oxidative damage was observed in livers from Ppargc1af/+Alb-cre+/0 mice of each sex, in a cell-autonomous manner, but was greater in livers from the female mice. Expression of PGC1A in H2.35 cells coactivated estrogen receptor 1 and was required for estrogen-dependent expression of genes that encode antioxidant proteins. These findings could account for the increased liver damage observed in female Ppargc1af/+Alb-cre+/0 mice; while, compensatory increases in PPARG coactivator 1ß could prevent oxidative damage associated with complete loss of PGC1A expression in Ppargc1af/fAlb-cre+/0 female mice. CONCLUSIONS: In mice, loss of estrogen signaling contributes to oxidative damage caused by low levels of PGC1A in liver, exacerbating steatohepatitis associated with diets high in fructose and fat.
Asunto(s)
Estrógenos/metabolismo , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Estrés Oxidativo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Grasas de la Dieta/administración & dosificación , Receptor alfa de Estrógeno/metabolismo , Femenino , Fructosa/administración & dosificación , Expresión Génica , Glutatión Peroxidasa/metabolismo , Hemicigoto , Células Hep G2 , Hepatitis/genética , Hepatitis/metabolismo , Hepatocitos , Humanos , Insulina/metabolismo , Integrasas/genética , Masculino , Ratones , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/patología , Proteínas Nucleares/metabolismo , Peroxirredoxinas/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Polimorfismo de Nucleótido Simple , Factores Sexuales , Transducción de Señal , Superóxido Dismutasa/metabolismo , Factores de Transcripción/metabolismo , Transfección , Glutatión Peroxidasa GPX1RESUMEN
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that binds and escorts the low density lipoprotein receptor (LDLR) into the lysosomal degradation pathway. Prescribed monoclonal antibodies (mAbs) against PCSK9 prevent its binding to the LDLR, and result in ~60% lower LDL cholesterol (LDLc) levels. Although efficient, mAbs are expensive. Hence other PCSK9 inhibitors are needed. For screening purpose, we developed C57BL/6J mice expressing the human PCSK9 gene under the control of its own promoter, but lacking endogenous mouse PCSK9. All lines recapitulate the endogenous PCSK9 expression pattern. The Tg2 line that expresses physiological levels of human PCSK9 (hPCSK9) was selected to characterize the inhibitory properties of a previously reported single domain antibody (sdAb), PKF8-mFc, which binds the C-terminal domain of PCSK9. Upon intraveinous injection of 10 mg/kg, PKF8-mFc and the mAb evolocumab neutralized ~50% and 100% of the hPCSK9 impact on total cholesterol (TC) levels, respectively, but PKF8-mFc had a more sustained effect. PKF8-mFc barely affected hPCSK9 levels, whereas evolocumab promoted a 4-fold increase 3 days post-injection, suggesting very different inhibitory mechanisms. The present study also shows that the new transgenic mice are well suited to screen a variety of hPCSK9 inhibitors.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Cisteína/antagonistas & inhibidores , Histidina/antagonistas & inhibidores , Inhibidores de PCSK9 , Animales , Anticuerpos Monoclonales Humanizados , Cisteína/metabolismo , Genotipo , Histidina/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proproteína Convertasa 9/deficiencia , Proproteína Convertasa 9/metabolismoRESUMEN
The clinical efficacy and safety of a drug is determined by its activity profile across many proteins in the proteome. However, designing drugs with a specific multi-target profile is both complex and difficult. Therefore methods to design drugs rationally a priori against profiles of several proteins would have immense value in drug discovery. Here we describe a new approach for the automated design of ligands against profiles of multiple drug targets. The method is demonstrated by the evolution of an approved acetylcholinesterase inhibitor drug into brain-penetrable ligands with either specific polypharmacology or exquisite selectivity profiles for G-protein-coupled receptors. Overall, 800 ligand-target predictions of prospectively designed ligands were tested experimentally, of which 75% were confirmed to be correct. We also demonstrate target engagement in vivo. The approach can be a useful source of drug leads when multi-target profiles are required to achieve either selectivity over other drug targets or a desired polypharmacology.
Asunto(s)
Diseño de Fármacos , Ligandos , Animales , Automatización , Sistemas de Liberación de Medicamentos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Teóricos , Fenómenos Farmacológicos , Reproducibilidad de los ResultadosRESUMEN
Single domain antibodies (sdAbs) correspond to the antigen-binding domains of camelid antibodies. They have the same antigen-binding properties and specificity as monoclonal antibodies (mAbs) but are easier and cheaper to produce. We report here the development of sdAbs targeting human PCSK9 (proprotein convertase subtilisin/kexin type 9) as an alternative to anti-PCSK9 mAbs. After immunizing a llama with human PCSK9, we selected four sdAbs that bind PCSK9 with a high affinity and produced them as fusion proteins with a mouse Fc. All four sdAb-Fcs recognize the C-terminal Cys-His-rich domain of PCSK9. We performed multiple cellular assays and demonstrated that the selected sdAbs efficiently blocked PCSK9-mediated low density lipoprotein receptor (LDLR) degradation in cell lines, in human hepatocytes, and in mouse primary hepatocytes. We further showed that the sdAb-Fcs do not affect binding of PCSK9 to the LDLR but rather block its induced cellular LDLR degradation. Pcsk9 knock-out mice expressing a human bacterial artificial chromosome (BAC) transgene were generated, resulting in plasma levels of â¼300 ng/ml human PCSK9. Mice were singly or doubly injected with the best sdAb-Fc and analyzed at day 4 or 11, respectively. After 4 days, mice exhibited a 32 and 44% decrease in the levels of total cholesterol and apolipoprotein B and â¼1.8-fold higher liver LDLR protein levels. At 11 days, the equivalent values were 24 and 46% and â¼2.3-fold higher LDLR proteins. These data constitute a proof-of-principle for the future usage of sdAbs as PCSK9-targeting drugs that can efficiently reduce LDL-cholesterol, and as tools to study the Cys-His-rich domain-dependent sorting the PCSK9-LDLR complex to lysosomes.
Asunto(s)
LDL-Colesterol/metabolismo , Proproteína Convertasa 9/metabolismo , Proteolisis/efectos de los fármacos , Receptores de LDL/metabolismo , Anticuerpos de Dominio Único/farmacología , Animales , LDL-Colesterol/genética , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Inhibidores de PCSK9 , Proproteína Convertasa 9/genética , Receptores de LDL/genéticaRESUMEN
BACKGROUND: Loss of proprotein convertase subtilisin/kexin type 5 (Pcsk5) results in multiple developmental anomalies including cardiac malformations, caudal regression, pre-sacral mass, renal agenesis, anteroposterior patterning defects, and tracheo-oesophageal and anorectal malformations, and is a model for VACTERL/caudal regression/Currarino syndromes (VACTERL association - Vertebral anomalies, Anal atresia, Cardiac defects, Tracheoesophageal fistula and/or Esophageal atresia, Renal & Radial anomalies and Limb defects). RESULTS: Using magnetic resonance imaging (MRI), we examined heart development in mouse embryos with zygotic and cardiac specific deletion of Pcsk5. We show that conditional deletion of Pcsk5 in all epiblastic lineages recapitulates all developmental malformations except for tracheo-esophageal malformations. Using a conditional deletion strategy, we find that there is an essential and specific requirement for Pcsk5 in the cranio-cardiac mesoderm for cardiogenesis, but not for conotruncal septation or any other aspect of embryonic development. Surprisingly, deletion of Pcsk5 in cardiogenic or pharyngeal mesodermal progenitors that form later from the cranio-cardiac mesoderm does not affect heart development. Neither is Pcsk5 essential in the neural crest, which drives conotruncal septation. CONCLUSIONS: Our results suggest that Pcsk5 may have an essential and early role in the cranio-cardiac mesoderm for heart development. Alternatively, it is possible that Pcsk5 may still play a critical role in Nkx2.5-expressing cardiac progenitors, with persistence of mRNA or protein accounting for the lack of effect of deletion on heart development.
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Corazón/embriología , Mesodermo/embriología , Organogénesis/genética , Proproteína Convertasa 5/genética , Proproteína Convertasa 5/metabolismo , Animales , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Ratones , Ratones Noqueados , MutaciónRESUMEN
PURPOSE: Cystic Fibrosis (CF) is the most common genetic disorder and, with improved survival, glucose abnormalities have emerged as a major comorbidity. Proprotein convertase subtilisin/kexin type 9 (PCSK9), a regulator of plasma LDL-cholesterol homeostasis, is associated with lipid and glucose metabolism in healthy individuals. Here we report on the link between PCSK9 and markers of metabolism in CF. METHODS: Cross-sectional analysis was performed on CF patients (≥ 18 years, N=94) from the Montreal Cohort, without known diabetes, and on healthy individuals (N=19). The levels of PCSK9 and lipid markers were quantified and all subjects underwent a 2 h oral glucose tolerance test. RESULTS: No significant differences in PCSK9 levels were found between healthy individuals and patients with CF, or between the groups with different degrees of glucose tolerance. No association was found between PCSK9 and markers of lipid metabolism; however, a positive correlation was found between PCSK9 and total insulin secretion and a negative one with insulin sensitivity in CF patients who had normal glucose tolerance. CONCLUSION: Circulating levels of PCSK9 in the CF population are comparable to those in the healthy population. There are no associations between PCSK9 levels and either glucose or lipid homeostasis parameters. Nevertheless, a statistically significant link was observed between PCSK9 and markers of insulin homeostasis, solely in CF patients who presented normal glucose tolerance. Further exploration of the relationship between PCSK9 and insulin homeostasis in CF patients with normal glucose tolerance is warranted.
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Fibrosis Quística/sangre , Fibrosis Quística/metabolismo , Insulina/sangre , Metabolismo de los Lípidos/fisiología , Proproteína Convertasa 9/sangre , Adulto , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Estudios Transversales , Femenino , Glucosa/metabolismo , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Amyloid precursor-like protein 2 (APLP2) and sortilin were reported to individually bind the proprotein convertase subtilisin/kexin type 9 (PCSK9) and regulate its activity on the low-density lipoprotein receptor (LDLR). The data presented herein demonstrate that mRNA knockdowns of APLP2, sortilin, or both in the human hepatocyte cell lines HepG2 and Huh7 do not affect the ability of extracellular PCSK9 to enhance the degradation of the LDLR. Furthermore, mice deficient in APLP2 or sortilin do not exhibit significant changes in liver LDLR or plasma total cholesterol levels. Moreover, cellular overexpression of one or both proteins does not alter PCSK9 secretion, or its activity on the LDLR. We conclude that PCSK9 enhances the degradation of the LDLR independently of either APLP2 or sortilin both ex vivo and in mice. Interestingly, when co-expressed with PCSK9, both APLP2 and sortilin were targeted for lysosomal degradation. Using chemiluminescence proximity and co-immunoprecipitation assays, as well as biosynthetic analysis, we discovered that sortilin binds and stabilizes APLP2, and hence could regulate its intracellular functions on other targets.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proproteína Convertasas/metabolismo , Proteolisis , Receptores de LDL/biosíntesis , Serina Endopeptidasas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/biosíntesis , Proteínas Adaptadoras del Transporte Vesicular/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Regulación de la Expresión Génica , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Ratones , Proteínas del Tejido Nervioso/genética , Proproteína Convertasa 9 , Proproteína Convertasas/genética , Receptores de LDL/genética , Serina Endopeptidasas/genéticaRESUMEN
PC7 belongs to the proprotein convertase family, whose members are implicated in the cleavage of secretory precursors. The in vivo function of PC7 is unknown. Herein, we find that the precursor proBDNF is processed into mature BDNF in COS-1 cells coexpressing proBDNF with either PC7 or Furin. Conversely, the processing of proBDNF into BDNF is markedly reduced in the absence of either Furin or PC7 in mouse primary hepatocytes. In vivo we observe that BDNF and PC7 mRNAs are colocalized in mouse hippocampus and amygdala and that mature BDNF protein levels are reduced in these brain areas in PC7 KO mice but not in the hippocampus of PC1/3 KO mice. Various behavioral tests reveal that in PC7 KO mice spatial memory is intact and plasticity of responding is mildly abnormal. Episodic and emotional memories are severely impaired, but both are rescued with the tyrosine receptor kinase B agonist 7,8-dihydroxyflavone. Altogether, these results support an in vivo role for PC7 in the regulation of certain types of cognitive performance, in part via proBDNF processing. Because polymorphic variants of human PC7 are being characterized, it will be important in future studies to determine their effects on additional physiological and behavioral processes.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Aprendizaje por Laberinto/fisiología , Memoria/fisiología , Subtilisinas/metabolismo , Amígdala del Cerebelo/metabolismo , Animales , Western Blotting , Células COS , Células Cultivadas , Chlorocebus aethiops , Femenino , Flavanonas/farmacología , Expresión Génica , Células HEK293 , Hepatocitos/citología , Hepatocitos/metabolismo , Hipocampo/metabolismo , Humanos , Hibridación in Situ , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Memoria/efectos de los fármacos , Ratones , Ratones Noqueados , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Subtilisinas/genéticaRESUMEN
Proprotein convertase subtilisin kexin type 9 (PCSK9), the last member of the family of Proprotein Convertases related to Subtilisin and Kexin, regulates LDL-cholesterol by promoting the endosomal/lysosomal degradation of the LDL receptor (LDLR). Herein, we show that the LDLR cell surface levels dramatically increase in the liver and pancreatic islets of PCSK9 KO male but not female mice. In contrast, in KO female mice, the LDLR is more abundant at the cell surface enterocytes, as is the VLDL receptor (VLDLR) at the cell surface of adipocytes. Ovariectomy of KO female mice led to a typical KO male pattern, whereas 17ß-estradiol (E2) treatment restored the female pattern without concomitant changes in LDLR adaptor protein 1 (also known as ARH), disabled-2, or inducible degrader of the LDLR expression levels. We also show that this E2-mediated regulation, which is observed only in the absence of PCSK9, is abolished upon feeding the mice a high-cholesterol diet. The latter dramatically represses PCSK9 expression and leads to high surface levels of the LDLR in the hepatocytes of all sexes and genotypes. In conclusion, the absence of PCSK9 results in a sex- and tissue-specific subcellular distribution of the LDLR and VLDLR, which is determined by E2 levels.
Asunto(s)
Proproteína Convertasas/genética , Receptores de LDL/metabolismo , Serina Endopeptidasas/genética , Adiposidad , Animales , Estradiol/fisiología , Femenino , Grasa Intraabdominal/metabolismo , Hígado/enzimología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos , Proproteína Convertasa 9 , Proproteína Convertasas/sangre , Proproteína Convertasas/deficiencia , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serina Endopeptidasas/sangre , Serina Endopeptidasas/deficiencia , Caracteres SexualesRESUMEN
The proprotein convertases (PCs) furin, PC5/6, and PACE4 exhibit unique and/or complementary functions. Their knock-out (KO) in mice resulted in strong and specific phenotypes demonstrating that, in vivo, these PCs are unique and essential during development. However, they also exhibit redundant functions. Liver angiopoietin-like 3 (ANGPTL3) inhibits lipolysis by binding to lipoprotein lipases. It is found in the plasma as full length and truncated forms. The latter is more active and generated by cleavage at a furin-like site. Endothelial lipase (EL) binds heparin sulfate proteoglycans on cell surfaces and catalyzes the hydrolysis of HDL phospholipids. EL activity is regulated by two endogenous inhibitors, ANGPTL3 and ANGPTL4, and by PCs that inactivate EL through cleavage releasing the N-terminal catalytic and C-terminal lipid-binding domains. Herein, because furin and PC5/6 complete KOs are lethal, we used mice lacking furin or PC5/6 specifically in hepatocytes (hKO) or mice completely lacking PACE4. In primary hepatocytes, ANGPTL3 was processed into a shorter form of ANGPTL3 intracellularly by furin only, and extracellularly mainly by PACE4. In vivo, the absence of furin in hepatocytes reduced by â¼50% the circulating levels of cleaved ANGPTL3, while the lack of PACE4 had only a minor effect. Analysis of the EL processing in primary hepatocytes and in vivo revealed that it is mostly cleaved by furin. However, the lack of furin or PC5/6 in hepatocytes and complete PACE4 KO did not appreciably modify plasma HDL levels or EL activity. Thus, inhibition of furin in liver would not be expected to modify the plasma lipid profiles.
Asunto(s)
Angiopoyetinas/metabolismo , Furina/metabolismo , Hepatocitos/enzimología , Lipasa/metabolismo , Proteína 3 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina , Animales , Células COS , Chlorocebus aethiops , HDL-Colesterol/metabolismo , Furina/genética , Silenciador del Gen , Lipoproteínas HDL/metabolismo , Masculino , Ratones , Ratones Noqueados , Fosfolípidos/metabolismo , Proproteína Convertasa 5/genéticaRESUMEN
UNLABELLED: The first seven members of the proprotein convertase (PC) family activate protein precursors by cleavage after basic residues. While PC7 has no known specific substrates, it shows redundancy with other PCs. A genome-wide association study suggested that circulating levels of shed human transferrin receptor 1 (hTfR1) are regulated by PC7. We thus examined whether hTfR1 constitutes a specific substrate for PC7. Coexpression of hTfR1 with PCs in several cell lines indicated that PC7 is the only convertase that sheds this receptor into the medium. Site-directed mutagenesis showed that cleavage occurs at the unusual site KTECER100 ↓LA, in which the P1 Arg100 and P6 Lys95 are critical. Pharmacological treatments revealed that shedding of hTfR1 by PC7 requires endocytosis into acidic clathrin-coated vesicles. A PC7 chimera, in which the transmembrane domain and the cytosolic tail of PC7 were replaced by that of the convertase furin, lost its ability to cleave the receptor, demonstrating the importance of these domains in the regulation of PC7 function. Analysis of primary hepatocytes from mice lacking furin, PC5, PACE4, or PC7 revealed that hepcidin, which limits iron availability in the circulation, is specifically generated by furin and not by PC7. Finally, depletion of iron in the medium of hepatoma cell lines incubated with the iron chelator desferrioxamine resulted in PC7 down-regulation. CONCLUSION: Among the PC family members, only furin activates hepcidin in hepatocytes, and uniquely the full-length membrane-bound PC7 can directly shed hTfR1 by cleavage at Arg100 ↓. Our results support the notion that, when iron is limiting, hTfR1 levels increase at least in part by way of the down-regulation of PC7 expression. (HEPATOLOGY 2013;).