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1.
J Biol Chem ; 297(4): 101177, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34508778

RESUMEN

The hepatic carbohydrate-recognizing asialoglycoprotein receptor (ASGR1) mediates the endocytosis/lysosomal degradation of desialylated glycoproteins following binding to terminal galactose/N-acetylgalactosamine. Human heterozygote carriers of ASGR1 deletions exhibit ∼34% lower risk of coronary artery disease and ∼10% to 14% reduction of non-HDL cholesterol. Since the proprotein convertase PCSK9 is a major degrader of the low-density lipoprotein receptor (LDLR), we investigated the degradation and functionality of LDLR and/or PCSK9 by endogenous/overexpressed ASGR1 using Western blot and immunofluorescence in HepG2-naïve and HepG2-PCSK9-knockout cells. ASGR1, like PCSK9, targets LDLR, and both independently interact with/enhance the degradation of the receptor. This lack of cooperativity between PCSK9 and ASGR1 was confirmed in livers of wildtype (WT) and Pcsk9-/- mice. ASGR1 knockdown in HepG2-naïve cells significantly increased total (∼1.2-fold) and cell-surface (∼4-fold) LDLR protein. In HepG2-PCSK9-knockout cells, ASGR1 silencing led to ∼2-fold higher levels of LDLR protein and DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-LDL uptake associated with ∼9-fold increased cell-surface LDLR. Overexpression of WT-ASGR1/2 primarily reduced levels of immature non-O-glycosylated LDLR (∼110 kDa), whereas the triple Ala-mutant of Gln240/Trp244/Glu253 (characterized by loss of carbohydrate binding) reduced expression of the mature form of LDLR (∼150 kDa), suggesting that ASGR1 binds the LDLR in both a sugar-dependent and -independent fashion. The protease furin cleaves ASGR1 at the RKMK103↓ motif into a secreted form, likely resulting in a loss of function on LDLR. Altogether, we demonstrate that LDLR is the first example of a liver-receptor ligand of ASGR1. We conclude that silencing of ASGR1 and PCSK9 may lead to higher LDL uptake by hepatocytes, thereby providing a novel approach to further reduce LDL cholesterol levels.


Asunto(s)
Receptor de Asialoglicoproteína/metabolismo , Furina/metabolismo , Hígado/metabolismo , Proproteína Convertasa 9/metabolismo , Receptores de LDL/metabolismo , Animales , Receptor de Asialoglicoproteína/genética , Furina/genética , Células HEK293 , Células Hep G2 , Humanos , Ratones , Ratones Noqueados , Proproteína Convertasa 9/genética , Receptores de LDL/genética
2.
Arterioscler Thromb Vasc Biol ; 39(10): 1996-2013, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31553664

RESUMEN

OBJECTIVE: PCSK9 (proprotein convertase subtilisin-kexin 9) enhances the degradation of the LDLR (low-density lipoprotein receptor) in endosomes/lysosomes. This study aimed to determine the sites of PCSK9 phosphorylation at Ser-residues and the consequences of such posttranslational modification on the secretion and activity of PCSK9 on the LDLR. Approach and Results: Fam20C (family with sequence similarity 20, member C) phosphorylates serines in secretory proteins containing the motif S-X-E/phospho-Ser, including the cholesterol-regulating PCSK9. In situ hybridization of Fam20C mRNA during development and in adult mice revealed a wide tissue distribution, including liver, but not small intestine. Here, we show that Fam20C phosphorylates PCSK9 at Serines 47, 666, 668, and 688. In hepatocytes, phosphorylation enhances PCSK9 secretion and maximizes its induced degradation of the LDLR via the extracellular and intracellular pathways. Replacing any of the 4 Ser by the phosphomimetic Glu or Asp enhanced PCSK9 activity only when the other sites are phosphorylated, whereas Ala substitutions reduced it, as evidenced by Western blotting, Elisa, and LDLR-immunolabeling. This newly uncovered PCSK9/LDLR regulation mechanism refines our understanding of the implication of global PCSK9 phosphorylation in the modulation of LDL-cholesterol and rationalizes the consequence of natural mutations, for example, S668R and E670G. Finally, the relationship of Ser-phosphorylation to the implication of PCSK9 in regulating LDL-cholesterol in the neurological Fragile X-syndrome disorder was investigated. CONCLUSIONS: Ser-phosphorylation of PCSK9 maximizes both its secretion and activity on the LDLR. Mass spectrometric approaches to measure such modifications were developed and applied to quantify the levels of bioactive PCSK9 in human plasma under normal and pathological conditions.


Asunto(s)
Proteínas de Unión al Calcio/genética , Proteínas de la Matriz Extracelular/genética , Regulación de la Expresión Génica , Hiperlipoproteinemia Tipo II/genética , Proproteína Convertasa 9/metabolismo , Receptores de LDL/genética , Animales , Western Blotting , Células Cultivadas , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Hiperlipoproteinemia Tipo II/fisiopatología , Hibridación in Situ/métodos , Masculino , Ratones , Ratones Noqueados , Microscopía Confocal , Fosforilación/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Receptores de LDL/metabolismo , Sensibilidad y Especificidad
3.
Biol Chem ; 399(12): 1363-1374, 2018 11 27.
Artículo en Inglés | MEDLINE | ID: mdl-30044755

RESUMEN

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/metabolismo
4.
J Biol Chem ; 290(30): 18609-20, 2015 Jul 24.
Artículo en Inglés | MEDLINE | ID: mdl-26085104

RESUMEN

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/genética
5.
Proc Natl Acad Sci U S A ; 110(43): 17362-7, 2013 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-24101515

RESUMEN

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/genética
6.
J Lipid Res ; 56(11): 2133-42, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26323289

RESUMEN

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 Sexuales
7.
Circulation ; 125(7): 894-901, 2012 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-22261195

RESUMEN

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.


Asunto(s)
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 Sexuales
8.
Arterioscler Thromb Vasc Biol ; 31(4): 785-91, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21273557

RESUMEN

OBJECTIVE: Proprotein convertase subtilisin/kexin 9 (PCSK9) promotes the degradation of the low-density lipoprotein receptor (LDLR), and its gene is the third locus implicated in familial hypercholesterolemia. Herein, we investigated the role of PCSK9 in adipose tissue metabolism. METHODS AND RESULTS: At 6 months of age, Pcsk9(-/-) mice accumulated ≈80% more visceral adipose tissue than wild-type mice. This was associated with adipocyte hypertrophy and increased in vivo fatty acid uptake and ex vivo triglyceride synthesis. Moreover, adipocyte hypertrophy was also observed in Pcsk9(-/-) Ldlr(-/-) mice, indicating that the LDLR is not implicated. Rather, we show here by immunohistochemistry that Pcsk9(-/-) males and females exhibit 4- and ≈ 40-fold higher cell surface levels of very-low-density lipoprotein receptor (VLDLR) in perigonadal depots, respectively. Expression of PCSK9 in the liver of Pcsk9(-/-) females reestablished both circulating PCSK9 and normal VLDLR levels. In contrast, specific inactivation of PCSK9 in the liver of wild-type females led to ≈ 50-fold higher levels of perigonadal VLDLR. CONCLUSIONS: In vivo, endogenous PCSK9 regulates VLDLR protein levels in adipose tissue. This regulation is achieved by circulating PCSK9 that originates entirely in the liver. PCSK9 is thus pivotal in fat metabolism: it maintains high circulating cholesterol levels via hepatic LDLR degradation, but it also limits visceral adipogenesis likely via adipose VLDLR regulation.


Asunto(s)
Adipocitos/enzimología , Adiposidad , Grasa Intraabdominal/enzimología , Receptores de LDL/metabolismo , Serina Endopeptidasas/metabolismo , Triglicéridos/metabolismo , Adipocitos/patología , Adiposidad/genética , Factores de Edad , Animales , Colesterol/metabolismo , Femenino , Homeostasis , Hidrólisis , Hipertrofia , Inmunohistoquímica , Grasa Intraabdominal/patología , Hígado/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ácido Oléico/metabolismo , Proproteína Convertasa 9 , Proproteína Convertasas , Receptores de LDL/deficiencia , Receptores de LDL/genética , Serina Endopeptidasas/sangre , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/genética , Factores Sexuales
9.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1867(12): 159217, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-35985474

RESUMEN

PCSK9 promotes the lysosomal degradation of cell surface LDL receptor (LDLR). We analyzed how excess LDLR generated by PCSK9 deficiency is differently handled in male and female mice to possibly unveil the mechanism leading to the lower efficacy of PCSK9 mAb on LDL-cholesterol levels in women. Analysis of intact or ovariectomized PCSK9 knockout (KO) mice supplemented with placebo or 17ß-estradiol (E2) demonstrated that female, but not male mice massively shed the soluble ectodomain of the LDLR in the plasma. Liver-specific PCSK9 KO or alirocumab-treated WT mice exhibit the same pattern. This shedding is distinct from the basal one and is inhibited by ZLDI-8, a metalloprotease inhibitor pointing at ADAM10/ADAM17. In PCSK9 KO female mice, ZLDI-8 raises by 80 % the LDLR liver content in a few hours. This specific shedding is likely cholesterol-dependent: it is prevented in PCSK9 KO male mice that exhibit low intra-hepatic cholesterol levels without activating SREBP-2, and enhanced by mevalonate or high cholesterol feeding, or by E2 known to stimulate cholesterol synthesis via the estrogen receptor-α. Liver transcriptomics demonstrates that critically low liver cholesterol in ovariectomized female or knockout male mice also hampers the cholesterol-dependent G2/M transition of the cell cycle. Finally, higher levels of shed LDLR were measured in the plasma of women treated with PCSK9 mAb. PCSK9 knockout female mice hormonally sustain cholesterol synthesis and shed excess LDLR, seemingly like women. In contrast, male mice rely on high surface LDLR to replenish their stocks, despite 80 % lower circulating LDL.


Asunto(s)
Ácido Mevalónico , Proproteína Convertasa 9 , Animales , Colesterol/metabolismo , Estradiol/metabolismo , Estradiol/farmacología , Femenino , Hígado/metabolismo , Metaloproteasas/metabolismo , Ácido Mevalónico/metabolismo , Ratones , Ratones Noqueados , Proproteína Convertasa 9/metabolismo , Receptores de Superficie Celular , Receptores de Estrógenos , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo
10.
J Lipid Res ; 52(7): 1383-91, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21518694

RESUMEN

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a major role in cholesterol homeostasis through enhanced degradation of the LDL receptor (LDLR) in liver. As novel inhibitors/silencers of PCSK9 are now being tested in clinical trials to treat hypercholesterolemia, it is crucial to define the physiological consequences of the lack of PCSK9 in various organs. LDLR regulation by PCSK9 has not been extensively described during mouse brain development and injury. Herein, we show that PCSK9 and LDLR are co-expressed in mouse brain during development and at adulthood. Although the protein levels of LDLR and apolipoprotein E (apoE) in the adult brain of Pcsk9(-/-) mice are similar to those of wild-type (WT) mice, LDLR levels increased and were accompanied by a reduction of apoE levels during development. This suggests that the upregulation of LDLR protein levels in Pcsk9(-/-) mice enhances apoE degradation. Upon ischemic stroke, PCSK9 was expressed in the dentate gyrus between 24 h and 72 h following brain reperfusion. Although mouse behavior and lesion volume were similar, LDLR protein levels dropped ∼2-fold less in the Pcsk9(-/-)-lesioned hippocampus, without affecting apoE levels and neurogenesis. Thus, PCSK9 downregulates LDLR levels during brain development and following transient ischemic stroke in adult mice.


Asunto(s)
Isquemia Encefálica/metabolismo , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Apolipoproteínas E/metabolismo , Isquemia Encefálica/complicaciones , Isquemia Encefálica/genética , Cerebelo/metabolismo , Giro Dentado/metabolismo , Ratones , Proproteína Convertasa 9 , Proproteína Convertasas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/genética , Accidente Cerebrovascular/complicaciones , Telencéfalo/metabolismo , Factores de Tiempo , Regulación hacia Arriba
11.
Proc Natl Acad Sci U S A ; 105(15): 5750-5, 2008 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-18378898

RESUMEN

The proprotein convertase PC5/6 cleaves protein precursors after basic amino acids and is essential for implantation in CD1/129/Sv/C57BL/6 mixed-background mice. Conditional inactivation of Pcsk5 in the epiblast but not in the extraembryonic tissue bypassed early embryonic lethality but resulted in death at birth. PC5/6-deficient embryos exhibited Gdf11-related phenotypes such as altered anteroposterior patterning with extra vertebrae and lack of tail and kidney agenesis. They also exhibited Gdf11-independent phenotypes, such as a smaller size, multiple hemorrhages, collapsed alveoli, and retarded ossification. In situ hybridization revealed overlapping PC5/6 and Gdf11 mRNA expression patterns. In vitro and ex vivo analyses showed that the selectivity of PC5/6 for Gdf11 essentially resides in the presence of a P1' Asn in the RSRR downward arrowN cleavage motif. This work identifies Gdf11 as a likely in vivo specific substrate of PC5/6 and opens the way to the identification of other key substrates of this convertase.


Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Crecimiento y Desarrollo , Proproteína Convertasa 5/fisiología , Animales , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Factores de Diferenciación de Crecimiento , Ratones , Fenotipo , Proproteína Convertasa 5/genética , Proproteína Convertasa 5/metabolismo , ARN Mensajero/análisis , Especificidad por Sustrato
12.
Hepatology ; 48(2): 646-54, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18666258

RESUMEN

UNLABELLED: The gene encoding the proprotein convertase subtilisin/kexin type 9 (PCSK9) is linked to familial hypercholesterolemia, as are those of the low-density lipoprotein receptor (LDLR) and apolipoprotein B. PCSK9 enhances LDLR degradation, resulting in low-density lipoprotein accumulation in plasma. To analyze the role of hepatic PCSK9, total and hepatocyte-specific knockout mice were generated. They exhibit 42% and 27% less circulating cholesterol, respectively, showing that liver PCSK9 was responsible for two thirds of the phenotype. We also demonstrated that, in liver, PCSK9 is exclusively expressed in hepatocytes, representing the main source of circulating PCSK9. The data suggest that local but not circulating PCSK9 regulates cholesterol levels. Although transgenic mice overexpressing high levels of liver and circulating PCSK9 led to the almost complete disappearance of the hepatic LDLR, they did not recapitulate the plasma cholesterol levels observed in LDLR-deficient mice. Single LDLR or double LDLR/PCSK9 knockout mice exhibited similar cholesterol profiles, indicating that PCSK9 regulates cholesterol homeostasis exclusively through the LDLR. Finally, the regenerating liver of PCSK9-deficient mice exhibited necrotic lesions, which were prevented by a high-cholesterol diet. However, lipid accumulation in hepatocytes of these mice was markedly reduced under both chow and high-cholesterol diets, revealing that PCSK9 deficiency confers resistance to liver steatosis. CONCLUSION: Although PCSK9 is a target for controlling hypercholesterolemia, our data indicate that upon hepatic damage, patients lacking PCSK9 could be at risk.


Asunto(s)
Hepatocitos/metabolismo , Regeneración Hepática/fisiología , Receptores de LDL/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Colesterol/sangre , Colesterol en la Dieta/administración & dosificación , Hígado Graso/prevención & control , Hepatectomía/métodos , Inmunidad Innata , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Noqueados , Ratones Transgénicos , Necrosis , Proproteína Convertasa 9 , Proproteína Convertasas , Receptores de LDL/deficiencia , Serina Endopeptidasas/sangre , Serina Endopeptidasas/deficiencia , Distribución Tisular , Regulación hacia Arriba
13.
Mol Cell Biol ; 26(1): 354-61, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16354705

RESUMEN

PC5 belongs to the proprotein convertase family and activates precursor proteins by cleavage at basic sites during their transit through the secretory pathway and/or at the cell surface. These precursors include prohormones, proreceptors, growth factors, adhesion molecules, and viral glycoproteins. The Pcsk5 gene encodes two alternatively spliced isoforms, the soluble PC5A and transmembrane PC5B. We have carefully analyzed the expression of PC5 in the mouse during development and in adulthood by in situ hybridization, as well as in mouse tissues and various cell lines by quantitative reverse transcription-PCR. The data show that adrenal cortex and intestine are the richest sources of PC5A and PC5B, respectively. To better define the specific physiological roles of PC5, we have generated a mouse Pcsk5(Delta4)-deficient allele missing exon 4 that encodes the catalytic Asp173. While Delta4/+ heterozygotes were healthy and fertile, genotyping of progeny obtained from Delta4/+ interbreeding indicated that Delta4/Delta4 embryos died between embryonic days 4.5 and 7.5. These data demonstrate that Pcsk5 is an essential gene.


Asunto(s)
Embrión de Mamíferos/enzimología , Desarrollo Embrionario/genética , Genes Letales , Proproteína Convertasa 5/genética , Corteza Suprarrenal/enzimología , Animales , Células Cultivadas , Femenino , Eliminación de Gen , Humanos , Intestinos/enzimología , Isoenzimas/análisis , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Noqueados , Proproteína Convertasa 5/análisis , Proproteína Convertasa 5/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Distribución Tisular
14.
J Clin Invest ; 111(11): 1723-32, 2003 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-12782675

RESUMEN

The secretory factor VEGF-C has been directly implicated in various physiological processes during embryogenesis and human cancers. However, the importance of the conversion of its precursor proVEGF-C to mature VEGF-C in tumorigenesis, and vessel formation and the identity of the protease(s) that regulate these processes is/are not known. The intracellular processing of proVEGF-C that occurs within the dibasic motif HSIIRR(227)SL suggests the involvement of the proprotein convertases (PCs) in this process. In addition, furin and VEGF-C were found to be coordinately expressed in adult mouse tissues. Cotransfection of the furin-deficient colon carcinoma cell line LoVo with proVEGF-C and different PC members revealed that furin, PC5, and PC7 are candidate VEGF-C convertases. This finding is consistent with the in vitro digestions of an internally quenched synthetic fluorogenic peptide mimicking the cleavage site of proVEGF-C ((220)Q-VHSIIRR downward arrow SLP(230)). The processing of proVEGF-C is blocked by the inhibitory prosegments of furin, PC5, and PACE4, as well as by furin-motif variants of alpha2-macroglobulin and alpha1-antitrypsin. Subcutaneous injection of CHO cells stably expressing VEGF-C into nude mice enhanced angiogenesis and lymphangiogenesis, but not tumor growth. In contrast, expression of proVEGF-C obtained following mutation of the cleavage site (HSIIRR(227)SL to HSIISS(227)SL) inhibits angiogenesis and lymphangiogenesis as well as tumor growth. Our findings demonstrate the processing of proVEGF-C by PCs and highlight the potential use of PC inhibitors as agents for inhibiting malignancies induced by VEGF-C.


Asunto(s)
Carbamatos/metabolismo , Transformación Celular Neoplásica , Factores de Crecimiento Endotelial/metabolismo , Neoplasias/metabolismo , Oligopéptidos/metabolismo , Subtilisinas/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Western Blotting , Células CHO , Cricetinae , Relación Dosis-Respuesta a Droga , Furina , Humanos , Inmunohistoquímica , Cinética , Mutación , Biosíntesis de Péptidos , Pruebas de Precipitina , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transfección , Células Tumorales Cultivadas , Factor C de Crecimiento Endotelial Vascular
15.
Angiology ; 67(2): 157-67, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25904765

RESUMEN

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 ∼ 75% by X-ray and ∼ 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.


Asunto(s)
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 X
16.
J Biol Chem ; 283(4): 2363-72, 2008 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-18039658

RESUMEN

The proprotein convertase PCSK9 gene is the third locus implicated in familial hypercholesterolemia, emphasizing its role in cardiovascular diseases. Loss of function mutations and gene disruption of PCSK9 resulted in a higher clearance of plasma low density lipoprotein cholesterol, likely due to a reduced degradation of the liver low density lipoprotein receptor (LDLR). In this study, we show that two of the closest family members to LDLR are also PCSK9 targets. These include the very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) implicated in neuronal development and lipid metabolism. Our results show that wild type PCSK9 and more so its natural gain of function mutant D374Y can efficiently degrade the LDLR, VLDLR, and ApoER2 either following cellular co-expression or re-internalization of secreted human PCSK9. Such PCSK9-induced degradation does not require its catalytic activity. Membrane-bound PCSK9 chimeras enhanced the intracellular targeting of PCSK9 to late endosomes/lysosomes and resulted in a much more efficient degradation of the three receptors. We also demonstrate that the activity of PCSK9 and its binding affinity on VLDLR and ApoER2 does not depend on the presence of LDLR. Finally, in situ hybridization show close localization of PCSK9 mRNA expression to that of VLDLR in mouse postnatal day 1 cerebellum. Thus, this study demonstrates a more general effect of PCSK9 on the degradation of the LDLR family that emphasizes its major role in cholesterol and lipid homeostasis as well as brain development.


Asunto(s)
Hipercolesterolemia/metabolismo , Metabolismo de los Lípidos/fisiología , Receptores de LDL/metabolismo , Receptores de Lipoproteína/metabolismo , Serina Endopeptidasas/metabolismo , Sustitución de Aminoácidos , Animales , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Células CHO , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/metabolismo , LDL-Colesterol/genética , LDL-Colesterol/metabolismo , Cricetinae , Cricetulus , Homeostasis/fisiología , Humanos , Hipercolesterolemia/complicaciones , Hipercolesterolemia/genética , Proteínas Relacionadas con Receptor de LDL , Hígado/metabolismo , Ratones , Mutación Missense , Células 3T3 NIH , Neuronas/metabolismo , Proproteína Convertasa 9 , Proproteína Convertasas , Transporte de Proteínas/fisiología , Receptores de LDL/genética , Receptores de Lipoproteína/genética , Serina Endopeptidasas/genética
17.
Proc Natl Acad Sci U S A ; 100(3): 928-33, 2003 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-12552133

RESUMEN

Seven secretory mammalian kexin-like subtilases have been identified that cleave a variety of precursor proteins at monobasic and dibasic residues. The recently characterized pyrolysin-like subtilase SKI-1 cleaves proproteins at nonbasic residues. In this work we describe the properties of a proteinase K-like subtilase, neural apoptosis-regulated convertase 1 (NARC-1), representing the ninth member of the secretory subtilase family. Biosynthetic and microsequencing analyses of WT and mutant enzyme revealed that human and mouse pro-NARC-1 are autocatalytically and intramolecularly processed into NARC-1 at the (Y,I)VV(V,L)(L,M) downward arrow motif, a site that is representative of its enzymic specificity. In vitro peptide processing studies andor Ala substitutions of the P1-P5 sites suggested that hydrophobicaliphatic residues are more critical at P1, P3, and P5 than at P2 or P4. NARC-1 expression is highest in neuroepithelioma SK-N-MCIXC, hepatic BRL-3A, and in colon carcinoma LoVo-C5 cell lines. In situ hybridization and Northern blot analyses of NARC-1 expression during development in the adult and after partial hepatectomy revealed that it is expressed in cells that have the capacity to proliferate and differentiate. These include hepatocytes, kidney mesenchymal cells, intestinal ileum, and colon epithelia as well as embryonic brain telencephalon neurons. Accordingly, transfection of NARC-1 in primary cultures of embryonic day 13.5 telencephalon cells led to enhanced recruitment of undifferentiated neural progenitor cells into the neuronal lineage, suggesting that NARC-1 is implicated in the differentiation of cortical neurons.


Asunto(s)
Apoptosis , Hígado/metabolismo , Hígado/fisiología , Neuronas/metabolismo , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/fisiología , Animales , Northern Blotting , Células CHO , Diferenciación Celular , Células Cultivadas , Cricetinae , Electroforesis en Gel de Poliacrilamida , Hepatocitos/citología , Humanos , Hibridación in Situ , Ratones , Modelos Genéticos , Mutación , Neuronas/fisiología , Proproteína Convertasa 9 , Proproteína Convertasas , Ratas , Regeneración , Factores de Tiempo , Distribución Tisular , Transfección , Células Tumorales Cultivadas
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