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1.
Cell ; 175(1): 133-145.e15, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30220454

RESUMO

Nonalcoholic fatty liver disease (NAFLD) progresses to nonalcoholic steatohepatitis (NASH) in response to elevated endoplasmic reticulum (ER) stress. Whereas the onset of simple steatosis requires elevated de novo lipogenesis, progression to NASH is triggered by accumulation of hepatocyte-free cholesterol. We now show that caspase-2, whose expression is ER-stress inducible and elevated in human and mouse NASH, controls the buildup of hepatic-free cholesterol and triglycerides by activating sterol regulatory element-binding proteins (SREBP) in a manner refractory to feedback inhibition. Caspase-2 colocalizes with site 1 protease (S1P) and cleaves it to generate a soluble active fragment that initiates SCAP-independent SREBP1/2 activation in the ER. Caspase-2 ablation or pharmacological inhibition prevents diet-induced steatosis and NASH progression in ER-stress-prone mice. Caspase-2 inhibition offers a specific and effective strategy for preventing or treating stress-driven fatty liver diseases, whereas caspase-2-generated S1P proteolytic fragments, which enter the secretory pathway, are potential NASH biomarkers.


Assuntos
Caspase 2/fisiologia , Lipogênese/fisiologia , Pró-Proteína Convertases/fisiologia , Serina Endopeptidases/fisiologia , Animais , Colesterol/metabolismo , Retículo Endoplasmático/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Fígado Gorduroso/fisiopatologia , Células HEK293 , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pró-Proteína Convertases/metabolismo , Serina Endopeptidases/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos/metabolismo
2.
Am J Physiol Cell Physiol ; 313(6): C632-C643, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28903918

RESUMO

Proteinuria is a characteristic of chronic kidney disease and also a causative factor that promotes the disease progression, in part, via activation of the intrarenal renin-angiotensin system (RAS). (Pro)renin receptor (PRR), a newly discovered component of the RAS, binds renin and (pro)renin to promote angiotensin I generation. The present study was performed to test the role of soluble PRR (sPRR) in albumin overload-induced responses in cultured human renal proximal tubular cell line human kidney 2 (HK-2) cells. Bovine serum albmuin (BSA) treatment for 24 h at 20 mg/ml induced renin activity and inflammation, both of which were attenuated by a PRR decoy inhibitor PRO20. BSA treatment induced a more than fivefold increase in medium sPRR due to enhanced cleavage of PRR. Surprisingly, this cleavage event was unaffected by inhibition of furin or a disintegrin and metalloproteinase 19. Screening for a novel cleavage enzyme led to the identification of site-1 protease (S1P). Inhibition of S1P with PF-429242 or siRNA remarkably suppressed BSA-induced sPRR production, renin activity, and inflammatory response. Administration of a recombinant sPRR, termed sPRR-His, reversed the effects of S1P inhibition. In HK-2 cells overexpressing PRR, mutagenesis of the S1P, but not furin cleavage site, reduced sPRR levels. Together, these results suggest that PRR mediates albumin-induced cellular responses through S1P-derived sPRR.


Assuntos
Células Epiteliais/metabolismo , Rim/metabolismo , Pró-Proteína Convertases/fisiologia , Receptores de Superfície Celular/fisiologia , Serina Endopeptidases/fisiologia , Albumina Sérica Humana/farmacologia , ATPases Vacuolares Próton-Translocadoras/fisiologia , Linhagem Celular , Linhagem Celular Transformada , Células Epiteliais/efeitos dos fármacos , Humanos , Rim/citologia , Rim/efeitos dos fármacos , Masculino , Pró-Proteína Convertases/antagonistas & inibidores , RNA Interferente Pequeno/farmacologia
3.
J Biol Chem ; 291(5): 2055-66, 2016 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-26645686

RESUMO

The proprotein convertase subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is implicated in lipid homeostasis, the unfolded protein response, and lysosome biogenesis. The protease is further hijacked by highly pathogenic emerging viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P requires removal of an N-terminal prodomain, by a multistep process, generating the mature enzyme. Here, we uncover a modular structure of the human SKI-1/S1P prodomain and define its function in folding and activation. We provide evidence that the N-terminal AB fragment of the prodomain represents an autonomous structural and functional unit that is necessary and sufficient for folding and partial activation. In contrast, the C-terminal BC fragment lacks a defined structure but is crucial for autoprocessing and full catalytic activity. Phylogenetic analysis revealed that the sequence of the AB domain is highly conserved, whereas the BC fragment shows considerable variation and seems even absent in some species. Notably, SKI-1/S1P of arthropods, like the fruit fly Drosophila melanogaster, contains a shorter prodomain comprised of full-length AB and truncated BC regions. Swapping the prodomain fragments between fly and human resulted in a fully mature and active SKI-1/S1P chimera. Our study suggests that primordial SKI-1/S1P likely contained a simpler prodomain consisting of the highly conserved AB fragment that represents an independent folding unit. The BC region appears as a later evolutionary acquisition, possibly allowing more subtle fine-tuning of the maturation process.


Assuntos
Pró-Proteína Convertases/fisiologia , Dobramento de Proteína , Serina Endopeptidases/fisiologia , Sequência de Aminoácidos , Animais , Catálise , Dicroísmo Circular , Drosophila melanogaster , Deleção de Genes , Teste de Complementação Genética , Células HEK293 , Homeostase , Humanos , Isoenzimas/química , Lipídeos/química , Dados de Sequência Molecular , Filogenia , Pró-Proteína Convertases/química , Desnaturação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/química , Transdução de Sinais , Transfecção
4.
Circulation ; 132(17): 1648-66, 2015 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-26503748

RESUMO

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an important role in the regulation of cholesterol homeostasis. By binding to hepatic low-density lipoprotein (LDL) receptors and promoting their lysosomal degradation, PCSK9 reduces LDL uptake, leading to an increase in LDL cholesterol concentrations. Gain-of-function mutations in PCSK9 associated with high LDL cholesterol and premature cardiovascular disease have been causally implicated in the pathophysiology of autosomal-dominant familial hypercholesterolemia. In contrast, the more commonly expressed loss-of-function mutations in PCSK9 are associated with reduced LDL cholesterol and cardiovascular disease risk. The development of therapeutic approaches that inhibit PCSK9 function has therefore attracted considerable attention from clinicians and the pharmaceutical industry for the management of hypercholesterolemia and its associated cardiovascular disease risk. This review summarizes the effects of PCSK9 on hepatic and intestinal lipid metabolism and the more recently explored functions of PCSK9 in extrahepatic tissues. Therapeutic approaches that prevent interaction of PCSK9 with hepatic LDL receptors (monoclonal antibodies, mimetic peptides), inhibit PCSK9 synthesis in the endoplasmic reticulum (antisense oligonucleotides, siRNAs), and interfere with PCSK9 function (small molecules) are also described. Finally, clinical trials testing the safety and efficacy of monoclonal antibodies to PCSK9 are reviewed. These have shown dose-dependent decreases in LDL cholesterol (44%-65%), apolipoprotein B (48%-59%), and lipoprotein(a) (27%-50%) without major adverse effects in various high-risk patient categories, including those with statin intolerance. Initial reports from 2 of these trials have indicated the expected reduction in cardiovascular events. Hence, inhibition of PCSK9 holds considerable promise as a therapeutic option for decreasing cardiovascular disease risk.


Assuntos
Doenças Cardiovasculares/prevenção & controle , Terapia de Alvo Molecular , Pró-Proteína Convertases/antagonistas & inibidores , Adipócitos/metabolismo , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Encéfalo/metabolismo , Doenças Cardiovasculares/enzimologia , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/genética , LDL-Colesterol/metabolismo , Ensaios Clínicos como Assunto , Predisposição Genética para Doença , Humanos , Hiperlipoproteinemias/genética , Incidência , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Camundongos , Mutação , Oligonucleotídeos Antissenso/uso terapêutico , Fragmentos de Peptídeos/uso terapêutico , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/química , Pró-Proteína Convertases/deficiência , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/fisiologia , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/uso terapêutico , Receptores de LDL/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Serina Endopeptidases/fisiologia , Relação Estrutura-Atividade
5.
Circ Res ; 114(6): 1022-36, 2014 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-24625727

RESUMO

Since the discovery of proprotein convertase subtilisin kexin 9 (PCSK9) in 2003, this PC has attracted a lot of attention from the scientific community and pharmaceutical companies. Secreted into the plasma by the liver, the proteinase K-like serine protease PCSK9 binds the low-density lipoprotein (LDL) receptor at the surface of hepatocytes, thereby preventing its recycling and enhancing its degradation in endosomes/lysosomes, resulting in reduced LDL-cholesterol clearance. Surprisingly, in a nonenzymatic fashion, PCSK9 enhances the intracellular degradation of all its target proteins. Rare gain-of-function PCSK9 variants lead to higher levels of LDL-cholesterol and increased risk of cardiovascular disease; more common loss-of-function PCSK9 variants are associated with reductions in both LDL-cholesterol and risk of cardiovascular disease. It took 9 years to elaborate powerful new PCSK9-based therapeutic approaches to reduce circulating levels of LDL-cholesterol. Presently, PCSK9 monoclonal antibodies that inhibit its function on the LDL receptor are evaluated in phase III clinical trials. This review will address the biochemical, genetic, and clinical aspects associated with PCSK9's biology and pathophysiology in cells, rodent and human, with emphasis on the clinical benefits of silencing the expression/activity of PCSK9 as a new modality in the treatment of hypercholesterolemia and associated pathologies.


Assuntos
Hepatócitos/metabolismo , Pró-Proteína Convertases/fisiologia , Receptores de LDL/metabolismo , Serina Endopeptidases/fisiologia , Adulto , Envelhecimento/metabolismo , Animais , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Criança , LDL-Colesterol/sangue , Cromossomos Humanos Par 1/genética , Ensaios Clínicos Fase III como Assunto , Dieta , Modelos Animais de Doenças , Endossomos/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Predisposição Genética para Doença , Hepatite C/metabolismo , Humanos , Hiperlipoproteinemia Tipo II/tratamento farmacológico , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo II/metabolismo , Resistência à Insulina/fisiologia , Lipoproteínas LDL/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Lisossomos/metabolismo , Masculino , Terapia de Alvo Molecular , Mutação , Especificidade de Órgãos , Gravidez , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/antagonistas & inibidores , Pró-Proteína Convertases/química , Pró-Proteína Convertases/deficiência , Pró-Proteína Convertases/genética , Processamento de Proteína Pós-Traducional , Risco , Roedores , Serina Endopeptidases/química , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Vertebrados/genética
6.
Postepy Biochem ; 62(4): 472-481, 2016.
Artigo em Polonês | MEDLINE | ID: mdl-28132449

RESUMO

A large group of secretory proteins involved in proper functioning of living organisms, is synthesized as inactive precursor molecules. Their biologically active forms are obtained as a result of numerous post-translational modifications. Some of these processes occur irreversibly, permanently changing the initial compound structure. An example of such modifications is catalytic treatment of proteins performed by proteolytic enzymes. Among five separate classes of these enzymes, the most numerous are serine proteases. Mammalian proprotein convertases (PCs), which include: furin, PC1/3, PC2, PACE4, PC4, PC5/6, PC7, PCSK9, SKI-1, represent serine endoproteases family. PCs play a key role in the activation of a number of precursor proteins causing formation of biologically active forms of enzymes, hormones, signaling molecules, transcription and growth factors. This article summarizes current state of knowledge on biosynthesis, structure and substrate specificity of PCs, identifies the relationship between location and intracellular activity of these enzymes, and their physiological functions in mammals.


Assuntos
Pró-Proteína Convertases/fisiologia , Animais , Humanos , Pró-Proteína Convertases/metabolismo , Conformação Proteica , Especificidade por Substrato
7.
Curr Opin Lipidol ; 26(3): 155-61, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25887680

RESUMO

PURPOSE OF REVIEW: Proprotein convertase subtilisin kexin type 9 (PCSK9) acts as an endogenous natural inhibitor of the LDL receptor pathway, by targeting the receptor to lysosomes for degradation. Beside the liver, PCSK9 is also expressed at significant levels in other tissues, where its function remains unclear. The current review focuses on the extrahepatic actions of PCSK9. RECENT FINDINGS: The generation of liver-specific PCSK9 knockout mice has clearly indicated that PCSK9 affects cholesterol homeostasis via its action on extrahepatic organs. PCSK9 is highly expressed in the intestine, where it controls the production of triglyceride-rich lipoproteins and the transintestinal cholesterol excretion. The role of PCSK9 in the endocrine pancreas and glucose homeostasis remains unclear because conflicting data exist concerning the metabolic phenotype of PCSK9-deficient mice. Sparse data suggest that PCSK9 might also play a role in kidneys, vascular smooth muscle cells, and neurons. SUMMARY: Based on the virtuous combination of genetic and pharmacological approaches, the major function of PCSK9 as a key regulator of hepatic LDL receptor metabolism had quickly emerged. Accumulating evidence indicates that intestinal PCSK9 is also involved in the modulation of lipid homeostasis. Additional studies are warranted to decipher the physiological function of PCSK9 in other extrahepatic tissues and thus to better assess the safety of PCSK9 inhibitors.


Assuntos
Fígado/enzimologia , Pró-Proteína Convertases/fisiologia , Serina Endopeptidases/fisiologia , Animais , Humanos , Intestino Delgado/enzimologia , Ilhotas Pancreáticas/enzimologia , Metabolismo dos Lipídeos , Especificidade de Órgãos , Pró-Proteína Convertase 9
8.
Basic Res Cardiol ; 110(2): 4, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25600226

RESUMO

The proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a promising treatment target to lower serum cholesterol, a major risk factor of cardiovascular diseases. Gain-of-function mutations of PCSK9 are associated with hypercholesterolemia and increased risk of cardiovascular events. Conversely, loss-of-function mutations cause low-plasma LDL-C levels and a reduction of cardiovascular risk without known unwanted effects on individual health. Experimental studies have revealed that PCSK9 reduces the hepatic uptake of LDL-C by increasing the endosomal and lysosomal degradation of LDL receptors (LDLR). A number of clinical studies have demonstrated that inhibition of PCSK9 alone and in addition to statins potently reduces serum LDL-C concentrations. This review summarizes the current data on the regulation of PCSK9, its molecular function in lipid homeostasis and the emerging evidence on the extra-hepatic effects of PCSK9.


Assuntos
Doenças Cardiovasculares/fisiopatologia , Pró-Proteína Convertases/fisiologia , Serina Endopeptidases/fisiologia , Animais , Homeostase , Humanos , Pró-Proteína Convertase 9
9.
Clin Sci (Lond) ; 128(6): 379-85, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25291660

RESUMO

Postprandial lipaemia, due to elevated plasma apolipoprotein (apo) B-48 concentrations, contributes to increased cardiovascular (CV) risk in obesity. Proprotein convertase subtilisin/kexin type 9 (PCSK9) and apoC-III may play a role in regulating triacylglycerol-rich lipoprotein (TRL)-apoB-48 metabolism. We investigated the associations between plasma PCSK9 and apoC-III concentrations and the kinetics of apoB-48 in obese subjects. Seventeen obese subjects were given an oral fat load. ApoB-48 tracer/tracee ratios were measured after an intravenous 2H3-leucine administration using GC-MS. Kinetic parameters, including secretion and fractional catabolic rates (FCRs), were derived using a multi-compartmental model. Plasma PCSK9 and apoC-III concentrations were significantly and positively (P<0.05 in all) associated with the total area-under-curve (AUC) and incremental AUC for apoB-48 and inversely with TRL-apoB-48 FCR. Plasma PCSK9 and apoC-III concentrations were not correlated (P>0.05 in all) with basal secretion or the number of TRL-apoB-48 secreted over the postprandial period. In the stepwise regression analysis, plasma PCSK9 was the best predictor of the total and incremental AUCs for plasma apoB-48 and the FCR of TRL-apoB-48. The association between plasma PCSK9 and apoC-III and TRL-apoB-48 FCR remained significant (P<0.05 in all) after adjusting for age, homoeostasis model assessment (HOMA) score, hepatic lipase or lipoprotein lipase (LPL). In a multiple regression model, 31% of variance in TRL-apoB-48 FCR was accounted for by plasma PCSK9 and apoC-III concentrations (adjusted R2=0.306, P<0.05). However, their associations with TRL-apoB-48 FCR were not independent of each other. Our results suggest that the catabolism of TRL-apoB-48 in the postprandial state may be co-ordinated by PCSK9 and apoC-III in obese individuals.


Assuntos
Apolipoproteína B-48/sangue , Apolipoproteína C-III/sangue , Obesidade/sangue , Período Pós-Prandial/fisiologia , Pró-Proteína Convertases/sangue , Serina Endopeptidases/sangue , Idoso , Apolipoproteína C-III/fisiologia , Transporte Biológico/fisiologia , Deutério , Gorduras na Dieta/administração & dosagem , Feminino , Humanos , Lipoproteínas/sangue , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Obesidade/fisiopatologia , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/fisiologia , Serina Endopeptidases/fisiologia
10.
Arterioscler Thromb Vasc Biol ; 34(6): 1171-8, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24675665

RESUMO

OBJECTIVE: Low-density lipoprotein receptor (LDLR) is degraded by inducible degrader of LDLR (Idol) and protein convertase subtilisin/kexin type 9 (PCSK9), thereby regulating circulating LDL levels. However, it remains unclear whether, and if so how, these LDLR degraders affect each other. We therefore investigated effects of liver-specific expression of Idol on LDL/PCSK9 metabolism in mice and hamsters. APPROACH AND RESULTS: Injection of adenoviral vector expressing Idol (Ad-Idol) induced a liver-specific reduction in LDLR expression which, in turn, increased very-low-density lipoprotein/LDL cholesterol levels in wild-type mice because of delayed LDL catabolism. Interestingly, hepatic Idol overexpression markedly increased plasma PCSK9 levels. In LDLR-deficient mice, plasma PCSK9 levels were already elevated at baseline and unchanged by Idol overexpression, which was comparable with the observation for Ad-Idol-injected wild-type mice, indicating that Idol-induced PCSK9 elevation depended on LDLR. In wild-type mice, but not in LDLR-deficient mice, Ad-Idol enhanced hepatic PCSK9 expression, with activation of sterol regulatory element-binding protein 2 and subsequently increased expression of its target genes. Supporting in vivo findings, Idol transactivated PCSK9/LDLR in sterol regulatory element-binding protein 2/LDLR-dependent manners in vitro. Furthermore, an in vivo kinetic study using (125)I-labeled PCSK9 revealed delayed clearance of circulating PCSK9, which could be another mechanism. Finally, to extend these findings into cholesteryl ester transfer protein-expressing animals, we repeated the above in vivo experiments in hamsters and obtained similar results. CONCLUSIONS: A vicious cycle in LDLR degradation might be generated by PCSK9 induced by hepatic Idol overexpression via dual mechanisms: sterol regulatory element-binding protein 2/LDLR. Furthermore, these effects would be independent of cholesteryl ester transfer protein expression.


Assuntos
Fígado/metabolismo , Pró-Proteína Convertases/sangue , Receptores de LDL/fisiologia , Serina Endopeptidases/sangue , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 2/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Proteínas de Transferência de Ésteres de Colesterol/fisiologia , Cricetinae , Células Hep G2 , Humanos , Lipoproteínas LDL/metabolismo , Receptores X do Fígado , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Receptores Nucleares Órfãos/fisiologia , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/fisiologia , Serina Endopeptidases/fisiologia
11.
J Immunol ; 191(1): 97-109, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23740954

RESUMO

Marginal zone (MZ) B cells are an innate-like population that oscillates between MZ and follicular areas of the splenic white pulp. Differentiation of B cells into the MZ subset is governed by BCR signal strength and specificity, NF-κB activation through the B cell-activating factor belonging to the TNF family (BAFF) receptor, Notch2 signaling, and migration signals mediated by chemokine, integrin, and sphingosine-1-phosphate receptors. An imbalance in splenic B cell development resulting in expansion of the MZ subset has been associated with autoimmune pathogenesis in various murine models. One example is the NOD inbred mouse strain, in which MZ B cell expansion has been linked to development of type 1 diabetes and Sjögren's syndrome. However, the cause of MZ B cell expansion in this strain remains poorly understood. We have determined that increased MZ B cell development in NOD mice is independent of T cell autoimmunity, BCR specificity, BCR signal strength, and increased exposure to BAFF. Rather, mixed bone marrow chimeras showed that the factor(s) responsible for expansion of the NOD MZ subset is B cell intrinsic. Analysis of microarray expression data indicated that NOD MZ and precursor transitional 2-MZ subsets were particularly dysregulated for genes controlling cellular trafficking, including Apoe, Ccbp2, Cxcr7, Lgals1, Pla2g7, Rgs13, S1pr3, Spn, Bid, Cd55, Prf1, and Tlr3. Furthermore, these B cell subsets exhibited an increased steady state dwell time within splenic MZ areas. Our data therefore reveal that precursors of mature B cells in NOD mice exhibit an altered migration set point, allowing increased occupation of the MZ, a niche favoring MZ B cell differentiation.


Assuntos
Fator Ativador de Células B/fisiologia , Subpopulações de Linfócitos B/imunologia , Diferenciação Celular/imunologia , Receptor Notch2/fisiologia , Receptores de Antígenos de Linfócitos B/fisiologia , Receptores de Lisoesfingolipídeo/fisiologia , Baço/imunologia , Animais , Fator Ativador de Células B/deficiência , Receptor do Fator Ativador de Células B/fisiologia , Subpopulações de Linfócitos B/metabolismo , Subpopulações de Linfócitos B/patologia , Diferenciação Celular/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Pró-Proteína Convertases/fisiologia , Serina Endopeptidases/fisiologia , Baço/metabolismo , Baço/patologia
12.
Biochem J ; 457(1): 99-105, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24144304

RESUMO

PCSK9 (proprotein convertase subtilisin/kexin type 9) binds to the LDLR (low-density lipoprotein receptor) at the cell surface and disrupts recycling of the LDLR. However, PCSK9 also interacts with the LDLR in the ER (endoplasmic reticulum). In the present study we have investigated the role of PCSK9 for the transport of the LDLR from the ER to the cell membrane. A truncated LDLR consisting of the ectodomain (ED-LDLR) was used for these studies to avoid PCSK9-mediated degradation of the LDLR. The amount of secreted ED-LDLR was used as a measure of the amount of ED-LDLR transported from the ER. From co-transfection experiments of various PCSK9 and ED-LDLR plasmids, PCSK9 increased the amount of WT (wild-type) ED-LDLR in the medium, but not of an ED-LDLR lacking the EGF (epidermal growth factor)-A repeat or of a Class 2a mutant ED-LDLR which fails to exit the ER. Mutant PCSK9s which failed to undergo autocatalytic cleavage or failed to exit the ER, failed to increase the amount of WT-ED-LDLR in the medium. These mutants also reduced the amount of WT-ED-LDLR intracellularly, which could partly be prevented by the proteasome inhibitor lactacystine. WT-ED-LDLR promoted autocatalytic cleavage of pro-PCSK9. The findings of the present study indicate that the binding of WT-ED-LDLR to pro-PCSK9 in the ER promotes autocatalytic cleavage of PCSK9, and autocatalytically cleaved PCSK9 acts as a chaperone to promote the exit of WT-ED-LDLR from the ER.


Assuntos
Retículo Endoplasmático/metabolismo , Chaperonas Moleculares/fisiologia , Pró-Proteína Convertases/fisiologia , Receptores de LDL/metabolismo , Serina Endopeptidases/fisiologia , Catálise , Membrana Celular/metabolismo , Células Hep G2 , Homeostase , Humanos , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/química , Domínios e Motivos de Interação entre Proteínas/fisiologia , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiologia , Precursores de Proteínas/química , Precursores de Proteínas/fisiologia , Transporte Proteico , Proteólise , Receptores de LDL/química , Serina Endopeptidases/química
13.
Rev Med Liege ; 70(5-6): 292-8, 2015.
Artigo em Francês | MEDLINE | ID: mdl-26285455

RESUMO

Individualized therapeutic strategy of dyslipidemias, classically relies upon a phenotypic approach. The pattern of lipid profile allows the choice of the best pharmacological option (statin, fibrate) and the patient's clinical risk profile allows the definition of therapeutic goals, especially LDL cholesterol target levels. Dyslipidemias have a major genetic component, which is best illustrated by familial hypercholesterolemia, with its two heterozygous and homozygous forms. There is a huge between-subject variability in the response to lipid-lowering therapies (especially to statins) and ongoing pharmacogenetic and pharmacogenomic studies should help to better understand this inter-individual heterogeneity. The recent discovery of mutations in the PCSK9 rene opened new perspectives regarding the understanding of some forms of familial hypercholesterolemia and led to the development of monoclonal antibodies that selectively inhibit PCSK9. These PCSK9 inhibitors allow, when combined to a statin, drastic reductions in LDL cholesterol concentrations, even when familial hypercholesterolemia is present. They are currently tested in large prospective controlled trials aiming to demonstrate a significant reduction in the residual cardiovascular risk in statin-treated patients.


Assuntos
Hipolipemiantes/uso terapêutico , Medicina de Precisão/métodos , Dislipidemias/tratamento farmacológico , Dislipidemias/genética , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Individualidade , Lipídeos/sangue , Fenótipo , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/fisiologia , Serina Endopeptidases/fisiologia
14.
Circulation ; 127(24): 2403-13, 2013 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-23690465

RESUMO

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) modulates low-density lipoprotein (LDL) receptor (LDLR) degradation, thus influencing serum cholesterol levels. However, dysfunctional LDLR causes hypercholesterolemia without affecting PCSK9 clearance from the circulation. METHODS AND RESULTS: To study the reciprocal effects of PCSK9 and LDLR and the resultant effects on serum cholesterol, we produced transgenic mice expressing human (h) PCSK9. Although hPCSK9 was expressed mainly in the kidney, LDLR degradation was more evident in the liver. Adrenal LDLR levels were not affected, likely because of the impaired PCSK9 retention in this tissue. In addition, hPCSK9 expression increased hepatic secretion of apolipoprotein B-containing lipoproteins in an LDLR-independent fashion. Expression of hPCSK9 raised serum murine PCSK9 levels by 4.3-fold in wild-type mice and not at all in LDLR(-/-) mice, in which murine PCSK9 levels were already 10-fold higher than in wild-type mice. In addition, LDLR(+/-) mice had a 2.7-fold elevation in murine PCSK9 levels and no elevation in cholesterol levels. Conversely, acute expression of human LDLR in transgenic mice caused a 70% decrease in serum murine PCSK9 levels. Turnover studies using physiological levels of hPCSK9 showed rapid clearance in wild-type mice (half-life, 5.2 minutes), faster clearance in human LDLR transgenics (2.9 minutes), and much slower clearance in LDLR(-/-) recipients (50.5 minutes). Supportive results were obtained with an in vitro system. Finally, up to 30% of serum hPCSK9 was associated with LDL regardless of LDLR expression. CONCLUSIONS: Our results support a scenario in which LDLR represents the main route of elimination of PCSK9 and a reciprocal regulation between these 2 proteins controls serum PCSK9 levels, hepatic LDLR expression, and serum LDL levels.


Assuntos
Colesterol/sangue , Pró-Proteína Convertases/sangue , Receptores de LDL/sangue , Serina Endopeptidases/sangue , Animais , Células Cultivadas , Modelos Animais de Doenças , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/fisiopatologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/fisiologia , Receptores de LDL/deficiência , Receptores de LDL/genética , Serina Endopeptidases/genética , Serina Endopeptidases/fisiologia , Transfecção
15.
Clin Calcium ; 24(6): 853-61, 2014 Jun.
Artigo em Japonês | MEDLINE | ID: mdl-24870836

RESUMO

Bone is constantly renewed by the balanced action of osteoblastic bone formation and osteoclastic bone resorption both of which mainly occur at the bone surface. This restructuring process called "bone remodeling" is important not only for normal bone mass and strength, but also for mineral homeostasis. Coupling has been understood as a balanced induction of osteoblastic bone formation in response to osteoclastic bone resorption. An imbalance of this coupling is often linked to various bone diseases. TGF-ß and IGF released from bone matrix during osteoclastic bone resorption are the favored candidates as classical coupling factor. Recently, several reports suggest that osteoclast-derived molecules/cytokines (clastokine) mediate directional signaling between osteoblasts and osteoclasts into the bone microenvironment. Thus, the elucidation of the regulatory mechanisms involved in bone cell communication and coupling is critical for a deeper understanding of the skeletal system in health and disease.


Assuntos
Osso e Ossos/citologia , Osso e Ossos/fisiologia , Comunicação Celular/genética , Comunicação Celular/fisiologia , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Animais , Antígenos CD/fisiologia , Proteína Morfogenética Óssea 6/fisiologia , Remodelação Óssea/genética , Remodelação Óssea/fisiologia , Microambiente Celular/genética , Microambiente Celular/fisiologia , Homeostase/genética , Homeostase/fisiologia , Humanos , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/fisiologia , Camundongos , Fatores de Transcrição NFATC/metabolismo , Osteoprotegerina/fisiologia , Pró-Proteína Convertases/fisiologia , Ligante RANK/fisiologia , Semaforina-3A/fisiologia , Semaforinas/fisiologia , Serina Endopeptidases/fisiologia , Somatomedinas/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Via de Sinalização Wnt/fisiologia
16.
J Biol Chem ; 287(40): 33745-55, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-22875854

RESUMO

The proprotein convertase PCSK9 is a major target in the treatment of hypercholesterolemia because of its ability bind the LDL receptor (LDLR) and enhance its degradation in endosomes/lysosomes. In the endoplasmic reticulum, the zymogen pro-PCSK9 is first autocatalytically cleaved at its internal Gln(152)↓, resulting in a secreted enzymatically inactive complex of PCSK9 with its inhibitory prosegment (prosegment·PCSK9), which is the active form of PCSK9 on the LDLR. We mutagenized the P1 cleavage site Gln(152) into all other residues except Cys and analyzed the expression and secretion of the resulting mutants. The data demonstrated the following. 1) The only P1 residues recognized by PCSK9 are Gln > Met > Ala > Ser > Thr ≈ Asn, revealing an unsuspected specificity. 2) All other mutations led to the formation of an unprocessed zymogen that acted as a dominant negative retaining the native protein in the endoplasmic reticulum. Analysis of a large panoply of known natural and artificial point mutants revealed that this general dominant negative observation applies to all PCSK9 mutations that result in the inability of the protein to exit the endoplasmic reticulum. Such a tight quality control property of the endoplasmic reticulum may lead to the development of specific PCSK9 small molecule inhibitors that block its autocatalytic processing. Finally, inspired by the most active gain-of-function mutant, D374Y, we evaluated the LDLR degradation activity of 18 Asp(374) variants of PCSK9. All Asp(374) mutations resulted in similar gain-of-function activity on the LDLR except that D374E was as active as native PCSK9, D374G was relatively less active, and D374N and D374P were completely inactive.


Assuntos
Mutação , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/fisiologia , Receptores de LDL/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/fisiologia , Processamento Alternativo , Catálise , Colesterol/metabolismo , LDL-Colesterol/metabolismo , Precursores Enzimáticos/química , Citometria de Fluxo , Genes Dominantes , Células HEK293 , Humanos , Hipercolesterolemia/genética , Modelos Biológicos , Pró-Proteína Convertase 9 , Receptores de LDL/química , Transfecção
17.
Pharmacol Res ; 73: 27-34, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23578522

RESUMO

Proprotein convertase subtilisin/kexin 9 (PCSK9) has been shown to degrade hepatic low-density lipoprotein receptors (LDLR). Gain-of-function mutations promote the development of familial hypercholesterolemia, whereas loss-of-function mutations are associated with lower levels of circulating low-density lipoprotein cholesterol (LDL-C) and significant protection against coronary heart disease. The major classes of commonly prescribed lipid-lowering medications, such as statins, increase serum PCSK9 levels, thus PCSK9 inhibition would increase the efficacy of statins on LDL-C lowering. Therefore, PCSK9 is an attractive therapeutic target for the new generation of cholesterol-lowering drugs. Here, we present a brief overview of the development of PCSK9 inhibitors and highlight the effect of currently prescribed LDL-C-lowering drugs on PCSK9, and the strategies that are being explored for its therapeutic inhibition. Current research and clinical trial results indicate that a PCSK9 inhibitor may be an exciting new therapeutic drug for the treatment of dyslipidemia and relevant cardiovascular diseases.


Assuntos
Anticolesterolemiantes/uso terapêutico , Hipercolesterolemia/tratamento farmacológico , Pró-Proteína Convertases/antagonistas & inibidores , Inibidores de Serina Proteinase/uso terapêutico , Animais , LDL-Colesterol/sangue , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/genética , Mutação , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/fisiologia , Serina Endopeptidases/fisiologia
18.
Arterioscler Thromb Vasc Biol ; 32(7): 1585-95, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22580899

RESUMO

OBJECTIVE: proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively regulates the low-density lipoprotein (LDL) receptor (LDLR) in hepatocytes and therefore plays an important role in controlling circulating levels of LDL-cholesterol. To date, the relationship between PCSK9 and metabolism of apolipoprotein B (apoB), the structural protein of LDL, has been controversial and remains to be clarified. METHODS AND RESULTS: We assessed the impact of PCSK9 overexpression (≈400-fold above baseline) on apoB synthesis and secretion in 3 mouse models: wild-type C57BL/6 mice and LDLR-null mice (Ldlr(-/-) and Ldlr(-/-)Apobec1(-/-)). Irrespective of LDLR expression, mice transduced with the PCSK9 gene invariably exhibited increased levels of plasma cholesterol, triacylglycerol, and apoB. Consistent with these findings, the levels of very-low-density lipoprotein and LDL were also increased whereas high-density lipoprotein levels were unchanged. Importantly, we demonstrated that endogenous PCSK9 interacted with apoB in hepatocytes. The PCSK9/apoB interaction resulted in increased production of apoB, possibly through the inhibition of intracellular apoB degradation via the autophagosome/lysosome pathway. CONCLUSIONS: We propose a new role for PCSK9 that involves shuttling between apoB and LDLR. The present study thus provides new insights into the action of PCSK9 in regulating apoB metabolism. Furthermore, our results indicate that targeting PCSK9 expression represents a new paradigm in therapeutic intervention against hyperlipidemia.


Assuntos
Apolipoproteínas B/fisiologia , Pró-Proteína Convertases/fisiologia , Receptores de LDL/fisiologia , Serina Endopeptidases/fisiologia , Animais , Apolipoproteínas B/sangue , Autofagia , Colesterol/sangue , Células Hep G2 , Humanos , Lipoproteínas VLDL/sangue , Lisossomos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9 , Triglicerídeos/sangue
19.
Cell Mol Life Sci ; 69(11): 1903-16, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22481440

RESUMO

The secreted protease proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to low-density lipid (LDL) receptor family members LDLR, very low density lipoprotein receptor (VLDLR) and apolipoprotein receptor 2 (ApoER2), and promotes their degradation in intracellular acidic compartments. In the liver, LDLR is a major controller of blood LDL levels, whereas VLDLR and ApoER2 in the brain mediate Reelin signaling, a critical pathway for proper development of the nervous system. Expression level of PCSK9 in the brain is highest in the cerebellum during perinatal development, but is also increased in the adult brain after ischemia. The mechanism of PCSK9 function and its involvement in neuronal apoptosis is poorly understood. We show here that RNAi-mediated knockdown of PCSK9 significantly reduced the death of potassium-deprived cerebellar granule neurons (CGN), as shown by reduced levels of nuclear phosphorylated c-Jun and activated caspase-3, as well as condensed apoptotic nuclei. ApoER2 protein levels were increased in PCSK9 RNAi cells. Knockdown of ApoER2 but not of VLDLR was sufficient to reverse the protection provided by PCSK9 RNAi, suggesting that proapoptotic signaling of PCSK9 is mediated by altered ApoER2 function. Pharmacological inhibition of signaling pathways associated with lipoprotein receptors suggested that PCSK9 regulates neuronal apoptosis independently of NMDA receptor function but in concert with ERK and JNK signaling pathways. PCSK9 RNAi also reduced staurosporine-induced CGN apoptosis and axonal degeneration in the nerve growth factor-deprived dorsal root ganglion neurons. We conclude that PCSK9 potentiates neuronal apoptosis via modulation of ApoER2 levels and related anti-apoptotic signaling pathways.


Assuntos
Apoptose/fisiologia , Proteínas Relacionadas a Receptor de LDL/metabolismo , Neurônios/citologia , Pró-Proteína Convertases/fisiologia , Serina Endopeptidases/fisiologia , Animais , Caspase 3/metabolismo , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas Relacionadas a Receptor de LDL/genética , Camundongos , Fosforilação , Potássio/metabolismo , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Interferência de RNA , Proteína Reelina , Serina Endopeptidases/genética , Transdução de Sinais
20.
PLoS Genet ; 6(5): e1000967, 2010 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-20523747

RESUMO

Peptide hormones are potent signaling molecules that coordinate animal physiology, behavior, and development. A key step in activation of these peptide signals is their proteolytic processing from propeptide precursors by a family of proteases, the subtilisin-like proprotein convertases (PCs). Here, we report the functional dissection of amontillado (amon), which encodes the Drosophila homolog of the mammalian PC2 protein, using cell-type specific inactivation and rescue experiments, and we show that amon is required in the islet-like adipokinetic hormone (AKH)-producing cells that regulate sugar homeostasis. In Drosophila, AKH acts analogously to vertebrate glucagon to increase circulating sugar levels from energy stores, while insulin-like peptides (DILPs) act to decrease sugar levels. amon mutant larvae have significantly reduced hemolymph sugar levels, and thus phenocopy larvae where the AKH-producing cells in the corpora cardiaca have been ablated. Reduction of amon expression in these cells via cell-specific RNA inactivation also results in larvae with reduced sugar levels while expression of amon in AKH cells in an amon mutant background rescues hypoglycemia. Hypoglycemia in larvae resulting from amon RNA inactivation in the AKH cells can be rescued by global expression of the akh gene. Finally, mass spectrometric profiling shows that the production of mature AKH is inhibited in amon mutants. Our data indicate that amon function in the AKH cells is necessary to maintain normal sugar homeostasis, that amon functions upstream of akh, and that loss of mature AKH is correlated with loss of amon activity. These observations indicate that the AKH propeptide is a proteolytic target of the amon proprotein convertase and provide evidence for a conserved role of PC2 in processing metabolic peptide hormones.


Assuntos
Drosophila/fisiologia , Células Endócrinas/metabolismo , Glucose/metabolismo , Hormônios de Inseto/biossíntese , Pró-Proteína Convertases/fisiologia , Animais , Animais Geneticamente Modificados , Sequência de Bases , Primers do DNA , Imuno-Histoquímica , Mutação , Pró-Proteína Convertases/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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