RESUMO
The pro-protein convertase FURIN (PCSK3) is implicated in a wide range of normal and pathological biological processes such as infectious diseases, cancer and cardiovascular diseases. Previously, we performed a systemic inhibition of FURIN in a mouse model of atherosclerosis and demonstrated significant plaque reduction and alterations in macrophage function. To understand the cellular mechanisms affected by FURIN inhibition in myeloid cells, we optimized a CRISPR-mediated gene deletion protocol for successfully deriving hemizygous (HZ) and nullizygous (NZ) FURIN knockout clones in U937 monocytic cells using lipotransfection-based procedures and a dual guide RNA delivery strategy. We observed differences in monocyte and macrophage functions involving phagocytosis, lipid accumulation, cell migration, inflammatory gene expression, cytokine release patterns, secreted proteomics (cytokines) and whole-genome transcriptomics between wild-type, HZ and NZ FURIN clones. These studies provide a mechanistic basis on the possible roles of myeloid cell FURIN in cardiovascular disorders.
Assuntos
Furina , Edição de Genes , Monócitos , Animais , Humanos , Camundongos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Citocinas/genética , Furina/genética , Furina/metabolismo , Monócitos/metabolismo , Multiômica , RNA Guia de Sistemas CRISPR-Cas , Células U937RESUMO
Vascular restenosis remains a major problem in patients with coronary artery disease (CAD) and peripheral artery disease (PAD). Neointimal hyperplasia, defined by post-procedure proliferation and migration of vascular smooth muscle cells (VSMCs) is a key underlying pathology. Here we investigated the role of Interleukin 11 (IL-11) in a mouse model of injury-related plaque development. Apoe-/- mice were fed a hyperlipidaemic diet and subjected to carotid wire injury of the right carotid. Mice were injected with an anti-IL11 antibody (X203), IgG control antibody or buffer. We performed ultrasound analysis to assess vessel wall thickness and blood velocity. Using histology and immunofluorescence approaches, we determined the effects of IL-11 inhibition on VSMC and macrophages phenotypes and fibrosis. Treatment of mice with carotid wire injury using X203 significantly reduced post-endothelial injury vessel wall thickness, and injury-related plaque, when compared to control. Immunofluorescence staining of the injury-related plaque showed that X203 treatment did not reduce macrophage numbers, but reduced the number of VSMCs and lowered matrix metalloproteinase 2 (MMP2) levels and collagen content in comparison to control. X203 treatment was associated with a significant increase in smooth muscle protein 22α (SM22α) positive cells in injury-related plaque compared to control, suggesting preservation of the contractile VSMC phenotype. Interestingly, X203 also reduced the collagen content of uninjured carotid arteries as compared to IgG, showing an additional effect on hyperlipidemia-induced arterial remodeling in the absence of mechanical injury. Therapeutic inhibition of IL-11 reduced vessel wall thickness, attenuated neointimal hyperplasia, and has favorable effects on vascular remodeling following wire-induced endothelial injury. This suggests IL-11 inhibition as a potential novel therapeutic approach to reduce arterial stenosis following revascularization in CAD and PAD patients.
Assuntos
Anticorpos Neutralizantes/farmacologia , Artérias Carótidas/efeitos dos fármacos , Lesões das Artérias Carótidas/tratamento farmacológico , Hiperplasia/tratamento farmacológico , Interleucina-11/metabolismo , Animais , Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno/metabolismo , Modelos Animais de Doenças , Hiperplasia/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Neointima/tratamento farmacológico , Neointima/metabolismo , Remodelação Vascular/efeitos dos fármacosRESUMO
Bile acids are signalling hormones involved in the regulation of several metabolic pathways. The ability of bile acids to bind and signal through their receptors is modulated by the gut microbiome, since the microbiome contributes to the regulation and synthesis of bile acids as well to their physiochemical properties. From the gut, bacteria have been shown to send signals to the central nervous system via their metabolites, thus affecting the behaviour and brain function of the host organism. In the last years it has become increasingly evident that bile acids affect brain function, during normal physiological and pathological conditions. Although bile acids may be synthesized locally in the brain, the majority of brain bile acids are taken up from the systemic circulation. Since the composition of the brain bile acid pool may be regulated by the action of intestinal bacteria, it is possible that bile acids function as a communication bridge between the gut microbiome and the brain. However, little is known about the molecular mechanisms and the physiological roles of bile acids in the central nervous system. The possibility that bile acids may be a direct link between the intestinal microbiome and the brain is also an understudied subject. Here we review the influence of gut bacteria on the bile acid pool composition and properties, as well as striking evidence showing the role of bile acids as neuroactive molecules.
Assuntos
Ácidos e Sais Biliares/metabolismo , Encéfalo/metabolismo , Microbioma Gastrointestinal , Animais , Colesterol/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Ingestão de Alimentos , Células Enterocromafins/metabolismo , Fermentação , Vesícula Biliar/metabolismo , Vida Livre de Germes , Humanos , Fígado/metabolismo , Camundongos , Doenças Neurodegenerativas/metabolismo , Neurotransmissores/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Acidente Vascular Cerebral/metabolismo , Xantomatose Cerebrotendinosa/metabolismoRESUMO
Objective- Atherosclerotic coronary artery disease is the leading cause of death worldwide, and current treatment options are insufficient. Using systems-level network cluster analyses on a large coronary artery disease case-control cohort, we previously identified PCSK3 (proprotein convertase subtilisin/kexin family member 3; FURIN) as a member of several coronary artery disease-associated pathways. Thus, our objective is to determine the role of FURIN in atherosclerosis. Approach and Results- In vitro, FURIN inhibitor treatment resulted in reduced monocyte migration and reduced macrophage and vascular endothelial cell inflammatory and cytokine gene expression. In vivo, administration of an irreversible inhibitor of FURIN, α-1-PDX (α1-antitrypsin Portland), to hyperlipidemic Ldlr-/- mice resulted in lower atherosclerotic lesion area and a specific reduction in severe lesions. Significantly lower lesional macrophage and collagen area, as well as systemic inflammatory markers, were observed. MMP2 (matrix metallopeptidase 2), an effector of endothelial function and atherosclerotic lesion progression, and a FURIN substrate was significantly reduced in the aorta of inhibitor-treated mice. To determine FURIN's role in vascular endothelial function, we administered α-1-PDX to Apoe-/- mice harboring a wire injury in the common carotid artery. We observed significantly decreased carotid intimal thickness and lower plaque cellularity, smooth muscle cell, macrophage, and inflammatory marker content, suggesting protection against vascular remodeling. Overexpression of FURIN in this model resulted in a significant 67% increase in intimal plaque thickness, confirming that FURIN levels directly correlate with atherosclerosis. Conclusions- We show that systemic inhibition of FURIN in mice decreases vascular remodeling and atherosclerosis. FURIN-mediated modulation of MMP2 activity may contribute to the atheroprotection observed in these mice.
Assuntos
Aterosclerose/prevenção & controle , Furina/antagonistas & inibidores , Placa Aterosclerótica/tratamento farmacológico , alfa 1-Antitripsina/uso terapêutico , Animais , Aorta/enzimologia , Aterosclerose/genética , Aterosclerose/patologia , Artéria Carótida Primitiva , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos , Indução Enzimática/efeitos dos fármacos , Furina/genética , Furina/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/fisiologia , Masculino , Metaloproteinase 2 da Matriz/análise , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/fisiologia , Placa Aterosclerótica/patologia , Receptores de LDL/deficiência , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/patologia , Remodelação Vascular , alfa 1-Antitripsina/farmacologiaRESUMO
OBJECTIVE: High-density lipoproteins (HDL) are considered to protect against atherosclerosis in part by facilitating the removal of cholesterol from peripheral tissues. However, factors regulating lipid efflux are incompletely understood. We previously identified a variant in adenosine triphosphate-binding cassette transporter A8 (ABCA8) in an individual with low HDL cholesterol (HDLc). Here, we investigate the role of ABCA8 in cholesterol efflux and in regulating HDLc levels. APPROACH AND RESULTS: We sequenced ABCA8 in individuals with low and high HDLc and identified, exclusively in low HDLc probands, 3 predicted deleterious heterozygous ABCA8 mutations (p.Pro609Arg [P609R], IVS17-2 A>G and p.Thr741Stop [T741X]). HDLc levels were lower in heterozygous mutation carriers compared with first-degree family controls (0.86±0.34 versus 1.17±0.26 mmol/L; P=0.005). HDLc levels were significantly decreased by 29% (P=0.01) in Abca8b-/- mice on a high-cholesterol diet compared with wild-type mice, whereas hepatic overexpression of human ABCA8 in mice resulted in significant increases in plasma HDLc and the first steps of macrophage-to-feces reverse cholesterol transport. Overexpression of wild-type but not mutant ABCA8 resulted in a significant increase (1.8-fold; P=0.01) of cholesterol efflux to apolipoprotein AI in vitro. ABCA8 colocalizes and interacts with adenosine triphosphate-binding cassette transporter A1 and further potentiates adenosine triphosphate-binding cassette transporter A1-mediated cholesterol efflux. CONCLUSIONS: ABCA8 facilitates cholesterol efflux and modulates HDLc levels in humans and mice.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Colesterol na Dieta/sangue , HDL-Colesterol/sangue , Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/genética , Adulto , Idoso , Animais , Apolipoproteína A-I/sangue , Apolipoproteína B-100/sangue , Transporte Biológico , Biomarcadores/sangue , Células COS , Estudos de Casos e Controles , Chlorocebus aethiops , Análise Mutacional de DNA , Dieta Hiperlipídica , Fezes/química , Feminino , Células HEK293 , Hereditariedade , Heterozigoto , Humanos , Fígado/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Mutação , Linhagem , Fenótipo , TransfecçãoRESUMO
Besides their role in facilitating lipid absorption, bile acids are increasingly being recognized as signaling molecules that activate cell-signaling receptors. Targeted disruption of the sterol 12α-hydroxylase gene (Cyp8b1) results in complete absence of cholic acid (CA) and its derivatives. Here we investigate the effect of Cyp8b1 deletion on glucose homeostasis. Absence of Cyp8b1 results in improved glucose tolerance, insulin sensitivity, and ß-cell function, mediated by absence of CA in Cyp8b1(-/-) mice. In addition, we show that reduced intestinal fat absorption in the absence of biliary CA leads to increased free fatty acids reaching the ileal L cells. This correlates with increased secretion of the incretin hormone GLP-1. GLP-1, in turn, increases the biosynthesis and secretion of insulin from ß-cells, leading to the improved glucose tolerance observed in the Cyp8b1(-/-) mice. Thus, our data elucidate the importance of Cyp8b1 inhibition on the regulation of glucose metabolism.
Assuntos
Peptídeo 1 Semelhante ao Glucagon/metabolismo , Glucose/metabolismo , Homeostase/fisiologia , Resistência à Insulina/fisiologia , Insulina/metabolismo , Esteroide 12-alfa-Hidroxilase/metabolismo , Animais , Ácido Cólico/metabolismo , Polipeptídeo Inibidor Gástrico/metabolismo , Peptídeo 1 Semelhante ao Glucagon/genética , Células Secretoras de Insulina/metabolismo , Camundongos , Camundongos Knockout , Esteroide 12-alfa-Hidroxilase/genéticaRESUMO
While genetic determinants strongly influence HDL cholesterol (HDLc) levels, most genetic causes underlying variation in HDLc remain unknown. We aimed to identify novel rare mutations with large effects in candidate genes contributing to extreme HDLc in humans, utilizing family-based Mendelian genetics. We performed next-generation sequencing of 456 candidate HDLc-regulating genes in 200 unrelated probands with extremely low (≤10th percentile) or high (≥90th percentile) HDLc. Probands were excluded if known mutations existed in the established HDLc-regulating genes ABCA1, APOA1, LCAT, cholesteryl ester transfer protein (CETP), endothelial lipase (LIPG), and UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2). We identified 93 novel coding or splice-site variants in 72 candidate genes. Each variant was genotyped in the proband's family. Family-based association analyses were performed for variants with sufficient power to detect significance at P < 0.05 with a total of 627 family members being assessed. Mutations in the genes glucokinase regulatory protein (GCKR), RNase L (RNASEL), leukocyte immunoglobulin-like receptor 3 (LILRA3), and dynein axonemal heavy chain 10 (DNAH10) segregated with elevated HDLc levels in families, while no mutations associated with low HDLc. Taken together, we have identified mutations in four novel genes that may play a role in regulating HDLc levels in humans.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Dineínas do Axonema/genética , HDL-Colesterol/sangue , Endorribonucleases/genética , Mutação , Receptores Imunológicos/genética , Transportador 1 de Cassete de Ligação de ATP/genética , Adulto , Idoso , Apolipoproteína A-I/genética , Proteínas de Transferência de Ésteres de Colesterol/genética , HDL-Colesterol/genética , Feminino , Humanos , Lipase/genética , Masculino , Pessoa de Meia-Idade , N-Acetilgalactosaminiltransferases/genética , Fosfatidilcolina-Esterol O-Aciltransferase/genética , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
To date, few mutations are described to underlie highly-elevated HDLc levels in families. Here we sequenced the coding regions and adjacent sequence of the LIPG, CETP, and GALNT2 genes in 171 unrelated Dutch Caucasian probands with HDLc≥90th percentile and analyzed segregation of mutations with lipid phenotypes in family members. In these probands, mutations were most frequent in LIPG (12.9%) followed by GALNT2 (2.3%) and CETP (0.6%). A total of 6 of 10 mutations in these three genes were novel (60.0%), and mutations segregated with elevated HDLc in families. Interestingly, the LIPG mutations N396S and R476W, which usually result in elevated HDLc, were unexpectedly found in 6 probands with low HDLc (i.e., ≤10th percentile). However, 5 of these probands also carried mutations in ABCA1, LCAT, or LPL. Finally, no CETP and GALNT2 mutations were found in 136 unrelated probands with low HDLc. Taken together, we show that rare coding and splicing mutations in LIPG, CETP, and GALNT2 are enriched in persons with hyperalphalipoproteinemia and segregate with elevated HDLc in families. Moreover, LIPG mutations do not overcome low HDLc in individuals with ABCA1 and possibly LCAT and LPL mutations, indicating that LIPG affects HDLc levels downstream of these proteins.
Assuntos
Proteínas de Transferência de Ésteres de Colesterol/genética , HDL-Colesterol/genética , Hipercolesterolemia/genética , Lipase/genética , Mutação , N-Acetilgalactosaminiltransferases/genética , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Idoso , Processamento Alternativo , Estudos de Coortes , Doença da Artéria Coronariana/genética , Saúde da Família , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Análise de Sequência de DNA , População Branca , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Huntingtin interacting protein 14 (HIP14, ZDHHC17) is a huntingtin (HTT) interacting protein with palmitoyl transferase activity. In order to interrogate the function of Hip14, we generated mice with disruption in their Hip14 gene. Hip14-/- mice displayed behavioral, biochemical and neuropathological defects that are reminiscent of Huntington disease (HD). Palmitoylation of other HIP14 substrates, but not Htt, was reduced in the Hip14-/- mice. Hip14 is dysfunctional in the presence of mutant htt in the YAC128 mouse model of HD, suggesting that altered palmitoylation mediated by HIP14 may contribute to HD.
Assuntos
Aciltransferases/deficiência , Doença de Huntington/etiologia , Lipoilação/genética , Proteínas do Tecido Nervoso/deficiência , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Morte Celular/genética , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Encefalinas/metabolismo , Doença de Huntington/genética , Doença de Huntington/patologia , Camundongos , Camundongos Knockout , Atividade Motora/genética , Proteínas Mutantes/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Sinapses/metabolismoRESUMO
Huntington disease (HD) is caused by polyglutamine expansion in the huntingtin (HTT) protein. Huntingtin-interacting protein 14 (HIP14), one of 23 DHHC domain-containing palmitoyl acyl transferases (PATs), binds to HTT and robustly palmitoylates HTT at cysteine 214. Mutant HTT exhibits reduced palmitoylation and interaction with HIP14, contributing to the neuronal dysfunction associated with HD. In this study, we confirmed that, among 23 DHHC PATs, HIP14 and its homolog DHHC-13 (HIP14L) are the two major PATs that palmitoylate HTT. Wild-type HTT, in addition to serving as a palmitoylation substrate, also modulates the palmitoylation of HIP14 itself. In vivo, HIP14 palmitoylation is decreased in the brains of mice lacking one HTT allele (hdh+/-) and is further reduced in mouse cortical neurons treated with HTT antisense oligos (HTT-ASO) that knockdown HTT expression by â¼95%. Previously, it has been shown that palmitoylation of DHHC proteins may affect their enzymatic activity. Indeed, palmitoylation of SNAP25 by HIP14 is potentiated in vitro in the presence of wild-type HTT. This influence of HTT on HIP14 activity is lost in the presence of CAG expansion. Furthermore, in both brains of hdh+/- mice and neurons treated with HTT-ASO, we observe a significant reduction in palmitoylation of endogenous SNAP25 and GluR1, synaptic proteins that are substrates of HIP14, suggesting wild-type HTT also influences HIP14 enzymatic activity in vivo. This study describes an important biochemical function for wild-type HTT modulation of HIP14 palmitoylation and its enzymatic activity.
Assuntos
Aciltransferases/metabolismo , Doença de Huntington/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Aciltransferases/genética , Animais , Western Blotting , Células Cultivadas , Proteína Huntingtina , Doença de Huntington/genética , Lipoilação , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Ligação Proteica , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Atherosclerosis is a progressive disease in which macrophages play an essential role. Macrophage infiltration into the arterial wall induces the development of an early atherosclerotic lesion. However, the dynamics of macrophage infiltration into the arterial wall during lesion progression remain poorly understood. In this study, low-density lipoprotein receptor knockout mice were fed a Western-type diet for 3, 6, 9, and 12 weeks to induce the formation of atherosclerotic lesions with different degrees of complexity. Subsequently, these mice underwent transplantation with bone marrow-overexpressing enhanced green fluorescent protein to track donor-derived cells, including macrophages. After 8 weeks of Western-type diet feeding after transplantation, macrophage infiltration was evaluated by immunohistochemical staining of donor-derived macrophages (enhanced green fluorescent protein-positive F4/80(+)) in the aortic roots. We found that the growth of pre-existing initial lesions was mainly caused by continued recruitment of donor-derived macrophages into the arterial wall. Interestingly, macrophage infiltration into pre-existing more advanced lesions was largely impaired, likely because of the formation of fibrous caps. In addition, interference with the expression of macrophage ATP-binding cassette transporter 1, an ATP-binding cassette transporter involved in cellular cholesterol efflux and macrophage recruitment into tissues, affects the infiltration of macrophages into pre-existing early lesions but not into advanced lesions. In conclusion, our data suggest that the dynamics of macrophage infiltration into the arterial wall vary greatly during atherogenesis and, thus, may affect the efficiency of pharmaceutical interventions aimed at targeting macrophage infiltration into the arterial wall.
Assuntos
Artérias/patologia , Aterosclerose/patologia , Movimento Celular , Macrófagos/patologia , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Aterosclerose/genética , Transplante de Medula Óssea , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Fígado/patologia , Linfonodos/patologia , Macrófagos/fisiologia , Camundongos , Camundongos Knockout , Receptores de LDL/genética , Baço/patologiaRESUMO
Mutations in the adenosine-triphosphate-binding cassette transporter-1 (ABCA1) lead to Tangier disease, a genetic disorder characterized by an almost complete absence of plasma high-density lipoprotein cholesterol. Although the importance of ABCA1 localization to its cholesterol efflux function has been extensively characterized, the cellular itinerary of ABCA1 leading to the plasma membrane is not fully elucidated. This review will summarize the current knowledge of ABCA1 trafficking and its relationship to function. Understanding these crucial processes provides potential novel therapeutic targets to regulate high-density lipoprotein biogenesis through influencing pathways of ABCA1 trafficking.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Apolipoproteína A-I/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Colesterol/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Retículo Endoplasmático/metabolismo , Expressão Gênica , Complexo de Golgi/metabolismo , Humanos , Lipoproteínas HDL/metabolismo , Camundongos , Processamento de Proteína Pós-Traducional/fisiologiaRESUMO
Loss of ABCA1 activity in Tangier disease (TD) is associated with abnormal apoB lipoprotein (Lp) metabolism in addition to the complete absence of high density lipoprotein (HDL). We used hepatocyte-specific ABCA1 knock-out (HSKO) mice to test the hypothesis that hepatic ABCA1 plays dual roles in regulating Lp metabolism and nascent HDL formation. HSKO mice recapitulated the TD lipid phenotype with postprandial hypertriglyceridemia, markedly decreased LDL, and near absence of HDL. Triglyceride (TG) secretion was 2-fold higher in HSKO compared with wild type mice, primarily due to secretion of larger TG-enriched VLDL secondary to reduced hepatic phosphatidylinositol 3-kinase signaling. HSKO mice also displayed delayed clearance of postprandial TG and reduced post-heparin plasma lipolytic activity. In addition, hepatic LDLr expression and plasma LDL catabolism were increased 2-fold in HSKO compared with wild type mice. Last, adenoviral repletion of hepatic ABCA1 in HSKO mice normalized plasma VLDL TG and hepatic phosphatidylinositol 3-kinase signaling, with a partial recovery of HDL cholesterol levels, providing evidence that hepatic ABCA1 is involved in the reciprocal regulation of apoB Lp production and HDL formation. These findings suggest that altered apoB Lp metabolism in TD subjects may result from hepatic VLDL TG overproduction and increased hepatic LDLr expression and highlight hepatic ABCA1 as an important regulatory factor for apoB-containing Lp metabolism.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Hepatócitos/metabolismo , Lipoproteínas LDL/metabolismo , Lipoproteínas VLDL/biossíntese , Triglicerídeos/biossíntese , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenoviridae/genética , Animais , Apolipoproteínas B/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Marcação de Genes , Humanos , Hipertrigliceridemia/genética , Hipertrigliceridemia/metabolismo , Lipoproteínas HDL/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Doença de Tangier/genética , Doença de Tangier/metabolismoRESUMO
ATP-binding cassette transporter (ABC)A1 lipidates apolipoprotein A-I both directly at the plasma membrane and also uses lipids from the late endosomal or lysosomal compartment in the internal lipidation of apolipoprotein A-I. However, how ABCA1 targeting to these specific membranes is regulated remains unknown. Palmitoylation is a dynamically regulated lipid modification that targets many proteins to specific membrane domains. We hypothesized that palmitoylation may also regulate ABCA1 transport and function. Indeed, ABCA1 is robustly palmitoylated at cysteines 3, -23, -1110, and -1111. Abrogation of palmitoylation of ABCA1 by mutation of the cysteines results in a reduction of ABCA1 localization at the plasma membranes and a reduction in the ability of ABCA1 to efflux lipids to apolipoprotein A-I. ABCA1 is palmitoylated by the palmitoyl transferase DHHC8, and increasing DHHC8 protein results in increased ABCA1-mediated lipid efflux. Thus, palmitoylation regulates ABCA1 localization at the plasma membrane, and regulates its lipid efflux ability.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Processamento de Proteína Pós-Traducional , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Aciltransferases/genética , Aciltransferases/metabolismo , Sequência de Aminoácidos , Animais , Apolipoproteína A-I/metabolismo , Transporte Biológico , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Colesterol/metabolismo , Cisteína , Humanos , Lipoilação , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Palmitatos/metabolismo , Fosfolipídeos/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão , Relação Estrutura-Atividade , TransfecçãoRESUMO
Palmitoylation, a post-translational modification of cysteine residues with the lipid palmitate, has recently emerged as an important mechanism for regulating protein trafficking and function. With the identification of 23 DHHC mammalian palmitoyl acyl transferases (PATs), a key question was the nature of substrate-enzyme specificity for these PATs. Using the acyl-biotin exchange palmitoylation assay, we compared the substrate specificity of four neuronal PATs, namely DHHC-3, DHHC-8, HIP14L (DHHC-13), and HIP14 (DHHC-17). Exogenous expression of enzymes and substrates in COS cells reveals that HIP14L and HIP14 modulate huntingtin palmitoylation, DHHC-8 modulates paralemmin-1 palmitoylation, and DHHC-3 shows the least substrate specificity. These in vitro data were validated by lentiviral siRNA-mediated knockdown of endogenous HIP14 and DHHC-3 in cultured rat cortical neurons. PATs require the presence of palmitoylated cysteines in order to interact with their substrates. To understand the elements that influence enzyme/substrate specificity further, we fused the HIP14 ankryin repeat domain to the N terminus of DHHC-3, which is not a PAT for huntingtin. This modification enabled DHHC-3 to behave similarly to HIP14 by modulating palmitoylation and trafficking of huntingtin. Taken together, this study indicates that individual PATs have specific substrate preference, determined by regulatory domains outside the DHHC domain of the enzymes.
Assuntos
Aciltransferases/metabolismo , Lipoilação/fisiologia , Neurônios/enzimologia , Aciltransferases/antagonistas & inibidores , Aciltransferases/química , Aciltransferases/genética , Animais , Sequência de Bases , Células COS , Células Cultivadas , Chlorocebus aethiops , Complexo de Golgi/enzimologia , Proteína Huntingtina , Técnicas In Vitro , Proteínas de Membrana/metabolismo , Modelos Moleculares , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , RNA Interferente Pequeno/genética , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
During spermatogenesis, spermatids undergo a "down and up" translocation event in the seminiferous epithelium. This event has been proposed to result from the movement of ectoplasmic specializations, which are formed in Sertoli cells at sites of adhesion to spermatids, along adjacent microtubule tracts. To test the hypothesis that a kinesin is associated with ectoplasmic specializations, we generated antibodies to conserved kinesin sequences and detected kinesins on fixed frozen testis sections and fixed seminiferous epithelial fragments. The antibodies reacted with ectoplasmic specializations related to spermatids, in addition to reacting with other structures in the epithelium known to contain kinesins. At the electron microscopy level, the antibodies reacted with the cytoplasmic face of the endoplasmic reticulum component of ectoplasmic specializations. Based on mRNA transcript screens using mouse GeneChip arrays of testis and Sertoli cells, we identified KIF20 as a candidate kinesin at ectoplasmic specializations. Antibodies generated against a peptide sequence unique to this kinesin reacted at ectoplasmic specializations in testis sections and epithelial fragments, as well as with the endoplasmic reticulum component of ectoplasmic specializations when analyzed by electron microscopy. The antibody reacted on Western blots with full-length KIF20. On Western blots of testis lysates, the antibody reacted with a protein that is not present in other tissues and which migrates at a higher molecular weight than that predicted for KIF20. Our results demonstrate that a kinesin is associated with apical ectoplasmic specializations in Sertoli cells and that the motor may be an isoform of KIF20.
Assuntos
Junções Aderentes/metabolismo , Cinesinas/metabolismo , Células de Sertoli/metabolismo , Espermátides/metabolismo , Junções Aderentes/ultraestrutura , Animais , Anticorpos/metabolismo , Citoplasma/metabolismo , Cinesinas/imunologia , Masculino , Camundongos , Peptídeos/metabolismo , Ratos , Células de Sertoli/ultraestrutura , Especificidade da Espécie , Espermátides/ultraestruturaRESUMO
BACKGROUND: Extrahepatic tissues have long been considered critical contributors of cholesterol to nascent HDL particles in the reverse cholesterol transport pathway, in which ABCA1 plays the crucial role. Recent studies, however, including both overexpression and deletion of ABCA1 selectively in the liver, have highlighted the primary role of the liver in the maintenance of HDL levels in vivo. METHODS AND RESULTS: The availability of mice with complete deletion of ABCA1 (total knockout [TKO]) and with liver-specific deletion of ABCA1 (LSKO) has enabled us to dissect the discrete roles of hepatic relative to extrahepatic ABCA1 in HDL biogenesis. Delivery of adenoviral ABCA1 resulted in selective expression of physiological levels of ABCA1 in the livers of both LSKO and TKO mice, resulting in increased HDL cholesterol (HDL-C). Expression of ABCA1 in the liver of LSKO mice resulted in plasma HDL-C levels that were similar to those in wild-type mice and significantly above those seen in similarly treated TKO mice. HDL particles from ABCA1-expressing LSKO mice were larger and contained significantly increased cholesterol compared with TKO mice. Infusion of human apolipoprotein A-I/phospholipid reconstituted HDL particles normalized plasma HDL-C levels in LSKO mice but had no effect on HDL-C levels in TKO mice. CONCLUSIONS: Although hepatic ABCA1 appears crucial for phospholipid transport, extrahepatic tissues play an important role in cholesterol transfer to nascent HDL particles. These data highlight the discrete and specific roles of both liver and extrahepatic ABCA1 in HDL biogenesis in vivo and indicate that ABCA1 shows lipid cargo selectivity depending on its site of expression.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , HDL-Colesterol/sangue , Fígado/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenoviridae , Animais , Apolipoproteína A-I/farmacologia , HDL-Colesterol/genética , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Post-translational modification by the lipid palmitate is crucial for the correct targeting and function of many proteins. Here we show that huntingtin (htt) is normally palmitoylated at cysteine 214, which is essential for its trafficking and function. The palmitoylation and distribution of htt are regulated by the palmitoyl transferase huntingtin interacting protein 14 (HIP14). Expansion of the polyglutamine tract of htt, which causes Huntington disease, results in reduced interaction between mutant htt and HIP14 and consequently in a marked reduction in palmitoylation. Mutation of the palmitoylation site of htt, making it palmitoylation resistant, accelerates inclusion formation and increases neuronal toxicity. Downregulation of HIP14 in mouse neurons expressing wild-type and mutant htt increases inclusion formation, whereas overexpression of HIP14 substantially reduces inclusions. These results suggest that the expansion of the polyglutamine tract in htt results in decreased palmitoylation, which contributes to the formation of inclusion bodies and enhanced neuronal toxicity.
Assuntos
Proteínas de Transporte/metabolismo , Córtex Cerebral/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Ácido Palmítico/metabolismo , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos/fisiologia , Animais , Animais Recém-Nascidos , Células COS , Proteínas de Transporte/genética , Células Cultivadas , Córtex Cerebral/citologia , Chlorocebus aethiops , Cisteína/metabolismo , Regulação para Baixo/genética , Humanos , Proteína Huntingtina , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Camundongos , Camundongos Transgênicos , Mutação/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Proteínas Nucleares/química , Proteínas Nucleares/genética , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Transporte Proteico/fisiologia , Ratos , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
BACKGROUND: ATP-binding cassette transporter A1 (ABCA1) is a key regulator of cellular cholesterol and phospholipid transport. Previously, we have shown that inactivation of macrophage ABCA1 induces atherosclerosis in low-density lipoprotein receptor knockout (LDLr-/-) mice. However, the possibly beneficial effects of specific upregulation of macrophage ABCA1 on atherogenesis are still unknown. METHODS AND RESULTS: Chimeras that specifically overexpress ABCA1 in macrophages were generated by transplantation of bone marrow from human ABCA1 bacterial artificial chromosome (BAC) transgenic mice into LDLr-/- mice. Peritoneal macrophages isolated from the ABCA1 BAC --> LDLr-/- chimeras exhibited a 60% (P=0.0006) increase in cholesterol efflux to apolipoprotein AI. To induce atherosclerosis, the mice were fed a Western-type diet containing 0.25% cholesterol and 15% fat for 9, 12, and 15 weeks, allowing analysis of effects on initial lesion development as well as advanced lesions. No significant effect of macrophage ABCA1 overexpression was observed on atherosclerotic lesion size after 9 weeks on the Western-type diet (245+/-36x10(3) microm2 in ABCA1 BAC --> LDLr-/- mice versus 210+/-20x10(3) microm2 in controls). However, after 12 weeks, the mean atherosclerotic lesion area in ABCA1 BAC --> LDLr-/- mice remained only 164+/-15x10(3) microm2 (P=0.0008) compared with 513+/-56x10(3) microm2 in controls (3.1-fold lower). Also, after 15 weeks on the diet, lesions in mice transplanted with ABCA1 overexpressing bone marrow were still 1.6-fold smaller (393+/-27x10(3) microm2 compared with 640+/-59x10(3) microm2 in control transplanted mice; P=0.0015). CONCLUSIONS: ABCA1 upregulation in macrophages inhibits the progression of atherosclerotic lesions.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Aterosclerose/genética , Receptores de LDL/deficiência , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Animais , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Dieta Aterogênica , Regulação da Expressão Gênica , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Receptores de LDL/genética , Regulação para CimaRESUMO
ABCA1 is essential for the transport of lipids across plasma membranes and for the maintenance of plasma HDL-cholesterol levels. The transcriptional regulation of ABCA1 is complex and is currently poorly understood. We previously generated human ABCA1 bacterial artificial chromosome transgenic mice that expressed RNA and protein, which allowed us to identify three alternate ABCA1 transcripts. Each transcript arises from different exon 1 sequences (exon1b, exon1c, and exon1d) that are spliced directly into exon 2, which contains the ATG site, and all generate full-length protein. We have now determined the tissue-specific expression of each of these transcripts in humans and mice and have shown that their patterns of expression are similar. Exon1d transcript is predominantly expressed in liver and macrophages and is preferentially increased in the liver in response to a high-fat diet. The exon1b transcript is expressed predominantly in liver, testis, and macrophages, but it is only upregulated in macrophages in response to a high-fat diet. The exon1c transcript is ubiquitously expressed and is upregulated in the brain, stomach, and other tissues in mice on a high-fat diet. Our data indicate that specific transcripts in different tissues play key roles in alterations of ABCA1-mediated changes in HDL levels and atherosclerosis in response to environmental stimuli.