RESUMEN
Lipid homeostasis in animal cells is maintained by sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors whose proteolytic activation requires the cholesterol-sensing membrane protein Scap. In endoplasmic reticulum (ER) membranes, the carboxyl-terminal domain (CTD) of SREBPs binds to the CTD of Scap. When cholesterol levels are low, Scap escorts SREBPs from the ER to the Golgi, where the actions of two proteases release the amino-terminal domains of SREBPs that travel to the nucleus to up-regulate expression of lipogenic genes. The CTD of SREBP remains bound to Scap but must be eliminated so that Scap can be recycled to bind and transport additional SREBPs. Here, we provide insights into how this occurs by performing a detailed molecular dissection of the CTD of SREBP2, one of three SREBP isoforms expressed in mammals. We identify a degradation signal comprised of seven noncontiguous amino acids encoded in exon 19 that mediates SREBP2's proteasomal degradation in the absence of Scap. When bound to the CTD of Scap, this signal is masked and SREBP2 is stabilized. Binding to Scap requires an arginine residue in exon 18 of SREBP2. After SREBP2 is cleaved in Golgi, its CTD remains bound to Scap and returns to the ER with Scap where it is eliminated by proteasomal degradation. The Scap-binding motif, but not the degradation signal, is conserved in SREBP1. SREBP1's stability is determined by a degradation signal in a different region of its CTD. These findings highlight a previously unknown role for the CTD of SREBPs in regulating SREBP activity.
Asunto(s)
Colesterol/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles , Secuencias de Aminoácidos , Animales , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Unión Proteica , Dominios Proteicos , Proteolisis , Proteína 2 de Unión a Elementos Reguladores de Esteroles/química , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismoRESUMEN
Sterol regulatory element-binding protein (SREBP)-2 is a pivotal transcriptional factor in cholesterol metabolism. Factors interfering with the proper functioning of SREBP-2 potentially alter plasma lipid profiles. Phorbol 12-myristate 13-acetate (PMA), which is a common protein kinase C (PKC) activator, was shown to promote the post-translational processing and nuclear translocation of SREBP-2 in hepatic cells in the current study. Following SREBP-2 translocation, the transcripts of its target genes HMGCR and LDLR were upregulated as demonstrated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Electrophoretic mobility shift assays (EMSA) also demonstrated an induced DNA-binding activity on the sterol response element (SRE) domain under PMA treatment. The increase of activated Srebp-2 without the concurrent induced mRNA expression was also observed in an animal model. As the expression of SREBP-2 was not increased by PMA, the activation of PKC was the focus of investigation. Specific PKC isozyme inhibition and overexpression supported that PKCß was responsible for the promoting effect. Further studies showed that the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK), but not 5' adenosine monophosphate-activated protein kinase (AMPK), were the possible downstream signaling proteins of PKCß. In conclusion, this study illustrated that PKCß increased SREBP-2 nuclear translocation in a pathway mediated by MEK/ERK and JNK, rather than the one dictated by AMPK. These results revealed a novel signaling target of PKCß in the liver cells.
Asunto(s)
Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hidroximetilglutaril-CoA Reductasas/genética , Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de LDL/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/químicaRESUMEN
Somatic cell genetics is a powerful approach for unraveling the regulatory mechanism of cholesterol metabolism. However, it is difficult to identify the mutant gene(s) due to cells are usually mutagenized chemically or physically. To identify important genes controlling cholesterol biosynthesis, an unbiased forward genetics approach named validation-based insertional mutagenesis (VBIM) system was used to isolate and characterize the 25-hydroxycholesterol (25-HC)-resistant and SR-12813-resistant mutants. Here we report that five mutant cell lines were isolated. Among which, four sterol-resistant mutants either contain a truncated NH2-terminal domain of sterol regulatory element-binding protein (SREBP)-2 terminating at amino acids (aa) 400, or harbor an overexpressed SREBP cleavage-activating protein (SCAP). Besides, one SR-12813 resistant mutant was identified to contain a truncated COOH-terminal catalytic domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase). This study demonstrates that the VBIM system can be a powerful tool to screen novel regulatory genes in cholesterol biosynthesis.
Asunto(s)
Colesterol/biosíntesis , Hidroximetilglutaril-CoA Reductasas/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Mutagénesis Insercional/métodos , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Animales , Células CHO , Cricetulus , Difosfonatos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Pruebas Genéticas/métodos , Vectores Genéticos , Células HEK293 , Células HeLa , Humanos , Hidroxicolesteroles/farmacología , Hidroximetilglutaril-CoA Reductasas/química , Hidroximetilglutaril-CoA Reductasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lentivirus/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Estructura Terciaria de Proteína , Transducción de Señal/efectos de los fármacos , Proteína 2 de Unión a Elementos Reguladores de Esteroles/química , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismoRESUMEN
BACKGROUND: Aberrantly elevated sterol regulatory element binding protein (SREBP), the lipogenic transcription factor, contributes to the development of fatty liver and insulin resistance in animals. Our recent studies have discovered that AMP-activated protein kinase (AMPK) phosphorylates SREBP at Ser-327 and inhibits its activity, represses SREBP-dependent lipogenesis, and thereby ameliorates hepatic steatosis and atherosclerosis in insulin-resistant LDLR(-/-) mice. Chronic inflammation and activation of NLRP3 inflammasome have been implicated in atherosclerosis and fatty liver disease. However, whether SREBP is involved in vascular lipid accumulation and inflammation in atherosclerosis remains largely unknown. PRINCIPAL FINDINGS: The preclinical study with aortic pouch biopsy specimens from humans with atherosclerosis and diabetes shows intense immunostaining for SREBP-1 and the inflammatory marker VCAM-1 in atherosclerotic plaques. The cleavage processing of SREBP-1 and -2 and expression of their target genes are increased in the well-established porcine model of diabetes and atherosclerosis, which develops human-like, complex atherosclerotic plaques. Immunostaining analysis indicates an elevation in SREBP-1 that is primarily localized in endothelial cells and in infiltrated macrophages within fatty streaks, fibrous caps with necrotic cores, and cholesterol crystals in advanced lesions. Moreover, concomitant suppression of NAD-dependent deacetylase SIRT1 and AMPK is observed in atherosclerotic pigs, which leads to the proteolytic activation of SREBP-1 by diminishing the deacetylation and Ser-372 phosphorylation of SREBP-1. Aberrantly elevated NLRP3 inflammasome markers are evidenced by increased expression of inflammasome components including NLPR3, ASC, and IL-1ß. The increase in SREBP-1 activity and IL-1ß production in lesions is associated with vascular inflammation and endothelial dysfunction in atherosclerotic pig aorta, as demonstrated by the induction of NF-κB, VCAM-1, iNOS, and COX-2, as well as by the repression of eNOS. CONCLUSIONS: These translational studies provide in vivo evidence that the dysregulation of SIRT1-AMPK-SREBP and stimulation of NLRP3 inflammasome may contribute to vascular lipid deposition and inflammation in atherosclerosis.
Asunto(s)
Aterosclerosis/metabolismo , Proteínas Portadoras/metabolismo , Complicaciones de la Diabetes/metabolismo , Inflamasomas/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Porcinos , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Acetilación/efectos de los fármacos , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/patología , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Biomarcadores/metabolismo , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/patología , Enfermedad de la Arteria Coronaria/fisiopatología , Complicaciones de la Diabetes/patología , Complicaciones de la Diabetes/fisiopatología , Progresión de la Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-1beta/metabolismo , Masculino , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteolisis/efectos de los fármacos , Serina/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/química , Proteína 2 de Unión a Elementos Reguladores de Esteroles/química , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Although proteins are translated on cytoplasmic ribosomes, many of these proteins play essential roles in the nucleus, mediating key cellular processes including but not limited to DNA replication and repair as well as transcription and RNA processing. Thus, understanding how these critical nuclear proteins are accurately targeted to the nucleus is of paramount importance in biology. Interaction and structural studies in the recent years have jointly revealed some general rules on the specificity determinants of the recognition of nuclear targeting signals by their specific receptors, at least for two nuclear import pathways: (i) the classical pathway, which involves the classical nuclear localization sequences (cNLSs) and the receptors importin-α/karyopherin-α and importin-ß/karyopherin-ß1; and (ii) the karyopherin-ß2 pathway, which employs the proline-tyrosine (PY)-NLSs and the receptor transportin-1/karyopherin-ß2. The understanding of specificity rules allows the prediction of protein nuclear localization. We review the current understanding of the molecular determinants of the specificity of nuclear import, focusing on the importin-αâ¢cargo recognition, as well as the currently available databases and predictive tools relevant to nuclear localization. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Señales de Localización Nuclear/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bases de Datos de Proteínas , Humanos , Ratones , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Señales de Localización Nuclear/química , Señales de Localización Nuclear/genética , Proteína Relacionada con la Hormona Paratiroidea/química , Proteína Relacionada con la Hormona Paratiroidea/fisiología , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal/fisiología , Proteína 2 de Unión a Elementos Reguladores de Esteroles/química , Proteína 2 de Unión a Elementos Reguladores de Esteroles/fisiología , alfa Carioferinas/química , alfa Carioferinas/fisiología , beta Carioferinas/química , beta Carioferinas/fisiologíaRESUMEN
Recent studies have shown that cooperative interactions in endoplasmic reticulum (ER) membranes between Scap, cholesterol, and Insig result in switch-like control over activation of SREBP-2 transcription factors. This allows cells to rapidly adjust rates of cholesterol synthesis and uptake in response to even slight deviations from physiological set-point levels, thereby ensuring cholesterol homeostasis. In the present study we directly probe for the accessibility of cholesterol in purified ER membranes. Using a soluble cholesterol-binding bacterial toxin, perfringolysin O, we show that cholesterol accessibility increases abruptly at â¼5 mol % ER cholesterol, the same concentration at which SREBP-2 activation is halted. This switch-like change in cholesterol accessibility is observed not only in purified ER membranes but also in liposomes made from ER lipid extracts. The accessibility of cholesterol in membranes is related to its chemical activity. Complex formation between cholesterol and some ER phospholipids can result in sharp changes in cholesterol chemical activity and its accessibility to perfringolysin O or membrane sensors like Scap. The control of the availability of the cholesterol ligand to participate in cooperative Scap/cholesterol/Insig interactions further sharpens the sensitive switch that exerts precise control over cholesterol levels in cell membranes.
Asunto(s)
Colesterol/química , Retículo Endoplásmico/química , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Animales , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Células CHO , Cricetinae , Cricetulus , Retículo Endoplásmico/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Immunoblotting , Liposomas/química , Fosfolípidos/química , Unión Proteica , Proteína 2 de Unión a Elementos Reguladores de Esteroles/químicaRESUMEN
Nucleocytoplasmic transport of proteins in eukaryotic cells is a fundamental process for gene expression. The transport is regulated by posttranslational modifications of the proteins, such as ligand-binding, phosphorylation, and proteolysis. For monitoring the nuclear transport of proteins induced by a ligand binding, we have recently developed a genetically encoded bioluminescent indicator based on reconstitution of split fragments of Renilla reniformis (RLuc) by protein splicing with DnaE inteins. We herein describe that the technique is used for detecting phosphorylation- or proteolysis-induced nuclear transports of a target protein. Two model proteins, signal transducer and activator of transcription 3 (STAT3) and sterol-regulatory element binding protein-2 (SREBP-2), were exemplified as phosphorylation- and proteolysis-induced nuclear transport, respectively. Each STAT3 or SREBP-2 is connected with C-terminal halves of RLuc and DnaE. If the protein translocates into the nucleus, the C-terminal fragment of RLuc meets the N-terminal fragment of RLuc, and full-length RLuc is reconstituted by protein splicing in the nucleus. The indicator with SREBP-2 enabled us to quantify the intracellular concentrations of cholesterol. The indicator with STAT3 quantified the extent of the nuclear transport induced by representative cytokines. This simple assay based on protein nuclear transports allows the selection of suitable drugs among candidates and has significant potential for risk assessments, such as carcinogenic chemical screening in vitro and in vivo.