RESUMEN
The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is a key regulator in cholesterol biosynthesis and HMG CoA reductase inhibitors (statins) have become a widely prescribed family of lipid lowering agents. Cholesterol synthesis occurs predominantly in liver which is the target organ of statins. We studied the effects of fluvastatin (Lescol), a member of the statin family, on hepatic protein regulation. Male F344 rats treated with 0.8 mg/kg per day fluvastatin or 24 mg/kg per day fluvastatin for 7 days showed treatment-related changes in 58 liver proteins (P<0.005). Major effects were evident in the cholesterol biosynthesis pathway including the induction of enzymes upstream and downstream of the target enzyme HMG CoA reductase. Treatment also triggered alterations in key enzymes of carbohydrate metabolism and was associated with changes in a heterogeneous set of cellular stress proteins involved in cytoskeletal structure, calcium homeostasis and protease activity. The latter set of protein alterations indicates that hepatotoxicity is associated with high-dose treatment. Based on the results it is suggested that HMG-CoA synthase and isopentenyl-diphosphate delta-isomerase may be explored as alternative drug targets and that the induction levels of these enzymes may serve as a measure of potency of individual statin drugs. It is proposed that efficacy and cellular stress markers discovered in this study may be used in a high throughput screen (HTS) assay format to compare efficiently and accurately the therapeutic windows of different members of the statin family.
Asunto(s)
Colesterol/biosíntesis , Ácidos Grasos Monoinsaturados/toxicidad , Inhibidores de Hidroximetilglutaril-CoA Reductasas/toxicidad , Indoles/toxicidad , Hígado/efectos de los fármacos , Proteínas/metabolismo , Animales , Metabolismo de los Hidratos de Carbono , Fluvastatina , Metabolismo de los Lípidos , Hígado/metabolismo , Masculino , Proteoma , Ratas , Ratas Endogámicas F344RESUMEN
The Syrian hamster embryo (SHE) cell transformation assay is widely used to screen chemicals for carcinogenic potential. However, the biochemical mechanisms of transformation in SHE cells are incompletely understood relative to other rodent systems. Thus identification of proteins which change during transformation can provide clues to biochemical mechanisms. Previously, we published a map of SHE cell proteins based on comparisons to other maps. In this report we provide direct sequence analysis of numerous proteins which were previously identified solely by electrophoretic mobility. Protein sequencing verified original spot identifications and extended the range of identified proteins. The updated map will assist in evaluating biochemical mechanisms of morphological transformation in hamster cells.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Embrión de Mamíferos/química , Mapeo Peptídico/métodos , Proteínas/análisis , Análisis de Secuencia/métodos , Animales , Línea Celular , Cricetinae , Bases de Datos Factuales , Mesocricetus , Ratones , RatasRESUMEN
The liver is composed of a variety of cells that form a functional unit involved in uptake, synthesis, metabolism, and secretion. Until recently, most studies examining liver function did not analyze the specific proteins expressed or functions performed by the multiple individual cell types that constitute the hepatic mass. In the last decade, novel isolation methods have been developed that allow the purification of liver cell populations highly enriched in one type of liver cell. Here, we present a detailed two-dimensional (2-D) protein map of rat bile duct epithelial cells (i.e., cholangiocytes) using a recently developed isolation procedure. In addition, we identify 27 major cholangiocyte proteins either by comparison to maps of known rat liver proteins (based on pI and Mr) or by tryptic digestion and microsequencing. Finally, we compare the relative abundance of individual proteins present in cholangiocytes to whole liver as well as hepatocyte-specific proteins. Our results show that cholangiocytes express a unique array of individual proteins. The cholangiocyte 2-D protein pattern is markedly different from that of isolated rat hepatocytes or whole rat liver, with high levels of proteins previously known to be expressed by cholangiocytes (e.g., cytokeratins, actins) as well as protein not previously demonstrated to be expressed at high levels (e.g., annexin V, selenium binding protein). We conclude that this cholangiocyte-derived, 2-D protein map will be a crucial resource for studies directed at our understanding of cholangiocyte physiology and pathobiology.
Asunto(s)
Conductos Biliares Intrahepáticos/química , Bases de Datos Factuales , Electroforesis en Gel Bidimensional/métodos , Proteínas/análisis , Animales , Anexina A5/análisis , Conductos Biliares Intrahepáticos/citología , Proteínas Portadoras/análisis , Células Epiteliales/química , Queratinas/análisis , Mapeo Peptídico , Ratas , Ratas Endogámicas F344 , Proteínas de Unión al SelenioRESUMEN
We have investigated the effects of five peroxisome proliferators (PPs : clofibric acid, DEHP, WY14,643, nafenopin, and LY171883) on the abundances of a large number of proteins in the livers of treated mice at 5- and 35-day time points. LY171883 was investigated at a range of doses, and one of its close structural analogs that is not a peroxisome proliferator (LY163443) was included as a negative control compound. Liver samples were analyzed by quantitative 2-D electrophoresis. Data for a selected set of 107 liver protein spots that respond strongly to at least one of the test compounds was subjected to principal component analysis to search for global protein pattern changes. The first component (PC1) accounted for 51% of the total data variance and was identified as a global measure of peroxisome proliferation by its correlation with enzymatic peroxisomal beta-oxidation. Component PC2 (7%) separated 5- and 35-day exposures, and PC3 (5%) separated groups treated with LY163443 from the rest. We used PC1 as a surrogate for equivalent dose in order to examine the effects of diverse compounds, with widely differing potencies, on a common scale. Analyzed in this way, the data indicate that all the peroxisome proliferators tested produce effects over wide time and dose ranges that fall on or near a single curve. Examination of specific protein responses showed that many proteins individually show a unified response curve, but that curves for different proteins were different. In particular, it appears that some constitutive proteins showing modest inductions with a high dose plateau (such as cytosolic epoxide hydrolase) are inducible at lower doses than some proteins showing very strong, nonplateaued inductions (such as the 80-kDa peroxisomal bifunctional enzyme). The results provide support for a unified receptor-based mechanism controlling the main PP response, but demonstrate that individual responsive genes can show quite different dose-response curves.
Asunto(s)
Acetofenonas/toxicidad , Anticolesterolemiantes/toxicidad , Dietilhexil Ftalato/toxicidad , Hígado/efectos de los fármacos , Microcuerpos/efectos de los fármacos , Proteínas/metabolismo , Tetrazoles/toxicidad , Secuencia de Aminoácidos , Animales , Ácido Clofíbrico/toxicidad , Relación Dosis-Respuesta a Droga , Electroforesis en Gel Bidimensional , Procesamiento de Imagen Asistido por Computador , Hígado/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Nafenopina , Oxidación-Reducción , Pirimidinas/toxicidad , RatasRESUMEN
Despite the widespread use of cyclosporine A (CsA), its mechanism of action and side effects are not yet completely understood. There exists a large body of evidence suggesting that disturbance of calcium homeostasis is a critical step in the cascade of cellular and molecular events induced by the drug. As recently shown in our laboratory by two-dimensional protein gel electrophoresis (2-DE) analysis of kidney homogenates, CsA induced numerous changes in several kidney proteins. One kidney protein in particular was shown to be strongly down-regulated by the drug. In this work we report the identification of the strongly decreased kidney protein as calbindin-D 28kDa, a vitamin D-dependent calcium-binding protein associated with calcium handling by cells. The assignment of the down-regulated protein spot is based on its internal amino acid sequence analysis and its specific reaction with a monoclonal antibody raised against calbindin-D 28kDa. In kidney homogenates of male Wistar rats treated with 50 mg/kg/d CsA for up to 28 days, calbindin levels were measured by ELISA and were shown to be continuously decreased with prolonged CsA treatment. To our knowledge, this is the first report describing the effect of CsA on kidney calbindin-D 28kDa protein levels. Further studies are needed to elucidate whether the CsA-mediated down-regulation of the calcium-binding protein calbindin-D 28kDa may be a critical factor for the renal adverse effects induced by this drug.
Asunto(s)
Ciclosporina/farmacología , Inmunosupresores/farmacología , Riñón/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Secuencia de Aminoácidos , Animales , Calbindinas , Regulación hacia Abajo , Electroforesis en Gel Bidimensional , Riñón/efectos de los fármacos , Masculino , Datos de Secuencia Molecular , Mapeo Peptídico , Ratas , Ratas WistarRESUMEN
Proteins implement most biological functions at the molecular level. As one might expect based on this fact, it appears that the altered functional states associated with toxic effects involve changes in the abundance or structure of proteins. Although numerous specific assays exist to measure changes in the abundance of individual proteins, practical limitations have prevented widespread use of multiple protein assays for the global characterization of toxicity. Recent developments in protein analytical technology are rapidly changing this picture. Two-dimensional gel electrophoresis, a technique capable of resolving and quantitating hundreds of proteins simultaneously, is becoming an automated, high-throughput tool. In parallel, techniques have been developed that allow the resulting deluge of protein measurements to be organized into a prototype Molecular Effects Database describing xenobiotic effects in rodent liver. This database can detect, classify, and characterize a broad range of liver toxicity mechanisms. It currently contains approximately 10 million protein measurements, including data on the liver effects of 43 compounds, with a further 50 compounds to be added in 1995. Observed effects range from very broad (sex steroids alter levels of 45% of all liver proteins) to very specific (e.g., hepatic hydroxymethyl glutaryl coenzyme A reductase inhibitors). Companion 2-dimensional databases describing rodent brain and kidney have been initiated, as have linkages to the genomic sequence databases. Assimilation of this approach into research and regulatory toxicology poses an interesting challenge--one that is likely to lead to a radically more sophisticated understanding of toxicity and its biological basis.
Asunto(s)
Pruebas de Función Hepática/métodos , Hígado/química , Hígado/efectos de los fármacos , Animales , Electroforesis en Gel Bidimensional , Humanos , Hígado/fisiología , Relación Estructura-ActividadRESUMEN
We have improved upon the reference two-dimensional (2-D) electrophoretic map of rat liver proteins originally published in 1991 (N. L. Anderson et al., Electrophoresis 1991, 12, 907-930). A total of 53 proteins (102 spots) are now identified, many by microsequencing. In most cases, spots cut from wet, Coomassie Blue stained 2-D gels were submitted to internal tryptic digestion [2], and individual peptides, separated by high-performance liquid chromatography (HPLC), were sequenced using a Perkin-Elmer 477A sequenator. Additional spots were identified using specific antibodies.
Asunto(s)
Electroforesis en Gel Bidimensional , Sistemas de Información , Hígado/química , Proteínas/química , Animales , Cromatografía Líquida de Alta Presión , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Ratones , Peso Molecular , Fragmentos de Péptidos/química , Proteínas/análisis , Ratas , Análisis de Secuencia , TripsinaRESUMEN
A standard two-dimensional (2-D) protein map of Fischer 344 rat liver (F344MST3) is presented, with a tabular listing of more than 1200 protein species. Sodium dodecyl sulfate (SDS) molecular mass and isoelectric point have been established, based on positions of numerous internal standards. This map has been used to connect and compare hundreds of 2-D gels of rat liver samples from a variety of studies, and forms the nucleus of an expanding database describing rat liver proteins and their regulation by various drugs and toxic agents. An example of such a study, involving regulation of cholesterol synthesis by cholesterol-lowering drugs and a high-cholesterol diet, is presented. Since the map has been obtained with a widely used and highly reproducible 2-D gel system (the Iso-Dalt system), it can be directly related to an expanding body of work in other laboratories.