RESUMEN
The aetiology of SSc is subject to ongoing research, as the precise events that underlie the development of this disease remain unclear. The pathogenesis is known to involve endothelium, epithelium, fibroblasts, innate and adaptive immune systems and their component immunological mediators. Endothelial cell damage may be the initiating factor, but the precise triggering event(s) remain elusive. Angiogenesis also appears to be dysregulated. Vasculopathy shows similarities in different organs (e.g. pulmonary arterial hypertension, renal disease, digital tip ulcers). Endothelin-1 is a potent mediator of vasculopathy, and hence represents a highly relevant target for intervention of vascular features in SSc.
Asunto(s)
Esclerodermia Sistémica/etiología , Endotelio Vascular/inmunología , Endotelio Vascular/patología , Epitelio/inmunología , Epitelio/patología , Fibroblastos/inmunología , Fibroblastos/patología , Humanos , Pulmón/inmunología , Pulmón/patología , Esclerodermia Sistémica/inmunología , Piel/inmunología , Piel/patologíaRESUMEN
Multiplex protein technology has the potential to identify biomarkers for the differentiation, classification and improved understanding of the pathogenesis of interstitial lung disease. The aim of this study was to determine whether a 30-inflammatory biomarker panel could discriminate between healthy controls, sarcoidosis and systemic sclerosis (SSc) patients independently of other clinical indicators. We also evaluated whether a panel of biomarkers could differentiate between the presence or absence of lung fibrosis in SSc patients. We measured 30 circulating biomarkers in 20 SSc patients, 21 sarcoidosis patients and 20 healthy controls using Luminex bead technology and used Fisher's discriminant function analysis to establish the groups of classification mediators. There were significant differences in median concentration measurements between study groups for 20 of the mediators but with considerable range overlap between the groups, limiting group differentiation by single analyte measurements. However, a 17-analyte biomarker model correctly classified 90% of study individuals to their respective group and another 14-biomarker panel correctly identified the presence of lung fibrosis in SSc patients. These findings, if they are corroborated by independent studies in other centres, have potential for clinical application and may generate novel insights into the modulation of immune profiles during disease evolution.
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Biomarcadores/metabolismo , Neumología/métodos , Sarcoidosis/sangre , Esclerodermia Sistémica/sangre , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Fibrosis/sangre , Humanos , Sistema Inmunológico , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Persona de Mediana EdadRESUMEN
Scleroderma [systemic sclerosis (SSc)] is a spectrum of connective tissue diseases characterized by micro- and macro-vasculopathy, inflammation and autoimmunity and tissue remodelling that often leads to excessive scarring and fibrosis in both interstitial and vascular compartments. Pre-clinical investigations and gene association studies have led to improved understanding of the cell and molecular mechanisms underlying disease pathogenesis and to the identification of key molecular candidates that may represent potentially useful disease biomarkers and effective therapeutic targets. Studies on the endothelin (ET) system, pre-dominantly ET-1 and the cell surface receptors [type A (ET(A))] and type B (ET(B))], have provided evidence for an important role of this system in the vascular and fibrotic pathologies in SSc. To date, promising clinical results, utilizing dual/mixed ET receptor antagonism have been obtained in two of the vascular complications associated with SSc, ischaemic digital ulceration and pulmonary arterial hypertension. Evidence suggests that ET-1 is able to activate and re-program the functional phenotypes of vascular smooth muscle cells, microvascular pericytes and tissue fibroblasts into pro-fibrogenic cell populations with myofibroblasts-like properties. The impact of receptor-selective, over mixed-receptor, antagonism has also been studied in vitro with respect to cell differentiation and proliferation, extracellular matrix synthesis, production and deposition and in pathological cellular contraction. However, the complexity of the ET system, potential for receptor cross-talk, interactions with down-stream signal transduction cascades, as well as the potent inter-relationships with other important ligand-receptor pathways have made in vivo studies difficult to unravel. Moreover, little information is available on the role of the ET system and receptor selectivity in the recruitment and activation of mesenchymal progenitor cells in tissue remodelling and fibrosis or on the early inflammatory response. Here, we discuss the available pre-clinical evidence for the role of the ET system in tissue repair, scarring and fibrosis, using the connective tissue diseases SSc and model systems of fibrogenesis.
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Endotelina-1/fisiología , Receptores de Endotelina/fisiología , Esclerodermia Sistémica/fisiopatología , Animales , Tejido Conectivo/fisiología , Modelos Animales de Enfermedad , Fibrosis , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Esclerodermia Sistémica/genética , Transducción de SeñalRESUMEN
The CCN (cyr61, ctgf, nov) family of modular proteins regulate diverse biological affects including cell adhesion, matrix production, tissue remodelling, proliferation and differentiation. Recent targeted gene disruption studies have demonstrated the CCN family to be developmentally essential for chondrogenesis, osteogenesis and angiogenesis. CCN2 is induced by agents such as angiotensin II, endothelin-1, glucocorticoids, HGF, TGFbeta, and VEGF, and by hypoxia and biomechanical and shear stress. Dysregulated expression of CCN2 has also been widely documented in many fibroproliferative diseases. This mini-review will focus on CCN2, and the recent progress in understanding CCN2 gene regulation in health and disease. That CCN2 should be considered a novel and informative surrogate clinical bio-marker for fibroproliferative disease is discussed.
RESUMEN
OBJECTIVES: Nitric oxide (*NO) is an important physiological signalling molecule and a potent vasodilator. We have previously demonstrated abnormal *NO metabolism in the plasma of patients with systemic sclerosis (SSc; scleroderma), a disease that features vascular dysfunction as well as collagen overproduction and fibrosis. The aim of the present study was to examine nitric oxide synthase (NOS) expression and activity and assess the potential role of antioxidants in the scleroderma-like syndrome of the tight-skin 1 (TSK-1/+) mouse, an experimental animal model for fibrosis. METHODS: Skin, lung or plasma was taken from TSK-1/+ (n = 15) and wild-type (WT; n = 12) littermate mice. Type 1 collagen, endothelial NOS (eNOS), haemoxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) protein and gene expression were determined by western blot and reverse transcriptase-polymerase chain reaction. eNOS expression was further determined by immunohistochemistry. NOS activity was evaluated by conversion of [14C] L-arginine to [14C] L-citrulline. Levels of circulating plasma nitrite/nitrate (NO(x)) were also measured. Total antioxidant activity was evaluated by ABTS+ production (ABTS = 2,2'-azino-bis-[3-ethylbenz-thiazoline-6-sulphonic acid). RESULTS: In the skin, eNOS was present in the epidermal layer, hair follicles and also in the endothelial cells lining the blood vessels. Expression of both the eNOS protein and gene was significantly reduced in TSK-1/+ skin tissue, while type 1 collagen protein was elevated compared with WT. Furthermore, there was decreased NOS activity in TSK-1/+ skin tissue; however, there was no measurable difference in plasma NO(x). Correspondingly, the protective antioxidant enzyme HO-1 and the associated transcription factor Nrf2 showed reduced protein and gene expression levels in TSK-1/+ skin, while there was also less total antioxidant activity. In TSK-1/+ lung tissue, however, we observed no difference in collagen protein expression, *NO metabolism or HO-1 expression and total antioxidant activity compared with WT. CONCLUSIONS: The findings suggest that there is also abnormal *NO metabolism in the TSK-1/+ mouse model of fibrosis, particularly in the skin, while expression and activity of protective antioxidants are reduced. The TSK-1/+ mouse may also be useful for testing treatments that target vascular endothelial cell function in patients with SSc.
Asunto(s)
Antioxidantes/metabolismo , Modelos Animales de Enfermedad , Fibrosis/enzimología , Fibrosis/patología , Ratones Mutantes , Óxido Nítrico Sintasa/metabolismo , Análisis de Varianza , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Femenino , Inmunohistoquímica , Masculino , Ratones , Óxido Nítrico Sintasa/análisis , Probabilidad , ARN/metabolismo , Distribución Aleatoria , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esclerodermia Localizada/enzimología , Esclerodermia Localizada/patología , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Ischaemia is known to induce angiogenesis, but the effects of critical leg ischaemia (CLI) on angiogenesis remain unclear. The aim of this study was to examine the physiological angiogenic response in CLI by investigating the extent of neovascularization, characterizing microvessel subtypes and determining the microvessel ultrastructure. METHODS: Gastrocnemius muscles were biopsied from 12 patients with CLI and 12 without leg ischaemia. Microvessels were evaluated immunohistochemically using three endothelial markers (anti-CD31, anti-CD34 and PAL-E) and anti-alpha smooth muscle actin (SMA) as a mural cell marker to label arterioles. Ki67 was used to demonstrate active cell proliferation. Further microvessel ultrastructural characteristics were determined by transmission electron microscopy. RESULTS: The CLI group had significantly higher microvessel density and microvessel : muscle fibre ratio for all endothelial subtypes examined (P < 0.001). PAL-E staining demonstrated the highest increase: 4.7 times higher in CLI muscle. There was no significant difference in alphaSMA-positive microvessel density (P = 0.118) or microvessel: muscle fibre ratio (P = 0.214). Ki67 staining showed no active cell proliferation. Transmission electron microscopy showed CLI microvessels had abnormal morphology, mainly a thick basement membrane. CONCLUSION: A physiological angiogenic response was found in CLI, but the microvessels had an abnormal ultrastructure. A lack of active cell proliferation suggests that the angiogenic response may have been exhausted.
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Vasos Sanguíneos/ultraestructura , Isquemia/patología , Pierna/irrigación sanguínea , Neovascularización Patológica/patología , Anciano , Amputación Quirúrgica , Enfermedad Crítica , Femenino , Humanos , Inmunohistoquímica , Masculino , Microcirculación , Microscopía Electrónica de Transmisión de Rastreo , Músculo Esquelético/irrigación sanguínea , Músculo Liso Vascular/ultraestructuraRESUMEN
OBJECTIVES: Endothelial dysfunction is a primary event in systemic sclerosis; however, the aetiology of events and the role of nitric oxide (NO) is still unclear. The aim of the present study is to investigate whether there are abnormalities in NO metabolism in plasma from patients with primary Raynaud's phenomenon (RP) and in the pathogenesis of systemic sclerosis (SSc): limited SSc (lSSc) and diffuse (dSSc). We also wanted to investigate the effect of factors within patients' SSc serum on NO metabolism in human microvascular endothelial cells (HMECs). METHODS: Plasma (n=89) or serum (n=80) was assayed for total nitrate and nitrite (NOx), nitration of proteins and the NO inhibitor asymmetric dimethylarginine (ADMA). HMECs were treated with patients' SSc serum and assayed for indicators of NO metabolism. RESULTS: Plasma NOx was elevated in patients with RP or lSSc (P<0.002), but not in patients with dSSc, compared with controls. Nitrated proteins in plasma, however, were found to be very high in dSSc patients (P<0.03), compared with RP, lSSc or controls. Patients with dSSc also showed increased levels of serum ADMA (P<0.05). The high level of nitrated proteins in dSSc was strongly associated with the severity and duration of dSSc disease. Skin biopsy sections from dSSc patients also showed enhanced nitrotyrosine staining compared with controls. In HMECs, pre-incubation with SSc serum impaired the activity of nitric oxide synthase (NOS) but not the expression of inducible or endothelial NOS. SSc serum also induced a reduction in intracellular cGMP synthesis, and NOx production in the cell culture medium, but was not associated with increased cell cytotoxicity. CONCLUSIONS: NO formation is increased in patients with primary RP or lSSc, but nitration of proteins and elevated ADMA is a particular feature of dSSc and may reflect abnormal NO regulation and/or contribute to endothelial dysfunction in SSc.
Asunto(s)
Arginina/análogos & derivados , Óxido Nítrico/sangre , Esclerodermia Sistémica/sangre , Adulto , Arginina/sangre , Células Cultivadas , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nitratos/sangre , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Nitritos/sangre , Enfermedad de Raynaud/sangre , Esclerodermia Difusa/sangre , Esclerodermia Limitada/sangre , Índice de Severidad de la EnfermedadRESUMEN
OBJECTIVES: To determine the relationship between clinical features and circulating levels of active transforming growth factor (TGF) beta1 in the major subsets of systemic sclerosis (SSc). METHODS: In a cross-sectional study cases of diffuse cutaneous SSc (dose) (n = 27) or limited cutaneous SSc (dose) (n = 20) were compared with healthy controls (n = 22). Active and total TGFbeta1 was measured in serum and plasma by a high-sensitivity enzyme-linked immunosorbent assay. RESULTS: There were no significant differences between levels of total serum TGFbeta1. However, cases of dcSSc had lower levels of active TGFbeta1 than cases of lcSSc or controls. In addition, more cases of dcSSc (18/27; 66%, P < 0.025) had no detectable active TGFbeta1 than controls (7/22, 32%) or lcSSc (7/20, 35%). In dcSSc, serum active TGFbeta1 levels correlated negatively with skin score and positively with disease duration. CONCLUSIONS: Contrary to expectation, levels of active TGFbeta1 are reduced in dcSSc and this correlates with two variables known to associate with disease activity, shorter duration and more extensive skin sclerosis. This suggests that active TGFbeta1 may be sequestered in active involved SSc skin and that serum levels are reduced despite strong evidence implicating TGFbeta isoforms in the pathogenesis of fibrosis. Our findings may have implications for systemic TGFbeta-trapping therapies in this disease.
Asunto(s)
Esclerodermia Difusa/sangre , Factor de Crecimiento Transformador beta/sangre , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Esclerodermia Difusa/patología , Esclerodermia Limitada/sangre , Esclerodermia Limitada/patología , Índice de Severidad de la Enfermedad , Factores de Tiempo , Factor de Crecimiento Transformador beta1RESUMEN
The liganded hemoglobin (Hb) high-salt crystallization condition described by Max Perutz has generated three different crystals of human adult carbonmonoxy hemoglobin (COHbA). The first crystal is isomorphous with the "classical" liganded or R Hb structure. The second crystal reveals a new liganded Hb quaternary structure, RR2, that assumes an intermediate conformation between the R form and another liganded Hb quaternary structure, R2, which was discovered more than a decade ago. Like the R2 structure, the diagnostic R state hydrogen bond between beta2His97 and alpha1Thr38 is missing in the RR2 structure. The third crystal adopts a novel liganded Hb conformation, which we have termed R3, and it shows substantial quaternary structural differences from the R, RR2, and R2 structures. The quaternary structure differences between T and R3 are as large as those between T and R2; however, the T --> R3 and T --> R2 transitions are in different directions as defined by rigid-body screw rotation. Moreover, R3 represents an end state. Compared to all known liganded Hb structures, R3 shows remarkably reduced strain at the alpha-heme, reduced steric contact between the beta-heme ligand and the distal residues, smaller alpha- and beta-clefts, and reduced alpha1-alpha2 and beta1-beta2 iron-iron distances. Together, these unique structural features in R3 should make it the most relaxed and/or greatly enhance its affinity for oxygen compared to the other liganded Hbs. The current Hb structure-function relationships that are now based on T --> R, T -->R --> R2, or T --> R2 --> R transitions may have to be reexamined to take into account the RR2 and R3 liganded structures.
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Carboxihemoglobina/química , Hemoglobinas/química , Modelos Moleculares , Estructura Cuaternaria de Proteína , Adulto , Regulación Alostérica/fisiología , Carboxihemoglobina/fisiología , Cristalización , Cristalografía por Rayos X , Hemo/química , Hemoglobinas/fisiología , Humanos , Hierro/química , Ligandos , Modelos QuímicosRESUMEN
RATIONALE: The aim of the study was to evaluate the validity of collagen type I metabolites as markers of disease activity in scleroderma (SSc), through a systematic review of the literature and by validating the results by measuring collagen type I metabolites in well characterized patients with scleroderma spectrum disorders and in Raynaud's phenomenon. METHODS: A systematic review was performed of studies of collagen type I metabolites in scleroderma spectrum disorders published from 1980 to 2003. The collected results from the literature were compared with our own measurements of collagen type I metabolites (PINP and ICTP) in a small number of well characterized patients within the scleroderma spectrum and in patients with primary and "autoimmune" Raynaud's phenomenon. Peptide concentrations from all sources, including the present study, were compared. Reported correlations between peptide concentrations and clinical variables were also analysed. RESULTS: Of 19 papers identified by an extensive Medline search, 12 were eligible for systematic analysis. There was a considerable heterogeneity in the results with a wide range of metabolite concentrations. Values from disease groups and healthy controls overlapped. These findings were confirmed by our study where, similarly, there was a large range of values in all groups, but particularly in the diffuse SSc subset. When the correlation between peptide levels and clinical variables was assessed, large discordance between the studies was observed. CONCLUSIONS: We have not found sufficient evidence to support the use of serum markers of collagen turnover in the assessment of scleroderma activity and severity, in view of their low specificity and the heterogeneity of the results of various studies. Lack of standardized routine evaluation of SSc patients in clinical studies might have accounted for the variability of the findings. However, due to the small sizes of most published studies, demonstration of no effect should come from large-scale randomised trials. Longitudinal serial analysis of these molecules in individual patients may play a future role in the evaluation of the response to fibroblast-targeting therapeutic strategies in scleroderma patients.
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Colágeno Tipo I/sangre , Esclerodermia Sistémica/diagnóstico , Pruebas Serológicas , Biomarcadores/análisis , Humanos , Enfermedad de Raynaud/sangre , Enfermedad de Raynaud/diagnóstico , Esclerodermia Sistémica/sangreRESUMEN
OBJECTIVE: As an initial approach to understanding the basis of the systemic sclerosis (SSc; scleroderma) phenotype, we sought to identify genes in the transforming growth factor beta (TGF beta) signaling pathway that are up-regulated in lesional SSc fibroblasts relative to their normal counterparts. METHODS: We used gene chip, differential display, fluorescence-activated cell sorter, and overexpression analyses to assess the potential role of TGF beta signaling components in fibrosis. Fibroblasts were obtained by punch biopsy from patients with diffuse cutaneous SSc of 2-14 months' duration (mean 8 months) and from age- and sex-matched healthy control subjects. RESULTS: Unexpectedly, we found that fibroblasts from SSc patients showed elevated expression of the endothelial cell-enriched TGF beta receptor endoglin. Endoglin is a member of the nonsignaling high-affinity TGF beta receptor type III family. The expression of endoglin increased with progression of disease. Transfection of endoglin in fibroblasts suppressed the TGF beta-mediated induction of connective tissue growth factor promoter activity. CONCLUSION: SSc is characterized by overproduction of matrix; that is, genes that are targets of TGF beta signaling in normal fibroblasts. Our findings suggest that lesional SSc fibroblasts may overexpress endoglin as a negative feedback mechanism in an attempt to block further induction of profibrotic genes by TGF beta.
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Fibroblastos/metabolismo , Esclerodermia Sistémica/metabolismo , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/fisiología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Antígenos CD , Endoglina , Femenino , Humanos , Técnicas In Vitro , Receptores de Superficie Celular , Esclerodermia Sistémica/genética , Factor de Crecimiento Transformador beta/genética , Regulación hacia ArribaRESUMEN
Human erythrocyte pyruvate kinase plays an important role in erythrocyte metabolism. Mutation on the gene results in pyruvate kinase deficiency and is an important cause of hereditary nonspherocytic hemolytic anemia. Because of difficulties in isolating the mutant enzymes from patients, these mutations have not been fully studied. In this study, a complementary DNA (cDNA) encoding the human erythrocyte pyruvate kinase was generated. The cDNA was cloned into several expression vectors, and the protein was expressed and purified. The tetrameric protein exhibited properties characteristic of authentic human erythrocyte pyruvate kinase, including response to substrate, phosphoenolpyruvate, activation by fructose 1,6-bisphosphate, and inhibition by adenosine triphosphate (ATP). The N-terminal segment of the protein was highly susceptible to proteolysis, but only 2 of the 4 subunits were cleaved and lacked 47 N-terminal amino acid residues. A mutant protein, R510Q, which is the most frequently occurring mutation among Northern European population, was also generated and purified. The mutant protein retained its binding capacity to and could be activated by fructose 1,6-bisphosphate and showed similar kinetics toward phosphoenolpyruvate and adenosine diphosphate as for the wild-type enzyme. Conversely, the mutant protein has a dramatically decreased stability toward heat and is more susceptible to ATP inhibition. The enzyme instability decreases the enzyme level in the cell, accounting for the clinically observed "pyruvate kinase deficiency" of patients who are homozygous for this mutation. This study provides the first detailed functional characterization of human erythrocyte pyruvate kinase. These findings will allow the establishment of a fine correlation between molecular abnormalities and the clinical expression of the disease.
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Sustitución de Aminoácidos , Anemia Hemolítica Congénita no Esferocítica/genética , Eritrocitos/enzimología , Isoenzimas/genética , Mutación Missense , Mutación Puntual , Piruvato Quinasa/genética , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos , Anemia Hemolítica Congénita no Esferocítica/enzimología , Clonación Molecular , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Activación Enzimática/efectos de los fármacos , Fructosadifosfatos/farmacología , Calor , Humanos , Isoenzimas/química , Isoenzimas/deficiencia , Cinética , Datos de Secuencia Molecular , Peso Molecular , Fosfoenolpiruvato/metabolismo , Desnaturalización Proteica , Subunidades de Proteína , Piruvato Quinasa/química , Piruvato Quinasa/deficiencia , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Evidence for a role for members of the transforming growth factor beta (TGF-beta) family of cytokines in the pathogensis of systemic sclerosis and other fibrotic conditions is provided from studies of TGF-beta protein and gene expression in lesional biopsy specimens, from altered responses of explanted fibroblasts to TGF-beta stimulation which are associated with increased receptor expression on these cells and from genetic data linking TGF-beta gene loci to the disease. Of the many effects of TGF-beta on fibroblast properties induction of the connective tissue growth factor/Cyr61/NOV (CCN) family members, connective tissue growth factor (CTGF) may be particularly relevant to fibrosis. Moreover, systemic sclerosis (SSc) fibroblasts demonstrate constitutive over expression of CTGF that promotes migration, proliferation and matrix production. Studies of mechanisms regulating constitutive expression of CTGF by SSc fibroblasts are currently being undertaken and indicate that a TGF-beta responsive element in the CTGF promoter is involved, although this appears to function independent of the Smad proteins, suggesting that other TGF-beta-regulated pathways may be involved. TGF-neutralizing strategies have now been shown to abrogate many animal models of fibrosis, and will soon reach the clinical arena for SSc. These agents will further clarify the role of this ligand in initiating or sustaining fibrosis and offer the exciting possibility of targeted therapy for this disease.
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Sustancias de Crecimiento/fisiología , Proteínas Inmediatas-Precoces/fisiología , Péptidos y Proteínas de Señalización Intercelular , Esclerodermia Sistémica/etiología , Factor de Crecimiento Transformador beta/fisiología , Factor de Crecimiento del Tejido Conjuntivo , Proteínas de Unión al ADN/fisiología , Humanos , Familia de Multigenes , Proteína Hiperexpresada del Nefroblastoma , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Esclerodermia Sistémica/tratamiento farmacológico , Proteína smad3 , Transactivadores/fisiología , Factor de Crecimiento Transformador beta/genéticaRESUMEN
To date, a number of p53-derived peptides have been evaluated in vitro for their ability to inhibit the carcinogenic p53-mdm2 interaction. Design of second-generation nonpeptidic compounds requires the reduction of large peptide structures down to small molecules maintaining the proper spatial arrangement of key functional groups. Molecular modeling software exists that can predict and rank intermolecular interactions from the p53-mdm2 complex crystal structure. Such analyses can yield a pharmacophore model suitable as a search query for a 3D chemical database to generate new lead compounds. As preliminary validation of this methodology, the Hydropathic INTeractions (HINT) program has been used to generate noncovalent interaction measurements between reported peptide inhibitors and mdm2. Quantitative structure-activity relationships were developed expressing peptide activity as a linear combination of hydropathic descriptors. In general, HINT measurements accurately modeled the effects of even single-atom alterations of the p53-peptide structure on activity, accounting for 70-90% of variation in experimental inhibition constants. These results surpassed those of a recently described molecular dynamics-based approach and required significantly less computation time. In conclusion, the HINT program can be integrated into the drug design cycle for next-generation p53-mdm2 complex inhibitors with confidence in its ability to simulate this noteworthy protein-protein interaction.
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Proteínas Nucleares , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Relación Estructura-Actividad Cuantitativa , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Cristalografía por Rayos X/métodos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis/genética , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Fragmentos de Péptidos/metabolismo , Biblioteca de Péptidos , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2 , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Agua/metabolismo , Xenopus laevisRESUMEN
We report the first protein map of human adult lung MRC-5 fibroblasts using isoelectric focusing-immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis. MRC-5 is an immortalised cell line used in a wide range of investigations. The two-dimensional gel pattern of proteins generated from any given cell system provides a fingerprint that is unique to those cells. Therefore, the establishment of a protein map for a particular cell system provides a useful reference tool as a "master map" for subsequent studies using those cells. In this map a total of 98 protein spots were identified by comparative searches of the nucleotide and protein database using peptide masses obtained by matrix-assisted laser desorption/ionization time of flight following trypsin digestion. To increase the utility of the reference map, cells were cultured in both Dulbecco's modified Eagle medium (DMEM), the standard medium, and Roswell Park Memorial Institute (RPMI)-1640. Two-dimensional gel protein patterns of MRC-5 cultures were shown to be largely unaffected by the use of RPMI compared to DMEM, respectively. In combination with the reference map, the standardised protocol described provides a tool for comparative studies involving MRC-5 cells in which nonspecific variation is minimized.
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Electroforesis en Gel Bidimensional/métodos , Focalización Isoeléctrica/métodos , Pulmón/química , Proteínas/química , Adulto , Fibroblastos/química , Humanos , Concentración de Iones de Hidrógeno , Pulmón/citologíaRESUMEN
A non-covalent interaction force field model derived from the partition coefficient of 1-octanol/water solubility is described. This model, HINT for Hydropathic INTeractions, is shown to include, in very empirical and approximate terms, all components of biomolecular associations, including hydrogen bonding, Coulombic interactions, hydrophobic interactions, entropy and solvation/desolvation. Particular emphasis is placed on: (1) demonstrating the relationship between the total empirical HINT score and free energy of association, deltaGinteraction; (2) showing that the HINT hydrophobic-polar interaction sub-score represents the energy cost of desolvation upon binding for interacting biomolecules; and (3) a new methodology for treating constrained water molecules as discrete independent small ligands. An example calculation is reported for dihydrofolate reductase (DHFR) bound with methotrexate (MTX). In that case the observed very tight binding, deltaGinteraction < or = -13.6 kcal mol(-1), is largely due to ten hydrogen bonds between the ligand and enzyme with estimated strength ranging between -0.4 and -2.3 kcal mol(-1). Four water molecules bridging between DHFR and MTX contribute an additional -1.7 kcal mol(-1) stability to the complex. The HINT estimate of the cost of desolvation is +13.9 kcal mol(-1).
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Diseño Asistido por Computadora , Diseño de Fármacos , 1-Octanol , Entropía , Escherichia coli/enzimología , Enlace de Hidrógeno , Ligandos , Metotrexato/química , Metotrexato/metabolismo , Modelos Químicos , Modelos Moleculares , Solubilidad , Solventes , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/metabolismo , Termodinámica , AguaRESUMEN
Crystallographic studies of the intermediate states between unliganded and fully liganded hemoglobin (Hb) have revealed a large range of subtle but functionally important structural differences. Only one T state has been reported, whereas three other quaternary states (the R state, B state, and R2 or Y state) for liganded Hb have been characterized; other studies have defined liganded Hbs that are intermediate between the T and R states. The high-salt crystal structure of bovine carbonmonoxy (CO bovine) Hb has been determined at a resolution of 2.1 A and is described here. A detailed comparison with other crystallographically solved Hb forms (T, R, R2 or Y) shows that the quaternary structure of CO bovine Hb closely resembles R state Hb. However, our analysis of these structures has identified several important differences between CO bovine Hb and R state Hb. Compared with the R state structures, the beta-subunit N-terminal region has shifted closer to the central water cavity in CO bovine Hb. In addition, both the alpha- and beta-subunits in CO bovine Hb have more constrained heme environments that appear to be intermediate between the T and R states. Moreover, the distal pocket of the beta-subunit heme in CO bovine Hb shows significantly closer interaction between the bound CO ligand and the Hb distal residues Val 63(E11) and His 63(E7). The constrained heme groups and the increased steric contact involving the CO ligand and the distal heme residues relative to human Hb may explain in part the low intrinsic oxygen affinity of bovine Hb.
Asunto(s)
Carboxihemoglobina/química , Hemoglobinas/química , Animales , Bovinos , Cristalografía por Rayos X/métodos , Hemo/química , Humanos , Ligandos , Modelos Moleculares , Oxígeno/metabolismo , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Although the role of transforming growth factor beta (TGFbeta) in initiating fibrosis is well established, the role that TGFbeta plays in maintaining fibrosis is unclear. The gene encoding connective tissue growth factor (ccn2; ctgf), which promotes fibrosis, is not normally expressed in dermal fibroblasts unless TGFbeta is present. However, in dermal fibroblasts cultured from lesional areas of scleroderma, ccn2 (ctgf) is expressed constitutively. The contribution of several elements in the ccn2 (ctgf) promoter to basal and TGFbeta induced ccn2 (ctgf) expression in normal and scleroderma fibroblasts has been investigated. A functional SMAD binding site in the ccn2 (ctgf) promoter that is necessary for the TGFbeta mediated induction of this gene has been identified. The previously termed TGFbeta responsive enhancer (TGFbetaRE) in the ccn2 (ctgf) promoter has been found to be necessary for basal promoter activity in normal fibroblasts. The SMAD element is not necessary for the high ccn2 (ctgf) promoter activity seen in scleroderma fibroblasts. However, mutation of the previously termed TGFbetaRE reduces ccn2 (ctgf) promoter activity in scleroderma fibroblasts to that seen in normal fibroblasts. Thus, the maintenance of the scleroderma phenotype, as assessed by a high degree of ccn2 (ctgf) promoter activity, appears to be relatively independent of SMAD action and seems to reflect increased basal promoter activity.
Asunto(s)
Fibroblastos/metabolismo , Sustancias de Crecimiento/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Esclerodermia Sistémica/genética , Estudios de Casos y Controles , Factor de Crecimiento del Tejido Conjuntivo , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Humanos , Mutación/genética , Fenotipo , Regiones Promotoras Genéticas , Esclerodermia Sistémica/patología , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/genéticaRESUMEN
The crystal structure of human deoxy hemoglobin (Hb) complexed with a potent allosteric effector (2-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy]-2-methylpropionic acid) = RSR-13) is reported at 1.85 A resolution. Analysis of the hemoglobin:effector complex indicates that two of these molecules bind to the central water cavity of deoxy Hb in a symmetrical fashion, and that each constrains the protein by engaging in hydrogen bonding and hydrophobic interactions with three of its four subunits. Interestingly, we also find that water-mediated interactions between the bound effectors and the protein make significant contributions to the overall binding. Physiologically, the interaction of RSR-13 with Hb results in increased oxygen delivery to peripheral tissues. Thus, this compound has potential therapeutic application in the treatment of hypoxia, ischemia, and trauma-related blood loss. Currently, RSR-13 is in phase III clinical trials as a radiosensitizing agent in the treatment of brain tumors. A detailed structural analysis of this compound complexed with deoxy Hb has important implications for the rational design of future analogs.