RESUMEN
Proteomics, the large-scale analysis of proteins, is a rapidly evolving field with an increasing number of key clinical applications, such as diagnosis, prognosis, and classification. In order to generate complete protein expression profiles, or protein atlases, any crude sample format must be addressable in a rapid, multiplex, and sensitive manner. A common and clinically central sample format, formalin-fixed, paraffin-embedded (FFPE) tissue material, holds great potential as a source for disease-associated biomarker signatures. However, despite major efforts, extraction and subsequent profiling of proteins from FFPE tissue has proven to be challenging. In this proof-of-concept study, we have demonstrated for the first time that proteins could be extracted, labeled, and subsequently profiled in a multiplex, sensitive, and reproducible manner using recombinant scFv antibody microarrays. Thus, we have added FFPE samples to the list of sample formats available for high-throughput analysis by affinity proteomics, paving the way for the next generation of biomarker-driven discovery projects.
Asunto(s)
Neoplasias de la Mama/genética , Región Variable de Inmunoglobulina , Linfoma Folicular/genética , Linfoma de Células del Manto/genética , Proteínas de Neoplasias/análisis , Análisis por Matrices de Proteínas/instrumentación , Anticuerpos de Cadena Única , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Fijadores , Formaldehído , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/inmunología , Límite de Detección , Linfoma Folicular/diagnóstico , Linfoma Folicular/inmunología , Linfoma de Células del Manto/diagnóstico , Linfoma de Células del Manto/inmunología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Adhesión en Parafina , Análisis por Matrices de Proteínas/métodos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Reproducibilidad de los Resultados , Anticuerpos de Cadena Única/biosíntesis , Anticuerpos de Cadena Única/inmunología , Fijación del TejidoRESUMEN
Schistosomiasis is a parasitic disease affecting over 200 million people currently treated with one drug, praziquantel. A possible drug target is the seleno-protein thioredoxin-glutathione reductase (TGR), a key enzyme in the pathway of the parasite for detoxification of reactive oxygen species. The enzyme is a unique fusion of a glutaredoxin domain with a thioredoxin reductase domain, which contains a selenocysteine (Sec) as the penultimate amino acid. Auranofin (AF), a gold-containing compound already in clinical use as an anti-arthritic drug, has been shown to inhibit TGR and to substantially reduce worm burden in mice. Using x-ray crystallography we solved (at 2.5 A resolution) the structure of wild type TGR incubated with AF. The electron density maps show that the actual inhibitor is gold, released from AF. Gold is bound at three different sites not directly involving the C-terminal Sec residue; however, because the C terminus in the electron density maps is disordered, we cannot exclude the possibility that gold may also bind to Sec. To investigate the possible role of Sec in the inactivation kinetics, we tested the effect of AF on a model enzyme of the same superfamily, i.e. the naturally Sec-lacking glutathione reductase, and on truncated TGR. We demonstrate that the role of selenium in the onset of inhibition by AF is catalytic and can be mimicked by an external source of selenium (benzeneselenol). Therefore, we propose that Sec mediates the transfer of gold from its ligands in AF to the redox-active Cys couples of TGR.
Asunto(s)
Antirreumáticos/química , Auranofina/química , Regulación de la Expresión Génica , Proteínas del Helminto/química , Complejos Multienzimáticos/química , NADH NADPH Oxidorreductasas/química , Schistosoma mansoni/metabolismo , Animales , Antirreumáticos/farmacología , Auranofina/farmacología , Catálisis , Cristalografía por Rayos X/métodos , Cisteína/química , Relación Dosis-Respuesta a Droga , Cinética , Modelos Moleculares , Complejos Multienzimáticos/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Oxidación-Reducción , Estructura Terciaria de Proteína , Selenio/químicaRESUMEN
B-cell lymphoma (BCL) heterogeneity represents a key issue, often making the classification and clinical management of these patients challenging. In this pilot study, we outlined the first resolved view of BCL disease heterogeneity on the protein level by deciphering disease-associated plasma biomarkers, specific for chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma, using recombinant antibody microarrays targeting mainly immunoregulatory proteins. The results showed the BCLs to be heterogeneous, and revealed potential novel subgroups of each BCL. In the case of diffuse large B-cell lymphoma, we also indicated a link between the novel subgroups and survival.