RESUMO
Ubiquitin plays an essential role in modulating protein function, and deregulation of the ubiquitin system leads to the development of a variety of human diseases. E3 Ubiquitin ligases that mediate ubiquitination and degradation of caspases prevent apoptosis, and as such belong to the family of inhibitors of apoptosis proteins (IAPs). Diablo is a substrate of IAPs but also a negative regulator of IAPs in apoptotic pathway as it blocks the interaction between IAPs and caspases. In efforts to identify IAP inhibitors, we developed sandwich immunoassays in conjunction with an electrochemical luminescence (ECL) platform for quantitation of total Diablo, ubiquitinated Diablo, and ubiquitinated Diablo with K48-specific linkage. The assay panel detects Diablo ubiquitination level changes in the presence of IAP inhibitor or proteasome inhibitor, demonstrating its potential as a cost-efficient high-throughput method for drug discovery involving IAP ubiquitination cascade. The ECL based sandwich assay panel performance was subsequently evaluated for precision, linearity, and limit of quantification.
Assuntos
Proteínas Reguladoras de Apoptose/isolamento & purificação , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Proteínas Mitocondriais/isolamento & purificação , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Humanos , Imunoensaio/métodos , Medições Luminescentes/métodos , Proteínas Mitocondriais/metabolismo , Inibidores de Proteassoma/farmacologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ubiquitinação/efeitos dos fármacos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
Coiled-Coil Domain Containing 47 (CCDC47) is an endoplasmic reticulum (ER) transmembrane protein involved in calcium signaling through utilization of its calcium binding-acidic luminal domain. CCDC47 also interacts with ERAD (endoplasmic reticulum-associated degradation) complex and is involved in ER stress relief. In this report, we developed human CCDC47 monoclonal antibodies and a sandwich immunoassay for CCDC47 measurement in biological matrices. Specificity of developed antibodies were confirmed by immunoblot and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated cell lysates. To achieve high analytical sensitivity, traditional colorimetric enzyme-linked immunosorbent assay (ELISA) and electrochemiluminescence (ECL) technology were compared, and 3 logs of increased sensitivity was observed with the use of ECL. A CCDC47 sandwich ECL assay was subsequently developed and performances evaluated for calibration curves, precision and accuracy, as well as selectivity and interferences for sample measurement. Sample stability was also characterized for freeze/thaw cycles and short/long term storage conditions.
