RESUMO
We have previously reported that Monad, a novel WD40 repeat protein, potentiates apoptosis induced by tumor necrosis factor-alpha(TNF-alpha) and cycloheximide (CHX). By affinity purification and mass spectrometry, we identified RNA polymerase II-associated protein 3 (RPAP3) as a binding protein of Monad. Overexpression of RPAP3 in HEK 293 potentiated caspase-3 activation and apoptosis induced by TNF-alpha and CHX. In addition, knockdown of RPAP3 by RNA interference resulted in a significant reduction of apoptosis induced by TNF-alpha and CHX in HEK293 and HeLa cells. These results raise the possibility that RPAP3, together with Monad, may function as a novel modulator of apoptosis pathway.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Proteínas de Transporte/fisiologia , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Sequências Repetitivas de Aminoácidos , Distribuição TecidualRESUMO
We have previously shown that procaspase-3 exists in a high molecular weight complex in neonatal rat brain. Here, we purify and identify the protein that interacts with procaspase-3 from rat neonatal cortex. We searched binding proteins to procaspase-3 from a cytosolic extract of neonatal rat brain using chromatogram, two-dimensional gel electrophoresis, and far Western immunoblot. Analysis by tandem mass spectrometry identified the protein as a regulatory subunit of calcineurin (calcineurin B). Overexpression of calcineurin B in HEK293 cells potentiated processing of caspase-3 and apoptosis triggered by tumor necrosis factor-alpha and cycloheximide treatment. In a cell-free system, overexpression of calcineurin B in HEK293 cells markedly increased processing of caspase-3 by cytochrome c. Immunodepletion of calcineurin B from cytosolic extracts from Jurkat cells decreased processing of caspase-3 by cytochrome c. Knockdown of calcineurin B by RNA interference resulted in reduced apoptosis in HEK293 cells but not in caspase-3-deficient MCF-7 cells. These results suggest that calcineurin B potentiates the activation of procaspase-3 by accelerating its proteolytic maturation.
Assuntos
Calcineurina/fisiologia , Caspase 3/metabolismo , Animais , Apoptose , Encéfalo/metabolismo , Calcineurina/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Citocromos c/metabolismo , Ativação Enzimática , Humanos , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/metabolismoRESUMO
WD40 repeat proteins have a wide range of diverse biological functions including signal transduction, cell cycle regulation, RNA splicing, and transcription. Here we report the identification and characterization of a novel human WD40 repeat protein, Monad. Monad is unique, since it contains only two WD40 repeats. Monad is widely expressed in human tissues with the highest expression in testis. Overexpression of Monad in HEK293 cells potentiated apoptosis and caspase-3 activation induced by tumor necrosis factor-alpha and cycloheximide. These results raise the possibility that Monad may function as a novel modulator of apoptosis pathway.