Article pubs.acs.org/jpr
Low Dose Proteasome Inhibition Affects Alternative Splicing Sven Bieler,† Elke Hammer,‡ Manuela Gesell-Salazar,‡ Uwe Völker,‡ Karl Stangl,† and Silke Meiners*,§ †
Medizinische Klinik mit Schwerpunkt Kardiologie und Angiologie, Charité-Universitätsmedizin, Berlin, Germany Interfakultäres Institut für Genetik und Funktionelle Genomforschung, Ernst Moritz Arndt Universität, Greifswald, Germany § Comprehensive Pneumology Center, Ludwig Maximilians Universität München and Helmholtz Zentrum, München, Germany ‡
S Supporting Information *
ABSTRACT: Protein degradation by the ubiquitin proteasome system ensures controlled degradation of structural proteins, signaling mediators, and transcription factors. Inhibition of proteasome function by specific proteasome inhibitors results in dose-dependent cellular effects ranging from induction of apoptosis to protective stress responses. The present study seeks to identify nuclear regulators mediating the protective stress response to low dose proteasome inhibition. Primary human endothelial cells were treated with low doses of the proteasome inhibitor MG132 for 2 h, and proteomic analysis of nuclear extracts was performed. Using a 2-D differential in gel electrophoresis (DIGE) approach, we identified more than 24 splice factors to be differentially regulated by low dose proteasome inhibition. In particular, several isoforms of hnRNPA1 were shown to be increased, pointing toward altered posttranslational modification of hnRNPA1 upon proteasome inhibition. Elevated levels of splice factors were associated with a different alternative splicing pattern in response to proteasome inhibition as determined by Affymetrix exon array profiling. Of note, we observed alternative RNA processing for stress associated genes such as caspases and heat shock proteins. Our study provides first evidence that low dose proteasome inhibition affects posttranscriptional regulation of splice factors and early alternative splicing events. KEYWORDS: endothelial cell, proteomics, splicing, ubiquitin proteasome system, 2-D-DIGE
1. INTRODUCTION Protein degradation by the ubiquitin proteasome system is essential for proper cell function. Proteasome function ensures the controlled and timely degradation of structural proteins, signaling mediators, and transcription factors.1 Proteins are first tagged with multiple ubiquitin molecules and are then degraded by the 26S proteasome. This 26S proteasome particle consists of two 19S regulatory complexes and a 20S catalytic core. While the 19S subunits are involved in substrate recognition, ubiquitin removal, and protein unfolding, the 20S core contains three distinct proteolytic activities for hydrolysis of the protein chain into small peptides.2 Inhibitors of the proteasome have been developed that specifically interact with the N-terminal threonine residues of the catalytic active sites thereby blocking peptide chain hydrolysis and protein degradation.3 Proteasome inhibition results in the accumulation of cell cycle regulators and signaling mediators inducing cell cycle arrest and apoptosis.4 Accordingly, the specific proteasome inhibitor Bortezomib has been FDA-approved in 2003 for the treatment of patients with multiple myeloma. Several other inhibitors with distinct chemical reactivities have since then been developed and are currently tested in clinical trials for treatment of hematologic and solid tumors.5 However, beyond their cytotoxic potential, proteasome inhibitors exert pronounced anti-inflammatory and also protective effects in different cell types and animal models.6 We and others have previously demonstrated that low and nontoxic proteasome inhibition © 2012 American Chemical Society
protects vascular cells from oxidative stress and inflammatory signaling thereby contributing to improved vascular function.7−11 To reconcile the divergent cellular effects of proteasome inhibitors, we have recently proposed a unifying concept of proteasome inhibitors acting as poisons or remedies depending on the cell type and degree of proteasome inhibition:12 while high inhibitor doses efficiently block protein degradation by the proteasome and induce apoptosis in proliferating cells, low doses of proteasome inhibitors only partially inhibit the three proteasomal proteolytic activities and mediate a protective cellular stress response. Using combined proteomic, transcriptomic, and functional approaches, we have since then provided evidence for a differential stress response to low- and high-dose proteasome inhibition: Low-dose proteasome inhibition induces a transcriptional profile reminiscent of a physiological stress response that preconditions and protects endothelial cells from oxidative stress. In contrast, high inhibitor doses mediate massive transcriptional dysregulation and pronounced oxidative stress, which commits endothelial cells to apoptosis.13−15 In our present study, we focused on the analysis of nuclear regulators that may mediate the protective stress response to low dose proteasome inhibition. Using a 2-DDIGE approach on nuclear extracts, we identified several splice factors to be differentially regulated by low doses of proteasome Received: August 4, 2011 Published: June 16, 2012 3947
dx.doi.org/10.1021/pr300435c | J. Proteome Res. 2012, 11, 3947−3954
Journal of Proteome Research
Article
displayed different intensities in cells treated with low dose proteasome inhibitor compared to those treated with DMSO only (pairwise t-test p-value
