A SILAC-based Approach Identifies Substrates of Caspase-dependent Cleavage upon TRAIL-induced Apoptosis

Gabriele Stoehr,Christoph Schaab,Johannes Graumann,Matthias Mann

doi:10.1074/mcp.m112.024679

Abstract

The extracellular ligand-induced extrinsic pathway of apoptosis is executed via caspase protease cascades that activate downstream effectors by means of site-directed proteolysis. Here we identify proteome changes upon the induction of apoptosis by the cytokine tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in a Jurkat T cell line. We detected caspase-dependent cleavage substrates by quantifying protein intensities before and after TRAIL induction in SDS gel slices. Apoptotic protein cleavage events are identified by a characteristic stable isotope labeling with amino acids in cell culture (SILAC) ratio pattern across gel slices that results from differential migration of the cleaved and uncleaved proteins. We applied a statistical test to define apoptotic substrates in the proteome. Our approach identified more than 650 of these cleaved proteins in response to TRAIL-induced apoptosis, including many previously unknown substrates and cleavage sites. Inhibitor treatment combined with triple SILAC demonstrated that the detected cleavage events were caspase dependent. Proteins located in the lumina of organelles such as mitochondria and endoplasmic reticulum were significantly underrepresented in the substrate population. Interestingly, caspase cleavage is generally observed in not only one but several members of stable complexes, but often with lower stoichiometry. For instance, all five proteins of the condensin I complex were cleaved upon TRAIL treatment. The apoptotic substrate proteome data can be accessed and visualized in the MaxQB database and might prove useful for basic and clinical research into TRAIL-induced apoptosis. The technology described here is extensible to a wide range of other proteolytic cleavage events.

Highlights

From the ‡Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany; §Evotec (Munich) GmbH, Am Klopferspitz 19a, D-82152 Martinsried, Germany; ¶Weill Cornell Medical College in Qatar, Qatar Foundation, Education City, Doha, State of Qatar
In our SILAC experiments, untreated cells were labeled with light RPMI medium (Arg0; Lys0) and cells used for treatment were labeled with heavy medium (Arg10; Lys8) before the induction of apoptosis (Fig. 1A)
We have described a quantitative SILAC-based approach for the identification of proteolytically cleaved substrates and used it to investigate the events of apoptosis induced by the extrinsic stimulus tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)

Summary

Introduction

Several methods have been introduced to identify substrates cleaved in a caspase-dependent manner, as well as the exact location of cleavage sites. In vitro approaches such as the incubation of peptides or protein libraries with the active protease of interest have led to the identification of substrate motifs but do not necessarily represent in vivo events in the context of an intact cell [7]. Mass spectrometry (MS)-based methods employed may be divided into those directed toward the detection of the peptides cleaved by the protease and those applied at the proteome level for the identification of substrates and downstream effects. The approach was further extended to include the quantitative ratio information of the proteins in combination with offline LC-MALDI-MS/MS [22]

Objectives

Results

Conclusion