title: A SILAC-based approach identifies substrates of caspase-dependent cleavage upon TRAIL-induced apoptosis.
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.
authors: Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
authors: Gabriele
authors: G
authors: Stoehr
authors: Christoph
authors: C
authors: Schaab
authors: Johannes
authors: J
authors: Graumann
authors: Matthias
authors: M
authors: Mann
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D000071183:Autophagy-Related Proteins; D020169:Caspases / Q000378:metabolism*; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D046912:Multiprotein Complexes / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D059748:Proteolysis; D020543:Proteome / Q000378:metabolism; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000502:physiology*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; D000071183:Autophagy-Related Proteins; D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D020543:Proteome; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; C108312:condensin complexes; D044763:Ubiquitin-Conjugating Enzymes; D020169:Caspases; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; C455621:ATG3 protein, human