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Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection
Published on: July 21, 2022
A SILAC-based approach identifies substrates of caspase-dependent cleavage upon TRAIL-induced apoptosis
Gabriele Stoehr1, Christoph Schaab, Johannes Graumann
1Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
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.
Insights
This study identifies over 650 caspase-dependent protein cleavage events during tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. The findings reveal new substrates and cleavage sites, advancing our understanding of programmed cell death.
Area of Science:
- Proteomics
- Cell Biology
- Biochemistry
Background:
- Extracellular ligand-induced apoptosis involves caspase protease cascades.
- Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers the extrinsic apoptosis pathway.
- Understanding proteome changes during apoptosis is crucial for basic and clinical research.
Purpose of the Study:
- To identify proteome-wide changes, specifically caspase-dependent cleavage substrates, induced by TRAIL in Jurkat T cells.
- To characterize novel apoptotic substrates and cleavage sites.
- To investigate the distribution of apoptotic substrates within cellular compartments and protein complexes.
Main Methods:
- Quantitative proteomic analysis using Stable Isotope Labeling with Amino acids in Cell culture (SILAC).
- Differential protein intensity quantification across SDS-PAGE gel slices to detect cleavage events.
- Statistical analysis to identify apoptotic substrates and caspase-dependent cleavage.
- Inhibitor treatment and triple SILAC to confirm caspase dependency.
Main Results:
- Identified over 650 caspase-dependent protein cleavage substrates in response to TRAIL-induced apoptosis.
- Discovered numerous previously unknown substrates and cleavage sites.
- Observed underrepresentation of proteins from organelle lumens (mitochondria, ER) in the substrate population.
- Demonstrated cleavage of multiple subunits within stable protein complexes, such as the condensin I complex.
Conclusions:
- The developed proteomic approach effectively identifies extensive caspase-dependent cleavage events during TRAIL-induced apoptosis.
- The findings provide a comprehensive catalog of apoptotic substrates and cleavage sites, expanding knowledge of the apoptotic proteome.
- The technology is adaptable for studying other proteolytic cleavage events in various biological contexts.
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