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Updated: May 2, 2026
Exploring Caspase Mutations and Post-Translational Modification by Molecular Modeling Approaches
Published on: October 13, 2022
An unbiased proteomic screen reveals caspase cleavage is positively and negatively regulated by substrate
Jacob P Turowec1, Stephanie A Zukowski, James D R Knight
1Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario N6A 5C1, Canada;
Abstract:
Post-translational modifications of proteins regulate diverse cellular functions, with mounting evidence suggesting that hierarchical cross-talk between distinct modifications may fine-tune cellular responses. For example, in apoptosis, caspases promote cell death via cleavage of key structural and enzymatic proteins that in some instances is inhibited by phosphorylation near the scissile bond. In this study, we systematically investigated how protein phosphorylation affects susceptibility to caspase cleavage using an N-terminomic strategy, namely, a modified terminal amino isotopic labeling of substrates (TAILS) workflow, to identify proteins for which caspase-catalyzed cleavage is modulated by phosphatase treatment. We validated the effects of phosphorylation on three of the identified proteins and found that Yap1 and Golgin-160 exhibit decreased cleavage when phosphorylated, whereas cleavage of MST3 was promoted by phosphorylation. Furthermore, using synthetic peptides we systematically examined the influence of phosphoserine throughout the entirety of caspase-3, -7, and -8 recognition motifs and observed a general inhibitory effect of phosphorylation even at residues considered outside the classical consensus motif. Overall, our work demonstrates a role for phosphorylation in controlling caspase-mediated cleavage and shows that N-terminomic strategies can be tailored to study cross-talk between phosphorylation and proteolysis.
Insights
Protein phosphorylation can alter caspase cleavage, impacting cellular functions like apoptosis. This study reveals how phosphorylation modifies protein susceptibility to caspase proteolysis, offering insights into cellular signaling regulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Biology
Background:
- Post-translational modifications (PTMs) critically regulate protein function and cellular processes.
- Hierarchical cross-talk between different PTMs, such as phosphorylation and proteolysis, fine-tunes cellular responses.
- Caspase-mediated protein cleavage is a key event in apoptosis, and its regulation is crucial for cell fate determination.
Purpose of the Study:
- To systematically investigate the impact of protein phosphorylation on susceptibility to caspase cleavage.
- To identify proteins whose caspase cleavage is modulated by phosphorylation using an N-terminomic approach.
- To elucidate the general effect of phosphorylation on caspase recognition motifs.
Main Methods:
- Utilized a modified terminal amino isotopic labeling of substrates (TAILS) workflow for N-terminomic analysis.
- Employed phosphatase treatment to identify proteins with phosphorylation-dependent caspase cleavage.
- Validated findings on specific proteins (Yap1, Golgin-160, MST3) and analyzed synthetic peptides representing caspase recognition motifs.
Main Results:
- Identified several proteins whose caspase cleavage is modulated by phosphorylation.
- Yap1 and Golgin-160 showed decreased cleavage upon phosphorylation, while MST3 cleavage was promoted.
- Phosphorylation generally inhibited caspase cleavage, even outside canonical recognition sites.
Conclusions:
- Protein phosphorylation plays a significant role in regulating caspase-mediated proteolysis.
- N-terminomic strategies are effective for studying the cross-talk between phosphorylation and proteolysis.
- Understanding this cross-talk provides insights into the fine-tuning of cellular signaling pathways, particularly in apoptosis.
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