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Published on: March 5, 2018
A Site-Specific Photo-Crosslinking Proteomics Approach Provides Insights into Noncanonical Pyroptotic Caspase-4
Yi Zhou1, Xinyu Zhang1, Hang Yin1
1Key Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology, Department of Chemistry, School of Pharmaceutical Sciences, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, 100084, China.
Abstract:
Inflammatory caspases (1/4/5) are key effectors in the process of pyroptosis by cleaving and activating the pore-forming protein gasdermin D (GSDMD). Unlike other caspases whose substrates have been well characterized, the substrates for caspase-4, which mediate noncanonical pyroptosis, remain poorly understood. Here, we combined noncanonical amino acids, photo-crosslinking, and proteomics to profile caspase-4 substrates, enabling the capture of transient protein interactions with activated caspase-4. A set of new substrates were identified by photo-crosslinking mass spectrometry, revealing the signaling pathway and biological process affected by pyroptosis. Notably, we found that AKT1 is cleaved at D108, which removes its autoinhibition and membrane localization domain, resulting in the release of activated AKT1. Our results also showed the precursor of caspase-5/12 could be cleaved by caspase-4 to form the p20/p10 active conformation, uncovering a previously unrecognized pyroptotic caspase cascade. Overall, this study presents an approach for identifying caspase-4 substrates and offers further understanding of noncanonical pyroptosis.
Insights
This study identifies new substrates for caspase-4, a key protein in noncanonical pyroptosis. Findings reveal AKT1 cleavage and a novel caspase-5/12 activation pathway, advancing pyroptosis research.
Area of Science:
- Cellular Biology
- Immunology
- Biochemistry
Background:
- Inflammatory caspases (caspase-1/4/5) are crucial for pyroptosis, a programmed cell death pathway.
- Gasdermin D (GSDMD) is activated by inflammatory caspases, leading to cell membrane permeabilization.
- The substrates of caspase-4, involved in noncanonical pyroptosis, are not well understood.
Purpose of the Study:
- To identify novel substrates of caspase-4 using advanced proteomic techniques.
- To elucidate the signaling pathways and biological processes influenced by caspase-4 activity.
- To gain a deeper understanding of noncanonical pyroptosis mechanisms.
Main Methods:
- Utilized noncanonical amino acid tagging, photo-crosslinking, and mass spectrometry.
- Employed a proteomic approach to capture transient interactions with activated caspase-4.
- Investigated the cleavage of specific proteins, including AKT1 and caspase-5/12 precursors.
Main Results:
- Identified a set of previously unknown caspase-4 substrates.
- Demonstrated that caspase-4 cleaves AKT1 at D108, releasing activated AKT1.
- Revealed that caspase-4 processes caspase-5/12 precursors into their active forms, uncovering a new pyroptotic cascade.
Conclusions:
- Developed a robust method for profiling caspase-4 substrates.
- Provided new insights into the molecular mechanisms of noncanonical pyroptosis.
- Highlighted the role of caspase-4 in regulating AKT signaling and activating other caspases.
