Visualization of calpain-1 activation during cell death and its role in GSDMD cleavage using chemical probes
Natalia Horbach1, Małgorzata Kalinka1, Natalia Ćwilichowska-Puślecka1
1Faculty of Chemistry, Wroclaw University of Science and Technology, 50-370 Wroclaw, Poland.
Abstract:
Calpain-1, a calcium-dependent cysteine protease, plays a vital role in cellular processes such as cell death, cytoskeletal remodeling, signal transduction, and cell cycle progression. While its role in apoptosis, including substrate cleavage for orderly disassembly, is well established, its involvement in pyroptosis remains less understood. This study focused on developing chemical tools to detect calpain-1 activity. Using the hybrid combinatorial substrate library (HyCoSuL) approach with unnatural amino acids, we designed fluorescent substrates, inhibitors, and fluorescent activity-based probe (ABP) specific to calpain-1, enabling its visualization in living cells. We further investigated calpain-1's expression alongside cell death proteins in immune cells using mass cytometry and observed strong colocalization with gasdermin D (GSDMD). Additionally, we demonstrated that calpain-1 can hydrolyze GSDMD in vitro. Through fluorescence-based substrate assays and mass spectrometry, we identified putative cleavage sites within the GSDMD sequence that may promote pyroptosis. These findings underscore calpain-1's multifaceted role in cell death pathways, extending beyond apoptosis.
Insights
Calpain-1 protease activity was visualized in living cells using novel chemical tools. This protease was found to cleave gasdermin D, suggesting a role in pyroptosis beyond its known function in apoptosis.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Calpain-1 is a calcium-dependent cysteine protease crucial for cellular processes like cell death and signaling.
- Its role in apoptosis is established, but involvement in pyroptosis is less understood.
Purpose of the Study:
- To develop chemical tools for detecting calpain-1 activity.
- To investigate calpain-1's role in pyroptosis.
Main Methods:
- Utilized the hybrid combinatorial substrate library (HyCoSuL) approach with unnatural amino acids.
- Designed fluorescent substrates, inhibitors, and activity-based probes (ABPs) for calpain-1.
- Employed mass cytometry to analyze immune cells and mass spectrometry to identify cleavage sites.
Main Results:
- Developed specific chemical tools enabling calpain-1 visualization in living cells.
- Observed strong colocalization of calpain-1 with gasdermin D (GSDMD) in immune cells.
- Demonstrated in vitro hydrolysis of GSDMD by calpain-1 and identified potential pyroptosis-promoting cleavage sites.
Conclusions:
- Calpain-1's role in cell death pathways extends beyond apoptosis to include pyroptosis.
- Novel chemical probes facilitate the study of calpain-1 in cellular contexts.
- Calpain-1's interaction with GSDMD provides new insights into pyroptosis regulation.
