Visualization of calpain-1 activation during cell death and its role in GSDMD cleavage using chemical probes

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.