Biology subjects

Exconde, P. M.

Publications and source records attributed to Exconde, P. M..

3 recordsLinked to original sources

bioRxiv2025

Human non-canonical inflammasomes activate CASP3 to limit intracellular Salmonella replication in macrophages.

Inflammasomes are multiprotein signaling platforms that activate inflammatory caspases to initiate innate immune signaling. In humans, canonical inflammasomes activate CASP1, which cleaves the pore-forming protein gasdermin D (GSDMD) and the cytokines IL-1{beta} and IL-18. In contrast, the non-canonical inflammasome detects bacterial lipopolysaccharide (LPS) through CASP4/5, which cleave GSDMD to drive pyroptosis. While CASP1 substrates are well characterized, CASP4/5 substrates remain less defined. Here, we show that in response to intracellular LPS and gram-negative bacterial infection, CASP4/5 directly cleave and activate the executioner caspases CASP3/7. CASP3 in turn cleaves and activates gasdermin E (GSDME). Surprisingly, CASP3, but not GSDME, was required for restricting intracellular Salmonella replication, suggesting that CASP4/5-induced apoptosis contributes to host defense. We further show that most GSDMD cleavage during non-canonical inflammasome activation is mediated by CASP1, and that GSDMD is the primary driver of pyroptosis. Finally, we confirm that CASP4/5 activate CASP3/7 and GSDME in human primary macrophages. These findings establish CASP4/5 as dual apoptotic initiator and inflammatory caspases and reveal a central role for the apoptotic signaling cascade in non-canonical inflammasome-mediated immunity.

cell biology↗

bioRxiv2025

Chemical Tools Based on the Tetrapeptide Sequence of IL-18 Reveals Shared Specificities between Inflammatory and Apoptotic Initiator Caspases

Caspases are a family of cysteine proteases that act as molecular scissors to cleave substrates and regulate biological processes such as programmed cell death and inflammation. Extensive efforts have been made to identify caspase substrates and to determine factors that dictate substrate specificity. We recently discovered that that the human inflammatory caspases (caspases-1, -4, and -5) cleave the cytokines IL-1{beta} and IL-18 in a sequence-dependent manner. Here, we report the development of a new peptide-based probe and inhibitor based on the tetrapeptide sequence of IL-18 (LESD). We found that this inhibitor was most selective and potent at inhibiting caspase-8 activity (IC50 = 50 nM). We also discovered that our LESD-based inhibitor is more potent than the currently used z-IETD-FMK inhibitor that is thought to be the most selective and potent inhibitor of caspase-8. Accordingly, we demonstrate that the LESD based inhibitor prevents caspase-8 activation during Yersinia pseudotuberculosis infection in primary bone-marrow derived macrophages. Furthermore, we characterize the selectivity and potency of currently known substrates and inhibitors for the apoptotic and inflammatory caspases using the same activity units of each caspase. Our findings reveal that VX-765, a known caspase-1 inhibitor, also inhibits caspase-8 (IC50 = 1 {micro}M) and even when specificities are shared, the caspases have different efficiencies and potencies for shared substrates and inhibitors. Altogether, we report the development of new tools that will facilitate the study of caspases and their roles in biology.

biochemistry↗

bioRxiv2023

The tetrapeptide sequence of IL-1β regulates its recruitment and activation by inflammatory caspases

The mammalian innate immune system uses germline-encoded cytosolic pattern-recognition receptors (PRRs) to detect intracellular danger signals. At least six of these PRRs are known to form multiprotein complexes called inflammasomes which activate cysteine proteases known as caspases. Canonical inflammasomes recruit and activate caspase-1 (CASP1), which in turn cleaves and activates inflammatory cytokines such as IL-1{beta} and IL-18, as well as the pore forming protein, gasdermin D (GSDMD), to induce pyroptotic cell death. In contrast, non-canonical inflammasomes, caspases-4/-5 (CASP4/5) in humans and caspase-11 (CASP11) in mice, are activated by intracellular LPS to cleave GSDMD, but their role in direct processing of inflammatory cytokines has not been established. Here we show that active CASP4/5 directly cleave IL-18 to generate the active species. Surprisingly, we also discovered that CASP4/5/11 cleave IL-1{beta} at D27 to generate a 27 kDa fragment that is predicted to be inactive and cannot signal to the IL-1 receptor. Mechanistically, we discovered that the sequence identity of the P4-P1 tetrapeptide sequence adjacent to the caspase cleavage site (D116) regulates the recruitment and processing of IL-1{beta} by inflammatory caspases to generate the bioactive species. Thus, we have identified new substrates of the non-canonical inflammasomes and reveal key mechanistic details regulating inflammation.

immunology↗