Biology subjects

Taabazuing, C. Y.

Publications and source records attributed to Taabazuing, C. Y..

8 recordsLinked to original sources

bioRxiv2026

Human caspase-4 and caspase-5 assemble into non-canonical inflammasomes in response to overlapping and distinct LPS structures

Innate immune sensing of bacterial lipopolysaccharide (LPS) is critical to host defense, but a dysregulated response can lead to sepsis, a life-threatening condition associated with multiple organ failure and shock. LPS is sensed at the cell surface by Toll-Like Receptor 4 (TLR4) and in the cytosol by mouse caspase-11 (Casp11) or its human orthologs, caspase-4 and 5 (CASP4/5). In response to cytosolic LPS, Casp11 oligomerizes in a catalytically dependent manner into supramolecular organizing centers (SMOCs). We demonstrate that CASP4 and CASP5 also form catalytically dependent SMOCs in response to bacterial LPS in macrophages. Furthermore, we find that CASP4 and CASP5 differentially respond to distinct LPS structural variants that fail to activate CASP11 and that only activating LPS structures induce SMOC formation by the corresponding caspase. Notably, transgenic mice expressing CASP4 in lieu of Casp11 exert more effective control of Francisella novicida, a rodent pathogen whose LPS evades TLR4 and Casp11 detection. Conversely, CASP4-expressing mice are more susceptible to sepsis when exposed to E. coli expressing a lipid A variant that fails to activate TLR4 and Casp11. Together, these findings reveal human CASP4 and CASP5 have evolved to assemble into SMOCs in response to a broader range of LPS structures than mouse Casp11 and highlight the potential tradeoff between systemic inflammation and control of bacterial infection that may underline these differences. Our findings may inform the development of targeted therapeutic strategies for gram-negative bacterial infection and sepsis that are effective in humans.

immunology↗

bioRxiv2026

DIPTAR: A synthetic biology platform for functional interrogation of protein degradation

Protein degradation regulates cellular homeostasis, yet many degradation events are difficult to study because they lack a readily selectable phenotype. Here, we develop Degradation-Induced Pyroptosis TArgeting Receptors (DIPTAR), a modular synthetic biology platform that couples protein degradation to CARD8-mediated pyroptosis. Using HIF-1 as a model substrate, we show that DIPTAR faithfully reports oxygen-dependent VHL-mediated degradation and enables pooled CRISPR screening to identify established and previously unrecognized regulators of HIF-1 stability. DIPTAR is functional across multiple cell types and can be programmed with diverse proteins, including BRD4, I{kappa}B, and p53, to convert distinct degradation stimuli into a common pyroptotic output. DIPTAR also detects pathogen-mediated perturbations of host degradation pathways, including both inhibition and induction of degradation-dependent signaling. By converting protein degradation into a robust selectable phenotype, DIPTAR provides a scalable platform for functional genetic discovery, interrogation of degradation pathways, degrader characterization, and investigation of host-pathogen interactions.

synthetic biology↗

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↗

bioRxiv2022

Noncanonical inflammasome assembly requires caspase-11 catalytic activity and intra-molecular autoprocessing

Inflammatory caspases are cysteine protease zymogens whose activation following infection or cellular damage occurs within supramolecular organizing centers (SMOCs) known as inflammsasomes. Inflammasomes are large oligomeric complexes that recruit caspases to undergo proximity-induced autoprocessing, leading to formation of the enzymatically active form that cleaves downstream targets. Binding of bacterial LPS to its cytosolic sensor, caspase-11 (Casp11), promotes Casp11 aggregation within a high molecular weight complex known as the noncanonical inflammasome, where it is activated to cleave gasdermin D and induce pyroptosis. However, the cellular correlates of Casp11 oligomerization and whether Casp11 forms an LPS-induced SMOC within cells remain unknown. Using fluorescently labeled Casp11, we found that LPS transfection of macrophages induced Casp11 speck formation, providing direct evidence that Casp11 forms LPS-induced specks in macrophages. Unexpectedly, we found that catalytic activity was required for Casp11 to form LPS-induced specks in macrophages. Importantly, both catalytic activity and autoprocessing were required for Casp11 speck formation in an ectopic expression system, and Casp11 processing via an exogenous protease was sufficient to induce Casp11 speck formation. These data reveal a previously-undescribed role for Casp11 catalytic activity and self-cleavage in non-canonical inflammasome assembly and indicate that Casp11 catalytic activity and autoprocessing occur upstream of, and mediate, Casp11 oligomerization. Our data provide new insight into the molecular requirements for assembly of Casp11 noncanonical inflammasome complexes.

immunology↗

bioRxiv2022

A ubiquitin-independent proteasome pathway controls the CARD8 inflammasome

CARD8 is a pattern-recognition receptor that forms a caspase-1-activating inflammasome. CARD8 undergoes autoproteolysis, generating an N-terminal (NT) fragment with a disordered region and a ZU5 domain and a C-terminal (CT) fragment with UPA and CARD domains. DPP8 and DPP9 (DPP8/9) inhibitors, including Val-boroPro (VbP), accelerate the degradation of the NT fragment via a poorly characterized proteasome-mediated pathway, thereby releasing the inflammatory CT fragment from autoinhibition. Here, we show that the core 20S proteasome, which degrades disordered and misfolded proteins independent of ubiquitin, controls CARD8 activation. In unstressed cells, the 20S proteasome degrades just the NT disordered region, leaving behind the folded ZU5, UPA, and CARD domains to act as an inhibitor of inflammasome assembly. In VbP-stressed cells, the 20S proteasome degrades the entire NT fragment, perhaps due to ZU5 domain unfolding, freeing the CT fragment from autoinhibition. Overall, this work shows that CARD8 NTs susceptibility to 20S proteasome-mediated degradation controls inflammasome activation.

immunology↗

bioRxiv2019

Human caspase-1 autoproteolysis is required for ASC-dependent and -independent inflammasome activation

Pathogen-related signals induce a number of cytosolic pattern-recognition receptors (PRRs) to form canonical inflammasomes, which activate pro-caspase-1 and trigger pyroptotic cell death. All well-studied PRRs oligomerize with the pro-caspase-1-adapter protein ASC to generate a single large structure in the cytosol, which induces the autoproteolysis and activation of the pro-caspase-1 zymogen. However, several PRRs can also directly interact with pro-caspase-1 without ASC, forming much smaller \"ASC-independent\" inflammasomes. It is currently thought that pro-caspase-1 autoproteolysis does not occur during, and is not required for, ASC-independent inflammasome activation. Here, we show that the related human PRRs NLRP1 and CARD8 exclusively form ASC-dependent and ASC-independent inflammasomes, respectively, identifying CARD8 as the first PRR that cannot form an ASC-containing signaling platform. Despite their different structures, we discovered that both the NLRP1 and CARD8 inflammasomes require pro-caspase-1 autoproteolysis between the small and large catalytic subunits to induce pyroptosis. Thus, pro-caspase-1 self-cleavage is an obligate regulatory step in the activation of human canonical inflammasomes.

immunology↗