Fluorochrome‐labeled inhibitors of caspase‐1 require membrane permeabilization to efficiently access caspase‐1 in macrophages

• DOI: 10.1002/eji.202350515
• Publication Date: 2024-02-16T04:39:40Z
• Authors: Sara J Thygesen; Sabrina S Burgener; Prerna Mudai; Mercedes Monteleone; Dave Boucher; Vitaliya Sagulenko; Kate Schroder; Katryn J Stacey
• Keywords: Caspase 8

Caspase-1 location in cells has been studied with fluorochrome-labeled inhibitors of caspase-1 (FLICA reagents). We report that FLICA reagents have limited cell-membrane permeability. This impacts experimental design as intact membranes, including knockout cells, are not appropriate controls for inflammasome-induced gasdermin D membrane pores. is an inflammatory initiator caspase responsible the maturation pro-inflammatory cytokines IL-1β and IL-18 pyroptotic cell death. Inhibitors important tools studying mechanism activation its role inflammation Here we caspases (FLICA) target very must be considered interpreting results; unstimulated or membranes activated by multi-protein complexes called inflammasomes response to pathogen- host-derived danger signals [1]. Inflammasome formation begins clustering protein following cellular stress microbial infection. In canonical inflammasome activation, sensor molecule typically recruits procaspase-1 help adaptor ASC (apoptosis-associated speck-like containing a recruitment domain). N-terminal pyrin domain interacts nucleated form filament [2], C-terminal both procaspase-1, mediates condensation filaments into tight speck structure [3]. Active cleaves activates (GSDMD) which initiates death [4]. The cleavage fragment GSDMD oligomerizes forms pores plasma inner diameter ∼215 Å [5]. Accumulation results release mature IL-18, ultimately lysis. first were carboxyfluorescein (FAM) derivatives inhibitors, such pan-caspase inhibitor benzyloxycarbonyl-valine-alanine-aspartic acid-fluoromethylketone (zVAD-FMK) developed enable detection activity living study apoptosis [6, 7]. FAM-VAD-FMK was reported enter bind irreversibly [6]. peptide moiety constructs conveys degree specificity while FMK leaving group facilitates covalent binding fluorescent active site [7]. Covalently bound reagent retained after fixation washing. FAM-YVAD-FMK 660-YVAD-FMK marketed cell-permeable caspase-1-specific reagents. Both use YVAD tetrapeptide sequence based on recognition O-methylated aspartic acid residue improve permeability; they differ only moiety. These used quantify assess within cell. Our suggest poorly permeable require efficiently caspase-1. Following FLICA-labeled co-localizes specks [8]. recreated typical experiment uses visualize NLRP3 inflammasome. LPS-primed WT bone marrow-derived macrophages (BMM) incubated prior treatment nigericin trigger (Fig. 1A). Cells fixed 30 min immunolabeled ASC. observed puncta colocalized 1A, short exposure). However, Gsdmd−/− BMM, do express activate [4], lacked staining at exposure cells. ASC-associated could seen BMM using longer exposure, although this gave much stronger saturated signal long problem restricted FAM-YVAD-FMK. At from but Casp1−/− 1B). detected completely white image. Considering quantification strength length imaging allows us estimate labeling approximately 30-fold than 660-YVAD-FMK, sevenfold To confirm presence caspase-1, assessed 2A). p33, [8], further cleaved inactivated product p20 present similar amount BMM. This, along Gsdmd-dependence readily signal, suggests Initial claims ability non-fluorochrome-containing live inhibit early work showing inhibition reproducible 9]. also tested three commonly nonfluorescent their zVAD-FMK inhibitor, VX-765 prodrug VRT-043198, caspase-4 [10] MCC950 directly NACHT [11]. each added nigericin. prevented cleavage, trapped p33 2B). latter quantified increase decrease compared alone sample. MCC950, showed inhibited processing p20. Thus FMK-containing may Evidently, sufficient permeability death-dependent apoptotic [12], 10 μM preincubation, able adequately penetrate prevent rapid activation. applicability should carefully when planning experiments already existing data. Negative experiments, YVAD-FMK cross-react other cysteine proteases [13]. Published protocols untreated negative [14, 15]. pore thus control due access cytosol. Indeed, any inflammasome-deficient where inhibited, inadequate normal For those shown Fig. 1, these two types would expected equal FLICA. low severely limits sensitivity, will likely analysis earliest sites While focused pores, consider possibility differential reagent, particularly reduced result genotype treatment. supported National Health Medical Research Council Australia (fellowship 2009075 KS grant 2003688 KJS) Australian grants (DP180103983 KJS DP190102285 KS). SSB fellowship #21C133 Novartis Stiftung für Medizinisch-Biologische Forschung. Open publishing facilitated University Queensland, part Wiley - Queensland agreement via Librarians. co-inventor patent applications licensed Inflazome Ltd., company headquartered Dublin, Ireland. developing drugs address unmet clinical needs disease. served Scientific Advisory Board 2016–2017 serves consultant Quench Bio, USA Novartis, Switzerland. peer review history article available https://publons.com/publon/10.1002/eji.202350515 data support findings corresponding author upon reasonable request. Please note: publisher content functionality supporting information supplied authors. Any queries (other missing content) directed article.