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, 26 (2), 229-244

Caspase Selective Reagents for Diagnosing Apoptotic Mechanisms

Affiliations

Caspase Selective Reagents for Diagnosing Apoptotic Mechanisms

Marcin Poreba et al. Cell Death Differ.

Abstract

Apical caspases initiate and effector caspases execute apoptosis. Reagents that can distinguish between caspases, particularly apical caspases-8, 9, and 10 are scarce and generally nonspecific. Based upon a previously described large-scale screen of peptide-based caspase substrates termed HyCoSuL, we sought to develop reagents to distinguish between apical caspases in order to reveal their function in apoptotic cell death paradigms. To this end, we selected tetrapeptide-based sequences that deliver optimal substrate selectivity and converted them to inhibitors equipped with a detectable tag (activity-based probes-ABPs). We demonstrate a strong relationship between substrate kinetics and ABP kinetics. To evaluate the utility of selective substrates and ABPs, we examined distinct apoptosis pathways in Jurkat T lymphocyte and MDA-MB-231 breast cancer lines triggered to undergo cell death via extrinsic or intrinsic apoptosis. We report the first highly selective substrate appropriate for quantitation of caspase-8 activity during apoptosis. Converting substrates to ABPs promoted loss-of-activity and selectivity, thus we could not define a single ABP capable of detecting individual apical caspases in complex mixtures. To overcome this, we developed a panel strategy utilizing several caspase-selective ABPs to interrogate apoptosis, revealing the first chemistry-based approach to uncover the participation of caspase-8, but not caspase-9 or -10 in TRAIL-induced extrinsic apoptosis. We propose that using select panels of ABPs can provide information regarding caspase-8 apoptotic signaling more faithfully than can single, generally nonspecific reagents.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
ABPs used for caspase analysis. a Two initiation pathways triggered by separate events converge at a common point to execute apoptosis in mammals. The extrinsic apoptosis pathway is triggered by extracellular ligation of death receptors, resulting in receptor clustering, adapter recruitment, and activation of the apical protease caspase-8 and possibly its close paralog caspase-10 (active enzymes are shown in orange/pink ovals—reviewed in ref. [57]). The intrinsic pathway responds primarily to cellular stress and some neurodevelopmental cues, with mitochondria acting as important integrators. Pro-apoptotic and anti-apoptotic members of the Bcl2 family (Bax and Bcl2 serve as examples) regulate the lethal stress–response threshold (reviewed in [58]). Activation of the apical caspases-8, -9, and -10 occurs when they are driven into an active conformation by their activation platforms—the DISC (death-inducing signaling complex) or the Apaf-1 apoptosome [59, 60]. Both pathways activate the effector proteases, caspases-3 and -7. Relevant to the extrinsic pathway, signals from several receptors, in response to cognate ligands, converge to activate caspases-8 and -10 (reviewed in ref. [61]). CrmA is a viral inhibitor of caspase-8 and XBIR2 is the second BIR domain of XIAP (X-linked inhibitor of apoptosis protein), highly selective for of caspases-3 and -7 [–64]. b General structure of amino acids selected for synthesis of ABPs targeting the apoptotic apical caspases-8, -9, and -10. In each case, the P1 position was fixed as Asp
Fig. 2
Fig. 2
Distinct mechanisms of caspase-8 inhibition by AOMK ABPs. Progress curves show that inhibition is irreversible (a), reversible (b), or bimodal (c). In b,c, the switch between reversible and bimodal inhibition occurs following exchange of one amino acid (His → Thr) in the P2 position of the AOMK-activity-based probe
Fig. 3
Fig. 3
ABP selectivity toward five human apoptotic caspases. a Kinetic parameters (kobs/I and KI) of ABPs for caspases displayed as heatmaps. All ABPs were irreversible for caspase-2, -3, -6, -7, and -10. Caspase-8 and -9 displayed either irreversible or reversible inhibition, depending on the ABP tested. b ABP selectivity tested by labeling of recombinant caspases. Probes (100 nM) were incubated with active site-titrated caspases (50 nM) for 30 min, followed by SDS-PAGE, membrane transfer, and detection with fluorescent streptavidin. The lane on the left in each blot is a controlled broad-spectrum caspase probe (LEHD; biotin-ahx-LEHD-AOMK) used to ensure equivalent exposure between each blot
Fig. 4
Fig. 4
Kinetic parameters of ACC substrates and AOMK-activity-based probes. a General structure of the substrate-leaving group and ABP electrophile. Correlation between kobs/I and kcat/KM for effector caspases-3 and -7 (b) and apical caspases-8, -9 and -10 (c). Red points show values obtained in the presence of broad-spectrum probes with peptide sequences DEVD, IETD, LEHD, or LQnD (where n is norleucine); green points indicate values obtained in the presence of a MP-3.01, which are selective for effector caspases-3 and -7. Pearson correlation coefficients (ρ) were calculated from linear kinetic parameters, and data were plotted as log/log to demonstrate the spread of values. In the case of caspase-8 (d) and caspase-9 (e), His at P2 differentially influences the catalytic efficiency for a substrate versus an ABP
Fig. 5
Fig. 5
Use of small-molecule substrates and ABPs reveals caspase activation in TRAIL-stimulated Jurkat T cells. a The general scheme of caspase-8 activation upon TRAIL stimulation. Procaspase-8 (p55) is activated by dimerization at the DISC and undergoes auto-processing within the catalytic domain to generate p43/p12 fragments, which are further cleaved by the release of the DEDD recruitment domain into p18/p12 subunits [65] Jurkat T cells primarily express caspase-8 isoforms a and b, which differ by a short indel in the DED domain region, hence, the doublets seen in the 55 kDa and 43 kDa regions [66, 67] (of b,c) . b Western blots with specific antisera demonstrating the kinetics of caspase and PARP processing after TRAIL stimulation. The band around 20 kDa in the caspase-8 blot may be the p18 (large) subunit or a nonspecific band. c Cells were preincubated with indicated ABPs for 2 h and stimulated for 8 h with TRAIL. Cell lysates were then prepared and treated with streptavidin resin, the resin was washed, and the captured proteins were then eluted in boiling SDS buffer and run on SDS-PAGE for immunoblotting with caspase-8 antiserum. d Caspase activity in Jurkat cells at various time points after apoptosis induction and using indicated broad-spectrum and selective ACC substrates. e Caspase activity measured in Jurkat T cells at 8 h after apoptosis induction in the presence or absence of caspase inhibitors XBIR2 (1 µM; for caspase-3/-7) and CrmA (1 µM; for caspase-8). RFU relative fluorescence units. Western blots from b,c were performed twice, and the kinetic experiments in d,e were performed three times. Average values (with SD) are presented
Fig. 6
Fig. 6
Apoptosis induction in the Jurkat T cells as analyzed using a diverse set of ABPs. a Probes were delivered to cells by reversible permeabilization, followed by apoptosis induction by TRAIL or etoposide. After 24 h, cell survival was measured relative to untreated controls using MTS assay. b Color-coded index of cell survival outcomes in the presence of a set diverse ABPs targeting caspases-8,- 9, and -10 tested in TRAIL- or etoposide-stimulated Jurkat T cells. c Correlation of probe inhibition potency (kobs/I) and cell survival (%, ratio to control). Red points show values obtained in the presence of broad-spectrum probes with peptide sequences DEVD, IETD, LEHD, or LQnD (where n is norleucine); green points indicate values obtained in the presence of a probe 3.01, which is selective for effector caspases-3 and -7. Each analysis contained the same probe panel set. Regression coefficients (R2) were calculated from semi-log plots, with dotted lines indicating 95% confidence interval

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