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Comparative Study
, 49 (38), 8307-15

Activation and Specificity of Human caspase-10

Comparative Study

Activation and Specificity of Human caspase-10

Katherine Wachmann et al. Biochemistry.


Two apical caspases, caspase-8 and -10, are involved in the extrinsic death receptor pathway in humans, but it is mainly caspase-8 in its apoptotic and nonapoptotic functions that has been an intense research focus. In this study we concentrate on caspase-10, its mechanism of activation, and the role of the intersubunit cleavage. Our data obtained through in vitro dimerization assays strongly suggest that caspase-10 follows the proximity-induced dimerization model for apical caspases. Furthermore, we compare the specificity and activity of the wild-type protease with a mutant incapable of autoprocessing by using positional scanning substrate analysis and cleavage of natural protein substrates. These experiments reveal a striking difference between the wild type and the mutant, leading us to hypothesize that the single chain enzyme has restricted activity on most proteins but high activity on the proapoptotic protein Bid, potentially supporting a prodeath role for both cleaved and uncleaved caspase-10.


(A) SDS-PAGE of caspase-10 purification. Proteins were expressed in E.coli, purified by affinity chromatography and visualized on an SDS-PAGE gel. The wildtype enzyme is autocleaved into large (LS) and small subunits (SS) during expression in E. coli. The cleavage site mutant D297A remains as a single chain of 35 kDa. (B) Structural organization and sequence alignment of caspase-8 and caspase-10 from various organisms. Numbering is according to the caspase-1 numbering convention. Putative conserved cleavage sites between large and small subunit are highlighted. Caspase-10 has been deleted in the rodent lineage. (C) Model of procaspase-10 showing the potential cleavage sites between the large and small subunit. Procaspase-10 is autocatalytically processed into large and small subunits. The model shows the two conserved potential cleavage sites at D297 (magenta) and D319 (cyan). In this model D297 is presented as an extended loop, whereas D319 seems to be closer to the core of the monomer (modeled on caspase-8 zymogen PDB: 2K7Z).
(A) Titration with dimerizer AP20187 induces activity of the wildtype but not the cleavage site mutant. Fusion proteins [30 nM] were incubated with a dilution series of the dimerizer AP20187 in the absence of sodium citrate for 30 min at room temperature prior to adding Ac-IETD-AFC [100μM]. Activity in 1.0 M sodium citrate was used as a positive control (right). (B) and (C) The compound AP20187 dimerizes Fv-caspase-10. The enzyme was incubated with dimerizer for 30 min at 25°C before performing size-exclusion chromatography. The fusion proteins run at their monomeric mass in the absence of AP20187 (full line). The Fv-caspase-10 (B) consists of a mixture of monomeric and dimeric species in the absence of AP20187. Upon addition of the compound all proteins run according to their dimeric mass (dotted line).
(A) Caspase-10 activation in sodium citrate. Caspase-10 [10nM] wildtype or cleavage site mutant was incubated with a dilution series of sodium citrate buffer for 30 min at 37°C before monitoring activity by hydrolysis of Ac-IETD-AFC [100 μM]. (B) and (C) Dimerization induced by AP20187 enhances overall activity of caspase-10. Protease [30nM] was incubated with dimerizer (open circles) and without dimerizer (closed circles) at an optimal ratio for 30 min at room temperature. Sodium citrate buffer was added and the mix was incubated another 30 min at room temperature before monitoring substrate hydrolysis of Ac-IETD-AFC [100μM]. The activity of the wildtype (B) was enhanced about 30% in the presence of dimerizer AP20187. The mutant (C) required less kosmotropic salt to gain activity indicated by the arrow in the figure. Experiments were repeated 3 times and data presented as mean +/− standard deviation.
Fig.4. Cleaved and non-cleavable caspase-10 reveal similar subsite preferences
Wildtype (cleaved) caspase-10 (black bars) and non-cleavable caspase-10 (white bars) were tested on a P1 Asp-fixed positional scanning substrate library in the presence of 1.0 M sodium citrate. The y-axis represents the rate of hydrolysis as a percentage of the maximum measured rate. The x-axis shows the tested amino acids in single letter code, with norleucine (O) substituting for methionine.
Fig.5. Cleavage site mutant shows restricted specificity on protein substrates
A constant amount of substrate was incubated with increasing amounts of enzyme and disappearance of the full length substrate was determined by quantifying the fluorescence signal of the secondary antibody with an Odyssey infrared scanner (LI-COR Biosciences). These values were then used to calculate kcat/KM as described in Methods. The tested substrates were catalytically inactive C/A pro-caspase-3 and pro-caspase-7 (c-3 C/A; c-7 C/A) as well as Bid and RIPK1. Note that there is already some cleaved RIPK1 present in the starting material, possibly processed by endogenous caspases in the cells wherein the protein was expressed.
Fig.6. Restricted substrate specificity of the cleavage site mutant
Comparison of cleavage site mutant to wildtype in respect to substrate preference (fractional activity of the cleavage site mutant with wildtype rates set as one). The cleavage sites (P4-P1') identified in the protein substrates are indicated above the bars.

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