Fibrils colocalize caspase-3 with procaspase-3 to foster maturation

Abstract

Most proteases are expressed as inactive precursors, or zymogens, that become activated by limited proteolysis. We previously identified a small molecule, termed 1541, that dramatically promotes the maturation of the zymogen, procaspase-3, to its mature form, caspase-3. Surprisingly, compound 1541 self-assembles into nanofibrils, and localization of procaspase-3 to the fibrils promotes activation. Here, we interrogate the biochemical mechanism of procaspase-3 activation on 1541 fibrils in addition to proteogenic amyloid-β(1-40) fibrils. In contrast to previous reports, we find no evidence that procaspase-3 alone is capable of self-activation, consistent with its fate-determining role in executing apoptosis. In fact, mature caspase-3 is >10(7)-fold more active than procaspase-3, making this proenzyme a remarkably inactive zymogen. However, we also show that fibril-induced colocalization of trace amounts of caspase-3 or other initiator proteases with procaspase-3 dramatically stimulates maturation of the proenzyme in vitro. Thus, similar to known cellular signaling complexes, these synthetic or natural fibrils can serve as platforms to concentrate procaspase-3 for trans-activation by upstream proteases.

FIGURE 1.

General model for procaspase-3 activation. A, procaspase-3 variants used to explore potential activation mechanisms. Wild-type procaspase-3 is cleaved at Asp-9, Asp-28, and Asp-175. Cleavage in the intersubunit linker (Asp-175, red arrow) is essential to generate mature caspase-3. Cleavages in the prodomain (Asp-9 and Asp-28, black arrows) do not impact the in vitro activity of caspase-3. Note, caspases are expressed as constitutive dimers but illustrated here as monomers for simplicity. B, structure of compound 1541, which assembles into nanofibrils to activate procaspase-3. C, procaspase-3 activation may proceed through an initiation event that forms the first mature caspase-3 molecule, which can rapidly feedback to process additional procaspase-3 molecules in a propagation phase.

FIGURE 2.

Mature caspase-3 drives activation of procaspase-3 in the presence of 1541. A, activation was monitored for 100 nm procaspase-3 alone (dark circles), 100 nm procaspase-3 with granzyme B (open circles), and 100 nm procaspase-3 with 25 μm 1541 (purple circles). The tetrapeptide substrate, Ac-DEVD-afc, was added at the indicated time points, and initial rates were plotted as a function of time. B, activities were monitored in the presence of 1 nm caspase-3, procaspase-3 (dark squares), procaspase-3 with granzyme B (open squares), and procaspase-3 with 25 μm 1541 (green squares). C, enzyme activity was measured in the presence of 1 nm Ac-DEVD-cmk, procaspase-3 (dark diamonds), procaspase-3 with granzyme B (open diamonds), and procaspase-3 with 25 μm 1541 (blue diamonds).

FIGURE 3.

Irreversible inhibitor of mature caspase-3. A, structure of Ac-DEVD-cmk. B, 5 or 500 μm Ac-DEVD-cmk was added to 5 μm caspase-3 or 5 μm wild-type procaspase-3. After a 24-h incubation at 37 °C, covalent modification was evaluated by mass spectrometry.

FIGURE 4.

Effective change in the catalytic efficiency of upstream proteases due to 1541 fibrils. A, dilution series of caspase-3 was added to the truncated, inactive procaspase-3 (29-277/C163A) in the absence (DMSO) or presence of 10 μm 1541. At 60 min, samples were quenched with LDS loading buffer, analyzed by SDS-PAGE, silver-stained, and band intensities quantified. B, processing of the inactive procaspase-3 (29–277/C163A) by a dilution series of mature caspase-8 with and without 10 μm 1541 was assessed at 20 min. C, processing of the inactive procaspase-3 by a dilution series of granzyme B with and without 10 μm 1541 was evaluated at 90 min. Replicate gels are not shown for clarity. The asterisks indicate roughly 50% cleavage of procaspase-3.

FIGURE 5.

Activities of caspase-3, caspase-8, and granzyme B against tetrapeptide substrates are similar with or without 1541. A, activity of caspase-3 (5 nm) against Ac-DEVD-afc was measured in the presence and absence of 10 μm 1541. B, activity of capsase-8 (20 nm) against Ac-IETD-afc was monitored with and without 10 μm 1541. Notably, inhibition of caspase-3 and caspase-8 was observed at higher concentrations of 1541 (>30 μm); however, compound concentration remained below this value to focus on activation alone. C, activity of granzyme B against Ac-IETD-afc was measured with and without 25 μm 1541.

FIGURE 6.

Rate of procaspase-3 cleavage by an active protease depends on its catalytic efficiency and the extent of binding to fibrils. A, 0.001 mg/ml (upper panel) and 0.01 mg/ml (lower panel) thermolysin were added to 1 μm procaspase-3 (29-277/C163A) in the presence of 50 μm 1541 or DMSO alone. Samples were quenched at the indicated time points and analyzed by SDS-PAGE. B, 10 μm 1541 was added to a dilution series of procaspase-3, caspase-3, caspase-8, TEV protease, thermolysin, and granzyme B, followed by centrifugation at 16,100 × g for 15 min. The pellet was analyzed by SDS-PAGE followed by Coomassie staining to determine the amount of enzyme that bound to 1541 nanofibrils. C, 2 nm caspase-3, caspase-8, or granzyme B was added to 200 nm truncated, dead procaspase-3 (29-277/C163A) in the presence of 10 μm 1541 or DMSO. Samples were collected at the indicated time points and quenched with LDS loading buffer. After analysis by SDS-PAGE and silver stain, percent cleavage of the procaspase was determined by quantifying band intensities. D, TEV cleavage site was engineered into the inactive procaspase-3, rendering it susceptible to proteolysis and activation by only TEV protease. 200 nm of the inactive TEV-cleavable procaspase-3 (D175ENLYFQ/C163A) was incubated with 10 μm 1541 or DMSO alone in the presence of 20 or 200 nm TEV protease. Processing was monitored as described above. Replicate gels are not shown for clarity.

FIGURE 7.

Two possible models for initiation of procaspase-3 activation. A, initial event derives from a procaspase-3 molecule either cutting itself in cis (intramolecular) or cutting another procaspase-3 molecule in trans (intermolecular) upon interaction with the fibrils. B, initial event involves a trace amount of active caspase-3 that cleaves procaspase-3 upon colocalization to the nanofibrils. In either model, propagation is greatly accelerated by the accumulation of active caspase-3. Note that procaspase-3 molecules are only illustrated on one face of 1541 nanofibrils for simplicity; however, previous studies show that they coat the full surface (23).

FIGURE 8.

Enhanced susceptibility of procaspase-3 to processing by mature caspase-3 in the presence of 1541 fibrils. A, activation as a function of time for 250 nm wild-type procaspase-3 in the absence (−) or presence (+) of 10 μm 1541. Procaspase-3 is processed at three sites (Asp-9, Asp-28, and Asp-175), which leads to multiple bands by SDS-PAGE. The final cleavage products are residues 29–175 (large subunit) and 176–277 (small subunit). B, uncleavable procaspase-3 (D9A/D28A/D175A, 250 nm) was incubated with a catalytically inactive procaspase-3 (C163A, 250 nm) with (+) or without (−) 10 μm 1541. C, higher concentration of uncleavable procaspase-3 (5 μm) was added to 5 μm dead procaspase-3 in the presence of 5% Ac-DEVD-cmk (250 nm). Either 50 μm 1541 or DMSO alone was added to the samples. Processing was monitored by Western blot with an antibody specific for the C terminus of the large subunit of cleaved caspase-3 (Cell Signaling , catalog no. 9664). D, 250 nm Ac-DEVD-cmk, an irreversible inhibitor that selectively labels mature caspase-3 under the assay conditions, was added to 250 nm wild-type procaspase-3. 1541 or DMSO alone was subsequently added. Processing was monitored by silver stain analysis. E, self-activation of a higher concentration of wild-type procaspase-3 (5 μm) in the presence of 20% (1 μm) Ac-DEVD-cmk was monitored by Western blot. F, processing was examined for the inactive procaspase-3 (C163A, 250 nm) upon addition of mature caspase-3 (5 nm).

FIGURE 9.

Wild-type procaspase-3 activation. Dilution series of wild-type procaspase-3 (starting at 10 μm) was incubated at 37 °C for 4 h (A) or 24 h (B). The reactions were quenched with LDS loading buffer, analyzed by SDS-PAGE, and visualized by Coomassie staining. Note the delay or lag in processing of the 2.5 and 1.25 μm procaspase-3 samples between the 4- and 24-h time points.

FIGURE 10.

Multiple mature caspase-3 products identified by active site titration with Ac-DEVD-cmk. An active site titration with Ac-DEVD-cmk was performed against 5 μm wild-type procaspase-3, which was expressed for 20 min (A), 5 μm uncleavable procaspase-3 (D9A/D28A/D175A), which was expressed for 8 h (B), or 10 μm uncleavable procaspase-3, which was expressed for 2.5 h (C). The titration was plotted on a log scale. The asterisk indicates stoichiometric concentrations of Ac-DEVD-cmk relative to enzyme concentrations. The initial drop in activity for all three procaspase preparations occurred at sub-stoichiometric concentrations. Note the distinct levels of maximum activity for each proenzyme batch. This indicates that increased expression times lead to increased levels of contaminating active enzyme. Also note the second species detected in the uncleavable procaspase-3 expressed for 8 h. Based on the kinetic measurements described in the text, this second species weakly interacts with Ac-DEVD-cmk and is most likely an alternate cleavage product or a hemi-cleaved dimer.

FIGURE 11.

Wild-type procaspase-3 and uncleavable procaspase-3 (D9A/D28A/D175A) have very small levels of mature caspase-3 in the preparations. A, after ion-exchange chromatography on wild-type procaspase-3, fractions were collected. Aliquots of each fraction were analyzed by SDS-PAGE followed by Coomassie stain. B, similar procedure was performed on the uncleavable procaspase-3 (D9A/D28A/D175A). C, uncleavable procaspase-3 from batches expressed for 8 h (red) and 2.5 h (blue) was run on a gel filtration column. No differences or impurities were detected by gel filtration. D, 15 μl of 5 μm samples of the uncleavable procaspase-3 (D9A/D28A/D175A) from different batches expressed for different times were analyzed by SDS-PAGE followed by Western blot with an antibody that recognizes the C terminus of the cleaved caspase-3 large subunit (Cell Signaling, catalog no. 9664). Because a large excess of the full-length procaspase-3 is loaded relative to the trace caspase-3 contaminants in each sample, the antibody nonspecifically recognizes the full-length band as well.

FIGURE 12.

Amyloid-β(1–40) fibrils bind and enhance procaspase-3 activation. A, 50 μm amyloid-β(1–40) was incubated at 37 °C for 6 h to form fibrils. In 500 μl of buffer, 20 μm Aβ(1–40) fibrils and 0.1 mg/ml procaspase-3 or caspase-3 was added, incubated at room temperature for 5 min, and centrifuged at 20,800 × g for 20 min. The pellets were analyzed for the amounts of the respective enzymes. B, after a 4-h incubation at 37 °C, amyloid-β(1–40) or buffer alone was added to 250 nm wild-type procaspase-3. Processing was evaluated at the indicated time points. C, processing of 250 nm wild-type procaspase-3 with or without amyloid-β(1–40) fibrils was evaluated in the presence of 50 nm Ac-DEVD-cmk. D, similar preparation of amyloid-β(1–40) or buffer alone was incubated with 200 nm truncated inactive procaspase-3 (29-277/C163A) and 5 nm caspase-3. Processing was again evaluated at the indicated time points.