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, 19 (3), 340-52

An Optimized Activity-Based Probe for the Study of caspase-6 Activation

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An Optimized Activity-Based Probe for the Study of caspase-6 Activation

Laura E Edgington et al. Chem Biol.

Abstract

Although significant efforts have been made to understand the mechanisms of caspase activation during apoptosis, many questions remain regarding how and when executioner caspases get activated. We describe the design and synthesis of an activity-based probe that labels caspase-3/-6/-7, allowing direct monitoring of all executioner caspases simultaneously. This probe has enhanced in vivo properties and reduced cross-reactivity compared to our previously reported probe, AB50. Using this probe, we find that caspase-6 undergoes a conformational change and can bind substrates even in the absence of cleavage of the proenzyme. We also demonstrate that caspase-6 activation does not require active caspase-3/-7, suggesting that it may autoactivate or be cleaved by other proteases. Together, our results suggest that caspase-6 activation proceeds through a unique mechanism that may be important for its diverse biological functions.

Figures

Figure 1
Figure 1. Direct comparison of LE22 and AB50 labeling in intact apoptotic cells
(A) Structure of the previous generation caspase probe, AB50, and the optimal probe from the new series, LE22. (B) Fluorescent SDS-PAGE comparing LE22 and AB50 labeling of apoptotic Human colorectal cancer COLO205 cells. Cells were induced to undergo apoptosis using anti-DR5 and intact cells were labeled with each probe at the indicated concentrations. Where indicated, the caspase-3 and -7 specific inhibitor, AB13 was added prior to labeling with the probes. Total protein lysates were analyzed by SDS-PAGE followed by scanning for Cy5 fluorescence using a flatbed laser scanner. An autofluorescent protein is indicated with a #. Arrows highlight caspase-6 species that are labeled by LE22 but not by AB50. The bottom panel shows enhanced contrast of the boxed region for easier viewing of the high molecular weight bands. (C) Immunoprecipitations using the indicated cleaved caspase antibodies to confirm the identity of labeled proteins in the 5 μM samples in (B). Faint bands in the pulldowns that are difficult to see are noted with an asterisk, and the bottom panel shows enhanced contrast for easier viewing. (See also Figure S1 and S2)
Figure 2
Figure 2. Cross-reactivity of probes towards other cysteine proteases
Fluorescence SDS-PAGE showing labeling in RAW cells. Intact RAW cell mouse macrophages were labeled with the indicated concentrations of LE22 or AB50. Total protein lysates were harvested and resolved by SDS-PAGE followed by scanning on a flatbed laser scanner for Cy5 fluorescence. An autofluorescent background protein in the no-probe control tumors is marked by an `#' (B) Images of kidney and liver fluorescence after probe injection and corresponding SDS-PAGE analysis. LE22 or AB50 was injected by tail vein into normal BALB/c mice. Kidney and liver were harvested after four hours of circulation and probe fluorescence in the tissues was imaged using a CCD camera. Note differences in scale. Tissues were then homogenized and analyzed by SDS-PAGE followed by fluorescence scanning. Labeled proteases are indicated, and an autofluorescent background protein in the no-probe control tumors is marked by an `#'.
Figure 3
Figure 3. Comparison of LE22 and AB50 in dexamethasone-induced thymocyte apoptosis
(A) Fluorescent images of apoptotic and control thymi and corresponding SDS-PAGE analysis. BALB/c mice were injected with dexamethasone or vehicle for 12 hours followed by injection of either LE22 or AB50 by tail vein. A subset of the dex-treated mice were pretreated with a caspase inhibitor, AB46, prior to probe injection. After four hours, thymi were harvested and imaged ex vivo for probe accumulation. Thymus proteins were then resolved by SDS-PAGE and scanned for Cy5 fluoresence using a flatbed scanner. An autofluorescent background protein in the no-probe control tumors is marked by an `#' (B) Immunoprecipitations of samples shown in (A). The first sample in each dex-treated lane for LE22 and AB50 was immunoprecipitated with the indicated antibody and analyzed by SDS-PAGE.
Figure 4
Figure 4. Comparison of LE22 and AB50 in COLO205 tumors treated with anti-DR5 anti-body
(A) Noninvasive imaging of apoptosis in tumor bearing mice. Nude mice bearing COLO205 tumors were treated with anti-DR5 antibody or vehicle for 12 hours followed by intravenous injection of either LE22 or AB50. After one hour of circulation, live mice were imaged using an IVIS 100. Red arrows indicate the location of the tumors. (B) Ex vivo imaging of tumors shown in (A) and corresponding biochemical analysis. Tumors were imaged ex vivo using epifluorescence on a Fluorescence Mediated Tomography (FMT) machine and then homogenized. Total protein lysates were analyzed by SDS-PAGE and scanned for Cy5 fluorescence. An autofluorescent background protein in the no-probe control tumors is marked by an `#' (C) Fluorescent SDS-PAGE analysis of tumors labeled ex vivo. Apoptosis was induced in a COLO205 tumor as in (A) however tumor was removed prior to probe administration and tumor lysates were labeled with LE22 or AB50 at the indicated concentration. Where noted the AB13 inhibitor was added to block caspase-3 and -7 labeling. Vehicle-treated control tumors were also included in the analysis. (D) Immunoprecipitations of tumor lysates labeled with 1 μM LE22 with the indicated antibodies to confirm the identity of labeled caspases. Weak bands in the immunoprecipitations are indicated by an asterisk.
Figure 5
Figure 5. Direct comparison of LE22 and AB50 labeling in apoptotic lysates
(A) Fluorescent SDS-PAGE of labeled COLO205 lysates. Apoptosis was induced in COLO205 cells with an anti-DR5 antibody and cells were harvested. Lysates were then labeled with LE22 or AB50 at the indicated concentrations. AB13 was added to samples as indicated to selectively block caspase-3 and -7 labeling. Protein was resolved by SDS-PAGE and scanned for Cy5 fluorescence. (B) Schematic of the proposed forms of caspase-6 based on known cleavage sites. The pro-domain is indicated in red, the large subunit is green, and the small subunit is blue. The intersubunit linker is depicted by a curved line, and the active site cysteine (the site at which the probe binds) is marked by a yellow star. Predicted sizes are listed at the left. (C) Immunoprecipitations of lysates labeled with 1 μM LE22. Five times as much protein was used as in (A) to ensure adequate pulldown of less abundantly labeled proteins. Faintly labeled species that are difficult to see in the pulldowns are marked by an asterisk. Ctrl indicates an immunoprecipitation that was performed in the absence of antibody to account for nonspecific sticking of proteins to the Protein A/G beads. (D) Immunoprecipations of LE22 labeled lysates with antibodies for the cleaved and full-length caspase-6 species in their native, folded state (left lanes) or denatured by boiling in SDS (right lanes). (E) Full-length capase-6 labeling by LE22 in intact apoptotic cells was blocked by pretreatment with AB46 and biotinylated LE22 (LE33), but not by pretreatment with AB13. An autofluorescent band is denoted with a #.
Figure 6
Figure 6. Caspase-6 undergoes a conformational change upon activation
(A and B) Recombinant caspase-6 as well as wild type and the active-site mutant (C/A) of caspase-3 were incubated with an excess of LE22 (4 μM) for 3 hours and analyzed by SDS-PAGE (A) or on pore limit native PAGE (B). D193A mutants of caspase-3 were cleaved with 1% caspase-3 overnight where indicated (+C3). The gel was first scanned on a fluorescent scanner to detect the probe signal and then stained with coomassie and re-scanned to detect total protein. The two figures were then overlaid to visualize the species of caspase-6 that were active (i.e. bound the probe; red) relative to species that were not catalytically active (i.e. only stained blue; blue). (C and D) Hypotonic lysates from 293T cells were activated by the addition of cytochrome c and dATP. Samples were taken before, after 30 minutes and after 60 minutes of cytochrome c addition, labeled with LE22 for 30 minutes at 37°C and analyzed by Western blot after separating the proteins either by SDS-PAGE (upper panels) or by pore limit native PAGE (lower panels). Blots were probed for total and active caspase-3 (C) or caspase-6 (D). The signal obtained for the caspase blots is displayed in green, signal from LE22 in red. Full-length and cleaved caspase species are indicated by arrows. Notice that on the pore limit gel, full length and cleaved caspase-3 run at the same molecular weight. (See also Figure S3, S4 and ST1)
Figure 7
Figure 7. Monitoring maturation of executioner caspases during apoptosis
(A) COLO205 cells were stimulated with anti-DR5 to initiate apoptosis for the indicated period of time. During the last 30 minutes of treatment, intact cells were labeled with 1 μM LE22 or AB50. Cells were then lysed and subject to SDS-PAGE analysis. The bottom panel shows enhanced contrast of the boxed region for easier viewing of the high molecular weight proteins. (B) Cells were treated in parallel to those in (A) however, labeling with 1μM LE22 was performed post-lysis. In the right panel, 10 μM AB13 was added prior to LE22 to allow for specific labeling of caspase-6. (C) Cells were treated as above, except AB13 was added at the time of anti-DR5 stimulation to block caspase-3/-7 activity throughout the course of the experiment. The bottom panel shows enhanced contrast of the boxed region for easier viewing of the high molecular weight bands. (See also Figure S5)

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