Caspase Activation Inhibits Proteasome Function during Apoptosis Xiao-Ming Sun, Michael Butterworth, Marion MacFarlane, Wolfgang Dubiel, Aaron Ciechanover, Gerald M Cohen  Molecular Cell  Volume 14, Issue 1, Pages 81-93 (April 2004) DOI: 10.1016/S1097-2765(04)00156-X

Figure 1 Mitochondrial Release of Smac and Omi Is Caspase Independent (A) Jurkat T cells were treated for the indicated times with etoposide (50 μM) or MG132 (5 μM) either alone or in the presence of Z-VAD.fmk (25 μM) (Z-VAD) or DEVD.fmk (50 μM) (DEVD), a more specific caspase-3 inhibitor. At the indicated times, apoptosis was assessed by measuring phosphatidylserine externalization using Annexin V labeling. Cells were treated with digitonin (0.01%), and cytosol (Cyto) and mitochondria (Mito) were separated by centrifugation. Proteins in these fractions were separated and analyzed by Western blotting for cytochrome c, Smac, and Omi. Cells were also analyzed by Western blotting for the processing of caspase-3. In the presence of DEVD.fmk, caspase-3 was only processed to its p20 form, as its catalytic activity was inhibited so preventing its autoprocessing to its p19 and p17 forms. (B) Freshly isolated and purified chronic lymphocytic leukemic (CLL) cells from patients were incubated for 0–8 hr either alone (Control), with MG132 (1 μM), or with Bis IX (0.4 μM) for 8 hr in the presence of Z-VAD (200 μM). At the indicated times, cells were analyzed for apoptosis or for the presence of proteins associated with the mitochondrial (Mito) or cytosol (Cyto) fractions as described above. Molecular Cell 2004 14, 81-93DOI: (10.1016/S1097-2765(04)00156-X)

Figure 2 Cleavage of Subunits in 19S RP but Not in 20S CP during Apoptosis Jurkat T cells were incubated for 6 hr either alone (lane 1) or with etoposide (50 μM) in the absence (lane 2) or in the presence of the caspase inhibitor Z-VAD.fmk (25 μM) (lane 3). Cells were then examined for the cleavage/loss of subunits of the 26S proteasome, including the 20S CP (20S core particle), 19S RP (19S regulator complex), and the 11S regulator complex. Molecular Cell 2004 14, 81-93DOI: (10.1016/S1097-2765(04)00156-X)

Figure 3 Cleavage of RP Subunits Occurs following Multiple Apoptotic Stimuli (A) Jurkat T cells were treated for the indicated times with apoptotic stimuli with different mechanisms of action––CD95 (50 ng ml−1), etoposide (50 μM), Bis IX (1 μM), or MG132 (5 μM)––either alone or in the presence of Z-VAD.fmk (25 μM, except 10 μM was used for CD95) (Z-VAD) or DEVD.fmk (50 μM) (DEVD). At the indicated times, cells were analyzed for apoptosis by Annexin V binding or the cleavage/loss of S6′, S1, and S5a. The cleavage/loss was caspase dependent, as it was prevented by either Z-VAD or DEVD. (B) Lysate from Jurkat cells was incubated in the presence of dATP (2 mM), MgCl2 (2 mM), and cytochrome c (0.5 mg ml−1) for the indicated times, either alone or in the presence of Z-VAD.fmk (25 μM) (Z-VAD) or DEVD.aldehyde (25 μM) (DEVD). Lysates were examined by Western blot analysis and revealed a similar time-dependent caspase-mediated cleavage/loss of S6′, S1, and S5a but not of other subunits, such as S2 and S6b. Molecular Cell 2004 14, 81-93DOI: (10.1016/S1097-2765(04)00156-X)

Figure 4 Caspase-3 Cleaves S6′ at Asp 27 and S1 at Asp 857 (A) The subunit proteins S6′, S1, and S5a were generated by in vitro transcription translation (TNT) in the presence of [35S]-methionine and subsequently incubated for 90 min with active recombinant caspase-3 (50–200 nM), caspase-7 (50–200 nM), and caspase-8 (50–200 nM). The asterisk indicates a nonspecific product. (B) Asp 27 and Asp 49 in S6′ were mutated using site-directed mutagenesis. Following sequence verification, these mutants were used as templates for the TNT reaction and their susceptibility to cleavage by caspase-3 (200 nM) determined as above. (C) Asp 857 of S1 was mutated using the Quik-Change site-directed mutagenesis kit. [35S]-labeled wild-type and Asp/Ala mutant of S1 proteins were then expressed and incubated for 90 min either alone (−) or in the presence of caspase-3 (200 nM) (+). The products in (A)–(C) were resolved by SDS-PAGE autoradiography. The larger and smaller [35S]-methionine-labeled S1 products in (A) were separated on 7% and 14% agarose gels, respectively. Molecular Cell 2004 14, 81-93DOI: (10.1016/S1097-2765(04)00156-X)

Figure 5 Accumulation of Ubiquitinated Proteins at an Early Stage of Apoptosis (A) Jurkat cells were treated with etoposide (100 μM) for 6 hr. A pure (>95%) population of apoptotic cells was separated from nonapoptotic cells using Annexin V Microbead kit. Lysates from control (•-•) and apoptotic (■-■) cells were incubated with [35S]-pulse-labeled proteins, from control Jurkat cells, in the presence of ATP and the release of acid-soluble radioactivity monitored as an indicator of the cells ability to degrade short-lived proteins. Control (○-○) and apoptotic (□-□) lysates were also incubated in the presence of MG132 (50 μM). BSA (50 μg per reaction) in place of lysate was used as a negative control either alone (▴-▴) or in the presence of MG132 (Δ-Δ). (B) Jurkat cells were treated with etoposide (50 μM) or CD95 (50 ng ml−1) for the indicated times and the amount of ubiquitinated proteins assessed by Western blotting and quantified by densitometry. The fold increase of ubiquitinated proteins is shown. At the indicated times, apoptosis was measured by determining the percentage of cells with Annexin V binding as described in the Experimental Procedures. Caspase-3 and -7-like enzymic activities were assessed by measuring DEVDase activity. (C) In similar experiments to those described in (B), the amount of ubiquitinated proteins present in MCF-7 cells exposed for the indicated times to TRAIL (500 ng ml−1) was determined by Western blotting using a ubiquitin antibody. (D) Lysates were prepared from Jurkat cells exposed, as in (B), to either etoposide (50 μM) or CD95 (50 ng ml−1) for the indicated times. The lysates (1 mg ml−1) were then incubated with biotinylated ubiquitin for 15 min in the presence of MG132 (50 μM) to inhibit the proteasome. The ability of the lysates to form de novo biotinylated-ubiquitin conjugates (Biotin-Ub) was then assessed as described in the Experimental Procedures. The numbers at the bottom of the figure represent the fold increase in de novo ubiquitination. Molecular Cell 2004 14, 81-93DOI: (10.1016/S1097-2765(04)00156-X)

Figure 6 Impaired Degradation of Smac and ODC in Apoptotic Lysates (A) MCF-7 and MCF-7 (casp3) cells were treated with TRAIL (1 μg ml−1) for the indicated times, and cytosolic fractions were examined by Western blot analysis for cleavage of S1, S2 (using Abcam antibody), S5a, S6′, and S6b. In these experiments an Ab to S5a, which detects a cleavage product(s), was used. (B) MCF-7 and MCF-7 (casp3) cells were treated with TRAIL for the indicated times either in the absence or presence of MG132 (1 μM) as indicated. Cytosolic fractions were then examined by Western blot analysis for cytochrome c, Smac, or Omi. The comparative levels of Smac and Omi released were examined by densitometric volume analysis. (C) Jurkat cells were exposed to etoposide for 6 or 16 hr, apoptotic cells purified using Annexin V Microbead kit, and lysates prepared as described in the Experimental Procedures. Control or apoptotic lysates (10 mg protein ml−1) were incubated with Smac (20 nM) at 37°C for the indicated times in the absence or presence of MG132 (50 μM). Smac was detected by Western blot analysis using an anti-penta-His antibody (Qiagen). The levels of Smac were examined by densitometric volume analysis. (D) Recombinant antizyme was produced in bacteria and [35S]-ornithine decarboxylase (ODC) was produced in a TNT system. ODC and antizyme were coincubated with either control (•-•) or apoptotic lysates (○-○) in the presence of ATP (2 mM). Control lysates were also incubated with ODC in the presence of ATP but no antizyme (■-■) or with ODC and antizyme in the presence of MG132 (50 μM) (▴-▴). Apoptotic lysates were also incubated with ODC in the presence of ATP but no antizyme (□-□) or with ODC and antizyme in the presence of MG132 (50 μM) (Δ-Δ). The apoptotic lysates were prepared from purified apoptotic Jurkat cells, prepared from cells exposed to etoposide for 6 hr, as described in Experimental Procedures. The percentage of ODC degradation was determined by measuring acid-soluble radioactivity as described in the Experimental Procedures. Molecular Cell 2004 14, 81-93DOI: (10.1016/S1097-2765(04)00156-X)

Figure 7 Loss of Functional Activity of Purified 26S Proteasome following Caspase Cleavage (A) Purified 26S proteasome (200 nM) was incubated for 4 hr in the presence or absence of active recombinant caspase-3 (200 nM) as indicated. Cleavage of the various proteasomal subunits was assessed by Western blotting. (B) After incubation for 4 hr with caspase-3, [35S]-methionine-labeled ornithine decarboxylase (ODC) degradation in the presence or absence of antizyme was determined by measuring acid-soluble radioactivity as described in the Experimental Procedures. ODC and antizyme were coincubated with either control (•-•) or caspase-3-treated (■-■) 26S purified proteasomes in the presence of ATP (1 mM). Control 26S proteasomes (○-○) and caspase-3-treated 26S proteasomes (□-□) were also incubated with ODC and antizyme in the presence of MG132 (50 μM). Control 26S proteasomes were incubated with ODC in the presence of ATP but no antizyme (♦-♦). Z-VAD.fmk (50 μM) was included in some studies to inhibit any residual active caspase-3 and so prevent any caspase-mediated nonspecific degradation of ODC or antizyme. Z-VAD.fmk alone did not affect antizyme-enhanced ODC degradation. Molecular Cell 2004 14, 81-93DOI: (10.1016/S1097-2765(04)00156-X)