Fibrils Colocalize Caspase-3 with Procaspase-3 to Foster
Julie A. Zorn‡, Dennis W. Wolan‡1, Nicholas J. Agard‡2, and James A. Wells‡§3
San Francisco, California 94158
Background: Procaspase-3 is a critical protease in apoptosis.
Results: Procaspase-3 has less than 1/10,000,000 the activity of mature caspase-3 and does not detectably autoprocess. Small
molecule and proteogenic fibrils promote procaspase-3 maturation through induced proximity to an active protease.
Conclusion: Fibrils enhance procaspase-3 maturation in vitro through colocalization with upstream proteases.
Significance: These studies demonstrate the importance of scaffolding and colocalization with active proteases for pro-
caspase-3 processing and activation.
gens, that become activated by limited proteolysis. We previ-
ously identified a small molecule, termed 1541, that dramati-
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 bio-
chemical mechanism of procaspase-3 activation on 1541 fibrils
in addition to proteogenic amyloid-?(1–40) fibrils. In contrast
role in executing apoptosis. In fact, mature caspase-3 is >107-
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 stimu-
lates 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.
Proteases catalyze the irreversible post-translational modifi-
cation of amide bond hydrolysis. Thus, cellular mechanisms
exist to restrict spurious activation (1). For example, cellular
inhibitors can often limit the activity of a mature protease.
known as zymogens, which require an external signal to gener-
ate the active enzyme (2, 3). Although the biological conse-
quences of zymogen maturation are evident, important mech-
anistic details are not well understood (4).
Caspases are cysteine-class aspartyl-specific proteases that
are expressed as inactive precursors, known as procaspases (5).
To generate the mature enzyme, procaspases require cleavage
after specific aspartic acid residues to remove an N-terminal
prodomain and to form the large and small subunit, the latter
processing event being critical for activation (Fig. 1A) (6, 7). In
cade (8). Upstream initiator procaspases-8, -9, and -10 are
recruited to scaffolding complexes, such as the apoptosome or
the death-inducing signaling complex, to generate active
enzymes (9–13). Subsequently, the initiator caspases target
10, 14–16). Together, these caspases can cleave ?1000 down-
stream substrates that drive the apoptotic phenotype (17–19).
Significant efforts have been directed toward identifying
potential chemotherapeutic agents (20–22). Our laboratory
previously discovered a small molecule, termed 1541, that pro-
molecule spontaneously forms nanofibrils (4–5 nm thin and
mote activation (23). Natural amyloid-? (residues 1–40) fibrils
also interact with procaspase-3 to increase its activity in vitro.
Importantly, such fibrous ?-sheet structures have been cor-
neurodegenerative diseases (24–30). Previous studies have
even observed localization of active caspases to fibrillar struc-
ing, evidence for direct procaspase activation on fibrils is lim-
ited. Furthermore, the mechanism by which procaspase
viously described. Such characterization would offer a better
procaspase maturation and may have significant implications
in drug discovery of procaspase activators.
amyloid-? fibrils to induce procaspase-3 activation and gain
Grant R01 CA136779, Grant F32 CA119641-03 from NCI (to D. W. W.), and
Grant F32 AI077177 (to N. J. A.). This work was also supported by an
J. A. Z.), a Scleroderma Research Foundation Evnin-Wright Fellowship (to
J. A. Z.), and the Multiple Myeloma Translational Initiative (MMTI) at UCSF.
1Present address: Dept. of Molecular and Experimental Medicine, Scripps
Research Institute, La, Jolla, CA 92037.
2Present address: Dept. of Biocatalyst Characterization and Design, Codexis
Inc., 200 Penobscot Dr., Redwood City, CA 94063-4718.
3To whom correspondence should be addressed: Depts. of Pharmaceutical
nia, San Francisco, 1700 4th St., San Francisco, CA 94158. Tel.: 415-514-
4498; Fax: 415-514-4507; E-mail: firstname.lastname@example.org.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 40, pp. 33781–33795, September 28, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc.Published in the U.S.A.
SEPTEMBER28,2012•VOLUME287•NUMBER40 JOURNALOFBIOLOGICALCHEMISTRY 33781
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insight into the mechanism of zymogen maturation. Previous
results hinted at an initiation and propagation phase to pro-
of procaspase-3 maturation is due to a trace amount of active
caspase-3 or other active proteases. In contrast to previous
reports (20–22, 33–35), we reveal that procaspase-3 has no
detectable activity, and it does not autoprocess by either an
intermolecular or intramolecular mechanism. Propagation of
procaspase-3 activation is due to autocatalytic maturation of
the proenzyme, whereby mature caspase-3 molecules that are
erate this process by concentrating or colocalizing mature
caspase-3 with its procaspase-3 substrate.
Materials—The mature caspase-3 antibody (9664) was
purchased from Cell Signaling. Thermolysin and 1,1,1,3,3,3-
hexafluoro-2-propanol were purchased from Sigma. Amyloid-
?(1–40) was purchased from AnaSpec. Acetyl-Asp-Glu-Val-
was purchased from SM Biochemicals LLC. Isopropyl ?-D-
ketone (Ac-DEVD-cmk) were purchased from Calbiochem.
Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, acetyl-
IETD-afc), and 7-amino-4-trifluoromethylcoumarin were pur-
chased from Enzo Life Sciences. The pET23b and pET15b
erous gift from Dan Hostetter and Cheryl Tajon in the Craik
laboratory at the University of California, San Francisco.
Tobacco etch virus (TEV) protease was a generous gift from
Charlie Morgan, and procaspase-3 (D175ENLYFQ) that has a
TEV cleavage site inserted at the inter-subunit linker was a
of California, San Francisco. All additional reagents were pur-
chased from Sigma, unless otherwise noted.
Constructs—Full-length human procaspase-3 gene was
cloned into pET23b, as described previously (22, 36). Trun-
cated procaspase-8(217–479), lacking the death effector
domains, was cloned into pET15b. Truncated procaspase-3
(29–277), lacking the prodomain, was cloned into pET23b.
QuikChange mutagenesis (Stratagene) on the above constructs
was used to generate the catalytically inactive procaspase-3
(C163A), the catalytically inactive, truncated procaspase-3 (29-
277/C163A), the uncleavable procaspase-3 (D9A/D28A/D175A),
Expression/Purification—For expression, all plasmids were
transformed into the bacterial strain BL21 (DE3) pLysS. Wild-
type, full-length procaspase-3 was expressed and purified as
at 37 °C to an A600 nmof ?0.6, and subsequent overexpression
cell lysate was purified using a nickel-nitrilotriacetic acid affin-
ity column, followed by anion-exchange chromatography.
Purified procaspase-3 fractions were collected and stored at
formed with the pET23b containing full-length procaspase-3.
expressed at 30 °C overnight from the respective full-length
constructs. Caspase-8 was overexpressed at 12 °C over-
night from cells transformed with the pET15b containing
procaspase-8(217–479). All mature caspases were purified as
described for procaspase-3.
All other constructs followed the same expression and puri-
expression time modified as follows: catalytically inactive pro-
caspase-3 (C163A) for 15 h; catalytically inactive, truncated
procaspase-3 (29-277/C163A) for 5 h; catalytically dead, TEV-
cleavable procaspase-3 (D175ENLYFQ/C163A) for 5 h; and
uncleavable procaspase-3 (D9A/D28A/D175A) for 2–8 h
(exact conditions described in the text).
Synthesis of 1541—Synthesis, purification, and characteriza-
tion of 1541 were performed as described previously (22).
was agitated in a caspase activity buffer (50 mM HEPES, pH 7.4,
1541 or 2% DMSO alone for the indicated time intervals. Sub-
sequent assays with procaspase-3 also included a 15-min pre-
incubation with the irreversible inhibitor, Ac-DEVD-cmk, at
loading buffer and analyzed by SDS-PAGE. Protein bands were
visualized by either silver stain analysis or Western blot.
4The abbreviations used are: afc, amino-4-trifluoromethylcoumarin; A?(1–
40), amyloid-?(1–40); cmk, chloromethyl ketone; TEV, tobacco etch virus;
LDS, lithium dodecyl sulfate.
ants used to explore potential activation mechanisms. Wild-type pro-
unit 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
dimers but illustrated here as monomers for simplicity. B, structure of com-
pound 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.
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Procaspase-3 Trans-activation Assays (33, 34)—Uncleavable
procaspase-3 (D9A/D28A/D175A) or mature caspase-3 was
agitated at 37 °C with the catalytically inactive procaspase-3
(C163A) in a caspase activity buffer (50 mM HEPES, pH 7.4, 50
mM KCl, 0.1 mM EDTA, 10 mM DTT, and 0.1% CHAPS) in the
presence or absence of 1541. The irreversible inhibitor, Ac-
DEVD-cmk, was added under the designated conditions to
inhibit any cleaved caspase-3 impurity present in the uncleav-
able procaspase-3 expression. Samples were quenched with
LDS loading buffer and analyzed by SDS-PAGE. Protein bands
were visualized by either silver stain analysis or Western blot.
Procaspase-3 Self-activation Assays (22)—Wild-type pro-
caspase-3 was preincubated for 15 min with DMSO alone or 1
nM Ac-DEVD-cmk in a caspase activity buffer (50 mM HEPES,
pH 7.4, 50 mM KCl, 0.1 mM EDTA, 10 mM DTT, and 0.1%
ples and incubated at 37 °C. Granzyme B (2 nM) was added to
wild-type procaspase-3 to determine the maximal activation
levels possible. At the indicated time points, 50 ?M Ac-DEVD-
afc was added to each sample, and fluorescence intensity was
monitored on a SpectraMax M5 (Molecular Devices) plate
caspase-3 (D9A/D28A/D175A), caspase-3, caspase-3 (S205A),
and caspase-3 (T199A) were measured as described previously
(22, 37–40). Briefly, 5 ?M procaspase-3, 0.1, 10, 20, and 50 ?M
procaspase-3 (D9A/D28A/D175A), 5 and 10 nM caspase-3, 5
and 10 nM caspase-3 (S205A), and 5 and 10 nM caspase-3
(T199A) were incubated in a caspase activity buffer (50 mM
HEPES, pH 7.4, 50 mM KCl, 0.1 mM EDTA, 10 mM DTT, and
0.1% CHAPS). Processing of the fluorogenic substrate
centration in Prism Version 5.0c. Curves were fit using the
standard Michaelis-Menten equation. Vmaxvalues were trans-
formed into kcatvalues using a linear plot of the fluorescence
intensity of the 7-amino-4-trifluoromethylcoumarin standard
versus concentration. Substoichiometric concentrations of Ac-
DEVD-cmk were incorporated into subsequent assays to eval-
uate the impact of low levels of the cleaved caspase on pro-
caspase activity. Notably, peptidic chloromethyl ketones have
an ?15-min half-life in the buffers containing reducing agent
Covalent Modification of (Pro)Caspase-3—The extent of
covalent modification of procaspase-3 and caspase-3 by Ac-
DEVD-cmk was evaluated using an LCT-Premier LC/electros-
pray ionization-MS instrument (Waters) after 24 h of incuba-
tion in assay buffer (50 mM HEPES, pH 7.4, 50 mM KCl, 0.1 mM
EDTA, and 10 mM DTT).
autocatalytic conversion of procaspase-3 to active caspase-3,
we fit our experimental data to the classic Michaelis-Menten
equation: v ? (Vmax?[S])/(Km? [S]).
Iterative application of this equation every minute of the
caspase-3 and caspase-3. Additionally, we evaluated how these
factors would change in response to various catalytic efficien-
cies and different amounts of contaminating active caspase-3.
The assumptions we have input for this model are as follows:
kcat ? 23.5 min?1for mature caspase-3; kcat ? 23.5
min?1?(10?7) for procaspase-3; Km? 140 ?M for both
caspase-3 and procaspase-3, and [S0] ? 100 nM or 1 ?M (pro-
caspase-3). Initial levels of caspase-3 were set at 0 or 0.2% of
Cosedimentation Assays with 1541 (23, 42)—20 ?l of 0.5 mM
1541 or DMSO alone was added to 1 ml of 0.128, 0.064, 0.032,
0.016, 0.008, 0.004, 0.002, 0.001, and 0 mg/ml procaspase-3,
caspase-3, caspase-8, or TEV protease in a caspase assay buffer
(20 mM HEPES, pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT,
1.5% sucrose, and 0.1% CHAPS). 10 ?l of 0.5 mM 1541 was
mM KCl, 0.1 mM EDTA, 1 mM DTT, and 0.1% CHAPS). Ther-
molysin was dissolved in 2.5 M NaCl and 10 mM CaCl2to 1.28,
0.64, 0.32, 0.16, 0.08, 0.04, 0.02, 0.01, and 0 mg/ml, and diluted
10? into a caspase assay buffer (20 mM HEPES, pH 7.4, 10 mM
KCl, 1.5 mM MgCl2, 1 mM DTT, 1.5% sucrose, and 0.1%
CHAPS) to a final volume of 1 ml. 20 ?l of 0.5 mM 1541 or
DMSO was next added to the thermolysin mixtures. All sam-
ples were vortexed and immediately centrifuged at 16,200 ? g
for 15 min. The supernatant was aspirated, and 100 ?l assay
buffer was added to the pellet in each tube. Samples were
diluted with 4? LDS sample buffer (Invitrogen) and analyzed
by SDS-PAGE. Bands were visualized by Coomassie (Bio-Rad)
stain, imaged on a LI-COR Odyssey Infrared Imaging System,
and quantified by ImageJ. Granzyme B samples were normal-
ized for direct comparison with other enzymes.
Rate of Procaspase-3 Processing by Upstream Proteases—20
?l of 0.5 mM 1541 or DMSO alone was added to 1 ml of 200 nM
procaspase-3 (29-277/C163A) in a caspase activity buffer (20
sucrose, and 0.1% CHAPS). 2 nM caspase-8, 2 nM caspase-3, 20
each sample were taken at the indicated time points, quenched
with LDS loading buffer, and analyzed by SDS-PAGE. Protein
bands were visualized by silver stain and quantified by ImageJ.
Similarly, 20 ?l of 0.5 mM 1541 or DMSO was added to 1 ml
of 200 nM procaspase-3 (29-277/C163A) in a caspase activity
essing was monitored as described above.
essing by thermolysin was determined, as described previously
(43). Briefly, thermolysin was dissolved in 2.5 M NaCl and 10
mM CaCl2to 0.5 or 0.05 mg/ml. 100 ?l of 10 ?M procaspase-3
(29-277/C163A) was added to 860 ?l of caspase buffer. 20 ?l of
?l of the specified thermolysin stocks. Processing was moni-
tored over time as described above except using Sypro Ruby
protein gel stain (Invitrogen).
Catalytic Efficiency of Procaspase-3 Cleavage (44, 45)—50 ?l
of 2 ?M procaspase-3 (29-277/C163A) followed by 10 ?l of 0.5
mM 1541 or DMSO were added to 390 ?l of buffer (20 mM
HEPES, pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 1.5%
Sucrose, and 0.1% CHAPS) in 12 separate 1.5-ml Eppendorf
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tubes. 50 ?l of a dilution series of an upstream protease was
agitated the samples at 37 °C and quenched the reactions with
LDS loading buffer at the indicated time points. We subse-
quently analyzed the samples by SDS-PAGE followed by silver
the substrate with increasing enzyme concentrations and
following equation gives an approximate catalytic efficiency
(kcat/Km): kcat/Km? ln(2)/(E1⁄2?t).
formed as described previously (23, 46). Briefly, 250 ?l of a
caspase activity buffer (20 mM HEPES, pH 7.4, 10 mM KCl, 1.5
to the lyophilized solid to 50 ?M. The peptide was agitated at
37 °C for 4–6 h. Fibril formation was confirmed by an increase
250 ?l 5 ?M thioflavin T (Sigma).
Procaspase-3 Processing with A?(1–40)—250 nM pro-
caspase-3 was agitated in a caspase activity buffer (20 mM
DTT, 0.1% CHAPS) with 20 ?M A?(1–40) fibrils (4 h preincu-
ing was monitored by SDS-PAGE as described above.
Trans-activation of Procaspase-3 with A?(1–40)—5 nM
caspase-3 was agitated with 200 nM procaspase-3 (29-277/
C163A) in a caspase activity buffer (20 mM HEPES, pH 7.4, 10
mM KCl, 1.5 mM MgCl2, 1.5% sucrose, 10 mM DTT, 0.1%
CHAPS) with 20 ?M A?(1–40) fibrils (4 h of preincubation) or
buffer alone for the indicated time intervals. Processing was
monitored by SDS-PAGE as described above.
Cosedimentation of Caspases with A?(1–40)—50 ?M A?(1–
preincubation). The samples were vortexed briefly and centri-
fuged at 20,817 ? g for 20 min. The supernatant was aspirated,
and 100 ?l of assay buffer was added to the pellet in each tube.
for 1541 samples.
1541 Nanofibrils Enhance Autocatalytic Maturation of
Procaspase-3—Previously, we reported that 1541 nanofibrils
induce procaspase-3 activation to form mature caspase-3 (23).
The addition of 25 ?M 1541 to 100 nM wild-type procaspase-3
results in a burst of activity after a 2-h incubation, using cleav-
age of Ac-DEVD-afc as a reporter substrate (Fig. 2A) (22). This
procaspase-3 activation curve is similar to the classic S-shaped
curve obtained for auto-activation of other zymogens, such as
trypsinogen (47, 48). Based on our data, we suspected an auto-
caspase-3 rapidly processes its zymogen substrate, pro-
dicts an initially accelerating activity curve that saturates as the
procaspase-3 substrate is depleted to give the observed
To confirm the role of active caspase-3 in accelerating wild-
type procaspase-3 activation, we explored how changing the
mature caspase-3 concentration affects the kinetics of wild-
type procaspase-3 activation. The rate of procaspase-3 activa-
tion upon inclusion of even a 1% stoichiometric amount of
active caspase-3 drastically shortens the lag period by 2.5-fold
an irreversible active site inhibitor, Ac-DEVD-cmk, to wild-
Ac-DEVD-cmk preferentially labels and inactivates mature
caspase-3 relative to procaspase-3 (Fig. 3). These data high-
light the importance of mature caspase-3 in the procaspase-3
activation process and support an autocatalytic activation
ence of 1541. A, activation was monitored for 100 nM procaspase-3 alone
procaspase-3 with 25 ?M 1541 (purple circles). The tetrapeptide substrate,
Ac-DEVD-afc, was added at the indicated time points, and initial rates were
nM caspase-3, procaspase-3 (dark squares), procaspase-3 with granzyme B
activity was measured in the presence of 1 nM Ac-DEVD-cmk, procaspase-3
(dark diamonds), procaspase-3 with granzyme B (open diamonds), and pro-
caspase-3 with 25 ?M 1541 (blue diamonds).
FIGURE 3. Irreversible inhibitor of mature caspase-3. A, structure of Ac-
wild-type procaspase-3. After a 24-h incubation at 37 °C, covalent modifica-
tion was evaluated by mass spectrometry.
33784 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER40•SEPTEMBER28,2012
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1541 Fibrils Enhance Procaspase-3 Processing by Several
Upstream Proteases—The results above demonstrate that the
fibrils plus trace amounts of active caspase-3 increase pro-
caspase-3 activation kinetics. To measure the extent to which
the nanofibrils enhance the natural zymogen maturation proc-
ess, we further quantified the magnitude of the acceleration.
We first characterized the apparent increase in caspase-3 cata-
lytic efficiency in the presence of 1541 fibrils. The catalytically
inactive procaspase-3 (C163A) lacking its 28-amino acid
prodomain (29-277) was used as the substrate (Fig. 1A). This
variant permits evaluation of the processing rate at the single
critical activating cut site between the large and small subunit
without the confounding effects of procaspase-3 autocatalytic
activation (Fig. 1C). In a preliminary competition assay with
Ac-DEVD-afc, we found that the Kmvalue for cleaving this
zymogen substrate is greater than 100 ?M (data not shown).
well below its Kmvalue. Using these experimental conditions,
we found caspase-3 to show an ?17-fold increase in catalytic
efficiency (kcat/Km) for cleaving this zymogen variant in the
presence of 1541 (Fig. 4A and Table 1).
We next evaluated whether this increase in activity is gener-
alizable to other known upstream proteases, such as caspase-8
and granzyme B. Interestingly, the catalytic efficiency for
caspase-8 cleaving the zymogen was enhanced 28-fold in the
presence compared with absence of 1541 (Fig. 4C and Table 1).
These results reinforce the importance of an active upstream
protease in promoting procaspase-3 activation and further
quantify the impact of 1541 nanofibrils on procaspase-3 matu-
ration by active proteases, including caspase-3.
Fibrils Concentrate Procaspase-3 with Mature Proteases to
Enhance Processing—1541 nanofibrils increase procaspase-3
processing by caspase-3 as well as other upstream proteases.
This enhancement in procaspase-3 maturation could result
from three possible mechanistic effects as follows: an increase
of the intrinsic activity of the upstream proteases, increased
susceptibility of procaspase-3 to processing, or increased prox-
imity of procaspase-3 to these active proteases.
of some known activating proteases, such as caspase-3,
substrates, Ac-DEVD-afc, Ac-IETD-afc, and Ac-IETD-afc,
respectively. No change in the catalytic efficiencies was
observed upon the addition of fibrils (Fig. 5, A–C). These data
show that the fibrils do not directly impact the intrinsic activi-
ties of these activating proteases. The unaltered activities fur-
ther suggest that the fibrils do not globally alter the conforma-
tion of the mature enzymes.
Although 1541 does not directly impact the mature enzyme
conformation or activity, it is possible that the nanofibrils alter
the conformation of the precursors to make the cleavage site
more accessible to proteolysis. Pulse proteolysis with the
broadly specific protease, thermolysin, has previously been
used to assess conformational changes and protein stability
(49). 1541 fibrils did not affect the rate of processing of the
inactive procaspase-3 (C163A) by thermolysin (Fig. 6A). These
data suggest that the fibrils do not alter the conformation of
procaspase-3 to make it generally more susceptible to
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
10 ?M 1541 was evaluated at 90 min. Replicate gels are not shown for clarity. The asterisks indicate roughly 50% cleavage of procaspase-3.
1541 fibrils increase the catalytic efficiency of upstream proteases
against the inactive procaspase-3 (C163A) substrate
Granzyme B0 90
Granzyme B 1090
aSubstrate ? inactive procaspase-3 (29–277/C163A).
bRatio ? (catalytic efficiency with 1541)/(catalytic efficiency without 1541).
2.8 ? 103
4.8 ? 104
2.7 ? 104
7.5 ? 105
8.3 ? 104
4.1 ? 105
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Finally, we explored if the fibrils promoted activation
through colocalization or concentration of procaspase-3 with
the activating proteases. We previously showed a direct associ-
ation between procaspase-3 and the 1541 nanofibrils using
ciate directly with the fibrils using the cosedimentation assays.
As seen in Fig. 6B, caspase-3 binds and saturates the fibrils at
roughly the same concentration as procaspase-3. Caspase-8
6B) consistent with the greater enhancement in catalytic effi-
and shows the smallest change in activity in the presence of the
fibrils. These studies support the notion that colocalization of
an active protease with the procaspase-3 substrate is critically
important for activation to occur.
This colocalization mechanism was further established by
caspase-3 (29-277/C163A) by low concentrations of caspase-3,
caspase-8, and granzyme B (Fig. 6C). As expected, very little
cleavage of the inactive zymogen was observed by caspase-3 or
caspase-8 in the absence of 1541. However, upon the inclusion
of 1541, the half-lives (t1⁄2) were reduced from undetectable lev-
els to 220 and 8 min by caspase-3 and caspase-8, respectively.
presence of 1541 are consistent with the relative catalytic effi-
ciencies of the respective enzymes.
In contrast, granzyme B does bind but not well to the fibrils,
which provides an important distinction in comparison with
granzyme B processing of procaspase-3, reducing the t1⁄2from
77 to 21 min. Although granzyme B alone cleaves procaspase-3
much more rapidly than caspase-8, granzyme B processes pro-
caspase-3 roughly 3-fold slower than caspase-8 in the presence
of 1541. This is also reflected by the much smaller change in
catalytic efficiency seen for granzyme B compared with
caspase-3 and caspase-8 against the procaspase-3 substrate in
the presence of the fibrils (Table 1). Both observations are con-
sistent with colocalization of the upstream protease with
its procaspase-3 substrate being especially important for
We subsequently explored the ability of 1541 nanofibrils to
tobacco etch virus (TEV) protease and thermolysin to evaluate
if procaspase-3 was more susceptible to proteolysis in general.
rapeptide 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 ?M 1541. B, activity of capsase-8 (20 nM) against Ac-IETD-afc
tion alone. C, activity of granzyme B against Ac-IETD-afc was measured with
and without 25 ?M 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 ?M procaspase-3 (29-277/C163A) in the presence of 50 ?M 1541 or DMSO alone.
Samples were quenched at the indicated time points and analyzed by SDS-PAGE. B, 10 ?M 1541 was added to a dilution series of procaspase-3, caspase-3,
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
LDS loading buffer. After analysis by SDS-PAGE and silver stain, percent cleavage of the procaspase was determined by quantifying band intensities. D, TEV
monitored as described above. Replicate gels are not shown for clarity.
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1541 nanofibrils do not significantly affect the rate of pro-
caspase-3 processing by thermolysin (Fig. 6A). To enable TEV
to cleave procaspase-3, we replaced the caspase cleavage site in
the inter-subunit linker with the recognition sequence for TEV
protease (D175ENLYFQ). Previous experiments showed this
procaspase-3 mutant could be cleaved by TEV (7). However,
(Fig. 6D). Correspondingly, both TEV and thermolysin do not
interact with the 1541 fibrils to a significant degree (Fig. 6B).
showed dramatic enhancement in processing of procaspase-3.
Those that cannot bind to the fibrils showed no enhancement
in the rate at which they cleaved procaspase-3.
Decreased Catalytic Efficiency of Resistance Mutants—In
previous studies (22), we identified point mutations in pro-
caspase-3 (S205A or T199A) that reduced activation by 1541
but could still be cleaved by granzyme B to generate an active
enzyme. We wished to test if the resistance to activation of
these variants could result from either decreased interaction
with the fibrils or from diminished catalytic efficiency relative
to the wild-type enzyme.
cleaving the preferred substrate Ac-DEVD-afc for caspase-3
respectively. This is mostly due to a significant decrease in the
inter-subunit linker of procaspase-3. The catalytic efficiency
against this substrate was reduced by 4- and 14-fold for the
S205A and T199A variants, respectively. These reductions in
kcat/Kmoffset the apparent 17-fold increase in activity that
caspase-3-resistant mutants are susceptible to enhanced proc-
essing by wild-type caspase-3 in the presence of 1541, which
would indicate that the mutant proenzymes still interact with
the fibrils (data not shown). Thus, the reduction in catalytic
efficiency for these variant enzymes, largely explains why these
mutations lead to greater resistance to 1541-stimulated
Procaspase-3 Does Not Self-activate by an Intermolecular or
Intramolecular Mechanism but Requires an Active Protease to
Initiate Processing—The above results elucidated the impor-
tance of trace levels of mature caspase-3 in rapidly activating
procaspase-3 and described how the fibrils act to enhance this
process. However, a detailed mechanistic understanding of the
initial event that leads to the first caspase-3 molecules was still
lacking. This initiating event could originate from one of two
sources as follows: procaspase-3 could cleave itself by either a
initially present in the procaspase-3 preparations could cleave
procaspase-3 to generate more mature caspase-3 (Fig. 7B).
Concurrent with the burst in activation observed in Fig. 2A,
wild-type procaspase-3 processing to the large and small sub-
unit of active caspase-3 in the presence of 1541 nanofibrils
occurs after a lag period of roughly 2 h (Fig. 8A) (22). Either
consistent with this lag or delay (Fig. 7). Thus, we first assessed
if concentrating procaspase-3 on the fibrils is sufficient to pro-
mote an initial trans-activation event, where one proenzyme
molecule, termed “the initiator,” cleaves another proenzyme
molecule, termed “the substrate” (Fig. 7A). The catalytically
inactive mutant of procaspase-3 (C163A) served as the sub-
strate, because it can be cleaved but cannot initiate. The cata-
lytically competent, yet uncleavable procaspase-3 served as the
initiator; here, the critical aspartic acid residues recognized by
the protease are mutated to alanines (D9A/D28A/D175A, Fig.
mixtures of the initiator and the substrate procaspase-3 (250
nM). At the indicated time points, samples were quenched with
LDS loading buffer, analyzed by SDS-PAGE, and visualized by
silver stain (Fig. 8B). No processing of the substrate variant
(C163A) was observed in the presence or absence of 1541. Fur-
thermore, even a 4-fold stoichiometric excess of the uncleav-
able initiator procaspase-3 did not promote processing of the
proenzyme (data not shown).
To detect any trace cleavage of the inactive procaspase-3 (5
?M) by the uncleavable proenzyme (5 ?M), we further analyzed
processing at extended times using more sensitive Western
blots. Weadded sub-stoichiometric
Ac-DEVD-cmk to ensure no feedback activation by mature
caspase-3. Still, no detectable change in the amount of pro-
Decreased catalytic efficiency of caspase-3 resistance mutations
tested on two synthetic peptide substrates
Wild-type12.0 ? 0.79.11 ? 0.15
T199A16.2 ? 0.9 1.46 ? 0.02
S205A5.9 ? 0.30.63 ? 0.01
Wild-type 140 ? 102.25 ? 0.04
T199A100 ? 100.12 ? 0.01
S205A56 ? 30.25 ? 0.01
aRatio ? (catalytic efficiency of mutant)/(catalytic efficiency of wild type).
7.6 ? 105
9.1 ? 104
1.1 ? 105
1.6 ? 104
1.2 ? 103
4.4 ? 103
FIGURE 7. Two possible models for initiation of procaspase-3 activation.
lecular) upon interaction with the fibrils. B, initial event involves a trace
the nanofibrils. In either model, propagation is greatly accelerated by the
illustrated on one face of 1541 nanofibrils for simplicity; however, previous
studies show that they coat the full surface (23).
SEPTEMBER28,2012•VOLUME287•NUMBER40 JOURNALOFBIOLOGICALCHEMISTRY 33787
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caspase-3 or its cleavage products was observed with an anti-
body that preferentially recognizes the large subunit of mature
caspase-3 (Fig. 8C). This indicates that 1541 fibrils do not pro-
mote trans-activation of procaspase-3 by another proenzyme
molecule. Moreover, trans-activation of procaspase-3 alone is
not detectable at physiologically relevant concentrations of the
proenzyme (?100 nM) or even at much higher concentrations
Nonetheless, we noted that incubation of wild-type pro-
caspase-3 at high concentrations (?1 ?M) could generate the
large and small subunit of the mature enzyme, even in the
absence of 1541 (Fig. 9). These results in addition to previous
reports would suggest that the proenzyme can auto-activate
(20, 21, 33–35); however, the above proteolytic susceptibility
assay shows that intermolecular processing between two pro-
caspase-3 molecules is restricted. Therefore, we assessed if
intramolecular processing of procaspase-3 can generate a trace
amount of mature caspase-3, which can then rapidly feedback
to promote autocatalytic maturation of the proenzyme (Fig.
To evaluate this mechanism, we needed to isolate wild-type
newly generated caspase-3 to the activation process. We used
the covalent inhibitor, Ac-DEVD-cmk, to preferentially label
and inactivate any mature caspase-3 while preserving pro-
caspase-3 (Fig. 3A). At stoichiometric concentrations (5 ?M),
the inhibitor covalently modifies mature caspase-3 (5 ?M) to
we see only ?70% single-site modification with some nonspe-
cific covalent modification of surface-exposed cysteine resi-
dues. Thus, although procaspase-3 can bind and react with Ac-
DEVD-cmk, it is much slower and less specific than labeling of
the active site of mature caspase-3.
We next added stoichiometric amounts of Ac-DEVD-cmk
(250 nM) to wild-type procaspase-3 (250 nM) with and without
essing to the large and small subunit of the mature caspase
higher concentrations of wild-type procaspase-3 (5 ?M) with
sub-stoichiometric amounts of Ac-DEVD-cmk (1 ?M), proc-
essing over time was still not observed with or without 1541 by
Western blot analysis (Fig. 8E). Thus, the proenzyme alone
appears to be incapable of intramolecular auto-activation.
These results in conjunction with the lack of trans-processing
argue strongly against a model whereby procaspase-3 activa-
tion occurs due to auto-proteolysis (Fig. 7A).
Thus, the data indicate that trace amounts of active
caspase-3 are present in standard procaspase-3 preparations
that facilitate procaspase-3 activation (Fig. 7B). Indeed, West-
before the start of our activation assays (Fig. 8, C and E).
This begs the following question. Can trace levels of
our fibrils? A sub-stoichiometric amount of active caspase-3
to markedly increase proenzyme processing in the presence of
age products generated from procaspase-3 activation. SDS-
for 250 nM wild-type procaspase-3 in the absence (?) or presence (?) of 10 ?M 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 ?M 1541.
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 ?M) in the presence of 20% (1 ?M) 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
The reactions were quenched with LDS loading buffer, analyzed by SDS-
ing of the 2.5 and 1.25 ?M procaspase-3 samples between the 4- and 24-h
by guest on November 19, 2015
PAGE and mass spectrometry analyses both show the same
processed products of procaspase-3 irrespective of the pres-
ence of the fibrils (data not shown). We also demonstrated
above that the activity of mature caspase-3 against a tetrapep-
the activity of mature caspase-3 but rather promote enhanced
cleavage of procaspase-3 by the active enzyme when concen-
trated on the nanofibrils.
Procaspase-3 Is an Extremely Inactive Zymogen—Given the
fact that we were unable to see any evidence for processing
driven in cis or in trans by the proenzyme form, we were moti-
vated to compare the basal activities of procaspase-3 and
Ac-DEVD-afc, we found that mature caspase-3 has a catalytic
agreement with previous reports (Table 3) (52–54).
We found that the catalytic efficiency of our standard prep-
15,000-fold less than the mature enzyme (Table 3). However,
the Kmvalue measured (9.8 ?M) is suspiciously similar to the
present, as shown in Fig. 8E. These data prompted us to deter-
mine whether the procaspase-3 activity that we observed was
1% stoichiometric equivalent of Ac-DEVD-cmk inhibitor to 5
?M procaspase-3 reduced the activity against Ac-DEVD-afc to
undetectable levels. Control studies show that the addition of
5% of Ac-DEVD-cmk to caspase-3 minimally impacted its cat-
alytic efficiency, showing that sub-stoichiometric concentra-
effects on enzyme activity (Table 3).
To quantify the amount of caspase-3 contaminant in our
to each sample. Activity was monitored, and initial rates were
ing caspase-3, which we estimate at 10 nM active caspase-3 or
0.2% of the total amount of proenzyme (Fig. 10A).
As an orthogonal test of the intrinsic activity of the full-
length procaspase-3, we assayed the activity of the uncleavable
procaspase-3 (D9A/D28A/D175A), reasoning that it should be
incapable of autocatalytic maturation (34). Consistent with
previous reports, we found a catalytic efficiency roughly 3000-
fold lower for the triple mutant (2.8 ? 102M?1s?1) relative to
the mature enzyme (Table 3) (52–54). However, the Kmvalue
for the uncleavable procaspase-3 (11.2 ?M) was again suspi-
in the catalytic efficiency upon the addition of 1% Ac-DEVD-
cmk to 3.0 M?1s?1, due to both an increase in Kmand decrease
upon the inclusion of 5% Ac-DEVD-cmk (Table 3). Because
such low concentrations of Ac-DEVD-cmk do not covalently
modify procaspase-3 (Fig. 3), there appears to be at least two
contaminant populations present in the uncleavable pro-
caspase-3 preparations. The initial drop in activity by 90-fold
suggests a population of mature caspase-3 that binds
Ac-DEVD-cmk well, similar to the fully mature caspase-3.
ent that does not bind tightly to the inhibitor, as shown by the
subsequent smaller 3-fold drop in activity (Table 3). This pop-
ulation, which is resistant to inhibition by Ac-DEVD-cmk and
binant mature caspase-3 but not the inactive procaspase-3
(C163A) (data not shown). This indicates that the second con-
taminant is also a cleavage product of full-length proenzyme,
Contaminants of mature caspase-3 present with procaspase-3 dominates activity measurements
7.6 ? 105
7.0 ? 105
2.8 ? 102
4.9 ? 101
12.0 ? 0.7
13.3 ? 0.6
11.2 ? 0.4
370 ? 30
890 ? 190
9.8 ? 0.7
9.11 ? 0.15
9.34 ? 0.14
0.0031 ? (1.0 ? 10?4)
0.00098 ? (1.0 ? 10?4)
0.00069 ? (1.2 ? 10?4)
0.00048 ? (1 ? 10?5)
No activity observed
0.00033 ? (1 ? 10?5)
No activity observed
9.8 ? 0.6
3.4 ? 101
aI/E ? [Ac-DEVD-cmk]/[enzyme].
bExpression time ? 8 h.
cSaturation was not reached.
dExpression time ? 2.5 h. Pro-3 (D3A) ? uncleavable procaspase-3 (D9A/D28A/D75A). Pro-3 (WT) ? wild-type procaspase-3.
FIGURE 10. Multiple mature caspase-3 products identified by active site
min (A), 5 ?M uncleavable procaspase-3 (D9A/D28A/D175A), which was
expressed for 8 h (B), or 10 ?M 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 prepara-
tions occurred at sub-stoichiometric concentrations. Note the distinct levels
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.
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or a hemi-cleaved dimer generated during expression in Esch-
erichia coli (Fig. 11) (55–57). Indeed, shorter expression times
ity for the uncleavable procaspase-3 (D9A/D28A/D175A)
(Table 3). No activity was detectable for this protein upon the
addition of 1% Ac-DEVD-cmk.
We next estimated the amount of caspase-3 contaminant
in 10 ?M uncleavable procaspase-3 (D9A/D28A/D175A),
expressed for 2.5 h. Again, by dosing in Ac-DEVD-cmk to
determine where the activity plateaued, we estimate a 40 nM
contamination of caspase-3 (0.4%). Using a similar approach,
we observed at least two distinct contaminants in the batch of
kinetic observations described (Fig. 10, B and C).
High Zymogenicity of Procaspase-3—The ratio of the activity
of a mature enzyme to the activity of its precursor is referred to
as a zymogenicity value. Zymogenicity describes the extent to
values signifying a more restrained proenzyme. Previous meas-
urements for the zymogenicity of procaspase-3 have been esti-
mated at ?10,000 (39, 52, 58). However, we felt this value
should be revisited given the clear presence of active caspase-3
in procaspase-3 preparations, which we could eliminate using
Ac-DEVD-cmk. Our experiments indicate procaspase-3 is
restrained to an even greater extent with a zymogenicity ?107,
essentially the detection limit of the activity assay (Table 4).
This zymogenicity value was determined by comparing the
maximum concentration of procaspase-3 (50 ?M), where we
observed no activity, with the limit of detection in our catalytic
efficiency assays against Ac-DEVD-afc. Because we do not
observe any activity for the proenzyme, our assessment of pro-
caspase-3 zymogenicity includes the assumption that the Km
value is similar for both the mature enzyme and its precursor.
This approximation is a lower limit because our results above
suggest that the Kmvalue for procaspase-3 against Ac-DEVD-
afc is actually much greater than caspase-3. We next evaluated
the limit of detection by diluting caspase-3 below a concentra-
tion (5 pM) where no activity could be observed against Ac-
DEVD-afc. This is a conservative estimate because dilute
enzymes can associate with the surface of assay plates. Even
with the conservative approximations, the ratio of these two
values gives us a zymogenicity estimate of ?107.
Amyloid-? Fibrils and 1541 Nanofibrils Activate Pro-
caspase-3 by a Similar Mechanism—In previous studies, we
showed that the proteogenic amyloid-?(1–40) fibrils also pro-
moted procaspase-3 activation (23). Here, we sought to deter-
mine whether the activation mechanism by amyloid-? fibrils is
analogous to that of 1541.
procaspase-3, caspase-3, as well as caspase-8 can bind and
increased maturation of the wild-type precursor to a large and
small subunit. The amyloid-? fibrils stimulated processing of
procaspase-3 as a function of time; however, 1541 acts ?2–4-
fold faster (Fig. 12B). The incubation period before robust pro-
caspase-3 cleavage is more variable for the amyloid-? fibrils
than for 1541 nanofibrils, depending on the exact preparation
of the fibrils (data not shown). Transmission electron micros-
copy studies have shown that amyloid-?(1–40) can also form
small oligomers as well as longer fibrils (59, 60), whereas 1541
fibrils are longer and more regular (23). The heterogeneity in
the amyloid-?(1–40) fibrils may account for the differences
seen in procaspase-3 activation kinetics.
Similar to 1541, the activation of wild-type procaspase-3 on
ric concentrations of Ac-DEVD-cmk, suggesting a need for an
initiator protease (Fig. 12C). This is consistent with trace
amounts of mature caspase-3 being necessary to drive proen-
tions. 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 pro-
caspase-3 from batches expressed for 8 h (red) and 2.5 h (blue) was run on a
tion. D, 15 ?l of 5 ?M samples of the uncleavable procaspase-3 (D9A/D28A/
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.
cally recognizes the full-length band as well.
Summary of zymogenicity values
Tissue plasminogen activator
aZymogenicity ? mature protease activity/zymogen activity.
bSee Ref. 68.
cSee Refs. 39, 58.
33790 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER40•SEPTEMBER28,2012
by guest on November 19, 2015
zyme maturation. Furthermore, the amyloid-?(1–40) fibrils
ing by exogenous caspase-3 (Fig. 12D). The variability in the
in the catalytic efficiency of caspase-3 against the zymogen
in contrast to the 28-fold enhancement seen with 1541 fibrils
(Fig. 4B). A number of factors may contribute to the difference
in rate enhancement, including the orientation of caspase-8 on
the amyloid-? fibrils or its ability to interact with amyloid-?
oligomers versus fibrils. The data show amyloid-?(1–40) can
enhance processing by promoting colocalization of active
caspase-3 and procaspase-3, albeit more slowly than for 1541.
Procaspase-3 Is Activated by Initiator Proteases on Synthetic
with caspase-3 to promote explosive activation in vitro. A col-
1541 and amyloid-?(1–40) fibrils, mutational studies on pro-
caspase-3, and thorough biochemical characterization of pro-
caspase-3 activity, show that procaspase-3 activation does not
happen to any extent by procaspase-3 alone (Fig. 7A). Instead,
caspase to bind and colocalize with the procaspase-3 on an
ordered fibrillar scaffold (Fig. 7B).
on 1541 nanofibrils exhibits some protease specificity. For
example, the 1541 nanofibrils show the greatest enhancement
and binding for caspase-8, followed by caspase-3 and gran-
zyme-B. We show that this colocalization of procaspase-3 with
upstream proteases results in an effective change in the cata-
ing protease to cleave soluble synthetic substrates. However,
1541 nanofibrils do not enhance proteolysis by some noncog-
nate proteases like thermolysin or TEV protease, even when
presented to procaspase-3 variants containing consensus TEV
protease cleavage sites in place of the natural caspase cleavage
The specificity of this process seems to also be at the level of
the chemical composition of the fibril. Previous and unpub-
lished studies show fibril-forming variants of 1541 can be spe-
cific for activation of procaspases-3, -6, and -7 (22, 23).5We
show here that procaspase-3 can be activated on amyloid-?(1–
40) fibrils by a similar mechanism as on 1541 fibrils. Additional
studies to explore the influence of different fibrillar structures
procaspase-3 will be instructive in identifying biologically rele-
vant activation platforms.
Because the caspases initiate fate-determining transforma-
tions in the cells, it is not surprising that their activity would be
highly restricted in the absence of a signaling event (9). Pro-
caspases are classically recruited and activated on scaffolding
complexes, such as the apoptosome, the inflammasome, and
induced proximity model that describes clustering and activa-
tion of procaspase-8 and procaspase-9 upon localization to
such platforms provides a similar model for procaspase-3 acti-
vation (34, 35, 58, 62). Our data further suggests that pro-
caspase-3 recruitment alone is not sufficient for auto-activa-
maturation of the proenzyme on fibrils.
Previous confocal microscopy and immunoprecipitation
studies have illustrated a direct interaction between mature
caspases and specific intracellular fibrillar structures. For
example, caspase-9 and caspase-3 appear to colocalize with
cytokeratin 18 in epithelial cells upon the addition of an apo-
Huntington disease, caspases can also localize to aggregates or
fibrils associated with disease progression (26). Also, the death
effector domain motifs in the prodomain of caspase-8 can
assemble into filaments in cells to promote apoptosis (63, 64).
Although localization has been demonstrated previously, our
results show that fibrils can serve as a direct platform for pro-
caspase recruitment and maturation in vitro. Furthermore, we
detail the specific mechanism that results in procaspase activa-
tion upon recruitment to the fibrils in vitro. However, addi-
tional studies are necessary to understand if these mechanisms
apply to the cellular activities of 1541 (22), amyloid-? fibrils, or
other intracellular fibrillar structures (28, 65).
5J. A. Zorn, D. W. Wolan, and J. A. Wells, unpublished results.
500 ?l of buffer, 20 ?M A?(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
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
with 200 nM truncated inactive procaspase-3 (29-277/C163A) and 5 nM caspase-3. Processing was again evaluated at the indicated time points.
by guest on November 19, 2015
Previous studies demonstrate that enzymes can retain activ-
ity upon fibril formation or fibril association. Zymogens in the
blood coagulation cascade are also well known to be activated
on fibers. For example, tissue plasminogen activator is stimu-
lated by fibrinogen and more dramatically by fibrin (66).
Remarkably, amyloid-? fibrils also enable activation of tissue
plasminogen activator (67). Furthermore, distinct aggregation
states and cleavage products of the amyloid-? precursor pro-
tein have distinct impact on the tissue plasminogen activator
catalytic efficiency against its substrate plasminogen. This
observation has implicated amyloid-? fibrils in the pathogene-
sis of hereditary cerebral hemorrhage with amyloidosis-Dutch
Lag in Procaspase-3 Activation Is Due to a Second-order
Reaction Rate—Our results show that procaspase-3 lacks any
procaspase-3 and mature caspase-3 can also associate rapidly
with the fibrils. Thus, the lag observed for procaspase-3 activa-
tion in the presence of 1541 nanofibrils is attributable to the
rate of reaction of procaspase-3 cleavage by caspase-3. A dis-
tinct delay or lag in activation is also observed for wild-type
procaspase-3 activation alone at high concentrations of the
proenzyme (Fig. 9). The 1541 fibrils act to enhance the natural
activation process through concentration or colocalization of
procaspase-3 with mature caspase-3.
A significant lag in activation would be predicted to exist
even in the presence of a trace amount of mature enzyme. We
further confirmed this observation through calculation of the
equation, taking into account an autocatalytic maturation of
100 nM procaspase-3 upon the inclusion of a trace amount of
caspase-3 (0.2 nM). These initial parameters were based on our
typical assay conditions, which contain 100 nM procaspase-3.
We also assumed a Kmof 140 ?M, which represents the Km
value measured for caspase-3 against the Ac-IETD-afc sub-
strate. With these parameters, we calculated an S-shaped acti-
vation curve, including the lag, which closely matched our
experimental data (data not shown). Thus, the activity of
mature caspase-3 alone in our population of wild-type pro-
experimentally. Similar activation curves have been demon-
of trypsin (47, 48).
Importance of Zymogenicity in the Mechanism of Caspase
Activation—Numerous structural and biochemical studies
have delineated the unique mechanisms for maintaining pro-
teases as inactive zymogens (3). Such mechanisms include an
conformational change that may alter or disorder substrate
binding or catalytic residues, and oligomerization or interac-
almost all cases, a proteolytic processing event is critical for
activation of the proenzyme by another protease.
zymogen state to variable extents (58, 68). Table 4 summarizes
the zymogenicity values for some well characterized proteases.
For example, tissue plasminogen activator exhibits a zymoge-
nicity between 2 and 10, which reflects a relatively small differ-
precursor (69–72). In stark contrast, trypsinogen and chymot-
genicities on the order of 104to 106(73, 74). Although the
ity in vitro, their cellular regulation is due to processing from
upstream proteases. Other proteases have intermediate values
the in vivo function and activation of the proteases (76).
Our results suggest that procaspase-3 has a much greater
zymogenicity than other proteases and is at least 3 orders of
magnitude less active than previous reports, based upon the
activity of the uncleavable mutant (D9A/D28A/D175A) (39,
58). Key to this assessment was the unexpected discovery that
active caspase-3 is present in preparations of the “uncleavable”
procaspase-3 (D9A/D28A/D175A). The active product was
probably generated from noncaspase proteolysis during
expression in E. coli. When the trace contaminants were inac-
tivated using sub-stoichiometric amounts of the active site
limit of detection of the assay, we estimate the activity for pro-
caspase-3 to be 1/10,000,00 that of mature caspase-3.
The extreme zymogenicity of procaspase-3 is not surprising
given the fact that caspase-3 executes the final and fate-deter-
mining stages of apoptosis. Moreover, this high zymogenicity
barrier probably accounts for the lack of any evidence for pro-
caspase-3 auto-processing. Precursors that exhibit such high
to observe any activity. Studies with trypsinogen and chymot-
rypsinogen used high enzyme concentrations in the micromo-
lar to millimolar range, low pH conditions, high ionic strength
buffers, and long time courses on the orders of days to detect
any processing by these precursors (34, 48, 74, 77, 78).
Challenges for Small Molecule Discovery—Our results
strongly suggest that auto-activation of procaspase-3 is
extremely restricted or nonexistent under typical cellular
enzyme concentrations, ?100 nM, as well as at higher proen-
zyme concentrations. Activation almost certainly requires at
least trace amounts of active caspase or an initiator protease to
trigger processing. Based on the zymogenicity approximation
here and modeling activation curves using the Michaelis-Men-
ten equation, we estimate a small molecule activator would
need to stimulate the activity of the proenzyme over 1000-fold
to generate 0.2% fractional activity to promote autocatalytic
period. Even increasing the enzyme concentration 10-fold, to
rier to ?100-fold.
ing endeavor for a small molecule to directly bind and activate
procaspase-3. From our results, procaspase-3 maturation is
critically dependent on the presence of active caspase. Others
case of PAC-1, this small molecule is proposed to function by
removing an inhibitory Zn2?ion (33). Clark and co-workers
33792 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER40•SEPTEMBER28,2012
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This small molecule activator is proposed to bind to an allos-
teric site on procaspase-3 to promote activation. Additional
studies will be important to elucidate if these compounds work
by a direct activation of the proenzyme or possibly act on trace
contaminants of mature caspase-3 to initiate the process.
The above considerations do not mean that one cannot acti-
vate a protease precursor with a small molecule. In fact, an
endogenous small molecule, inositol hexaphosphate, is known
to bind an allosteric site on the cysteine protease domain of
MARTXVctoxin to promote activation (80). It would be inter-
esting to explore the zymogenicity of the cysteine protease
domain. Perhaps enzymes with low zymogenicity values would
be better candidates for small molecule activator discovery.
conformational flexibility of the mature caspases (81, 82). As
little as a 2-fold change in the catalytic efficiency of caspase-3
would be enough to promote autocatalytic maturation of the
proenzyme with a 0.2% contaminant. Our studies also suggest
procaspase-3 with the mature caspase-3 would also stimulate
activation. The latter two approaches, however, depend on the
cellular concentrations of both pro- and mature enzymes and
are not direct activators of the proenzyme per se. Importantly,
successful protein engineering approaches for procaspase acti-
vation have been described for both these strategies (7, 35, 79,
Acknowledgments—We thank O. Julien for support and for critically
reviewing the manuscript. We thank D. Hostetter and C. Tajon from
providing granzyme B. We thank the Wells laboratory and the Small
Molecule Discovery Center (University of California, San Francisco)
for useful suggestions.
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Julie A. Zorn, Dennis W. Wolan, Nicholas J.
Procaspase-3 to Foster Maturation
Fibrils Colocalize Caspase-3 with
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