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A constitutively active and uninhibitable caspase-3 zymogen efficiently induces apoptosis.

Walters J, Pop C, Scott FL, Drag M, Swartz P, Mattos C, Salvesen GS, Clark AC - Biochem. J. (2009)

Bottom Line: We show that low concentrations of the pseudo-activated procaspase-3 kill mammalian cells rapidly and, importantly, this protein is not cleaved nor is it inhibited efficiently by the endogenous regulator XIAP (X-linked inhibitor of apoptosis).The 1.63 A (1 A = 0.1 nm) structure of the variant demonstrates that the mutation is accommodated at the dimer interface to generate an enzyme with substantially the same activity and specificity as wild-type caspase-3.The direct activation of procaspase-3 through a conformational switch rather than by chain cleavage may lead to novel therapeutic strategies for inducing cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA.

ABSTRACT
The caspase-3 zymogen has essentially zero activity until it is cleaved by initiator caspases during apoptosis. However, a mutation of V266E in the dimer interface activates the protease in the absence of chain cleavage. We show that low concentrations of the pseudo-activated procaspase-3 kill mammalian cells rapidly and, importantly, this protein is not cleaved nor is it inhibited efficiently by the endogenous regulator XIAP (X-linked inhibitor of apoptosis). The 1.63 A (1 A = 0.1 nm) structure of the variant demonstrates that the mutation is accommodated at the dimer interface to generate an enzyme with substantially the same activity and specificity as wild-type caspase-3. Structural modelling predicts that the interface mutation prevents the intersubunit linker from binding in the dimer interface, allowing the active sites to form in the procaspase in the absence of cleavage. The direct activation of procaspase-3 through a conformational switch rather than by chain cleavage may lead to novel therapeutic strategies for inducing cell death.

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V266E mutants kill cells more efficiently than does the wild-type caspase-3(A) HEK-293A cells were transiently transfected with FLAG-tagged caspase-3 DNA (or 50 ng Bax/0.95 μg empty vector), and Annexin V-positive cells were quantified after 24 h. Z-VAD-FMK (100 μM) or DMSO was added to the cultures 2 h post-transfection. The values represent the means for three independent experiments±S.D. (B) Western blots of the cellular lysates from (A) against anti-full-length caspase-3 or anti-FLAG antibodies, for detection of the transfected constructs, or anti-cleaved PARP. The lower panel shows the loading control of Hsp90.
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Figure 2: V266E mutants kill cells more efficiently than does the wild-type caspase-3(A) HEK-293A cells were transiently transfected with FLAG-tagged caspase-3 DNA (or 50 ng Bax/0.95 μg empty vector), and Annexin V-positive cells were quantified after 24 h. Z-VAD-FMK (100 μM) or DMSO was added to the cultures 2 h post-transfection. The values represent the means for three independent experiments±S.D. (B) Western blots of the cellular lysates from (A) against anti-full-length caspase-3 or anti-FLAG antibodies, for detection of the transfected constructs, or anti-cleaved PARP. The lower panel shows the loading control of Hsp90.

Mentions: One predicts that expression of a constitutively active procaspase-3 should kill cells efficiently; indeed this has been shown previously for a circular permutation of the caspase that mimics IL cleavage [25]. However, it was not clear whether the ~ 3–4-fold lower activity of the V266E mutants compared with fully active wild-type caspase-3 represented sufficient activity for cell death. To examine this, we transiently transfected HEK-293A cells with various caspase-3 mutants and monitored cell viability by Annexin V staining (Figure 2A). Interestingly, both V266E and D3A,V266E mutants resulted in robust cell death (~ 50%), which exceeded that produced by WT and D3A (~ 20%). The loss in cell viability produced by the interface mutants was as pronounced as that produced by Bax, a cytotoxic protein that initiates the intrinsic apoptotic pathway at the mitochondrial level (Figure 2A). In all cases, the levels of apoptosis were decreased in the presence of a caspase inhibitor, Z-VAD-FMK, suggesting that the increased levels of cell death were dependent on caspase activity. When protein production was monitored by Western blot analysis, only the WT and D3A species were detected by their reactivity to anti-FLAG antibodies (Figure 2B). Compared with the endogenous protein, the levels of WT were approx. 5–10-fold higher in transfected cells, as judged by the anti-caspase-3 immunoblot. The interface mutants could not be detected even after prolonged exposures of the FLAG immunoblots, after immunoblotting with anti-cleaved caspase-3 antibodies or when the proteasome inhibitor MG132 was added (results not shown). Thus the full-length procaspase-3 observed in the cells transfected with the interface mutants (Figure 2B, top panel) represents the endogenous protein. Immunoblots using cell lysates prepared at earlier time points post-transfection gave the same results (results not shown). However, RT–PCR (reverse transcription–PCR) reactions showed the presence of mRNA for the caspase variants (Supplementary Figure S2 at http://www.BiochemJ.org/bj/424/bj4240335add.htm), demonstrating that the genes were transcribed. The parsimonious explanation for the lack of immunostaining of transfected V266E variants is that cells expressing them die before sufficient protein can accumulate, suggesting a lethal nature of the interface mutation. In support of this assertion, we replaced the catalytic cysteine residue in the V266E variants with a serine residue to remove activity. We find that the inactive V266E variants no longer support apoptosis (Supplementary Figure S3A at http://www.BiochemJ.org/bj/424/bj4240335add.htm). In addition, the proteins are observed by anti-FLAG antibody staining, although the accumulation remains lower than that for WT. These data show that the toxicity of the V266E variants requires catalytic activity.


A constitutively active and uninhibitable caspase-3 zymogen efficiently induces apoptosis.

Walters J, Pop C, Scott FL, Drag M, Swartz P, Mattos C, Salvesen GS, Clark AC - Biochem. J. (2009)

V266E mutants kill cells more efficiently than does the wild-type caspase-3(A) HEK-293A cells were transiently transfected with FLAG-tagged caspase-3 DNA (or 50 ng Bax/0.95 μg empty vector), and Annexin V-positive cells were quantified after 24 h. Z-VAD-FMK (100 μM) or DMSO was added to the cultures 2 h post-transfection. The values represent the means for three independent experiments±S.D. (B) Western blots of the cellular lysates from (A) against anti-full-length caspase-3 or anti-FLAG antibodies, for detection of the transfected constructs, or anti-cleaved PARP. The lower panel shows the loading control of Hsp90.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2805924&req=5

Figure 2: V266E mutants kill cells more efficiently than does the wild-type caspase-3(A) HEK-293A cells were transiently transfected with FLAG-tagged caspase-3 DNA (or 50 ng Bax/0.95 μg empty vector), and Annexin V-positive cells were quantified after 24 h. Z-VAD-FMK (100 μM) or DMSO was added to the cultures 2 h post-transfection. The values represent the means for three independent experiments±S.D. (B) Western blots of the cellular lysates from (A) against anti-full-length caspase-3 or anti-FLAG antibodies, for detection of the transfected constructs, or anti-cleaved PARP. The lower panel shows the loading control of Hsp90.
Mentions: One predicts that expression of a constitutively active procaspase-3 should kill cells efficiently; indeed this has been shown previously for a circular permutation of the caspase that mimics IL cleavage [25]. However, it was not clear whether the ~ 3–4-fold lower activity of the V266E mutants compared with fully active wild-type caspase-3 represented sufficient activity for cell death. To examine this, we transiently transfected HEK-293A cells with various caspase-3 mutants and monitored cell viability by Annexin V staining (Figure 2A). Interestingly, both V266E and D3A,V266E mutants resulted in robust cell death (~ 50%), which exceeded that produced by WT and D3A (~ 20%). The loss in cell viability produced by the interface mutants was as pronounced as that produced by Bax, a cytotoxic protein that initiates the intrinsic apoptotic pathway at the mitochondrial level (Figure 2A). In all cases, the levels of apoptosis were decreased in the presence of a caspase inhibitor, Z-VAD-FMK, suggesting that the increased levels of cell death were dependent on caspase activity. When protein production was monitored by Western blot analysis, only the WT and D3A species were detected by their reactivity to anti-FLAG antibodies (Figure 2B). Compared with the endogenous protein, the levels of WT were approx. 5–10-fold higher in transfected cells, as judged by the anti-caspase-3 immunoblot. The interface mutants could not be detected even after prolonged exposures of the FLAG immunoblots, after immunoblotting with anti-cleaved caspase-3 antibodies or when the proteasome inhibitor MG132 was added (results not shown). Thus the full-length procaspase-3 observed in the cells transfected with the interface mutants (Figure 2B, top panel) represents the endogenous protein. Immunoblots using cell lysates prepared at earlier time points post-transfection gave the same results (results not shown). However, RT–PCR (reverse transcription–PCR) reactions showed the presence of mRNA for the caspase variants (Supplementary Figure S2 at http://www.BiochemJ.org/bj/424/bj4240335add.htm), demonstrating that the genes were transcribed. The parsimonious explanation for the lack of immunostaining of transfected V266E variants is that cells expressing them die before sufficient protein can accumulate, suggesting a lethal nature of the interface mutation. In support of this assertion, we replaced the catalytic cysteine residue in the V266E variants with a serine residue to remove activity. We find that the inactive V266E variants no longer support apoptosis (Supplementary Figure S3A at http://www.BiochemJ.org/bj/424/bj4240335add.htm). In addition, the proteins are observed by anti-FLAG antibody staining, although the accumulation remains lower than that for WT. These data show that the toxicity of the V266E variants requires catalytic activity.

Bottom Line: We show that low concentrations of the pseudo-activated procaspase-3 kill mammalian cells rapidly and, importantly, this protein is not cleaved nor is it inhibited efficiently by the endogenous regulator XIAP (X-linked inhibitor of apoptosis).The 1.63 A (1 A = 0.1 nm) structure of the variant demonstrates that the mutation is accommodated at the dimer interface to generate an enzyme with substantially the same activity and specificity as wild-type caspase-3.The direct activation of procaspase-3 through a conformational switch rather than by chain cleavage may lead to novel therapeutic strategies for inducing cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA.

ABSTRACT
The caspase-3 zymogen has essentially zero activity until it is cleaved by initiator caspases during apoptosis. However, a mutation of V266E in the dimer interface activates the protease in the absence of chain cleavage. We show that low concentrations of the pseudo-activated procaspase-3 kill mammalian cells rapidly and, importantly, this protein is not cleaved nor is it inhibited efficiently by the endogenous regulator XIAP (X-linked inhibitor of apoptosis). The 1.63 A (1 A = 0.1 nm) structure of the variant demonstrates that the mutation is accommodated at the dimer interface to generate an enzyme with substantially the same activity and specificity as wild-type caspase-3. Structural modelling predicts that the interface mutation prevents the intersubunit linker from binding in the dimer interface, allowing the active sites to form in the procaspase in the absence of cleavage. The direct activation of procaspase-3 through a conformational switch rather than by chain cleavage may lead to novel therapeutic strategies for inducing cell death.

Show MeSH
Related in: MedlinePlus