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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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Procaspase-3(D3A,V266E) is enzymatically active without cleavage of the IL(A) The interface mutation V266E was designed in the context of wild-type caspase-3 (WT) and the uncleavable procaspase-3(D9A,D28A,D175A), called D3A. Low expression generates ‘one-chain’ procaspase-3 (Pro-WT). Overexpression generates the ‘two-chain’ caspase-3 (WT or V266E) by automaturation. ‘Pro’ refers to the pro-domain. (B) Structure of WT caspase-3 (PDB code 2J30) highlighting the active site loops L1 (yellow), L2 (red), L3 (blue), L4 (brown) and L2′ (cyan). The prime (′) indicates residues from the second monomer. For clarity, only one active site is labelled. (C) Labelling the V266E mutants by affinity-based probes. Proteins labelled with bEVD-AOMK were probed by Western blot analysis with anti-biotin, anti-cleaved caspase-3, anti-full-length caspase-3 or anti-His-tag antibodies, or subjected to trichloroacetic acid precipitation and stained with Coomassie Blue. The positive control was WT, and the negative control was Pro-WT. The asterisk indicates a contaminating protein from E. coli expression. Molecular masses are shown to the left in kDa. (D) Determining the substrate specificity of the recombinant caspase-3 mutants. Activity of the caspase-3 mutants (10 nM) was measured using a tetrapeptide positional scanning library, with P1 fixed as an aspartate residue and 7-amino-4-carbamoylmethylcoumarin as the fluorophore group. Hydrolysis rates are presented as a percentage of the maximum rate for each subset (P2, P3 and P4).
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Figure 1: Procaspase-3(D3A,V266E) is enzymatically active without cleavage of the IL(A) The interface mutation V266E was designed in the context of wild-type caspase-3 (WT) and the uncleavable procaspase-3(D9A,D28A,D175A), called D3A. Low expression generates ‘one-chain’ procaspase-3 (Pro-WT). Overexpression generates the ‘two-chain’ caspase-3 (WT or V266E) by automaturation. ‘Pro’ refers to the pro-domain. (B) Structure of WT caspase-3 (PDB code 2J30) highlighting the active site loops L1 (yellow), L2 (red), L3 (blue), L4 (brown) and L2′ (cyan). The prime (′) indicates residues from the second monomer. For clarity, only one active site is labelled. (C) Labelling the V266E mutants by affinity-based probes. Proteins labelled with bEVD-AOMK were probed by Western blot analysis with anti-biotin, anti-cleaved caspase-3, anti-full-length caspase-3 or anti-His-tag antibodies, or subjected to trichloroacetic acid precipitation and stained with Coomassie Blue. The positive control was WT, and the negative control was Pro-WT. The asterisk indicates a contaminating protein from E. coli expression. Molecular masses are shown to the left in kDa. (D) Determining the substrate specificity of the recombinant caspase-3 mutants. Activity of the caspase-3 mutants (10 nM) was measured using a tetrapeptide positional scanning library, with P1 fixed as an aspartate residue and 7-amino-4-carbamoylmethylcoumarin as the fluorophore group. Hydrolysis rates are presented as a percentage of the maximum rate for each subset (P2, P3 and P4).

Mentions: Caspase activation, more than any other event, defines a cellular response to apoptosis. Synthesized initially as zymogens (procaspases) (Figure 1A), the cytosolic pool of inactive zymogen is converted rapidly into active protease upon the induction of apoptosis. A fundamental difference exists in the caspase subfamilies regarding maturation, and this difference is a key aspect for regulating apoptosis. Initiator procaspases are stable monomers in the cell, and dimerization is facilitated following recruitment to activation platforms [1]. Importantly, once dimerized, the initiator procaspases have high enzymatic activity, and subsequent chain cleavage simply stabilizes the active site [2,3]. In contrast, the effector procaspase-3 is a stable dimer, but has very low enzymatic activity (< 0.4% of that of the active protease) [4,5]. In this case, full activation occurs after cleavage of the IL (intersubunit linker) by initiator caspases, resulting in ordering of the active sites due to the release of two active site loops (L2 and L2′) from the IL and subsequent formation of the substrate-binding pocket (active site loop 3) (Figure 1B, and see Supplementary Movie S1 at http://www.BiochemJ.org/bj/424/0335/bj4240335add.htm). In short, the cell maintains a cytosolic pool of inactive procaspase-3 that is poised to carry out cell death.


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)

Procaspase-3(D3A,V266E) is enzymatically active without cleavage of the IL(A) The interface mutation V266E was designed in the context of wild-type caspase-3 (WT) and the uncleavable procaspase-3(D9A,D28A,D175A), called D3A. Low expression generates ‘one-chain’ procaspase-3 (Pro-WT). Overexpression generates the ‘two-chain’ caspase-3 (WT or V266E) by automaturation. ‘Pro’ refers to the pro-domain. (B) Structure of WT caspase-3 (PDB code 2J30) highlighting the active site loops L1 (yellow), L2 (red), L3 (blue), L4 (brown) and L2′ (cyan). The prime (′) indicates residues from the second monomer. For clarity, only one active site is labelled. (C) Labelling the V266E mutants by affinity-based probes. Proteins labelled with bEVD-AOMK were probed by Western blot analysis with anti-biotin, anti-cleaved caspase-3, anti-full-length caspase-3 or anti-His-tag antibodies, or subjected to trichloroacetic acid precipitation and stained with Coomassie Blue. The positive control was WT, and the negative control was Pro-WT. The asterisk indicates a contaminating protein from E. coli expression. Molecular masses are shown to the left in kDa. (D) Determining the substrate specificity of the recombinant caspase-3 mutants. Activity of the caspase-3 mutants (10 nM) was measured using a tetrapeptide positional scanning library, with P1 fixed as an aspartate residue and 7-amino-4-carbamoylmethylcoumarin as the fluorophore group. Hydrolysis rates are presented as a percentage of the maximum rate for each subset (P2, P3 and P4).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Procaspase-3(D3A,V266E) is enzymatically active without cleavage of the IL(A) The interface mutation V266E was designed in the context of wild-type caspase-3 (WT) and the uncleavable procaspase-3(D9A,D28A,D175A), called D3A. Low expression generates ‘one-chain’ procaspase-3 (Pro-WT). Overexpression generates the ‘two-chain’ caspase-3 (WT or V266E) by automaturation. ‘Pro’ refers to the pro-domain. (B) Structure of WT caspase-3 (PDB code 2J30) highlighting the active site loops L1 (yellow), L2 (red), L3 (blue), L4 (brown) and L2′ (cyan). The prime (′) indicates residues from the second monomer. For clarity, only one active site is labelled. (C) Labelling the V266E mutants by affinity-based probes. Proteins labelled with bEVD-AOMK were probed by Western blot analysis with anti-biotin, anti-cleaved caspase-3, anti-full-length caspase-3 or anti-His-tag antibodies, or subjected to trichloroacetic acid precipitation and stained with Coomassie Blue. The positive control was WT, and the negative control was Pro-WT. The asterisk indicates a contaminating protein from E. coli expression. Molecular masses are shown to the left in kDa. (D) Determining the substrate specificity of the recombinant caspase-3 mutants. Activity of the caspase-3 mutants (10 nM) was measured using a tetrapeptide positional scanning library, with P1 fixed as an aspartate residue and 7-amino-4-carbamoylmethylcoumarin as the fluorophore group. Hydrolysis rates are presented as a percentage of the maximum rate for each subset (P2, P3 and P4).
Mentions: Caspase activation, more than any other event, defines a cellular response to apoptosis. Synthesized initially as zymogens (procaspases) (Figure 1A), the cytosolic pool of inactive zymogen is converted rapidly into active protease upon the induction of apoptosis. A fundamental difference exists in the caspase subfamilies regarding maturation, and this difference is a key aspect for regulating apoptosis. Initiator procaspases are stable monomers in the cell, and dimerization is facilitated following recruitment to activation platforms [1]. Importantly, once dimerized, the initiator procaspases have high enzymatic activity, and subsequent chain cleavage simply stabilizes the active site [2,3]. In contrast, the effector procaspase-3 is a stable dimer, but has very low enzymatic activity (< 0.4% of that of the active protease) [4,5]. In this case, full activation occurs after cleavage of the IL (intersubunit linker) by initiator caspases, resulting in ordering of the active sites due to the release of two active site loops (L2 and L2′) from the IL and subsequent formation of the substrate-binding pocket (active site loop 3) (Figure 1B, and see Supplementary Movie S1 at http://www.BiochemJ.org/bj/424/0335/bj4240335add.htm). In short, the cell maintains a cytosolic pool of inactive procaspase-3 that is poised to carry out cell death.

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