Results: 5

1.
Fig. 3.

Fig. 3. From: Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysis.

The K38KKK motif is critical to the exosite function. (A–D) MCF-7sh7 detergent extracts were incubated for 30 min or the indicated time in the presence of 1 nM of the indicated caspases. Samples were analyzed by immunoblotting using an anti-PARP antibody recognizing the N terminus. Proteolysis quantification is presented in Fig. S2.

Dave Boucher, et al. Proc Natl Acad Sci U S A. 2012 April 10;109(15):5669-5674.
2.
Fig. 4.

Fig. 4. From: Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysis.

Efficient PARP proteolysis in apoptotic cells depends on the exosite. (A) 293sh7 cells were transfected with the indicated flag-tagged short-hairpin RNA-resistant caspase-7 cDNAs. Lysates were analyzed 24 h posttransfection with the indicated antibodies. (B) Trail (200 ng/mL)-induced PARP cleavage in cells expressing nonlethal levels of WT or caspase-7AAAA. Samples were harvested after 3 h. All lanes were from the same blot. (C) Time-course of Trail-induced PARP cleavage in cells expressing nonlethal levels of WT or caspase-7AAAA. (D) Hypotonic extracts from 293sh7 cells reconstituted with low amounts of WT or caspase-7AAAA and supplemented with PARP-containing extracts were activated with cytc and dATP. fl, full-length; -Npep, caspase-7 lacking the N-peptide.

Dave Boucher, et al. Proc Natl Acad Sci U S A. 2012 April 10;109(15):5669-5674.
3.
Fig. 1.

Fig. 1. From: Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysis.

Caspase-7 is better than caspase-3 at cleaving PARP and p23. (A–C) MCF-7sh7 extracts were incubated for the indicated period in the presence of 0, 5, 25, 50, or 100 nM recombinant caspase-7 or -3 in caspase buffer, then samples were analyzed by immunoblotting (IB) using the antibody indicated. (D and E) Recombinant full-length caspase-9 (1 μM) or p35-C2A (400 nM) was treated as in A. Samples were TCA-precipitated and analyzed by SDS/PAGE. (F) One nanomolar recombinant caspase-3 or -7 was incubated with immunoprecipitated flag-tagged PARP for the indicated period. Samples were analyzed as in A. Closed arrowhead, full-length protein; open arrowhead, cleaved fragment.

Dave Boucher, et al. Proc Natl Acad Sci U S A. 2012 April 10;109(15):5669-5674.
4.
Fig. 2.

Fig. 2. From: Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysis.

Caspase-7's N-terminal domain contains a transferable exosite to promote PARP cleavage. (A) The amino acid sequence of the N-terminal domain of caspase-7 and -3 are presented. Asp23 of caspase-7 or Asp28 of caspase-3 are the P1 cleavage site residue of the N-peptide that is removed during apoptosis. Mature caspase-7 and -3 start at Ala24 and Ser29, respectively. Key residues are underlined including the conserved methionine used as a convenient location for NTD deletion or chimera design. (B, D, and F) MCF-7sh7 detergent extracts were incubated for 30 min in the presence of 1 nM of the indicated caspases. Samples were analyzed by immunoblotting using an antibody recognizing the N-terminus of PARP. Quantification of PARP proteolysis is presented in Fig. S2. (C and E) Detergent extracts were incubated for the indicated period with twofold serial dilution of the indicated recombinant enzyme in caspase buffer starting at the indicated concentration. PARP hydrolysis rates were estimated as described in Materials and Methods. The enzyme concentration at which 50% of PARP is cleaved (arrow) was used to estimate rates. In C, the calculated rates are 6.2 × 105, <0.2, and 0.8 × 105 M-1⋅s−1 for caspase-7, M45-caspase-7, and caspase-3, respectively. In E, the calculated rates are 6.5 and 22.1 × 105 M-1⋅s−1 for caspase-7 and caspase-7:caspase-3 chimera, respectively.

Dave Boucher, et al. Proc Natl Acad Sci U S A. 2012 April 10;109(15):5669-5674.
5.
Fig. 5.

Fig. 5. From: Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysis.

The exosite promotes cleavage of p23. (A) Fluorescein-labeled p23 (50 nM) was incubated with 40 nM of the indicated caspase for 30 min and was analyzed by fluorescence imaging of gels. (B) Same as in A, with caspases from the caspase-7:caspase-3 library (Fig. S8). Mutants showing decrease (orange arrows) or increase (green arrows) in p23 processing are indicated. Mut8 was inactive, therefore not analyzed. (C) Surface model of caspase-7 [PDB: 1F1J (33)] presenting the 10 sets of residues identified in B. The dimeric caspase contains two catalytic units, each of them is constituted of a large (blue or gray) and a small (cyan or white) subunits. The active site (S) is identified in red. For each mutant, the relative specific activity (kcat/KM) vs. WT for the fluorogenic substrate and the percentage of p23 cleavage are presented. Mut2 lies in a crevasse on the side of the caspase. The NTD is attached at the yellow region. (D) Proposed model for the exosite mechanism. (i) Caspase-3 primes caspase-7 zymogen for activation by removing the negatively charged N-terminal peptide, simultaneously uncovering the basic exosite. (ii) Then, an initiator caspase activates caspase-7 by cleaving the linker that separates the large and small subunits. (iii) Up to two molecules of substrates bind the caspase dimer, either in a direct or in a crossed configuration. (iv) Exosite binding promotes cleavage of the aspartate-containing motif by the catalytic site.

Dave Boucher, et al. Proc Natl Acad Sci U S A. 2012 April 10;109(15):5669-5674.

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