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Search results for: Caspase 2 Substrate VDVAD pNA1000 assays

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#21903398   2011/08/16 To Up

Exploiting differences in caspase-2 and -3 S₂ subsites for selectivity: structure-based design, solid-phase synthesis and in vitro activity of novel substrate-based caspase-2 inhibitors.

Several caspases have been implicated in the pathogenesis of Huntington's disease (HD); however, existing caspase inhibitors lack the selectivity required to investigate the specific involvement of individual caspases in the neuronal cell death associated with HD. In order to explore the potential role played by caspase-2, the potent but non-selective canonical Ac-VDVAD-CHO caspase-2 inhibitor 1 was rationally modified at the P(2) residue in an attempt to decrease its activity against caspase-3. With the aid of structural information on the caspase-2, and -3 active sites and molecular modeling, a 3-(S)-substituted-l-proline along with four additional scaffold variants were selected as P(2) elements for their predicted ability to clash sterically with a residue of the caspase-3 S(2) pocket. These elements were then incorporated by solid-phase synthesis into pentapeptide aldehydes 33a-v. Proline-based compound 33h bearing a bulky 3-(S)-substituent displayed advantageous characteristics in biochemical and cellular assays with 20- to 60-fold increased selectivity for caspase-2 and ∼200-fold decreased caspase-3 potency compared to the reference inhibitor 1. Further optimization of this prototype compound may lead to the discovery of valuable pharmacological tools for the study of caspase-2 mediated cell death, particularly as it relates to HD.
Michel C Maillard, Frederick A Brookfield, Stephen M Courtney, Florence M Eustache, Mark J Gemkow, Rebecca K Handel, Laura C Johnson, Peter D Johnson, Mark A Kerry, Florian Krieger, Mirco Meniconi, Ignacio Muñoz-Sanjuán, Jordan J Palfrey, Hyunsun Park, Sabine Schaertl, Malcolm G Taylor, Derek Weddell, Celia Dominguez

1856 related Products with: Exploiting differences in caspase-2 and -3 S₂ subsites for selectivity: structure-based design, solid-phase synthesis and in vitro activity of novel substrate-based caspase-2 inhibitors.

3 mg1 mg5 mg20 µl (10 mM)100 µl (2 mM)20 20 ul (10 mM)20 µl (10 mM)12 x 25 ul2000 pcs

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#21828056   2011/08/02 To Up

Structural and enzymatic insights into caspase-2 protein substrate recognition and catalysis.

Caspase-2, the most evolutionarily conserved member in the human caspase family, may play important roles in stress-induced apoptosis, cell cycle regulation, and tumor suppression. In biochemical assays, caspase-2 uniquely prefers a pentapeptide (such as VDVAD) rather than a tetrapeptide, as required for efficient cleavage by other caspases. We investigated the molecular basis for pentapeptide specificity using peptide analog inhibitors and substrates that vary at the P5 position. We determined the crystal structures of apo caspase-2, caspase-2 in complex with peptide inhibitors VDVAD-CHO, ADVAD-CHO, and DVAD-CHO, and a T380A mutant of caspase-2 in complex with VDVAD-CHO. Two residues, Thr-380 and Tyr-420, are identified to be critical for the P5 residue recognition; mutation of the two residues reduces the catalytic efficiency by about 4- and 40-fold, respectively. The structures also provide a series of snapshots of caspase-2 in different catalytic states, shedding light on the mechanism of capase-2 activation, substrate binding, and catalysis. By comparing the apo and inhibited caspase-2 structures, we propose that the disruption of a non-conserved salt bridge between Glu-217 and the invariant Arg-378 is important for the activation of caspase-2. These findings broaden our understanding of caspase-2 substrate specificity and catalysis.
Yinyan Tang, James A Wells, Michelle R Arkin

1761 related Products with: Structural and enzymatic insights into caspase-2 protein substrate recognition and catalysis.

200ul200ug100ul10 mg200ul200ug1000 TESTS/0.65ml200 assays200 assays25mg250ul

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#12147221   // To Up

Bcl-2 blocks apoptosis caused by pierisin-1, a guanine-specific ADP-ribosylating toxin from the cabbage butterfly.

Pierisin-1, a 98-kDa protein that induces apoptosis in mammalian cell lines, is capable of being incorporated into cells where it ADP-ribosylates guanine residues in DNA. To investigate the apoptotic pathway induced by this unique protein, the bcl-2 gene was transfected into HeLa cells. Cy2-fluorescent pierisin-1 was incorporated into the resultant cells expressing Bcl-2 protein and ADP-ribosylated dG was detected to almost the same extent as in parent cells. However, bcl-2-transfected HeLa cells did not display apoptotic morphological changes, PARP cleavage, and DNA fragmentation, indicating acquisition of resistance. In parent HeLa cells, activation of caspase-9 and release of cytochrome c were observed after 8h treatment with 0.5ng/ml pierisin-1. Caspase substrate assays revealed further cleavage of Ac-DEVD-pNA, Ac-VDVAD-pNA, and Ac-VEID-pNA, suggesting activation of caspase-2, -3, and -6 in pierisin-1-treated HeLa cells. The caspase-3 inhibitor, Ac-DEVD-CHO, was also found to inhibit apoptosis. In contrast, this caspase activation was not observed in bcl-2-transfected HeLa cells. Our results thus indicate that pierisin-1-induced apoptosis is mediated primarily via a mitochondrial pathway involving Bcl-2 and caspases.
Takashi Kanazawa, Takuo Kono, Masahiko Watanabe, Yuko Matsushima-Hibiya, Tsuyoshi Nakano, Kotaro Koyama, Noriaki Tanaka, Takashi Sugimura, Keiji Wakabayashi

1095 related Products with: Bcl-2 blocks apoptosis caused by pierisin-1, a guanine-specific ADP-ribosylating toxin from the cabbage butterfly.

2 Pieces/Box2 Pieces/Box4 Membranes/Box4 Arrays/Slide2 Pieces/Box1200ul50 ug4 Arrays/Slide5 mg

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