Search results for: Caspase 2 Substrate VDVAD pNA1000 assays
#23747563 2013/06/06 To Up
Determining the contributions of caspase-2, caspase-8 and effector caspases to intracellular VDVADase activities during apoptosis initiation and execution.
Apoptosis signaling crucially depends on caspase activities. Caspase-2 shares features of both initiator and effector caspases. Opinions are divided on whether caspase-2 activity is established during apoptosis initiation or execution in response to DNA damage, death receptor stimulation, or heat shock. So far, approaches towards measuring caspase-2 activity were restricted to analyses in cell homogenates and extracts, yielded inconsistent results, and were often limited in sensitivity, thereby contributing to controversies surrounding the role of caspase-2 during apoptosis. Furthermore, caspases overlap in substrate specificities, and caspase-8 as well as effector caspases may cleave the optimal VDVAD recognition motif as well. We therefore generated a highly sensitive Förster resonance energy transfer (FRET) substrate to determine the relative contribution of these caspases to VDVADase activity non-invasively inside living cells. We observed limited proteolysis of the substrate during apoptosis initiation in response to death receptor stimulation by FasL, TNFα and TRAIL. However, this activity was attributable to caspase-8 rather than caspase-2. Likewise, no caspase-2-specific activity was detected during apoptosis initiation in response to genotoxic stress (cisplatin, 5-FU), microtubule destabilization (vincristine), or heat shock. The contribution of caspase-2 to proteolytic activities during apoptosis execution was insignificant. Since even residual, ectopically introduced caspase-2 activity could readily be detected inside living cells in our measurements, we conclude, in contrast to several previous studies, that caspase-2 activity does not contribute to apoptosis in the scenarios investigated, and that instead caspase-8 and effector caspases are the most significant VDVADases during canonical apoptosis signaling.M Eugenia Delgado, Magnus Olsson, Frank A Lincoln, Boris Zhivotovsky, Markus Rehm
1511 related Products with: Determining the contributions of caspase-2, caspase-8 and effector caspases to intracellular VDVADase activities during apoptosis initiation and execution.
100 Tests200 Tests100 units1 kit1 kit100 units1 kit100 Tests1 kit1 kit100 Tests1000 testsRelated Pathways
#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
2219 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 ul (10 mM)20 µl (10 mM)12 x 25 ul2000 pcs200ul100 assaysRelated Pathways
#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
2400 related Products with: Structural and enzymatic insights into caspase-2 protein substrate recognition and catalysis.
200ug200ul100ul10 mg200ug200ul1000 TESTS/0.65ml100 mg100 ug/vial250ul9 x 25 assaysRelated Pathways
#20405221 // To Up
The execution phase of autophagy associated PCD during insect metamorphosis.
DCaroline O B Facey, Richard A Lockshin
1851 related Products with: The execution phase of autophagy associated PCD during insect metamorphosis.
5500 Units10 ug1x10e7 cells10100 IU96testsRelated Pathways
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#17658284 2007/06/26 To Up
Specific inhibition of caspase-8 and -9 in CHO cells enhances cell viability in batch and fed-batch cultures.
IChee Yong Yun, Sen Liu, Sing Fee Lim, Tianhua Wang, Beatrice Y F Chung, Joong Jiat Teo, Kok Hwee Chuan, Allyson S C Soon, Keng Siong Goh, Zhiwei Song
1926 related Products with: Specific inhibition of caspase-8 and -9 in CHO cells enhances cell viability in batch and fed-batch cultures.
96 tests1.00 flask1 mg10 ug1x10e7 cells-96 wells1.00 flask1 mg2 Pieces/BoxRelated Pathways
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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
2806 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/Box1200ul100ug Lyophilized14 Arrays/SlideRelated Pathways
#11078888 // To Up
Mechanism of nitric oxide-induced apoptosis in human neuroblastoma SH-SY5Y cells.
We have attempted to elucidate the precise mechanism of nitric oxide (NO)-induced apoptotic neuronal cell death. Enzymatic cleavages of DEVD-AFC, VDVAD-AFC, and LEHD-AFC (specific substrates for caspase-3-like protease (caspase-3 and -7), caspase-2, and caspase-9, respectively) were observed by treatment with NO. Western blot analysis showed that pro-forms of caspase-2, -3, -6, and -7 are decreased during apoptosis. Interestingly, Ac-DEVD-CHO, a caspase-3-like protease inhibitor, blocked not only the decreases in caspase-2 and -7, but also the formation of p17 from p20 in caspase-3 induced by NO, suggesting that caspase-3 exists upstream of caspase-2 and -7. Bongkrekic acid, a potent inhibitor of mitochondrial permeability transition, specifically blocked both the loss of mitochondrial membrane potential and subsequent DNA fragmentation in response to NO. Thus, NO results in neuronal apoptosis through the sequential loss of mitochondrial membrane potential, caspase activation, and degradation of inhibitor of caspase-activated DNase (CAD) (CAD activation).R Moriya, T Uehara, Y Nomura
1375 related Products with: Mechanism of nitric oxide-induced apoptosis in human neuroblastoma SH-SY5Y cells.
1 mg1.00 flask10 ug1.00 flask4 Membranes/Box5ug96tests50 ul 100ul100 4 Arrays/Slide1.00 flaskRelated Pathways
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