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           Search results for: Caspase 3 proform, human recombinant   

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#11437602   2001/07/04 Save this To Up

[W206R]-procaspase 3: an inactivatable substrate for caspase 8.

We report here the cloning and high-level expression of a soluble proform of human caspase 3 (Ser(24)-H(277)) engineered to contain a short stretch of N-terminal sequence (MTISDSPREQD) from the prosegment of procaspase 8 and a C-terminal heptahistidine tag. The precursor protein isolated from extracts of recombinant Escherichia coli by immobilized metal-ion affinity chromatography was predominantly unprocessed and migrated as a 32-kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gels. Incubation of this protein with recombinant human caspase 8 produced fragments characteristic of the properly processed caspase 3, but the product was inactive. Amino-terminal sequence analysis of the caspase 3 polypeptides proved that caspase 8 had specifically cleaved the Asp(175)-Ser(176) bond to yield the expected p18 and p12 subunits, with partial cleavage at the Asp(28)-Ser(29) bond to release the prosegment. The lack of caspase 3 activity was found to be the result of a fortuitous mutation in which Trp(206) in the S4 subsite was replaced by arginine (W206R). This mutant procaspase 3, which we call m-pro3, serves as a useful reagent with which to test the efficacy of caspase 8 inhibitors in blocking processing of the natural polypeptide substrate of this enzyme and may be valuable as a source of "proenzyme" for crystallographic analysis.

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#11124942   2001/03/27 Save this To Up

Synergistic activation of caspase-3 by m-calpain after neonatal hypoxia-ischemia: a mechanism of "pathological apoptosis"?

The relative contributions of apoptosis and necrosis in brain injury have been a matter of much debate. Caspase-3 has been identified as a key protease in the execution of apoptosis, whereas calpains have mainly been implicated in excitotoxic neuronal injury. In a model of unilateral hypoxia-ischemia in 7-day-old rats, caspase-3-like activity increased 16-fold 24 h postinsult, coinciding with cleavage of the caspase-3 proenzyme and endogenous caspase-3 substrates. This activation was significantly decreased by pharmacological calpain inhibition, using CX295, a calpain inhibitor that did not inhibit purified caspase-3 in vitro. Activation of caspase-3 by m-calpain, but not mu-calpain, was facilitated in a dose-dependent manner in vitro by incubating cytosolic fractions, containing caspase-3 proform, with calpains. This facilitation required the presence of some active caspase-3 and could be abolished by including the specific calpain inhibitor calpastatin. This indicates that initial cleavage of caspase-3 by m-calpain, producing a 29-kDa fragment, facilitates the subsequent cleavage into active forms. This is the first report to our knowledge suggesting a direct link between the early, excitotoxic, calcium-mediated activation of calpain after cerebral hypoxia-ischemia and the subsequent activation of caspase-3, thus representing a tentative pathway of "pathological apoptosis."

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#9862464   1999/01/11 Save this To Up

The Drosophila inhibitor of apoptosis D-IAP1 suppresses cell death induced by the caspase drICE.

Many members of the Inhibitor of Apoptosis (IAP) family inhibit cell death and existing data suggest at least two mechanisms of action. Drosophila IAPs (D-IAP1 and D-IAP2) and a baculovirus-derived IAP, Op-IAP, physically interact with and inhibit the anti-apoptotic activity of Reaper, HID, and Grim, three genetically defined inducers of apoptosis in Drosophila, while human IAPs, c-IAP1, c-IAP2, and X-IAP interact with a number of different proteins including specific members of the caspase family of cysteine proteases which are crucial in the execution of cell death. We have examined whether insect-active IAPs can inhibit apoptosis induced by selected caspases, Drosophila drICE, Sf-caspase-1, and mammalian caspase-3, in insect SF-21 cells. D-IAP1 inhibited apoptosis induced by the active forms of all three caspases tested and physically interacted with the active, but not the proform of drICE. MIHA, the mouse homolog of X-IAP and an effective inhibitor of caspase-3, also interacted with and blocked apoptosis induced by active drICE but was relatively ineffective in blocking Sf-caspase-1. Op-IAP and D-IAP2 were unable to inhibit effectively any of the active caspases tested and failed to interact with drICE. The Drosophila IAPs and Op-IAP, but not MIHA, blocked HID-initiated activation of pro-drICE. We conclude that D-IAP1 is capable of inhibiting the activation of drICE as well as inhibiting apoptosis induced by the active form of drICE. In contrast, D-IAP2 and Op-IAP are more limited in their inhibitory targets and may be limited to inhibiting the activation of caspases.

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