A combinatorial approach for determining protease specificities: application to interleukin-1β converting enzyme (ICE)

Rano, Thomas A.; Timkey, Tracy; Peterson, Erin P.; Rotonda, J.; Nicholson, Donald W.; Becker, Joseph W.; Chapman, Kevin T.; Thornberry, Nancy A.

doi:10.1016/s1074-5521(97)90258-1

Cited by 251 publications

(199 citation statements)

References 29 publications

Supporting

Mentioning

192

Contrasting

Unclassified

Order By: Relevance

“…Expression and Purification of Caspase-1-Separate p20 and p10 subunits of caspase-1 were expressed in Escherichia coli BL21(DE3), harboring pET11a-ICEp20 or pET11a-ICEp10 as inclusion bodies in LB medium containing 150 g/ml ampicillin (17). Cells were grown at 37°C to A 600 ϭ 0.6, induced with 1 mM isopropyl 1-thio-␤-D-galactopyranoside, then further incubated overnight and harvested by centrifugation.…”

Section: Methodsmentioning

confidence: 99%

Cytokine Response Modifier A Inhibition of Initiator Caspases Results in Covalent Complex Formation and Dissociation of the Caspase Tetramer

Dobó

Swanson

Salvesen

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

Active caspases are generally composed of two catalytic domains, each containing a large (p20) and a small (p10) subunit so that a fully active caspase has the organization (p20-p10) 2 . The cowpox serpin crmA suppresses host apoptosis and inflammation by inhibiting endogenous caspases. We report on the mechanism crmA uses to inhibit caspases 1, 6, and 8. Native PAGE showed formation of tight crmA-caspase complexes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry provided evidence for a covalent crmA-p20 thioester linkage. SDS-PAGE of isolated complexes showed near complete loss of the p10 subunit from initiator caspases 1 and 8 but not from the executioner caspase-6. This was confirmed for caspase-1 by sequencing and Western blotting. Size exclusion chromatography indicated a size of ϳ60 kDa for complexes with caspases 1 and 8, consistent with a crmA⅐p20 species, suggesting that the p20-p10 interface and possibly the p10-p10 interface had been disrupted. In contrast, crmA⅐caspase-6 complex behaved as an equilibrium mixture of crmA 2 ⅐(p20-p10) 2 and crmA⅐(p20-p10). Complex deacylation rates were all slow, suggesting effective kinetic trapping of the covalent thioacyl intermediate. These results suggest a novel serpin inhibition mechanism through which crmA down-regulates apoptosis and inflammation. This involves (i) rapid formation of covalent complex with initiator caspases 8 or 1, (ii) very slow deacylation, and (iii) loss of the caspase p10 subunit for initiator but not for executioner caspases, so that any free p20 formed by deacylation of initiator caspases cannot reassociate to active heterotetramer, thus resulting in irreversible inhibition of apoptosis and inflammation.Caspases are intracellular cysteine proteinases involved in inflammation and apoptosis (1). Caspase-1, also known as interleukin-1␤ converting enzyme (ICE), 3 activates the pro-inflammatory cytokine interleukin-1␤ by proteolysis (2), whereas caspase-8, itself activated by dimerization through an extrinsic receptor-mediated pathway (3, 4), acts as an initiator of apoptosis through downstream proteolytic activation of so-called "executioner" caspases, such as caspases -3, -6, and -7 (5). Active caspases are obligate homodimers, with each monomer composed of a large (p20, 17-20 kDa) and a small (p10, 9 -12 kDa) subunit, which are formed after the activation cleavage of the procaspase (6). Structures of several caspases are known, and all show that two p20-p10 heterodimers are associated in an anti-parallel arrangement through the ␤-sheet of the two p10 subunits (7-11). The p20 subunit, however, contains both the catalytic cysteine and histidine (6).Orthopox viruses, such as the cowpox virus, enhance their infectivity through specific inhibition of caspases and consequent abrogation of the inflammatory response and of apoptosis (12). They do this using an inhibitor that is a member of the serpin family (13). In the case of cowpox virus, the serpin is crmA (14). crmA can inhibit a range of caspases, although at ver...

show abstract

Section: Methodsmentioning

confidence: 99%

Cytokine Response Modifier A Inhibition of Initiator Caspases Results in Covalent Complex Formation and Dissociation of the Caspase Tetramer

Dobó

Swanson

Salvesen

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Ac-WEHD-AMC and Ac-VEHD-AMC were prepared as previously described. 27 Preparation of recombinant caspase-1, -2, -3, -4, -5, -7, -8 and -9…”

Section: Methodsmentioning

confidence: 99%

“…Caspase-1 was purified to homogeneity by affinity chromatography as reported previously. 27 Preparation of recombinant caspases-6 and -10…”

Section: Puri®cation Of Recombinant Caspasesmentioning

confidence: 99%

Purification and catalytic properties of human caspase family members

et al. 1999

View full text Add to dashboard Cite

Members of the caspase family of cysteine proteases are known to be key mediators of mammalian inflammation and apoptosis. To better understand the catalytic properties of these enzymes, and to facilitate the identification of selective inhibitors, we have systematically purified and biochemically characterized ten homologues of human origin (caspases 1 ± 10). The method used for production of most of these enzymes involves folding of active enzymes from their constituent subunits which are expressed separately in E. coli, followed by ion exchange chromatography. In cases where it was not possible to use this method (caspase-6 and -10), the enzymes were instead expressed as soluble proteins in E. coli, and partially purified by ion exchange chromatography. Based on the optimal tetrapeptide recognition motif for each enzyme, substrates with the general structure Ac-XEXD-AMC were used to develop continuous fluorometric assays. In some cases, enzymes with virtually identical tetrapeptide specificities have k cat /K m values for fluorogenic substrates that differ by more than 1000-fold. Using these assays, we have investigated the effects of a variety of environmental factors (e.g. pH, NaCl, Ca 2+ ) on the activities of these enzymes. Some of these variables have a profound effect on the rate of catalysis, a finding that may have important biological implications.

show abstract

“…58 These inhibitors act as pseudosubstrates for active caspases ( Figure 1a) and are therefore competitive inhibitors. Peptides have been linked to chemical groups that serve to block the amino acids and to improve cell permeability, stability and efficacy.…”

Section: Synthetic Caspase Inhibitorsmentioning

confidence: 99%