Purification and catalytic properties of human caspase family members

Abstract

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.

Apoptosis of hematopoietic cells induced by growth factor withdrawal is associated with caspase-9 mediated cleavage of Raf-1

“…Caspase-9 cleaves Raf-1 at position D279, which is conserved in human and mouse Raf-1. D279 and its context do not fit the proposed consensus motif for a caspase-9 cleavage site (Thornberry et al, 1997;Garcia-Calvo et al, 1999), but the autocatalytic cleavage site (PEPDA) in caspase-9 itself also exhibits poor similarity with the consensus sequence (Stennicke et al, 1999). Previous work has shown that Raf-1 contains an autoinhibitory domain within the first 330 amino acids of its N-terminus (Cutler et al, 1998;.…”

Novel protein purification system utilizing an N-terminal fusion protein and a caspase-3 cleavable linker

“…In addition, other group III caspases, such as caspase-2 and -7, may be used with the present cleavage recognition site [17,18]. We note that other caspase proteins may be used as an alternative to caspase-3 if one determines that the aspartate in the P4 subsite (DXXD) results in a cleavage site in the target protein.…”

“…We note that other caspase proteins may be used as an alternative to caspase-3 if one determines that the aspartate in the P4 subsite (DXXD) results in a cleavage site in the target protein. For example, group II caspases, caspase-6, -8, -9, and -10 have a preference for branched apolar residues in the P4 position, whereas group I caspases, caspase-1, -4, and -5 have a preference for aromatic amino acids in the P4 position [17,18]. It is conceivable that mutations similar to those described here in procaspase-3 (D9A, D28A) may be made in any of the other 13 caspases, creating a myriad of protein purification systems that exploit the properties of caspase enzymes.…”

Regulation of gene expression by the amyloid precursor protein: inhibition of the JNK/c-Jun pathway

“…This substrate is preferentially cleaved by caspase 3, and to a lesser extent by caspases 6, 7, 8, 9, and 10. 51 Accumulation of AMC fluorescence was monitored over 1 h using an HTS fluorescent plate reader (excitation 380 nm, emission 465 nm). Fluorescence of blanks containing no cell lysate was subtracted from the values.…”