Enzymatic activity of two caspases related to interleukin-1beta-converting enzyme.
Interleukin-1beta-converting enzyme is a member of a family of human cysteine proteases with specificity for aspartic acid, which have been named caspases. Within this family of enzymes, transcript X (TX) and transcript Y (TY) (caspases 4 and 5, respectively) are very similar to ICE (caspase 1) and form the ICE subfamily. Given the high degree of conservation in the sequences of these proteases (more than 50% amino acid identity in the mature enzymes), it was of interest to examine whether they shared similar substrate specificities. The three enzymes, ICE, TX and TY, were therefore expressed in baculovirus-infected insect cells, as 30-kDa proteins lacking the propeptide. Automaturation into p20 and p10 subunits occurred within the cells. Active ICE, TX and TY were collected in the cell culture supernatants. In addition, their production induced the activation of an endogenous 32-kDa putative cysteine protease (CPP32) like caspase. T7-tagged ICE, TX and TY were purified by immunoaffinity and tested for their catalytic efficiency on YVAD-containing synthetic substrates and on the ICE natural substrate, pro-interleukin-1beta. TX cleaved the same synthetic substrates as ICE (Km of 90 microM and k(cat) of 0.4 s(-1) for Suc-YVAD-NH-Mec, where Suc represents succinyl and NH-Mec represents amino-4-methylcoumarin) and could cleave pro-interleukin-1beta into the same peptides as ICE but less efficiently. On the other hand, TY showed very little efficacy on the different ICE substrates (Km of 860 microM for Suc-YVAD-NH-Mec). These results show that the ICE/TX/TY subfamily has functional heterogeneity and that ICE remains the preferred enzyme for pro-interleukin-1beta cleavage.
Fassy
F
F
Hoechst Marion Roussel, Romainville, France. florence.fassy@hmrag.com
Krebs
O
O
Rey
H
H
Komara
B
B
Gillard
C
C
Capdevila
C
C
Yea
C
C
Faucheu
C
C
Blanchet
A M
AM
Miossec
C
C
Diu-Hercend
A
A
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D020170:Caspase 1; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007313:Insecta; D007375:Interleukin-1 / Q000378:metabolism; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D007375:Interleukin-1; D009842:Oligopeptides; D011498:Protein Precursors; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; D020170:Caspase 1