Caspase-1 (interleukin-1beta-converting enzyme) is inhibited by the human serpin analogue proteinase inhibitor 9.

The regulation of caspases, cysteine proteinases that cleave their substrates after aspartic residues, is poorly understood, even though they are involved in tightly regulated cellular processes. The recently discovered serpin analogue proteinase inhibitor 9 (PI9) is unique among human serpin analogues in that it has an acidic residue in the putative specificity-determining position of the reactive-site loop. We measured the ability of PI9 to inhibit the amidolytic activity of several caspases. The hydrolysis of peptide substrates by caspase-1 (interleukin-1beta-converting enzyme), caspase-4 and caspase-8 is inhibited by PI9 in a time-dependent manner. The rate of reaction of caspase-1 with PI9, as well as the rate of substrate hydrolysis of the initial caspase-PI9 complex, shows a hyperbolic dependence on the concentration of PI9, indicative of a two-step kinetic mechanism for inhibition with an apparent second-order rate constant of 7x10(2) M(-1).s(-1). The hydrolysis of a tetrapeptide substrate by caspase-3 is not inhibited by PI9. The complexes of caspase-1 and caspase-4 with PI9 can be immunoprecipitated but no complex with caspase-3 can be detected. No complex can be immunoprecipitated if the active site of the caspase is blocked with a covalent inhibitor. These results show that PI9 is an inhibitor of caspase-1 and to a smaller extent caspase-4 and caspase-8, but not of the more distantly related caspase-3. PI9 is the first example of a human serpin analogue that inhibits members of this class of cysteine proteinases.

Regulation of protein phosphatase 2A activity by caspase-3 during apoptosis.

Although the available evidence suggests that whereas the caspase family plays a major role in apoptosis, they are not the sole stimulators of death. A random yeast two-hybrid screen of a lymphocyte cDNA library (using caspase-3 as the bait) found an interaction between caspase-3 and the regulatory subunit Aalpha of protein phosphatase 2A. This protein was found to be a substrate for caspase-3, but not caspase-1, and could compete effectively against either a protein or synthetic peptide substrate. In Jurkat cells induced to undergo apoptosis with anti-Fas antibody, protein phosphatase 2A (PP2A) activity increased 4.5-fold after 6 h. By 12 h, the regulatory Aalpha subunit could no longer be detected in cell lysates. There was no change in the amount of the catalytic subunit. The effects on PP2A could be prevented by the caspase family inhibitors acetyl-Asp-Glu-Val-Asp (DEVD) aldehyde or Ac-DEVD fluoromethyl ketone. The mitogen-activated protein (MAP) kinase pathway is regulated by PP2A. At 12 h after the addition of anti-Fas antibody, a decrease in the amount of the phosphorylated forms of MAP kinase was observed. Again, this loss of activated MAP kinase could be prevented by the addition of DEVD-cho or DEVD-fmk. These data are consistent with a pathway whereby induction of apoptosis activates caspase-3. This enzyme then cleaves the regulatory Aalpha subunit of PP2A, increasing its activity. These data show that the activated PP2A will then effect a change in the phosphorylation state of the cell. These data provide a link between the caspases and signal transduction pathways.

Substrate specificities of caspase family proteases.

The caspase family represents a new class of intracellular cysteine proteases with known or suspected roles in cytokine maturation and apoptosis. These enzymes display a preference for Asp in the P1 position of substrates. To clarify differences in the biological roles of the interleukin-1beta converting enzyme (ICE) family proteases, we have examined in detail the specificities beyond the P1 position of caspase-1, -2, -3, -4, -6, and -7 toward minimal length peptide substrates in vitro. We find differences and similarities between the enzymes that suggest a functional subgrouping of the family different from that based on overall sequence alignment. The primary specificities of ICE homologs explain many observed enzyme preferences for macromolecular substrates and can be used to support predictions of their natural function(s). The results also suggest the design of optimal peptidic substrates and inhibitors.

Preparation of an autolysis-resistant interleukin-1 beta converting enzyme mutant.

We describe the expression, purification, and characterization of human interleukin-1 beta converting enzyme (ICE) containing an affinity tag and modified to resist autoproteolysis. The point mutation Asp381 to Glu was added to eliminate the major site of autolytic degradation while maintaining catalytic activity, and an N-terminal polyhistidine tag was added in place of the ICE pro-region to facilitate purification. N-His (D381E) ICE was expressed in Escherichia coli and purified by nickel-chelating Sepharose and size-exclusion chromatography (SEC). The enzyme was stabilized greater than 80-fold against autolytic degradation relative to wild-type N-His ICE. SDS-PAGE analysis with silver-staining revealed no impurities, and 85% of the protein was catalytically active as determined by titration with a novel titrant, PD 163594 (3-[2-(2-benzyloxycarbonylamino-3-methylbutyrylamino)prop ionylamino]-4- oxo-5-(2-oxo-2H-chromen-7-yloxypentanoic acid). An oxidized adduct of ICE with glutathione, formed by disulfide rearrangement with oxidized glutathione to inhibit and stabilize the enzyme during purification, was rapidly reduced upon exposure to 5 mM DTT. One mole of glutathione was released per mole of active enzyme. Of the nine cysteines in ICE, eight were present in their reduced form in the glutathione adduct. N-His (D381E) ICE cleaved Ac-YVAD-Amc with the Michaelis-Menten parameters K(M) = 14 microM and Kcat = 0.7 s-1, values essentially identical to those reported for enzyme from natural sources.

Stability and oligomeric equilibria of refolded interleukin-1beta converting enzyme.

We report the preparation and characterization of interleukin-1beta converting enzyme (ICE) refolded from its p20 and p10 protein fragments. Refolded ICE heterodimer (p20p10) was catalytically active but unstable, and in size exclusion chromatography eluted at an apparent molecular mass of 30 kDa. The mechanisms of the observed instability were pH-dependent dissociation at low enzyme concentrations, and autolytic degradation of the p10 subunit at high concentrations. Binding and subsequent removal of a high affinity peptidic inhibitor increased the apparent molecular mass to 43 kDa (by size exclusion chromatography), and significantly increased its stability and specific activity. Chemical cross-linking and SDS-polyacrylamide gel electrophoresis analysis of the 43-kDa size exclusion chromatography conformer revealed a 60-kDa species, which was absent in the 30-kDa conformer, suggesting that inhibitor binding caused formation of a (p20p10)2 homodimer. The observation of a reversible equilibrium between ICE (p20p10) and (p20p10)2 suggests that analogous associations, possibly between ICE and ICE homologs, can occur in vivo, resulting in novel oligomeric protease species.

Protease activity of in vitro transcribed and translated Caenorhabditis elegans cell death gene (ced-3) product.

The Caenorhabditis elegans cell death gene, ced-3, encodes one of the two proteins required for apoptosis in this organism. The primary sequence similarities between Ced-3 and the mammalian interleukin-1beta converting enzyme (ICE) suggest that these two proteins may have functionally similar active sites and that Ced-3 may function as a cysteine protease. Here we report that in vitro transcribed and translated Ced-3 protein (p56) underwent rapid processing to smaller fragments. Replacement of the predicted active site cysteine of Ced-3 with serine (C364S) prevented the generation of smaller proteolytic fragments, suggesting that the processing might be an autocatalytic process. Peptide aldehydes with aspartic acid at the P1 position blocked Ced-3 autocatalysis. Furthermore, the protease inhibition profile of Ced-3 was similar to the profile reported for ICE. These functional data demonstrate that Ced-3 is an Asp-dependent cysteine protease with substrate specificity similar to that of ICE. Aurintricarboxylic acid, an inhibitor of apoptosis in mammalian cells, blocked Ced-3 autocatalytic activity, suggesting that an aurintricarboxylic acid-sensitive Ced-3/ICE-related protease might be involved in the apoptosis pathway(s) in mammalian cells.

Nucleotide sequence of the Salmonella typhimurium mutL gene required for mismatch repair: homology of MutL to HexB of Streptococcus pneumoniae and to PMS1 of the yeast Saccharomyces cerevisiae.

The mutL gene of Salmonella typhimurium LT2 is required for dam-dependent methyl-directed DNA mismatch repair. We have cloned and sequenced the mutL gene of S. typhimurium LT2 and compared its sequence with those of the hexB gene product of the gram-positive bacterium Streptococcus pneumoniae and the PMS1 gene product of the yeast Saccharomyces cerevisiae. MutL was found to be quite similar to the HexB mismatch repair protein of S. pneumoniae and to the mismatch repair protein PMS1 of the yeast S. cerevisiae. The significant similarities among these proteins were confined to their amino-terminal regions and suggest common evolution of the mismatch repair machinery in those organisms. The DNA sequence for mutL predicted a gene encoding a protein of 618 amino acid residues with a molecular weight of 67,761. The assignment of reading frame was confirmed by the construction of a chimeric protein consisting of the first 30 amino acids of LacZ fused to residues 53 through 618 of MutL. Interestingly, the presence of excess amounts of this fusion protein in wild-type mutL+ cells resulted in a trans-dominant effect causing the cell to exhibit a high spontaneous mutation frequency.

Nucleotide sequence of the Salmonella typhimurium mutS gene required for mismatch repair: homology of MutS and HexA of Streptococcus pneumoniae.

The mutS gene product of Escherichia coli and Salmonella typhimurium is one of at least four proteins required for methyl-directed mismatch repair in these organisms. A functionally similar repair system in Streptococcus pneumoniae requires the hex genes. We have sequenced the S. typhimurium mutS gene, showing that it encodes a 96-kilodalton protein. Amino-terminal amino acid sequencing of purified S. typhimurium MutS protein confirmed the initial portion of the deduced amino acid sequence. The S. typhimurium MutS protein is homologous to the S. pneumoniae HexA protein, suggesting that they arose from a common ancestor before the gram-negative and gram-positive bacteria diverged. Overall, approximately 36% of the amino acids of the two proteins are identical when the sequences are optimally aligned, including regions of stronger homology which are of particular interest. One such region is close to the amino terminus. Another, located closer to the carboxy terminus, includes homology to a consensus sequence thought to be diagnostic of nucleotide-binding sites. A third one, adjacent to the second, is homologous to the consensus sequence for the helix-turn-helix motif found in many DNA-binding proteins. We found that the S. typhimurium MutS protein can substitute for the E. coli MutS protein in vitro as it can in vivo, but we have not yet been able to demonstrate a similar in vitro complementation by the S. pneumoniae HexA protein.

DNA and amino acid sequence analysis of structural and immunity genes of colicins Ia and Ib.

The nucleotide sequences for colicin Ia and colicin Ib structural and immunity genes were determined. The two colicins each consist of 626 amino acid residues. Comparison of the two sequences along their lengths revealed that the two colicins are nearly identical in the N-terminal 426 amino acid residues. The C-terminal 220 amino acid residues of the colicins are only 60% identical, suggesting that this is the region most likely recognized by their cognate immunity proteins. The predicted proteins for the colicin immunity proteins would contain 111 amino acids for the colicin Ia immunity protein and 115 amino acids for the colicin Ib immunity protein. The colicin immunity proteins have no detectable DNA or amino acid homology but do exhibit a conservation of overall hydrophobicity. The colicin immunity genes lie distal to and in opposite orientation to the colicin structural genes. The colicin Ia immunity protein was purified to apparent homogeneity by a combination of isoelectric focusing and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified Ia immunity protein was determined and was found to be in perfect agreement with that predicted from the DNA sequence of its structural gene. The Ia immunity protein is not a processed membrane protein.

Organization of the colicin Ib gene. Promoter structure and immunity domain.

The colicin Ib gene of the large low-copy-number plasmid Col Ib-P9 has been cloned into the plasmid vector pBR322. Observations of growth characteristics of strains carrying the cloned gene and analysis of insertion and deletion derivatives of the recombinant plasmid have led to the conclusion that colicin Ib production can occur without the colicin Ib immunity system. The direction of transcription for the colicin Ib gene was determined by use of an expression vector and subsequently confirmed by DNA-sequence analysis of the colicin Ib promoter. The physical maps of the cloned colicin Ia and colicin Ib genes exhibit a great deal of similarity which has enabled the construction of hybrid genes consisting of the N terminus of one colicin gene being linked to the C terminus of the other. From an analysis of the chimeric colicins and the locations of the fusions, we conclude that the information necessary for immunity recognition by colicin Ia and colicin Ib resides in the C-terminal half of the colicin proteins.