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

ABSTRACT: 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.

SUBMITTER: Annand RR

PROVIDER: S-EPMC1220507 | biostudies-other | 1999 Sep

REPOSITORIES: biostudies-other

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Project description:BackgroundSerine proteinase inhibitors (Serpins) are a large superfamily of structurally related, but functionally diverse proteins that control essential proteolytic pathways in most branches of life. Given their importance in the biology of many organisms, the concept that ticks might utilize serpins to evade host defenses and immunizing against or disrupting their functions as targets for tick control is an appealing option.ResultsA sequence homology search strategy has allowed us to identify at least 45 tick serpin genes in the Ixodes scapularis genome that are structurally segregated into 32 intronless and 13 intron-containing genes. Nine of the intron-containing serpins occur in a cluster of 11 genes that span 170 kb of DNA sequence. Based on consensus amino acid residues in the reactive center loop (RCL) and signal peptide scanning, 93% are putatively inhibitory while 82% are putatively extracellular. Among the 11 different amino acid residues that are predicted at the P1 sites, 16 sequences possess basic amino acid (R/K) residues. Temporal and spatial expression analyses revealed that 40 of the 45 serpins are differentially expressed in salivary glands (SG) and/or midguts (MG) of unfed and partially fed ticks. Ten of the 38 serpin genes were expressed from six to 24 hrs of feeding while six and fives genes each are predominantly or exclusively expressed in either MG and SG respectively.ConclusionGiven the diversity among tick species, sizes of tick serpin families are likely to be variable. However this study provides insight on the potential sizes of serpin protein families in ticks. Ticks must overcome inflammation, complement activation and blood coagulation to complete feeding. Since these pathways are regulated by serpins that have basic residues at their P1 sites, we speculate that I. scapularis may utilize some of the serpins reported in this study to manipulate host defense. We have discussed our data in the context of advances on the molecular physiology of I. scapularis. Although the paper is descriptive, this study provides the first step toward a comprehensive understanding of serpins in tick physiology.

Project description:Several members of the IL-1beta-converting enzyme (ICE) family of proteases recently have been implicated in the intracellular cascade mediating the apoptotic death of various cell types. It is unclear, however, whether ICE-related proteases are involved in apoptosis of mammalian neurons and, if so, how they are activated. Here we report the cloning of an ICE-related protease (IRP) from rat brain, which displays strong sequence identity to human CPP32. In situ hybridization histochemistry reveals that this IRP mRNA is expressed in neuron-enriched regions of the developing and adult rat brain but is profoundly downregulated in the adult (compared with developing) brain. To investigate whether this IRP is involved in the death of neurons in the developing brain, we studied IRP expression in cultured cerebellar granule neurons. In cultured cerebellar granule neurons, reduction of extracellular K+ reliably induces apoptosis and stimulates overexpression of IRP mRNA. The latter is especially prominent 4 hr after switching from high K+ to low K+ medium. The expression of IRP mRNA was maintained at this level for at least 8 hr and was followed by apoptotic death of these neurons. Induction of IRP mRNA and cell death are blocked completely by adding depolarizing concentrations of K+ </=90 min after switching to low K+ medium (i.e., before the commitment point for apoptosis) and partially blocked by brain-derived neurotrophic factor (BDNF), which also partially rescues granule neurons from low K+-induced apoptosis. In addition, overexpression of IRP cDNA in HeLa cells results in cell death accompanied by strong internucleosomal cleavage of DNA, a typical feature of apoptosis. Finally, we detected cleavage of the putative death substrate poly (ADP-ribose) polymerase (PARP), beginning 8 hr after changing from high K+ to low K+ medium, coinciding with the time course of induced expression of the IRP gene. Our data suggest that transcriptional activation of IRP could be one of the mechanisms involved in the apoptotic death of cerebellar granule neurons.

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

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