Dual role of caspase-11 in mediating activation of caspase-1 and caspase-3 under pathological conditions.

Caspase-11, a member of the murine caspase family, has been shown to be an upstream activator of caspase-1 in regulating cytokine maturation. We demonstrate here that in addition to its defect in cytokine maturation, caspase-11-deficient mice have a reduced number of apoptotic cells and a defect in caspase-3 activation after middle cerebral artery occlusion (MCAO), a mouse model of stroke. Recombinant procaspase-11 can autoprocess itself in vitro. Purified active recombinant caspase-11 cleaves and activates procaspase-3 very efficiently. Using a positional scanning combinatorial library method, we found that the optimal cleavage site of caspase-11 was (I/L/V/P)EHD, similar to that of upstream caspases such as caspase-8 and -9. Our results suggest that caspase-11 is a critical initiator caspase responsible for the activation of caspase-3, as well as caspase-1 under certain pathological conditions.

The Drosophila caspase DRONC cleaves following glutamate or aspartate and is regulated by DIAP1, HID, and GRIM.

The caspase family of cysteine proteases plays important roles in bringing about apoptotic cell death. All caspases studied to date cleave substrates COOH-terminal to an aspartate. Here we show that the Drosophila caspase DRONC cleaves COOH-terminal to glutamate as well as aspartate. DRONC autoprocesses itself following a glutamate residue, but processes a second caspase, drICE, following an aspartate. DRONC prefers tetrapeptide substrates in which aliphatic amino acids are present at the P2 position, and the P1 residue can be either aspartate or glutamate. Expression of a dominant negative form of DRONC blocks cell death induced by the Drosophila cell death activators reaper, hid, and grim, and DRONC overexpression in flies promotes cell death. Furthermore, the Drosophila cell death inhibitor DIAP1 inhibits DRONC activity in yeast, and DIAP1's ability to inhibit DRONC-dependent yeast cell death is suppressed by HID and GRIM. These observations suggest that DRONC acts to promote cell death. However, DRONC activity is not suppressed by the caspase inhibitor and cell death suppressor baculovirus p35. We discuss possible models for DRONC function as a cell death inhibitor.

Biochemical and genetic interactions between Drosophila caspases and the proapoptotic genes rpr, hid, and grim.

In Drosophila melanogaster, the induction of apoptosis requires three closely linked genes, reaper (rpr), head involution defective (hid), and grim. The products of these genes induce apoptosis by activating a caspase pathway. Two very similar Drosophila caspases, DCP-1 and drICE, have been previously identified. We now show that DCP-1 has a substrate specificity that is remarkably similar to those of human caspase 3 and Caenorhabditis elegans CED-3, suggesting that DCP-1 is a death effector caspase. drICE and DCP-1 have similar yet different enzymatic specificities. Although expression of either in cultured cells induces apoptosis, neither protein was able to induce DNA fragmentation in Drosophila SL2 cells. Ectopic expression of a truncated form of dcp-1 (DeltaN-dcp-1) in the developing Drosophila retina under an eye-specific promoter resulted in a small and rough eye phenotype, whereas expression of the full-length dcp-1 (fl-dcp-1) had little effect. On the other hand, expression of either full-length drICE (fl-drICE) or truncated drICE (DeltaN-drICE) in the retina showed no obvious eye phenotype. Although active DCP-1 protein cleaves full-length DCP-1 and full-length drICE in vitro, GMR-DeltaN-dcp-1 did not enhance the eye phenotype of GMR-fl-dcp-1 or GMR-fl-drICE flies. Significantly, GMR-rpr and GMR-grim, but not GMR-hid, dramatically enhanced the eye phenotype of GMR-fl-dcp-1 flies. These results indicate that Reaper and Grim, but not HID, can activate DCP-1 in vivo.

Purification and catalytic properties of human caspase family members.

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 kcat/Km 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, Ca2+) 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.

Well-differentiated endometrial adenocarcinoma in an infertility patient with later conception. A case report.

BACKGROUND: The incidence of well-differentiated endometrial adenocarcinoma in reproductive-age women is approximately 5%. When the women desires to retain her future fertility in light of this diagnosis, choices of surgery vs. medical therapy may present a dilemma for both the physician and patient. CASE: A young infertility patient with well-differentiated endometrial adenocarcinoma conceived by ovulation induction and intrauterine insemination after medical therapy. She subsequently delivered vaginally, and follow-up dilatation and curettage revealed no evidence of recurrent carcinoma. CONCLUSION: This case illustrates that with close observation by endometrial sampling for histologic diagnosis and follow-up, medical therapy can be an option for treating this condition to allow future fertility. The patient must be extensively counseled, however, concerning the nearly 33% chance of progression or recurrence of disease. One must also stress the importance of frequent evaluation of symptoms and endometrial pathology postpartum, with endometrial sampling as indicated and discussion of definitive surgical therapy once fertility is no longer desired.

Inhibition of human caspases by peptide-based and macromolecular inhibitors.

Studies with peptide-based and macromolecular inhibitors of the caspase family of cysteine proteases have helped to define a central role for these enzymes in inflammation and mammalian apoptosis. A clear interpretation of these studies has been compromised by an incomplete understanding of the selectivity of these molecules. Here we describe the selectivity of several peptide-based inhibitors and the coxpox serpin CrmA against 10 human caspases. The peptide aldehydes that were examined (Ac-WEHD-CHO, Ac-DEVD-CHO, Ac-YVAD-CHO, t-butoxycarbonyl-IETD-CHO, and t-butoxycarbonyl-AEVD-CHO) included several that contain the optimal tetrapeptide recognition motif for various caspases. These aldehydes display a wide range of selectivities and potencies against these enzymes, with dissociation constants ranging from 75 pM to >10 microM. The halomethyl ketone benzyloxycarbonyl-VAD fluoromethyl ketone is a broad specificity irreversible caspase inhibitor, with second-order inactivation rates that range from 2.9 x 10(2) M-1 s-1 for caspase-2 to 2.8 x 10(5) M-1 s-1 for caspase-1. The results obtained with peptide-based inhibitors are in accord with those predicted from the substrate specificity studies described earlier. The cowpox serpin CrmA is a potent (Ki < 20 nM) and selective inhibitor of Group I caspases (caspase-1, -4, and -5) and most Group III caspases (caspase-8, -9, and -10), suggesting that this virus facilitates infection through inhibition of both apoptosis and the host inflammatory response.

Definition and redesign of the extended substrate specificity of granzyme B.

Granzyme B is a protease involved in the induction of rapid target cell death by cytotoxic lymphocytes. Definition of the substrate specificity of granzyme B allows for the identification of in vivo substrates in this process. By using the combinatorial methods of synthetic substrate libraries and substrate-phage display, an optimal substrate for granzyme B that spans over six subsites was determined to be Ile-Glu-Xaa-(Asp downward arrowXaa)-Gly, with cleavage of the Asp downward arrowXaa peptide bond. Granzyme B proteolysis was shown to be highly dependent on the length and sequence of the substrate, supporting the role of granzyme B as a regulatory protease. Arginine 192 was identified as a determinant of P3-Glu and P1-Asp substrate specificity. Mutagenesis of arginine 192 to glutamate reversed the preference for negatively charged amino acids at P3 to positively charged amino acids. The preferred substrate sequence matches the activation sites of caspase 3 and caspase 7 and thus is consistent with the role of granzyme B in activation of these proteases during apoptosis. The caspase substrate poly(ADP)-ribose polymerase is cleaved by granzyme B in a cell-free assay at two sites that resemble the granzyme B specificity determined by the combinatorial methods. Many caspase substrates contain granzyme B cleavage sites and are proposed as potential granzyme B targets, suggesting a redundant function with certain caspases.

Efficacy of treatment for unexplained infertility.

OBJECTIVE: To analyze the efficacy and cost-effectiveness of alternative treatments for unexplained infertility. DESIGN: Retrospective analysis of 45 published reports. SETTING: Clinical practices. PATIENT(S): Couples who met criteria for unexplained infertility. Women with Stage I or Stage II endometriosis were included. INTERVENTION(S): Observation; clomiphene citrate (CC); gonadotropins (hMG); IUI; and GIFT and IVF. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate. RESULT(S): Combined pregnancy rates per initiated cycle, adjusted for study quality, were as follows: no treatment = 1.3%-4.1%; IUI = 3.8%; CC = 5.6%; CC + IUI = 8.3%; hMG = 7.7%; hMG + IUI = 17.1%; IVF = 20.7%; GIFT = 27.0%. The estimated cost per pregnancy was $10,000 for CC + IUI, $17,000 for hMG + IUI, and $50,000 for IVF. CONCLUSION(S): Clomiphene citrate + IUI is a cost-effective treatment for unexplained infertility. If this treatment fails, hMG + IUI and assisted reproduction are efficacious therapeutic options.

The caspase-3 precursor has a cytosolic and mitochondrial distribution: implications for apoptotic signaling.

Caspase-3-mediated proteolysis is a critical element of the apoptotic process. Recent studies have demonstrated a central role for mitochondrial proteins (e.g., Bcl-2 and cytochrome c) in the activation of caspase-3, by a process that involves interaction of several protein molecules. Using antibodies that specifically recognize the precursor form of caspase-3, we demonstrate that the caspase-3 proenzyme has a mitochondrial and cytosolic distribution in nonapoptotic cells. The mitochondrial caspase-3 precursor is contained in the intermembrane space. Delivery of a variety of apoptotic stimuli is accompanied by loss of mitochondrial caspase-3 precursor staining and appearance of caspase-3 proteolytic activity. We propose that the mitochondrial subpopulation of caspase-3 precursor molecules is coupled to a distinct subset of apoptotic signaling pathways that are Bcl-2 sensitive and that are transduced through multiple mitochondrion-specific protein interactions.

Cell death attenuation by 'Usurpin', a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by the CD-95 (Fas, APO-1) receptor complex.

Apoptotic cell suicide initiated by ligation of CD95 (Fas/APO-1) occurs through recruitment, oligomerization and autocatalytic activation of the cysteine protease, caspase-8 (MACH, FLICE, Mch5). An endogenous mammalian regulator of this process, named Usurpin, has been identified (aliases for Usurpin include CASH, Casper, CLARP, FLAME-1, FLIP, I-FLICE and MRIT). This protein is ubiquitously expressed and exists as at least three isoforms arising by alternative mRNA splicing. The Usurpin gene is comprised of 13 exons and is clustered within approximately 200 Kb with the caspase-8 and -10 genes on human chromosome 2q33-34. The Usurpin polypeptide has features in common with pro-caspase-8 and -10, including tandem 'death effector domains' on the N-terminus of a large subunit/small subunit caspase-like domain, but it lacks key residues that are necessary for caspase proteolytic activity, including the His and Cys which form the catalytic substrates diad, and residues that stabilize the P1 aspartic acid in substrates. Retro-mutation of these residues to functional caspase counterparts failed to restore proteolytic activity, indicating that other determinants also ensure the absence of catalytic potential. Usurpin heterodimerized with pro-caspase-8 in vitro and precluded pro-caspase-8 recruitment by the FADD/MORT1 adapter protein. Cell death induced by CD95 (Fas/APO-1) ligation was attenuated in cells transfected with Usurpin. In vivo, a Usurpin deficit was found in cardiac infarcts where TUNEL-positive myocytes and active caspase-3 expression were prominent following ischemia/reperfusion injury. In contrast, abundant Usurpin expression (and a caspase-3 deficit) occurred in surrounding unaffected cardiac tissue, suggesting reciprocal regulation of these pro- and anti-apoptotic molecules in vivo. Usurpin thus appears to be an endogenous modulator of apoptosis sensitivity in mammalian cells, including the susceptibility of cardiac myocytes to apoptotic death following ischemia/ reperfusion injury.

A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.

There is compelling evidence that members of the caspase (interleukin-1beta converting enzyme/CED-3) family of cysteine proteases and the cytotoxic lymphocyte-derived serine protease granzyme B play essential roles in mammalian apoptosis. Here we use a novel method employing a positional scanning substrate combinatorial library to rigorously define their individual specificities. The results divide these proteases into three distinct groups and suggest that several have redundant functions. The specificity of caspases 2, 3, and 7 and Caenorhabditis elegans CED-3 (DEXD) suggests that all of these enzymes function to incapacitate essential homeostatic pathways during the effector phase of apoptosis. In contrast, the optimal sequence for caspases 6, 8, and 9 and granzyme B ((I/L/V)EXD) resembles activation sites in effector caspase proenzymes, consistent with a role for these enzymes as upstream components in a proteolytic cascade that amplifies the death signal.

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

BACKGROUND: Interleukin-1beta converting enzyme (ICE/caspase-1) is the protease responsible for interleukin-1beta (IL-1beta) production in monocytes. It was the first member of a new cysteine protease family to be identified. Members of this family have functions in both inflammation and apoptosis. RESULTS: A novel method for identifying protease specificity, employing a positional-scanning substrate library, was used to determine the amino-acid preferences of ICE. Using this method, the complete specificity of a protease can be mapped in the time required to perform one assay. The results indicate that the optimal tetrapeptide recognition sequence for ICE is WEHD, not YVAD, as previously believed, and this led to the synthesis of an unusually potent aldehyde inhibitor, Ac-WEHD-CHO (Ki = 56 pM). The structural basis for this potent inhibition was determined by X-ray crystallography. CONCLUSIONS: The results presented in this study establish a positional-scanning library as a powerful tool for rapidly and accurately assessing protease specificity. The preferred sequence for ICE (WEHD) differs significantly from that found in human pro-interleukin-1beta (YVHD), which suggests that this protease may have additional endogenous substrates, consistent with evidence linking it to apoptosis and IL-1alpha production.

The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis.

Cysteine proteases related to mammalian interleukin-1 beta converting enzyme (ICE) and to its Caenorhabditis elegans homologue, CED-3, play a critical role in the biochemical events that culminate in apoptosis. We have determined the three-dimensional structure of a complex of the human CED-3 homologue CPP32/apopain with a potent tetrapeptide-aldehyde inhibitor. The protein resembles ICE in overall structure, but its S4 subsite is strikingly different in size and chemical composition. These differences account for the variation in specificity between the ICE- and CED-3-related proteases and enable the design of specific inhibitors that can probe the physiological functions of the proteins and disease states with which they are associated.

Human IL-1 beta processing and secretion in recombinant baculovirus-infected Sf9 cells is blocked by the cowpox virus serpin crmA.

Biologically active, mature IL-1 beta (mIL-1 beta) is released from activated monocytes after proteolytic processing from an inactive precursor (pIL-1 beta). IL-1 beta converting enzyme (ICE), the first member of a newly discovered family of cysteine proteinases, is required for this processing event. The cleaved cytokine is released from monocytes by an unknown mechanism which does not employ a standard hydrophobic signal sequence. As in mammalian fibroblasts, insect Sf9 cells do not normally process or secrete human IL-1 beta. The expression of active ICE enables Sf9 cells to process 31-kDa pIL-1 beta correctly at Asp27 and Asp116, and to export 17.5-kDa mIL-1 beta. The recombinant heterodimeric human enzyme purified from Sf9 cells possesses a sp. act. of 2.9 +/- 0.5 x 10(6) U/mg and is indistinguishable from native ICE with regard to its subunit composition and catalytic properties. In this system, co-expression of the cowpox virus crmA gene, an extremely potent serpin inhibitor of ICE (Ki < 7 pM), inhibits ICE activation completely and blocks pIL-1 beta processing and mIL-1 beta secretion by approximately 95%. The results indicate that ICE, in addition to its processing function, facilitates the transport of IL-1 beta across the plasma membrane.

A synthetic inhibitor of interleukin-1 beta converting enzyme prevents endotoxin-induced interleukin-1 beta production in vitro and in vivo.

A potent, reversible, tetrapeptide inhibitor of interleukin-1 beta converting enzyme (ICE), L-709,049, has been shown to suppress the in vitro production of mature IL-1 beta. We now report that this inhibitor also effectively suppresses the production of mature IL-1 beta in a murine model of endotoxic shock. Intraperitoneal administration of L-709,049 reduced the elevations of IL-1 beta in the plasma and peritoneal fluid of mice treated with LPS in a dose-related manner (ED50 = 2 +/- 0.9 mg/kg). LPS-induced elevations in IL-1 alpha and IL-6 in these mice were unaffected, indicating that the inhibitor specifically affected IL-1 beta production. Immunoblot analysis of plasma and peritoneal fluid indicated that L-709,049 suppressed the formation of mature IL-1 beta production in vivo. When mouse blood was incubated in vitro with LPS, IL-1 beta was released into the plasma. This assay was used to determine ex vivo the activity of an ICE inhibitor in the blood following its administration to mice. Blood obtained 15 minutes after ip administration of 10 mg/kg of L-709,049 to mice produced 80% less IL-1 beta than control blood, and IL-1 beta production returned to control levels in blood obtained 30 minutes after injection of this inhibitor. In addition, the capacity of the blood plasma obtained from these animals to prevent the cleavage of a synthetic substrate by ICE disappeared within 1 h of ip administration of 50 mg/kg of inhibitor.

Inhibition of interleukin-1 beta converting enzyme by the cowpox virus serpin CrmA. An example of cross-class inhibition.

We reported previously that human interleukin-1 beta converting enzyme (ICE) is regulated by the CrmA serpin encoded by cowpox virus. We now report the mechanism and kinetics of this unusual inhibition of a cysteine proteinase by a member of the serpin superfamily previously thought to inhibit serine proteinase only. CrmA possesses several characteristics typical of a number of inhibitory serpins. It is conformationally unstable, unfolding around 3 M urea, and stable to denaturation in 8 M urea upon complex formation with ICE. CrmA rapidly inhibits ICE with an association rate constant (kon) of 1.7 x 10(7) M-1 s-1, forming a tight complex with an equilibrium constant for inhibition (Ki) of less than 4 x 10(-12) M. These data indicate that CrmA is a potent inhibitor of ICE, consistent with the dramatic effects of CrmA on modifying host responses to virus infection. The inhibition of ICE by CrmA is an example of a "cross-class" interaction, in which a serpin inhibits a non-serine proteinase. Since CrmA possesses characteristics shared by inhibitors of serine proteinases, we presume that ICE, though it is a cysteine proteinase, has a substrate binding geometry strikingly close to that of serine proteinases. We reason that it is the substrate binding geometry, not the catalytic mechanism of a proteinase, that dictates its reactivity with protein inhibitors.

NIH 3T3 cells transfected with a yeast H(+)-ATPase have altered sensitivity to insulin, insulin growth factor-I, and platelet-derived growth factor-AA.

The role of intracellular pH (pHin) in the regulation of cell growth in both normal and transformed cells is a topic of considerable controversy. In an effort to study this relationship NIH 3T3 cells were stably transfected with the gene for the yeast H(+)-ATPase, constitutively elevating their pHin. The resulting cell line, RN1a, has a transformed phenotype: The cells are serum independent for growth, clone in soft agar, and form tumors in nude mice. In the present study, we further characterize this system in order to understand how transfection with this proton pump leads to serum-independent growth, using defined media to investigate the effects of specific growth factors on the transfected and parental NIH 3T3 cells. While both cell lines show similar growth increases in response to platelet-derived growth factor (PDGF)-BB and epidermal growth factor (EGF), they respond differently to insulin, insulin-like growth factor-I (IGF-I) and PDGF-AA. RN1a cells exhibit increased growth at nanomolar concentrations of insulin but the parental cells had only a relatively minor response to insulin at 10 microM. Both cell lines showed some response to IGF-I in the nanomolar range but the response of RN1a cells was much larger. Differences in insulin and IGF-I receptor number alone could not explain these results. The two cell lines also respond differently to PDGF-AA. RN1a cells are relatively insensitive to stimulation by PDGF-AA and express fewer PDGF alpha receptors as shown by Northern blots and receptor-binding studies. We propose a unifying hypothesis in which the H(+)-ATPase activates a downstream element in the PDGF-AA signal transduction pathway that complements insulin and IGF-I signals, while leading to downregulation of the PDGF alpha receptor.

Inactivation of interleukin-1 beta converting enzyme by peptide (acyloxy)methyl ketones.

Interleukin-1 beta converting enzyme (ICE) is a cysteine protease in monocytes that is essential for the proteolytic activation of interleukin-1 beta, an important mediator of inflammation. Peptide (acyloxy)methyl ketones designed with the appropriate peptide recognition sequence (Ac-Tyr-Val-Ala-Asp-CH2-OC(O)Ar) are potent, competitive, irreversible inhibitors. Mass spectrometry and sequence analysis indicate that inactivation proceeds through expulsion of the carboxylate leaving group to form a thiomethyl ketone with the active site Cys285. The second-order inactivation rate is independent of leaving group pKa, with an approximate value of 1 x 10(6) M-1 s-1. This rate constant is directly proportional to the reaction macroviscosity, indicating that the rate-limiting step in inactivation is association of enzyme and inhibitor, rather than any bond-forming reactions. Affinity labeling of THP.1 monocytic cell cytosol with a biotinylated tetrapeptide (acyloxy)methyl ketone for 28 half-lives resulted in labeling of only ICE, demonstrating the selectivity of these inhibitors. These inhibitors are relatively inert toward other bionucleophiles such as glutathione (< 5 x 10(-4) M-1 s-1), making them excellent candidates for in vivo studies of enzyme inhibition.

Interleukin 1 beta (IL-1 beta) processing in murine macrophages requires a structurally conserved homologue of human IL-1 beta converting enzyme.

Murine interleukin 1 beta (IL-1 beta) convertase (mICE) was identified in cytosolic extracts of peritoneal exudate cells (PECs) and macrophage cell lines. mICE cleaves both the human and mouse IL-1 beta precursors (pIL-1 beta) at sites 1 and 2 but fails to cleave a human pIL-1 beta (Asp116 to Ala) mutant at site 2, indicating that Asp is required to the left of the scissile bond. Ac-Tyr-Val-Ala-Asp-amino-4-methyl coumarin, patterned after site 2 of human pIL-1 beta, is a fluorogenic substrate for mICE, while the tetrapeptide aldehyde Ac-Tyr-Val-Ala-Asp-CHO is a potent inhibitor (Ki = 3 nM) that prevents generation and release of mature IL-1 beta by PECs (IC50 = 7 microM). Cloning of a full-length 1.4-kb cDNA shows that mICE is encoded as a 402-aa proenzyme (p45) that can be divided into a prodomain (Met1-Asp122), followed by a p20 subunit (Gly123-Asp296), a connecting peptide (Ser297-Asp314), and a p10 subunit (Gly315-His402). At the amino acid level, p45, p20, and p10 are 62%, 60%, and 81% identical with human IL-1 beta convertase (hICE). The active site Cys284 lies within a completely conserved stretch of 18 residues; however, Ser289 in hICE, which aligns with the catalytic region of serine and viral cysteinyl proteases, is absent from mICE. Expression in Escherichia coli of a truncated cDNA encoding Asn119-His402 generated active enzyme, which was autocatalytically processed at three internal Asp-Xaa bonds to generate a p20 subunit (Asn119-Asp296) complexed with either p11 (Ala309-His402) or p10. Recombinant mICE cleaves murine pIL-1 beta accurately at the Asp117-Val118 bond. The striking similarities of the human and murine enzymes will make it possible to assess the therapeutic potential of hICE inhibitors in murine models of disease.