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Caspases (aspartate-specific cysteine proteases) play a critical role in the execution of the mammalian apoptotic program. To address the regulation of human caspase activation, we used the yeast Saccharomyces cerevisiae, which is devoid of endogenous caspases. The apical procaspases, -8beta and -10, were efficiently processed and activated in yeast. Although protease activity, per se, was insufficient to drive cell death, caspase-10 activity had little effect on cell viability, whereas expression of caspase-8beta was cytotoxic. This lethal phenotype was abrogated by co-expression of the pan-caspase inhibitor, baculovirus p35, and by mutation of the active site cysteine of procaspase-8beta. In contrast, autoactivation of the executioner caspase-3 and -6 zymogens was not detected. Procaspase-3 activation required co-expression of procaspase-8 or -10. Surprisingly, activation of procaspase-6 required proteolytic activities other than caspase-8, -10, or -3. Caspase-8beta or -10 activity was insufficient to catalyze the maturation of procaspase-6. Moreover, a constitutively active caspase-3, although cytotoxic in its own right, was unable to induce the processing of wild-type procaspase-6 and vice versa. These results distinguish sequential modes of activation for different caspases in vivo and establish a yeast model system to examine the regulation of caspase cascades. Moreover, the distinct terminal phenotypes induced by various caspases attest to differences in the cellular targets of these apoptotic proteases, which may be defined using this system.

To understand the mechanism of interleukin-1 beta converting enzyme (ICE) activation in apoptosis, we analyzed the expression of ICE mRNA in two human cell lines by reverse transcription-polymerase chain reaction technique. This resulted in the identification and cloning of four alternatively spliced ICE mRNA isoforms. Although all the alternative splicing events were within the coding sequence of ICE, the four ICE isoforms maintained open reading frames and were designated as ICE beta, gamma, delta, and epsilon. In ICE gamma, most of the propeptide (amino acids 20-112) is deleted, which suggests that it may function as a catalyst for ICE autoprocessing in vivo. In ICE delta, amino acids 288-335, which contain the cleavage sites between the p20 and p10 subunits of ICE, are deleted thus resulting in its inactivation. Intriguingly, in ICE epsilon amino acids 20-335, which encompass most of the propeptide and the p20 subunit, are deleted resulting in the formation of a molecule that is homologous to the p10 subunit. Examination of the ability of these four ICE isoforms to cause apoptosis revealed that only the parental ICE alpha and isoforms beta and gamma, but not isoforms delta and epsilon, can induce apoptosis when overexpressed in Sf9 insect cells. In addition, coexpression of the p20 and p10 but not the p20 and ICE epsilon in Sf9 cells results in apoptosis. Interestingly, expression of ICE epsilon and to a lesser degree ICE delta resulted in extension of the survival of baculovirus-infected cells in a manner similar to expression of BCL2. The ability of ICE epsilon to extend the survival of Sf9 cells suggests that baculovirus-induced apoptosis in these cells is mediated by an ICE-like protease. We show that ICE epsilon can bind to the p20 subunit of ICE and potentially may compete with the p10 subunit to form an inactive ICE complex. Therefore, by acting as a dominant inhibitor of ICE activity, ICE epsilon may regulate ICE activation in vivo.

Physical measurements were made on the cytosolic form of the liver [3H]dexamethasone receptor. These include a Stokes radius of 3.5 nm, determined by gel filtration, and sedimentation coefficients of 5.1 and 7-8S, by sucrose-density-gradient centrifugation. From these measurements, the following physical properties were calculated: apparent mol. wt. 78000 (the 5.1 S form); D app. 6.1 × 10(-7) cm2-S-1; f/fo 1.25; axial ratio 4.7; these indicate a globular protein. Measurements of sedimentation coefficient of cytosol steroid-receptor complexes previously subjected to various activating conditions gave different values and lead to the conclusion that the mechanism of activation in vitro enabling the steroid-receptor complex to bind to DNA is more complex than simple disaggregation to a uniform size.

Expression of the human Na/K-ATPase beta1 subunit is regulated by a mineralocorticoid- and glucocorticoid-responsive elements. Here we identified an MR and GR responsive element, at positions -650 to -630, within the beta1 gene promoter that is required for both MR and GR activation. Independent expression of MR and GR activated by aldosterone or triamcinolone acetonide (TA) leads to significant transactivation of the beta1 promoter. Yet coexpression of both receptors activated by aldosterone plus TA or cortisol results in a much lower induction, indicating that coexpression of MR and GR is inhibitory. Gel shift mobility assay using an oligonucleotide including the 21-base pair MRE/GRE with whole cell extracts prepared from CV-1 cells overexpressing MR or GR showed specific MR and GR binding to this sequence. Additionally, antibodies to both MR and GR effectively supershifted the protein-DNA complexes, indicating that these receptors bound to the DNA sequence. Finally, the 21-base pair MRE/GRE was capable of activating transcription from a heterologous promoter in response to both aldosterone and TA. Together these data indicate that the 21-base pair sequence represents a true MRE/GRE and that optimal activation of the human Na/K-ATPase beta1 promoter is controlled by mineralocorticoid and glucocorticoid hormones. It appears that an interaction of MR with GR on the beta1 promoter effectively down-regulates transcription.

Previous studies have demonstrated the ATP-dependent formation of a complex containing the heat shock protein hsp90, the unique hsp90 binding protein p23, and one of three high molecular weight immunophilins. In the present study, hsp90 and p23 are shown to form a complex that requires elevated temperature and ATP/Mg2+. Complex formation is strongly promoted by molybdate and by the nonionic detergent Nonidet P-40. ADP and the benzoquinone ansamycin, geldanamycin, are potent inhibitors of complex formation. The ATP-dependent process alters the state of hsp90, not p23, and influences the ability of hsp90 to bind to phenyl-Sepharose. Conversion of hsp90 to the ATP-bound state lowers its affinity for phenyl-Sepharose. These results show that hsp90 can exist in at least two functional states, one able to bind p23 and the other with a high affinity for hydrophobic resins. A model is presented where these states are dictated by the binding of either ATP or ADP.

Studies with RU26988, a synthetic glucocorticoid which does not bind to aldosterone receptors, suggest glucocorticoid-induced colonic cation transport is affected through glucocorticoid-specific receptors. RU26988 produced a 700% increase in sodium absorption and doubled transmural potential difference in proximal and distal colon of adrenalectomized rats. Scatchard analysis suggested a single class of receptors with a KD of approximately 10(-9) M. Competition of unlabeled steroids for [3H]triamcinolone acetonide-binding sites paralleled the steroids' biologic potency as glucocorticoids. Heat treatment (25 degrees C, 30 min) markedly enhanced binding of the glucocorticoid-receptor complexes to DNA-cellulose. The activated receptor from both proximal and distal colon was eluted in the prewash from DEAE-Sephadex A-50 anion exchange columns both in the presence and absence of protease inhibitors and has an estimated molecular weight (Stokes radius) of 33,000-37,000 (25-26 A). These results identify the colonic receptor as glucocorticoid binder IB, a receptor previously identified as the major binder only in kidney cortex. The finding of an apparently unique receptor in the two tissues where glucocorticoids stimulate cation transport suggests that the phenotypic response mediated by glucocorticoids in different tissues might be determined by the structure of the receptor and that glucocorticoid binder IB is the glucocorticoid cation transport receptor.

Kinetic and binding studies with substrates, products, and a spin-labeled product analogue of glutathione (sl-glutathione) have been used to characterize the kinetic mechanism and properties of the catalytic site of the homodimer YaYa of glutathione S-transferase. Product inhibition studies and inhibition by sl-glutathione indicate the random addition of substrates. The kinetically determined dissociation constant for the product S-(2,4-dinitrophenyl)glutathione is approximately 7 microM. A newly described spin-labeled product analogue, S-[[(2,2,5,5,-tetramethyl-1-oxy-3-pyrrolidinyl)-carbamoyl]methyl] glutathione (sl-glutathione), acts as a competitive inhibitor with respect to both substrates (glutathione and 1-Cl-2,4-dinitrobenzene) with a kinetically determined dissociation constant of approximately 40 microM. Analysis of the glutathione S-transferase X sl-glutathione complex by EPR gives a rigid limit spectrum indicative of highly immobilized spin label. Kinetic and EPR results support the proposal that sl-glutathione binds as a bisubstrate or product analogue by occupying both the glutathione and hydrophobic substrate sites. Binding studies of sl-glutathione by EPR give a dissociation constant of 28 microM and a single binding site per homodimer. Displacement of sl-glutathione by substrates and product have been used to directly determine enzyme-ligand dissociation constants. Dissociation constants of 2.1 mM, 17 microM, and 25 microM were obtained for glutathione, 1-Cl-2,4-dinitrobenzene and S-(2,4-dinitrophenyl)glutathione when enzyme was added to a mixture of sl-glutathione and the competing ligand. The dissociation constants for glutathione and 1-Cl-2,4-dinitrobenzene but not for S-(2,4-dinitrophenyl) glutathione were dependent on the order of addition, consistent with the existence of several kinetically stable conformations for the enzyme. The sl-glutathione described here may provide a useful analogue for similar studies with other glutathione S-transferases or other enzymes which bind glutathione.

The unactivated, molybdate-stabilized rat hepatic glucocorticoid receptor has been purified approximately 4000-fold, as calculated by specific radioactivity, by affinity chromatography using a deoxycorticosterone-derivatized agarose, gel filtration on Bio-Gel A-1.5m agarose, and DEAE-cellulose chromatography. The final receptor sediments at 9-10 S in low salt (40 mM KCl) glycerol gradients containing molybdate. Elevated salt concentrations up to 1 M KCl reduce the sedimentation coefficient to 8-9 S. The final DEAE-cellulose eluted complexes exhibit a Stokes radius of 7.3 nm, a value similar to that reported for receptors in crude cytosol. From the hydrodynamic parameters an apparent Mr = 303,000 can be calculated for the steroid-receptor complex. Analysis of the receptor-containing fractions from DEAE-cellulose chromatography by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates the occurrence of a major Mr = 90,000 protein band which closely followed the distribution of bound radioactivity. Two other proteins corresponding to Mr = 41,000 and 40,000 also exhibit the same distribution pattern. Saturation of cytosolic specific binding sites with unlabeled triamcinolone acetonide prior to receptor purification results in the disappearance of these three proteins from the DEAE-cellulose chromatogram. Furthermore, a Mr = 24,000 component, which is eluted from DEAE-cellulose at a salt concentration higher than that of the bound radioactivity peak itself, also disappears. These observations argue that the Mr = 90,000, 41,000, 40,000, and 24,000 components are related as components or degradation products of the unactivated, molybdate-stabilized rat hepatic glucocorticoid-receptor complex. Studies on the in vitro activation of purified steroid-receptor complexes have revealed that Sephadex G-25 gel filtration and warming (25 degrees C for 30 min) enables purified receptors to become activated judged by ability to bind to DNA-cellulose but to a lower extent than observed for receptors in crude tissue homogenates. A DNA-cellulose binding capacity, similar to that shown by crude liver cytosolic receptor under the same conditions, can be conferred on the purified complexes only in a reconstituted system in which crude cytosol has been added. Molybdate is shown to completely inhibit activation induced by gel-filtration and offers significant protection against heat-induced activation both in highly purified and reconstituted systems. The activation inhibitory effect of molybdate has also been confirmed by DEAE-cellulose chromatography.

The mechanism of glucocorticoid-induced internucleosomal DNA cleavage and cytolysis of lymphatic cells is not known. Recent data (Compton, M. M., and Cidlowski, J. A. (1987) J. Biol. Chem. 262, 8288–8292) suggested that in vivotreatment of rat thymocytes with glucocorticoids induces a nucleolytic “lysis gene” product(s) responsible for lymphocytolysis. In this paper, the possibility that lymphocytolysis may result from glucocorticoid-induced nuclease(s) was examined. Using the rat thymocytes as a model system, we have shown by electrophoretic, enzymatic, and amino acid sequence analysis that the putative glucocorticoid-induced nucleases identified recently by Compton and Cidlowski are in fact H1, H1°, and core histones, and their gross appearance is not the result of new histone protein synthesis, but a result of the release of histone-containing nucleosomes during chromatin breakdown. Evidence presented here shows that the putative induced nuclease activity is an artifact of the assay system employed. Because our data do not support induction of a glucocorticoid-induced nuclease(s), we examined the possibility that DNA cleavage might be induced by activation of a constitutive endogenous endonuclease. We have shown that it is possible to produce characteristic internucleosomal DNA cleavage of rat thymocytes, merely by incubating intact nuclei from untreated adrenalectomized rat thymocytes with Ca2+and Mg2+for a short period of time. However, in glucocorticoid-sensitive human CEM-C7 lymphocytes activation of internucleosomal DNA cleavage was independent of calcium uptake. We conclude that glucocorticoid induction of internucleosomal DNA fragmentation does not necessarily require expression of a new nuclease(s), but is the result of the activation of a constitutive endogenous endonuclease(s). Also, our data suggest that the mechanism which controls activation of internucleosomal DNA cleavage in rat thymocytes differs from that which operates in CEM-C7 lymphocytes.

The Mr 94,000 steroid binding component of rat hepatic glucocorticoid receptor purified 5000-fold under-goes calcium-stimulated phosphorylation in vitro by [gamma-32P]ATP. Exogenous histones can be phosphorylated by this preparation without calcium. Calmodulin did not stimulate phosphorylation of the glucocorticoid receptor beyond that obtained with calcium alone. Although the specific calmodulin inhibitor calmidazolium had no effect, trifluoperazine and chlorpromazine, nonspecific calmodulin inhibitors, abolished the calcium-dependent phosphorylation of receptor. EGTA blocks the effect of calcium; magnesium cannot substitute for calcium. Cyclic nucleotides (cAMP or cGMP) do not stimulate phosphorylation of the receptor in the absence of calcium. Phosphorylation of the glucocorticoid receptor is steroid dependent. Triamcinolone acetonide elicited activation and phosphorylation of receptor in the presence of calcium, whereas the antagonists progesterone, cortexolone, and beta-lapachone did not. Sodium molybdate, which blocks the thermal activation step, inhibits phosphorylation of the receptor. The activated form of the glucocorticoid receptor is required for phosphorylation to occur. The ATP analogues 8-azido-ATP or fluorosulfonylbenzoyl adenosine, inhibit phosphorylation of the Mr 94,000 component, implying the presence of an ATP binding site inherent to the receptor.

Modulator is the low molecular weight heat-stable inhibitor of glucocorticoid-receptor complex activation. We have purified modulator to apparent homogeneity from heated rat liver cytosol. This was accomplished using Sephadex G-15 gel filtration, Dowex 1 anion-exchange chromatography, and preparative silica high-performance liquid chromatography. The modulator preparation was judged to be homogeneous by analytical silica high-performance liquid chromatography, two-dimensional silica thin-layer chromatography, and proton nuclear magnetic resonance spectroscopy. The apparent concentration of modulator in rat liver cytosol is 6.5 microM. The purified modulator inhibits heat activation of the rat liver glucocorticoid-receptor complex and stabilizes the steroid binding ability of the unoccupied rat liver glucocorticoid receptor in a dose-dependent manner. At a concentration of 5-6.5 microM, modulator inhibits receptor activation and stabilizes the unoccupied receptor by 50%. At a concentration of 500-630 microM, sodium molybdate also inhibits receptor activation and stabilizes the unoccupied receptor by 50%. Thus, modulator appears to be the endogenous factor that exogenous sodium molybdate mimics in vitro. Chemical analysis of the purified modulator following two-dimensional silica thin-layer chromatography indicates that modulator is an aminophospholipid. Physical analysis of the purified modulator by infrared and nuclear magnetic resonance spectroscopy, as well as mass spectrometry, demonstrates that modulator is an ether aminophosphoglyceride.

Monoclonal antibodies to the rat hepatic glucocorticoid receptor (GR) were produced by using 4000-fold-purified unactivated rat hepatic GR as the immunogen in an immunization in vitro. Hybridomas were screened for anti-GR antibody production by using an enzyme-linked immunosorbent assay. The antibody, 3A6, described here, is an IgM (lambda). The interaction of 3A6 with the purified GR was explored by sedimentation analysis, where a shift of the 9 S GR to a form with a higher s20,w value was demonstrated. Binding specificity and sensitivity were demonstrated by protein immunoblotting. 3A6 cross-reacted with all rat tissue glucocorticoid receptors (GRs) examined, except those of the brain. Species cross-reactivity was observed with other mammalian GRs (from human CEM-C7 cells and from pig and mouse liver). Immunocytochemical localization of the GR was assessed by indirect immunofluorescence in intact fixed cells, which demonstrated intense cytoplasmic staining in the absence of pretreatment with glucocorticoids and nuclear localization when cells were pretreated with glucocorticoids. This monoclonal antibody significantly inhibited steroid binding to unoccupied receptor and DNA binding of activated steroid-receptor complexes. Furthermore, preincubation of the purified activated GR complex with 3A6 prevented phosphorylation of the GR in vitro. Thus 3A6 differs from previous monoclonal antibodies to the GR in its capacity to cross-react with the human GR and by its specificity for an epitope on or near a functional domain of the GR.

Employing the degenerate primer-dependent polymerase chain reaction approach used recently to clone human Mch2, we have identified and cloned the insect Spodoptera frugiperda target of the baculovirus antiapoptotic protein p35. This protein named Sf caspase-1 belongs to the family of caspases and is highly related to human Mch3 and CPP32 in sequence and specific activity. The proenzyme of Sf caspase-1 is 299 amino acids in length and can undergo autocatalytic processing in Escherichia coli to an active enzyme heterocomplex. Autoprocessing occurs at Asp-28, Asp-184, and Asp-195 to generate the large p19/p18 and small p12 subunits. Sf caspase-1 is able to induce apoptosis in Sf9 cells and is capable of cleaving p35 to similar sized fragments as observed with extracts from p35 null mutant baculovirus-infected Sf9 cells. Sf caspase-1 activity is potently inhibited by p35, suggesting that it is an important target of this antiapoptotic protein. Finally, the Sf9 nuclear immunophilin FKBP46 was identified as a death-associated substrate for Sf caspase-1.

Apoptosis induced by a variety of agents results in the proteolytic cleavage of a number of cellular substrates by enzymes related to interleukin 1beta-converting enzyme (ICE). A small number of substrates for these enzymes have been identified to date, including enzymes involved in DNA repair processes: poly(ADP-ribose) polymerase and DNA-dependent protein kinase. We describe here for the first time the specific cleavage of the heteronuclear ribonucleoproteins (hnRNPs) C1 and C2 in apoptotic cells induced to undergo apoptosis by a variety of stimuli, including ionizing radiation, etoposide, and ceramide. No cleavage was observed in cells that are resistant to apoptosis induced by ionizing radiation. Protease inhibitor data implicate the involvement of an ICE-like protease in the cleavage of hnRNP C. Using recombinant ICE-like proteases and purified hnRNP C proteins in vitro, we show that the C proteins are cleaved by Mch3alpha and CPP32 and, to a lesser extent, by Mch2alpha, but not by ICE, Nedd2, Tx, or the cytotoxic T-cell protease granzyme B. The results described here demonstrate that the hnRNP C proteins, abundant nuclear proteins thought to be involved in RNA splicing, belong to a critical set of protein substrates that are cleaved by ICE-like proteases during apoptosis.

Recent evidence suggests that CPP32 is an essential component of an aspartate-specific cysteine protease (ASCP) cascade responsible for apoptosis execution in mammalian cells. Activation of CPP32 could lead to activation of other downstream ASCPs, resulting in late morphological changes such as lamin cleavage and DNA fragmentation, observed in cells undergoing apoptosis. Here we describe the identification and cloning of a novel human ASCP named Mch6 from Jurkat T lymphocytes. We demonstrate that the pro-enzymes of Mch6 and the lamin-cleaving enzyme Mch2alpha are substrates for mature CPP32. Site-directed mutagenesis revealed that CPP32 processes pro-Mch6 preferentially at Asp330 to generate two subunits of molecular masses 37 kDa (p37) and 10 kDa (p10). However, CPP32 processes pro-Mch2alpha at three aspartate processing sites (Asp23, Asp179, and Asp193) to produce the large (p18) and small (p11) subunits of the mature Mch2alpha enzyme. The CPP32-processed Mch2alpha is capable of cleaving the VEIDN lamin cleavage site, indicating that CPP32 can, in fact, activate pro-Mch2alpha. Granzyme B at a concentration that allows processing and activation of CPP32 failed to process pro-Mch2alpha. However, incubation of pro-Mch2alpha with granzyme B in the presence of a cellular extract containing pro-CPP32 resulted in activation of pro-CPP32 and subsequent processing of pro-Mch2alpha. Interestingly, granzyme B can also process pro-Mch6 but at a site N-terminal to that cleaved by CPP32. These data suggest that Mch2alpha and Mch6 are downstream proteases activated in CPP32- and granzyme B-mediated apoptosis. This is the first demonstration of a protease cascade involving granzyme B, CPP32, Mch2alpha, and Mch6 and evidence that the lamin-cleaving enzyme Mch2 is a target of mature CPP32.