Structures of the free and inhibitors-bound forms of bromelain and ananain from Ananas comosus stem and in vitro study of their cytotoxicity.

The Ananas comosus stem extract is a complex mixture containing various cysteine ​​proteases of the C1A subfamily, such as bromelain and ananain. This mixture used for centuries in Chinese medicine, has several potential therapeutic applications as anti-cancer, anti-inflammatory and ecchymosis degradation agent. In the present work we determined the structures of bromelain and ananain, both in their free forms and in complex with the inhibitors E64 and TLCK. These structures combined with protease-substrate complexes modeling clearly identified the Glu68 as responsible for the high discrimination of bromelain in favor of substrates with positively charged residues at P2, and unveil the reasons for its weak inhibition by cystatins and E64. Our results with purified and fully active bromelain, ananain and papain show a strong reduction of cell proliferation with MDA-MB231 and A2058 cancer cell lines at a concentration of about 1 µM, control experiments clearly emphasizing the need for proteolytic activity. In contrast, while bromelain and ananain had a strong effect on the proliferation of the OCI-LY19 and HL-60 non-adherent cell lines, papain, the archetypal member of the C1A subfamily, had none. This indicates that, in this case, sequence/structure identity beyond the active site of bromelain and ananain is more important than substrate specificity.

Synthesis and acrosin inhibitory activities of substituted ethyl 5-(4-aminophenyl)-1H-pyrazole-3-carboxylate derivatives.

A series of novel ethyl 5-(4-aminophenyl)-1H-pyrazole-3-carboxylate derivatives were designed and synthesized and their in vitro acrosin inhibitory activities were evaluated. Most of the compounds exhibited acrosin inhibitory activities. Among them, three compounds (5l, 5n, and 5v) were more potent than that of the control TLCK. These provide a new structural type for the development of novel contraceptive acrosin inhibitory agents.

Malaria proteases mediate inside-out egress of gametocytes from red blood cells following parasite transmission to the mosquito.

Malaria parasites reside in human erythrocytes within a parasitophorous vacuole. The parasites are transmitted from the human to the mosquito by the uptake of intraerythrocytic gametocytes during a blood meal, which in the midgut become activated by external stimuli and subsequently egress from the enveloping erythrocyte. Gametocyte egress is a crucial step for the parasite to prepare for fertilization, but the molecular mechanisms of egress are not well understood. Via electron microscopy, we show that Plasmodium falciparum gametocytes exit the erythrocyte by an inside-out type of egress. The parasitophorous vacuole membrane (PVM) ruptures at multiple sites within less than a minute following activation, a process that requires a temperature drop and parasite contact with xanthurenic acid. PVM rupture can also be triggered by the ionophore nigericin and is sensitive to the cysteine protease inhibitor E-64d. Following PVM rupture the subpellicular membrane begins to disintegrate. This membrane is specific to malaria gametocytes, and disintegration is impaired by the aspartic protease inhibitor EPNP and the cysteine/serine protease inhibitor TLCK. Approximately 15 min post activation, the erythrocyte membrane ruptures at a single breaking point, which can be inhibited by inhibitors TLCK and TPCK. In all cases inhibitor treatment results in interrupted gametogenesis.

Mechanisms of growth inhibition in human papillomavirus positive and negative cervical cancer cells by the chloromethyl ketone protease inhibitor, succinyl-alanine-alanine-proline-phenylalanine chloromethyl ketone.

The chymotrypsin-like serine protease inhibitor, succinyl-alanine-alanine-proline-phenylalanine chloromethyl ketone (AAPF(CMK)), has been shown to have anticarcinogenic activity in a number of model systems and to be relatively selective for a nuclear protease. This inhibitor also has substantial effects on growth of tumorigenic human papillomavirus (HPV)-infected keratinocytes in organotypic raft cultures. Here, we examined the effects of AAPF(CMK) on cell growth, cell-cycle kinetics, apoptosis induction, and DNA synthesis in two human cervical carcinoma cell lines: SiHa cells, which have integrated high-risk HPV-16; and C33a cells, which do not contain HPV DNA. AAPF(CMK) inhibited growth of both cell lines in a time- and dose-dependent manner. Apoptosis studies showed no significant difference in drug-treated versus vehicle-treated cells in the C33a cell line. However, a significant dose-dependent increase in apoptosis occurred at a late time point in SiHa cells. Cell-cycle progression and DNA synthesis assays showed that the cellular mechanisms of growth inhibition by AAPF(CMK) differ between the HPV16-positive and HPV-negative tumorigenic cell lines. Drug-treated C33a cells showed a significant accumulation of cells in the G(2) phase of the cell cycle. In SiHa cells, growth inhibition produced by AAPF(CMK) seemed to result from a global arrest of the cell cycle. Although the molecular mechanisms involved in AAPF(CMK)-induced growth inhibition are distinct between the two tumorigenic cell lines, such differences may ultimately prove to have therapeutic utility. Novel therapies for treating established HPV infections are needed, because HPV is a causative agent in the development of multiple types of cancer.

Sequential activation of caspases and serine proteases (serpases) during apoptosis.

Analogous to caspases, serine (Ser) proteases are involved in protein degradation during apoptosis. It is unknown, however, whether Ser proteases are activated concurrently, sequentially, or as an alternative to the activation of caspases. Using fluorescent inhibitors of caspases (FLICA) and Ser proteases (FLISP), novel methods to detect activation of these enzymes in apoptotic cells, we demonstrate that two types of Ser protease sites become accessible to these inhibitors during apoptosis of HL-60 cells. The prior exposure to caspases inhibitor Z-VAD-FMK markedly diminished activation of both Ser protease sites. However, the unlabeled inhibitor of Ser-proteases TPCK had modest suppressive effect- while TICK had no effect- on the activation of caspases. Activation of caspases, thus, appears to be an upstream event and likely a prerequisite for activation of FLISP-reactive sites. Differential labeling with the red fluorescing sulforhodamine-tagged VAD-FMK and the green fluorescing FLISP allowed us to discriminate, within the same cell, between activation of caspases and Ser protease sites. Despite a certain degree of co-localization, the pattern of intracellular caspase- vs FLISP- reactive sites, was different. Also different were relative proportions of activated caspases vs Ser protease sites in individual cells. The observed induction of FLISP-binding sites we interpret as revealing activation of at least two different apoptotic Ser proteases; by analogy to caspases we denote them serpases. Their apparent molecular weight (62-65 kD) suggests that they are novel enzymes.

Ethanol-induced apoptosis in hepatoma cells proceeds via intracellular Ca(2+) elevation, activation of TLCK-sensitive proteases, and cytochrome c release.

Ethanol is known to induce apoptosis in hepatocytes. However, intracellular signaling events of ethanol-induced death are still only partially understood. We studied such processes in ethanol-induced apoptosis in HepG2 cells as a model system for human liver cells. We determined the incidence of apoptosis by DNA fragmentation and tested the effects of various known inhibitors. Ethanol induces apoptosis in HepG2 cells in a dose- and time-dependent manner as well as in rat primary hepatocytes. This effect was not mediated through the death receptor CD95 and the tumor necrosis factor receptors. It was efficiently inhibited by the caspase inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zVAD-fmk), the Ca(2+) chelator EGTA, and the serine protease inhibitor N-p-tosyl-l-lysine chloromethyl ketone (TLCK). Upon ethanol treatment, the intracellular calcium ion concentration was increased and cytochrome c was released from the mitochondria, and caspases were activated. EGTA and TLCK could inhibit cytochrome c release from the mitochondria. Furthermore, overexpression of Bcl-x(L) saved cells from ethanol-induced apoptosis. These data suggest that ethanol-induced apoptosis in liver cells is initiated by the intracellular Ca(2+) elevation in the cytoplasm and activation of TLCK-sensitive serine proteases. Our data provide new insight into ethanol-induced apoptosis in liver cells and may lead to therapeutic strategies to prevent liver damage.

Comparison of cobalamin-independent and cobalamin-dependent methionine synthases from Escherichia coli: two solutions to the same chemical problem.

In Escherichia coli, two enzymes catalyze the synthesis of methionine from homocysteine using methyltetrahydrofolate as the donor of the required methyl group: cobalamin-dependent and cobalamin-independent methionine synthases. Comparison of the mechanisms of these two enzymes offers the opportunity to examine two different solutions to the same chemical problem. We initiated the research described here to determine whether the two enzymes were evolutionarily related by comparing the deduced amino acid sequences of the two proteins. We have determined the nucleotide sequence for the metE gene, encoding the cobalamin-independent methionine synthase. Our results reveal an absence of similarity between the deduced amino acid sequences of the cobalamin-dependent and cobalamin-independent proteins and suggest that the two have arisen by convergent evolution. We have developed a rapid one-step purification of the recombinant cobalamin-independent methionine synthase (MetE) that yields homogeneous protein in high yield for mechanistic and structural studies. In the course of these studies, we identified a highly reactive thiol in MetE that is alkylated by chloromethyl ketones and by iodoacetamide. We demonstrated that alkylation of this residue, shown to be cysteine 726, results in complete loss of activity. While we are unable to deduce the role of cysteine 726 in catalysis at this time, the identification of this reactive residue suggests the possibility that this thiol functions as an intermediate methyl acceptor in catalysis, analogous to the role of cobalamin in the reaction catalyzed by the cobalamin-dependent enzyme.

Complement factor D-like activity of Porphyromonas gingivalis W83.

Porphyromonas gingivalis is a proteolytic gram-negative anaerobic bacterium that is frequently isolated from lesions of human periodontal disease. Previous studies have shown that P. gingivalis strain W83 inactivates C3 in pooled normal human serum (NHS) by a mechanism that is inhibitable by EDTA, yet it degrades purified complement proteins by a mechanism that is not EDTA-inhibitable. Furthermore, during complement activation, only a small number of C3 molecules accumulate on the surface of this organism unless the bacteria are treated with the protease inhibitor TLCK prior to complement activation. The hypothesis was tested that P. gingivalis W83 contains protease activity mimicking that of complement factor D, thus enabling it to activate C3 in serum without significant C3 accumulation on the cell surface. It was first noted that incubation of P. gingivalis W83 in absorbed human serum that was depleted of factor D resulted in C3 consumption that was reversed in the presence of the protease inhibitor TLCK. To directly demonstrate that factor B-dependent C3 consumption occurs in the absence of factor D, P. gingivalis W83 was incubated with purified C3 or a mixture of C3 and B. Although some proteolysis of C3 was noted, increased C3 consumption was noted in mixtures containing both C3 and B. This increment in C3 consumption was inhibited by both EDTA and TLCK. Furthermore, the addition of purified factor H to this mixture inhibited the increment in C3 consumption, indicating that a C3 convertase was probably formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of platelets by alpha-thrombin is a receptor-mediated event. D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-thrombin, but not N alpha-tosyl-L-lysine chloromethyl ketone-thrombin, binds to the high affinity thrombin receptor.

Competition binding studies have been carried out to evaluate the antagonism of TLCK-thrombin (N alpha-tosyl-L-lysine chloromethyl ketone-treated thrombin) and PPACK-thrombin (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-treated thrombin) with alpha-thrombin using computer-assisted analysis of the binding isotherms (LIGAND). alpha-Thrombin bound to high, moderate, and low affinity sites as previously described (Harmon, J. T., and Jamieson, G. A. (1985) Biochemistry 24, 58-64). PPACK-thrombin bound to all three sites accessible to alpha-thrombin (K1, 7 nM; R1, 20 sites/platelet; K2, 3 nM; R2, 1800 sites/platelet; K3, 510 nM; R3, 84,000 sites/platelet) as well as to a separate fourth site (Kx, 0.4 nM; Rx, 20 sites/platelet) for PPACK-thrombin that was not accessible to alpha-thrombin. In contrast, TLCK-thrombin did not bind to the high affinity site for alpha-thrombin but bound to the moderate and low affinity sites for alpha-thrombin with similar affinity (K2, 2 nM; R2, 890 sites/platelet; K3, 900 nM; R3, 100,000 sites/platelet) and to another site (Ky, 0.03 nM; Ry, 10 sites/platelet) which was not accessible to alpha-thrombin. As predicted from these binding studies, TLCK-thrombin did not compete with alpha-thrombin for platelet activation at concentrations as high as 1000 nM (500-fold excess). In contrast a 300-fold excess of PPACK-thrombin (670 nM) totally inhibited platelet activation by 2 nM thrombin. These results demonstrate that the high affinity binding site for thrombin on human platelets is a classical receptor, occupancy of which is necessary for platelet activation by low concentrations of thrombin; that TLCK-thrombin does not occupy this high affinity site and hence cannot inhibit platelet activation by alpha-thrombin; and that PPACK-thrombin does compete with alpha-thrombin at the high affinity site and is an antagonist of alpha-thrombin induced activation.

Intestinal uptake of trypsin in newborn and weaned rabbits.

Earlier studies have shown that the uptake of intact proteins from the intestinal lumen into the systemic circulation is increased in neonates. The present experiments tested the uptake of trypsin in newborn compared with 4-wk-old weaned rabbits. Trypsin (200 mg/100 g body wt) was administered by gavage to newborn and 4-wk-old rabbits. Four hours later, the tryptic activity and immunoreactive trypsin (i-trypsin) content of serum from newborn rabbits exceeded that of the older animals. After Sephadex G-200 gel filtration of serum from animals gavaged with trypsin, tryptic activity was detected in the excluded volume (presumably reflecting trypsin bound to alpha 2-macroglobulin), and i-trypsin was detected in the included volume (presumably reflecting trypsin bound to alpha 1-antitrypsin). In vitro experiments demonstrated that large amounts of trypsin were required to overwhelm the antiprotease present in normal rabbit serum. We suggest that complete or partial deficiencies of serum protease inhibitors may permit proteases taken up from the intestinal lumen of the neonate to circulate, reach the liver, and induce tissue injury at this site.

Bovine alpha- and beta-thrombin. Reduced fibrinogen-clotting activity of beta-thrombin is not a consequence of reduced affinity for fibrinogen.

beta-Thrombin, a product of the limited proteolysis of alpha-thrombin, is characterized by greatly reduced fibrinogen-clotting activity as compared to alpha-thrombin but with unchanged activity toward ester substrates. The present study was designed to elucidate the basis for the changes in the catalytic activity resulting from the conversion of bovine alpha-thrombin to bovine beta-thrombin. Fibrinogen was utilized as a competitive inhibitor in the hydrolysis of a peptide nitroanilide substrate by bovine alpha- and beta-thrombin. The Ki values obtained for fibrinogen in these experiments were similar for alpha- and beta-thrombin (about 10 microM). Similar values for Ki were obtained when fibrinogen was used to inhibit the inactivation of bovine alpha- and beta-thrombin by diisopropylphosphorofluoridate. These experiments suggested that the conversion of bovine alpha- to beta-thrombin does not affect the fibrinogen-binding site on thrombin. Differences in the reactivity of functional groups at the active site were then explored. beta-Thrombin was observed to undergo modification at the active site histidine at a slower rate than that of alpha-thrombin when reacted with either tosyllysyl chloromethyl ketone or diethyl pyrocarbonate. It is suggested that the difference in the fibrinogen-clotting activity of these two forms of thrombin can result from changes in the reactivity of the active site histidine residue.

The site of action of N-alpha-tosyl-L-lysyl-chloromethyl-ketone (TLCK) on cloned cytotoxic T lymphocytes.

N-alpha-tosyl-L-lysyl-chloromethyl-ketone (TLCK), an irreversible inhibitor of trypsin-like serine proteases, is a potent, nontoxic inhibitor of cytotoxic T lymphocyte (CTL) activity with half-maximal inhibition of an alloreactive CTL clone occurring at [TLCK] = 30 microM. We have utilized TLCK as an affinity probe for functionally important CTL surface molecules by raising rabbit antibodies specific for the tosyl group and employing them as immunoprecipitating reagents. When 125I-labeled cloned CTL were treated with TLCK, immunoprecipitation with rabbit anti-tosyl antibodies and analysis by polyacrylamide gel electrophoresis revealed a small number of TLCK-binding proteins. Prior alkylation of radiolabeled CTL with iodoacetamide inhibited TLCK binding only slightly, suggesting that TLCK binding did not occur via free sulfhydryl groups. Thymocytes and a second CTL clone both had very similar patterns of TLCK-binding proteins; in contrast the TLCK-binding proteins of B cells differed greatly. Sequential immunoprecipitation experiments identified the predominant CTL TLCK-binding protein as T200. Lymphocyte function-associated antigen-1 also reacted with TLCK but to a lesser extent. The inhibitory role of cell-surface bound TLCK (vs intracellular TLCK) was demonstrated by protection experiments using Concanavalin A, a reversible ligand of the CTL cell surface. These experiments suggest that T200 may be required for cytotoxic activity of CTL.