N-alpha-Tosyl-L-lysine chloromethyl ketone and N-alpha-tosyl-L-phenylalanine chloromethyl ketone inhibit protein kinase C

TLCK (N-alpha-tosyl-L-lysine chloromethyl ketone) inhibits protein kinase C whether or not the enzyme is under the regulation of Ca2+ and phospholipid. TLCK (IC50 = 1 mM) is a much more potent inhibitor of protein kinase C than TPCK (N-alpha-tosyl-L-phenylalanine chloromethyl ketone) (IC50 = 8 mM), suggesting that the lysyl moiety of TLCK may be specifically recognized by the active site of protein kinase C. These results extend the evidence that the active site of protein kinase C recognizes basic amino acids, and suggest that the active sites of protein kinase C and the cAMP-dependent protein kinase, which is also inhibited by TLCK and TPCK, are structurally related.     

Protease Inhibitors Cause Necrotic Cell Death in Chlamydomonas reinhardtii by Inducing the Generation of Reactive Oxygen Species

Protease inhibitors affecting the activity of the proteasome were reported to induce programmed cell death (apoptosis) in some mammalian cell lines. Proteasome activity can be suppressed by specific peptide derivatives and by N‐tosyl‐lysine‐chloromethyl‐ketone (TLCK) and N‐tosyl‐phenylalanine‐chloromethyl‐ketone (TPCK), which affect the trypsine‐ and chymotrypsine‐like activities of the proteasome, respectively. Particularly TLCK and TPCK caused necrotic cell death in the unicellular green alga Chlamydomonas reinhardtii. As a control, the effects of these protease inhibitors on the survival of human WISH cells were also studied. Bleaching of the Chlamydomonas cells after addition of TLCK or TPCK indicated that reactive oxygen species (ROS) were involved in this process. Indeed, increased levels of ROS were detected in Chlamydomonas cells treated with TLCK or TPCK. Furthermore, cell death induced by these protease inhibitors was accelerated by illumination and prevented or slowed down by scavengers of ROS.     

N-α-Tosyl-L-lysine chloromethyl ketone and N-α-tosyl-L-phenylalanine chloromethyl ketone inhibit protein kinase C

TLCK (N-α-tosyl-L-lysine chloromethyl ketone) inhibits protein kinase C whether or not the enzyme is under the regulation of Ca²⁺ and phospholipid. TLCK (IC₅₀= 1 mM) is a much more potent inhibitor of protein kinase C than TPCK (N-α-tosyl-L-phenylalanine chloromethyl ketone) (IC₅₀=8 mM), suggesting that the lysyl moiety of TLCK may be specifically recognized by the active site of protein kinase C. These results extend the evidence that the active site of protein kinase C recognizes basic amino acids, and suggest that the active sites of protein kinase C and the cAMP-dependent protein kinase, which is also inhibited by TLCK and TPCK, are structurally related.Protein kinase CTumor promotionProtease inhibitor     

Down regulation of phorbol diester receptors by proteolytic degradation of protein kinase C in a cultured cell line of fetal rat skin keratinocytes

Down regulation of phorbol diester receptors was studied with respect to proteolysis of protein kinase C, which is activated by Ca2+, phospholipids, and diacylglycerols and which binds to phorbol diesters. We used FRSK cells, a cell line derived from fetal rat skin keratinocytes, because in these cells specific binding of phorbol 12,13-dibutyrate decreased rapidly (50% decrease in 30 min). This decrease (down regulation) was inhibited by some protease inhibitors, such as N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), N-p-tosyl-L-lysine chloromethyl ketone (TLCK), and leupeptin, but not by inhibitors of lysosomal hydrolases. On treatment with 12-O-tetradecanoylphorbol 13-acetate, protein kinase C was rapidly translocated from the cytosol to the membranes and then decreased. This decrease in protein kinase C was also inhibited by TPCK, TLCK, and leupeptin. The decrease in membrane activity of protein kinase C was associated with increase in cytosolic activity of a protein kinase that was smaller in molecular weight (Mr 40,000-60,000) than protein kinase C, did not depend on Ca2+/phosphatidylserine/diacylglycerol, and did not bind to phorbol 12,13-dibutyrate. These results indicate that down regulation of phorbol diester receptors is probably caused by nonlysosomal proteolysis of protein kinase C. The kinase formed by cleavage may be an active catalytic site of protein kinase C.     

Effects of proteinase inhibitors on fertilization in sea lamprey (Petromyzon marinus)

A search for alternative sterilants in parasitic fish encouraged us to explore the usefulness of proteinase inhibitors for this purpose. Fertilization in sea lamprey species (Petromyzon marinus L.) was inhibited by chymotrypsin and trypsin inhibitors 4'-acetamidophenyl 4-guanidinobenzoate (AGB), chymostatin, tosyl-L-lysine chloromethyl ketone (TLCK), and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) when these substances were added into a fertilization medium at the time of fertilization. Preincubation of eggs before fertilization with 100 microM TPCK, but not TLCK, resulted in inhibition of fertilization. Conversely, preincubation of spermatozoa with TLCK, but not TPCK, produced inhibition of fertilization. These data suggest the involvement of the chymotrypsin-like activity of eggs and trypsin-like activity of spermatozoa in fertilization. However, enzymes present in sperm suspensions were able to hydrolyze a chymotrypsin substrate N-glutaryl-L-phenylalanine-p-nitroanilide (GPNA) but not trypsin substrate N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA). The nature of this activity can be characterized as serine protease and our results indicate the involvement of serine proteinases in the fertilization of sea lamprey.     

Selective Inhibition of the Synthesis of Sindbis Virion Proteins by an Inhibitor of Chymotrypsin

Treatment of chick embryo fibroblasts infected with Sindbis virus with TPCK, the choloromethyl ketone derivative of tosyl-phenylalanine and an inhibitor of chymotrypsin, resulted in reduced synthesis of viral structural proteins and the accumulation of a high-molecular-weight polypeptide, thought to be a precursor. The analogous inhibitor of trypsin, TLCK, the chloromethyl ketone derivative of tosyllysine, had no such effect.     

The 32-kDa glycoprotein of Chlamydia trachomatis is an acidic protein that may be involved in the attachment process

Abstract The 32-kDa glycoprotein of Chlamydia trachomatis was shown to have a p I of 6.2 to 6.4 which distinguished this protein from the chlamydial histone-like protein of similar molecular mass that has a p I of > 10. The initial interaction of the glycan of 32 kDa glycoprotein and HeLa cells was also investigated. Glycan was cleaved from the protein backbone by N -glycanase and radiolabeled with tritium by sodium borohydride reduction. Competition assays showed the binding of glycan to HeLa cells was inhibited by galactose, mannose, and N -acetylglucosamine but not by sedoheptulose and fructose. Untreated and UV-treated organisms inhibited the binding, while heat-inactivated organisms did not. Binding was blocked by rabbit antiserum against whole organisms but not by rabbit anti-155-kDa antiserum or monoclonal antibodies against the lipopolysaccharide and major outer membrane protein.     

Chloromethyl ketones are insulin-like stimulators of lipid synthesis in locust fat body

Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK) stimulates lipid synthesis in locust fat body in vitro, and is able to reverse the inhibitory effects of AKH-I on lipid synthesis. Effective stimulatory concentrations of TLCK were in the range of 0.2–1.0 mM. Similar stimulatory effects were also achieved with phenylalanine chloromethyl ketone (PheCK) and leucine chloromethyl ketone (LeuCK), but not with tosyl-phenylalanine chloromethyl ketone (TPCK), dansyl-glu-gly-arg-CK, chloroacetone, chloroacetic acid, chloroacetamide, chloroacetaldehyde, chloroacetyl-L-leucine or acetylated or fluorescamine-labelled TLCK, PheCK, and LeuCK. The level of stimulation caused by TLCK was dependent on incubation time, so that after a 5-h preincubation of fat body tissue with TLCK the stimulated rate was severalfold higher than the control. TLCK also increased the rate of uptake of trehalose and uridine, but not glucose, deoxyglucose or glycine. Increasing concentrations of bovine serum albumin (BSA) in the incubation medium caused a reduction in the rate of TLCK-stimulated acetate uptake, such that levels of uptake were no higher with 1% BSA than in the controls. A range of more specific protease and kinase inhibitors was tested, but none caused stimulation; thus the mode of action of TLCK on the stimulation of acetate uptake has yet to be identified. Elucidation of the mode of action of TLCK may facilitate the development of novel compounds for insect pest control. Arch. Insect Biochem. Physiol. 39:9–17, 1998. © 1998 Wiley-Liss, Inc.     

Influence of the excision shock on the protein metabolism ofVicia faba L. meristematic root cells

UsingVicia faba root meristems we have shown that protein synthesis was dramatically changed after excision. The amino-acid incorporation dropped to 13% of the level in the unexcised control. This downshift was a direct consequence of the breakdown of polysomes which are converted into monosomes. In order to perform an analysis of the protein pattern by two-dimensional gel electrophoresis, endogenous proteolytic activity, which is high in broad bean root, had to be inhibited. Therefore, several protease inhibitors were tested and a very efficient inhibitor pool was obtained which could be used during the preparation of meristematic cell extracts. Protein-pattern analysis showed important differences between the unexcised control and excised apices. The number of proteins synthesized after excision droped from 250 in the control to 80, as a consequence of polysome breakdown. Futhermore, we present evidence that new and apparently specific proteins are synthesized in response to this excision shock.Abbreviations NEM N-ethylmaleimide - PMSF phenylmethylsulfonyl fluoride - TLCK N-tosyl-L-lysin chloromethyl ketone - TPCK N-tosyl-L-phenylalanine chloromethyl ketone     

The role of protease in streptolysin S formation

Production of streptolysin S by streptococci was found to be inhibited by treatment with protease inhibitors, tosylphenylalanine chloromethyl ketone (TPCK), tosyllysine chloromethyl ketone (TLCK), or phenylmethylsulfonyl fluoride (PMSF), even in the presence of the inducer oligonucleotides. Other protease inhibitors, antipain, leupeptin, or pepstatin were found to have little or no effect. Trypsin reversed the effect of TPCK or TLCK. The reversal was dependent upon the amount of added trypsin and the incubation time at 37 degrees C, suggesting that a protease activity was involved in the hemolysin formation. The effect of trypsin was not observed if chloramphenicol was also added, suggesting that a precursor of streptolysin S was processed as it was synthesized and released into medium as the active hemolysin, by the concerted action of a protease and inducer oligonucleotides. Experiments with the subcellular fractions of streptococci indicated that the streptolysin precursor was localized in the insoluble fraction and the "processing" protease in the supernatant fraction.     

The Characterization of Cell Death Induced by 1-(3-C-Ethynyl-β-D-ribo-Pentofuranosyl)Cytosine (ECyd) in FM3A Cells

Abstract

The characterization of cell death induced by 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine(ECyd), a potent inhibitor of RNA synthesis, was performed using mouse mammary tumor FM3A cells in vitro. Accompanied with the cell death induced by ECyd (3.0 μM)-treatment, about 100–200 kbp-sized and internucleosomal DNA fragmentation were observed by orthogonal-field-alternation gel electrophoresis (OFAGE) and conventional gel electrophoresis, respectively. Protease inhibitors, carbobenzoxy-L-aspart-1-yl[(2,6-dichlorobenzoyl)oxy]methane (Z-Asp-CH₂-DCB), Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK) and N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), effectively blocked the cell death, suggesting that the proteases inhibited by Z-Asp-CH₂-DCB, TLCK or TPCK were involved in the process of the cell death.     

Plasmodium knowlesi: studies on invasion of rhesus erythrocytes by merozoites in the presence of protease inhibitors

The effect of protease inhibitors on invasion of rhesus erythrocytes by Plasmodium knowlesi merozoites was evaluated. Chymostatin, N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), and L-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK) inhibited invasion. Leupeptin, antipain, pepstatin, and phenylmethylsulfonyl fluoride (PMSF) had no effect. TLCK and TPCK inhibited attachment of merozoites to host erythrocytes. Chymostatin had no adverse effect on attachment, and in its presence junction formation between the merozoite and host erythrocyte occurred. Both chymostatin and leupeptin inhibited normal rupture of schizont-infected erythrocytes. It is suggested that proteolytic activity may be important both in the rupture of schizont-infected erythrocytes and in the invasion of erythrocytes by malaria parasites.     

Inhibition of the transformation-specific kinase in ASV-transformed cells by N-alpha-tosyl-L-lysyl chloromethyl ketone.

We find that the protease inhibitor N-α-tosyl-L-lysyl chloromethyl ketone (TLCK) inhibits the transformation-specific kinase activity (Collett and Erikson, 1978) associated with p60^src, the avian sarcoma virus (ASV) gene product responsible for the transformation of fibroblasts. TLCK has been shown to induce the phenotypic reversion of ASV-transformed cells to normal (Weber, 1975). Kinase activity was measured in extracts of chick embryo fibroblasts (CEF) transformed by the Schmidt-Ruppin strain of ASV (SR-ASV) with antiserum from rabbits bearing ASV-induced tumors. The immunoprecipitates were incubated with γ-³²P-ATP under conditions in which the phosphorylation of the IgG heavy chain in the immunoprecipitate was directly proportional to the concentration of cell extract. When ASV-transformed CEF were treated with 0.1 mM TLCK, the kinase activity was reduced by 60% after 2 hr and by 80% after 6 hr, and continued to remain low for up to 40 hr when TLCK was present. When TLCK was removed, the kinase activity rose slowly over a period of many hours, suggesting that the enzyme is irreversibly inactivated by TLCK and new enzyme must be synthesized. The effect of TLCK in vivo is concentration-dependent and specific. Other serine protease inhibitors had no effect on kinase activity. At low concentrations (0.03 mM), TPCK produced partial inhibition (≤20%), but at higher concentrations TPCK was extremely toxic to the cells and therefore could not be tested. The inhibition by TLCK was not due to its ability to inhibit protein synthesis since cycloheximide treatment (1 μg/ml) did not significantly reduce kinase activity. TLCK also inhibited kinase activity when added directly to cell extracts, but about 5 times higher concentrations of TLCK were required to produce 50% inhibition. Under these conditions both TLCK and TPCK were comparable inhibitors, whereas PMSF had no effect. Our finding that the inhibition of the kinase by TLCK in vivo parallels the reversion of cell morphology to normal suggests that the kinase has an important role in transformation and offers a biochemical rationale for treatment of tumors with this agent.     

Reduction in cell entry of Eimeria tenella (Coccidia) sporozoites by protease inhibitors, and partial characterization of proteolytic activity associated with intact sporozoites and merozoites

The role of proteases in the invasion of host cells by Eimeria tenella (Wisconsin strain) was studied in vitro. Protease inhibitors were used to treat sporozoites before inoculation or were applied to cultured chicken kidney cells before infection. The inhibitors antipain, leupeptin, aprotinin, L-1-tosylamide-2-phenyl-ethyl chloromethyl ketone (TPCK), or N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) reduced parasite invasion to 16-66% of control after treatment of cultured cells or sporozoites with 5- or 50-micrograms/ml concentrations of inhibitors in the culture medium. Phenylmethylsulfonyl fluoride (PMSF) reduced invasion to 32-57.7% at concentrations of 1-4 mM. The optimum pH for hydrolysis of azocasein by intact sporozoites or merozoites was determined over a range of pH 5.0 to pH 9.0. Sporozoites were highly active over a broad range from pH 5.5 to pH 9.0, with an apparent optimum at pH 8.0. Merozoites had a much lower specific activity with pH optima at 7.0 and 8.5. The protease activity of sporozoites or merozoites could be inhibited completely by the addition of 50 micrograms/ml of leupeptin, TPCK, or TLCK or of 4 mM PMSF. Antipain inhibited proteases of sporozoites but not of merozoites. Pepstatin had little effect on either sporozoites or merozoites. The results suggest that parasite proteases of Eimeria may be necessary for invasion of host cells.     

A 43 kDa protein inserted at the plasma membrane-cytoskeleton interface in rumen entodiniomorphid ciliates

Summary The P-43 ofEudiplodinium and homologous proteins in three other entodiniomorphid species, free-living ciliates, flagellates, and HeLa cells, were identified at the plasma membrane-cytoskeleton interface. Proteins cross-reacting with MAb B6 were also located at the ciliary inner surface of the plasma membrane. Due to the strong adhesion of the plasma membrane to the underlying cytoskeleton, classical extraction with detergents, urea, NaOH, and PTA, failed to separate the two components completely. However, the extraction properties of P-43, associated with its membrane-cytoskeleton interactive functions, suggest that this unglycosylated protein may present some analogies with proteins of the intermediate filaments. Their ubiquity and localization suggest that P-43 and MAb B6 crossreacting proteins may not be strictly epiplasmic but could be amphitropic proteins, strongly anchored to both the plasma membrane and the underlying microfilament framework, via protein-protein binding or by direct insertion in the lipid bilayer.Abbreviations BSA bovine serum albumin - Con A concanavalin A - EDTA ethylene diamine tetraacetic acid - EM electron microscopy - IF intermediate filaments - MAb monoclonal antibody - MET 2-mercaptoethanol - MW molecular weight - PAb polyclonal antibody - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride - PTA phosphotungstic acid - SDS sodiumdodecyl sulfate - TAME Na-p-tosyl-arginine methyl ester - TLCK Na-p-tosyl-lysine chloromethyl ketone     

Suppression of phorbol ester-enhanced radiation-induced malignancy in vitro by protease inhibitors is independent of protein kinase C

X-irradiation and the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) act in a synergistic manner to increase the yield of transformed C3H10T1/2 cells in vitro. TPA modulated both translocation from the cytosol to the plasma membrane, and down regulation of protein kinase C (PKC) after prolonged (48 h) TPA exposure. N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), antipain, and soybean-derived Bowman-Birk inhibitor, protease inhibitors that suppress transformation of C3H10T1/2 cells, had no effect on these TPA-mediated alterations of PKC activity, suggesting that protease inhibitors suppress TPA-stimulated promotion in vitro via a PKC-independent pathway. Several experiments were performed to determine whether non-toxic concentrations of the PKC inhibitors, N-p-tosyl-L-lysine chloromethyl ketone (TLCK), TPCK, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), or 1-(5-isoquinoline-sulfonyl)-2-methyl-piperazine (H-7), modulated the movement of cells from a quiescent state into the cell cycle. TPCK and the combination of H-7 and W-7 lowered DNA synthesis when cells were stimulated to divide by TPA. Because other protease inhibitors that slow transformation in vitro did not have the same suppressive effect on DNA synthesis, the inhibitory pathway that suppresses carcinogenic activity is likely to be different from the suppression of DNA synthesis.     

Disulfide-mediated interactions of the chlamydial major outer membrane protein: role in the differentiation of chlamydiae?

The effects of exogenous reducing agents on a number of biological properties of purified Chlamydia trachomatis LGV-434 and Chlamydia psittaci meningopneumonitis elementary bodies (EBs) have been examined in an attempt to identify in vitro correlates of early events in the differentiation of the infectious EB to the replicative cell type, the reticulate body (RB). Treatment of EBs with dithiothreitol elicited a number of changes normally associated with differentiation to the RB. EBs in the presence of 10 mM dithiothreitol displayed enhanced rates of [14C]glutamate oxidation, reduced infectivity, and decreased osmotic stability, and their Machiavello staining properties changed to those characteristic of the RB. A true differentiation of EB to RB did not take place under these conditions, since EBs treated in this manner and examined by transmission electron microscopy did not demonstrate increased size or decreased electron density as do isolated RBs. Additional studies were initiated to identify the macromolecules involved in this process. With polyacrylamide gel electrophoresis and immunoblotting procedures with monoclonal and polyclonal monospecific antibodies, the chlamydial major outer membrane protein was found to be the predominant component that varied under reducing versus nonreducing conditions. Furthermore, the extent of disulfide-mediated cross-linking of the major outer membrane protein varied between the infective and replicative forms of the C. trachomatis LGV-434 life cycle. Implications of disulfide interactions in the life cycle of chlamydiae are discussed.     

Do Alkylating Agents Modify the Histidine Residue of the Desensitized Butyrylcholinesterase?

Tosylphenylalanine chloromethyl ketone (TPCK) and tosyllysine chloromethyl ketone (TLCK) are irreversible modifiers of histidine which is located in the catalytic triad of chymotrypsin and trypsin, respectively. The effects of TPCK and TLCK on the histidine in the catalytic triad of the desensitized butyrylcholinesterase (BChE), prepared from human serum by heating at 45°C for 24 h, were investigated in detail. It is found that these reagents do not modify, but reversibly inhibit the desensitized enzyme as a function of time. Just as it is for the native enzyme, TPCK is a hyperbolic mixed-type inhibitor of the desensitized BChE with K_i, a and ß values of 0.017 ± 0.003 mM, 3.942 ± 1.125 and 0.524 ± 0.070, respectively. However, TLCK is the pure competitive inhibitor of the desensitized BChE with a K_i value of 0.008 ± 0.000 mM, while it is hyperbolic mixed-type inhibitor of the native form. These findings show that the conformation of the active site cavity of desensitized BChE is different from that of the native enzyme.     

Evidence that CT694 is a novel Chlamydia trachomatis T3S substrate capable of functioning during invasion or early cycle development

Chlamydia trachomatis is an obligate intracellular parasite, occupies a membrane-bound vacuole throughout development and is capable of manipulating the eukaryotic host by translocating effector molecules via a type III secretion system (T3SS). The infectious chlamydial elementary body (EB) is metabolically inactive yet possesses a functional T3S apparatus capable of translocating effector proteins into the host cell to facilitate invasion and other early cycle events. We present evidence here that the C. trachomatis protein CT694 represents an early cycle-associated effector protein. CT694 is secreted by the Yersinia T3SS and immunodetection studies of infected HeLa cultures indicate that CT694-specific signal accumulates directly adjacent to, but not completely overlapping with EBs during invasion. Yeast two-hybrid analyses revealed an interaction of CT694 with the repeat region and C-terminus of human AHNAK. Immunolocalization studies of CT694 ectopically expressed in HeLa cells were consistent with an interaction with endogenous AHNAK. Additionally, expression of CT694 in HeLa cells resulted in alterations in the detection of stress fibres that correlated with the ability of CT694 to interact with AHNAK. These data indicate that CT694 is a novel T3S-dependent substrate unique to C. trachomatis , and that its interaction with host proteins such as AHNAK may be important for aspects of invasion or development particular to this species.