Serine Protease Inhibitor Diisopropylfluorophosphate Research Articles

Analysis of the Proteomes of Primary, De Novo Acute Myeloid Leukemia Cells from Adults

Introduction:Proteins, despite being the primary effectors of cellular processes, are often studied only indirectly through analysis of the transcriptome. However, it is clear that the relationship between mRNA expression and protein expression is approximate at best. In Acute Myeloid Leukemia (AML), the genome and transcriptome have been thoroughly characterized, but the proteome has been less well studied. Here, we present a deep-scale study of the proteomes of 44 primary AML bone marrow samples representing a wide range of AML across the spectrum of cytogenetic risk, common mutations, and driver fusions.Methods:Bone marrow samples were collected at presentation from 44 adult patients with de novo AML as part of an institutional banking protocol, and buffy coat cells were immediately cryopreserved without further manipulation. Cryovials were thawed in the presence of the cell permeable serine protease inhibitor diisopropyl fluorophosphate (DFP) to inactivate the abundant neutrophil serine proteases (ELANE, CTSG, PRTN3, and PRSS57), and further processed for nano-liquid chromatography mass spectrometry in the presence of an extensive cocktail of protease inhibitors. Both label-free quantification (LFQ) and tandem-mass-tag (TMT) deep-scale proteomics were performed on these 44 patient samples, as well as 3 lineage-depleted bone marrow samples from healthy adult donors. Matching RNA-seq and exome sequencing data were available for the same samples as part of The Cancer Genome Atlas (TCGA) AML project.Results:10,651 and 6,679 unique proteins were detected in the TMT and LFQ experiments, respectively. Correlations between measurements derived from the independent proteomic platforms (i.e. TMT and LFQ) is higher (mean Spearman correlation, 0.60, Figure 1A) than correlation between proteomic (TMT) and transcriptomic measurements from bulk RNA-seq data (Spearman 0.43, Figure 1B). Quality checks of the proteomic data strongly supported the reliability of quantification of protein measurements; for example, the mean ratio of beta globin protein (HBB) to alpha globin (HBA1) was 1.2 +/- 0.25 (Figure 1C), and several proteins known to be dysregulated by specific AML-initiating fusion proteins (for PML-RARA, HGF and RARA; for RUNX1-RUNX1T1, RUNX1T1; and for CBFB-MYH11, MYH11) were detected in the expected samples (Figure 1D). Globally, 1,364 proteins were differentially expressed in the AML samples (corrected p-value <0.05, fold change ≥ 1.5) compared to the lineage-depleted, healthy bone marrow samples. Globally overexpressed proteins were enriched for ribosomal RNA modification, mitochondrial protein import, nuclear export, and the mitochondrial electron transport chain, among others. These overexpressed proteins include 61 cell surface proteins that could potentially represent therapeutic targets (overexpressed on average in 82% of AML samples, range 25-97%). Globally downregulated proteins in AML samples were enriched for glycogen metabolism and protein groups associated with mature neutrophils (reflecting the expected maturation block in AML), among others. 771 of the 1364 differentially expressed proteins (56.5%) showed only minimal variability in mRNA expression levels (fold change of <1.1 between AML and normal marrow CD34 cell mRNA) that could not explain dysregulated protein expression. Several protein complexes likewise showed coordinated differential expression in the proteomic data, but no change in the transcriptome, including the THO complex (Figure 1E) and the phosphorylase kinase complex (Figure 1F), among others, indicating the presence of posttranscriptional regulation of the levels of many proteins in AML samples.Conclusion:We have created a deep-scale proteomic database from a set of well-characterized AML samples, allowing for a proteogenomic study of AML. We have identified many examples of post-transcriptional regulation of key metabolic pathways that may be relevant for better understanding AML cell metabolism and therapeutic vulnerabilities. Additional studies linking patterns of protein dysregulation with a variety of AML covariates are underway. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Design of a serine protease-like catalytic triad on an antibody light chain displayed on the yeast cell surface

Lc-WT, the wild-type light chain of antibody, and Lc-Triad, its double mutant with E1D and T27aS designing for the construction of catalytic triad within Asp1, Ser27a, and original His93 residues, were displayed on the cell surface of the protease-deficient yeast strain BJ2168. When each cell suspension was reacted with BODIPY FL casein and seven kinds of peptide-MCA substrates, respectively, a remarkable difference in hydrolytic activities toward Suc-GPLGP-MCA (succinyl-Gly-Pro-Leu-Gly-Pro-MCA), a substrate toward collagenase-like peptidase, was observed between the constructs: Lc-Triad-displaying cells showed higher catalytic activity than Lc-WT-displaying cells. The difference disappeared in the presence of the serine protease inhibitor diisopropylfluorophosphate, suggesting that the three amino acid residues, Ser27a, His93, and Asp1, functioned as a catalytic triad responsible for the proteolytic activity in a similar way to the anti-vasoactive intestinal peptide (VIP) antibody light chain. A serine protease-like catalytic triad (Ser, His, and Asp) is considered to be directly involved in the catalytic mechanism of the anti-VIP antibody light chain, which moderately catalyzes the hydrolysis of VIP. These results suggest the possibility of new approach for the creation of tailor-made proteases beyond limitations of the traditional immunization approach.

Anion Channels, Including ClC-3, Are Required for Normal Neutrophil Oxidative Function, Phagocytosis, and Transendothelial Migration

NADPH oxidase activity, phagocytosis, and cell migration are essential functions of polymorphonuclear leukocytes (PMNs) in host defense. The cytoskeletal reorganization necessary to perform these functions has been extensively studied, but the role of cell volume regulation, which is likely dependent upon anion channels, has not been defined. Mice lacking the anion channel ClC-3 (Clcn3(-/-)) died from presumed sepsis following intravascular catheter placement, whereas Clcn3(+/+) littermates survived. We hypothesized that ClC-3 has a critical role in host defense and reasoned that PMN function would be compromised in these mice. Clcn3(-/-) PMNs displayed markedly reduced NADPH oxidase activity in response to opsonized zymosan and modestly reduced activity after phorbol 12-myristate 13-acetate. Human PMNs treated with the anion channel inhibitors niflumic acid or 5-nitro-2-(3-phenylpropylamino)benzoic acid had a very similar defect. ClC-3 protein was detected in the secretory vesicles and secondary granules of resting PMNs and was up-regulated to the phagosomal membrane. Clcn3(-/-) PMNs and human PMNs lacking normal anion channel function both exhibited reduced uptake of opsonized zymosan at 1, 5, and 10 min in a synchronized phagocytosis assay. Niflumic acid-treated PMNs also had impaired transendothelial migration in vitro, whereas migration in vivo was not altered in Clcn3(-/-) PMNs. Selective inhibition of the swelling-activated chloride channel with tamoxifen profoundly reduced PMN migration but had no effect on NADPH oxidase activity. In summary, PMNs lacking normal anion channel function exhibited reduced NADPH oxidase activity, diminished phagocytosis, and impaired migration. ClC-3 was specifically involved in the respiratory burst and phagocytosis.

Evidence that elastase is the TNF-R75 shedding enzyme in resting human polymorphonuclear leukocytes

We previously showed that a metalloprotease and a serine protease mediate shedding of the TNF-R75 (75-kDa tumor necrosis factor receptor) in neutrophils. Here we show that elastase is the TNF-R75 solubilizing serine protease. Release of the TNF-R75 by resting cells was almost totally inhibited by the serine protease inhibitor diisopropylfluorophosphate (DFP), by two synthetic, chemically unrelated, elastase-specific inhibitors and by α1-protease inhibitor. Release after TNF or FMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) stimulation was blocked by DFP and a metalloprotease inhibitor used in combination. Supernatants from resting neutrophils contained a 28-kDa fragment of the receptor, compatible with that generated by elastase, whose appearance was inhibited by DFP. Upon FMLP stimulation, the release of 28-kDa and 40-kDa fragments was observed, which was inhibited by DFP and a metalloprotease inhibitor, respectively. We conclude that elastase is the TNF-R75 sheddase of resting neutrophils and that it contributes to shedding of this receptor in stimulated cells.

Functional dissociation between proforms and mature forms of proteinase 3, azurocidin, and granzyme B in regulation of granulopoiesis.

We previously demonstrated that secreted proform(s) of the neutrophil serine protease PR3 (proteinase 3) can down-modulate the fraction of normal human colony-forming unit granulocyte-macrophage (CFU-GM) in S-phase, whereas PR3 extracted from mature neutrophils lacks this ability. The objective of this study was to characterize the structural and functional dissociation between secreted proforms and granule-stored mature forms and to extend the investigation to other related hematopoietic serine proteases. Conditioned media containing secreted proteases from transfectant cell lines with stable expression of human PR3, neutrophil elastase, cathepsin G, azurocidin, and granzymes A, B, H, K, and M were tested for their ability to reduce the fraction of normal human CFU-GM in S phase. Furthermore, recombinant PR3, azurocidin, and granzyme B with defined N-terminal propeptides, and the respective mature forms without propeptide, were functionally characterized. In addition to PR3, secreted proforms of azurocidin and granzymes A, B, H, K, and M, but not cathepsin G or neutrophil elastase, have S-phase reducing activity. This activity is restricted to the dipeptide proforms, whereas mature forms without propeptide have no S-phase reducing activity. On the other hand, only the mature forms of PR3 and granzyme B could bind the serine protease inhibitor diisopropylfluorophosphate (DFP), or aprotinin in the case of azurocidin. We also demonstrate that granulocyte colony-stimulating factor-stimulated CD34+ cells and interleukin-2-stimulated lymphocytes secrete active proforms of PR3 and granzyme B, respectively. These results demonstrate distinctive functional and conformational differences between proforms and mature forms of these hematopoietic serine proteases and suggest novel growth regulatory mechanisms in granulopoiesis.

Sulfur mustard-stimulated protease: a target for antivesicant drugs.

One of the mechanisms of the skin blistering effect (vesication) of sulfur mustard (bis-(2-chloroethyl)sulfide, HD) is believed to be via the stimulation of specific protease(s) at the dermal-epidermal junction. Cultured normal human epidermal keratinocytes (NHEK) were used as a model to study and characterize protease stimulated by the mustards 2-chloroethyl ethyl sulfide (CEES), 2-chloro-N-(2-chloroethyl)-N-methylethanamine hydrochloride (nitrogen mustard, HN(2)) and HD. The results obtained using a chromozym (TRY) peptide substrate protease assay revealed the optimum mustard concentrations and time for protease stimulation to be about 200 microM (CEES), 100 microM (HN(2)) and 100 microM (HD) and 16 h. The mustard-stimulated protease was membrane bound and was inhibited by adding a Ca(2+) chelator (either 2 mM EGTA (ethylene glycol-bis(amino ethyl ether) N,N,N',N' tetraacetic acid) or 50 microM BAPTA AM (1,2-bis(z-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetraacetoxy methyl ester) alone or in combination), a serine protease inhibitor diisopropyl fluoro-phosphate (DFP, 1 mM), or a protein synthesis inhibitor cycloheximide (35 microM) in the extracellular medium. These results suggest that mustard toxicity may involve the stimulation of trypsin/chymotrypsin-like serine protease, dependent on Ca(2+) and new protein synthesis. Protein purification by gel exclusion and hydrophobic chromatography produced a 70-80 kDa protease, which had an amino acid sequence homologous with a mammalian-type bacterial serine endopeptidase. Based on this information, research is in progress to identify the protease stimulated by HD in NHEK and to determine whether its inhibitors are useful as prospective antivesicant drugs.

Ionomycin-Induced Neutrophil NADPH Oxidase Activity Is Selectively Inhibited by the Serine Protease Inhibitor Diisopropyl Fluorophosphate

The calcium-specific ionophore ionomycin triggers neutrophils to activate their NADPH oxidase and generate reactive oxygen species. This activation is restricted to intracellular sites and involves the neutrophil granules. Cells that have experienced an ionomycin-induced rise in intracellular calcium will also mobilize their intracellular granules and are primed to subsequent challenge with the chemoattractant formylmethionyl-leucyl-phenylalanine (fMLF), but have lost their ability to become desensitized to the same agonist. We have investigated the involvement of serine proteases in the calcium-induced effector functions using the inhibitor diisopropyl fluorophosphate (DFP). The ionomycin-induced NADPH oxidase activity was abrogated by the protease inhibitor, whereas the activity induced by fMLF was unaffected. The DFP-dependent inhibition was restricted to the NADPH oxidase activity, as all other ionomycin-induced cellular activities were largely unaffected. We thus suggest that a serine protease is of importance for the calcium ionophore-induced signal(s) to reach and activate the dormant NADPH oxidase in the neutrophil granules.

Purification of mast cell proteases from murine skin.

Different subpopulations of mast cells are characterized by their abundant contents of either tryptase or in addition chymase. These two neutral proteases are found in mast cells and may thus hold a key to the understanding of mast cell dependent reactions. Such studies are however hampered by the lack of readily available supplies of chymase. We have therefore studied the simultaneous purification of both proteases from hairless moro hr/hr mouse skin, using a sequence of salt extractions and chromatographic separations. After three steps of extraction, a 13-fold purification with an 82% yield was obtained for tryptase and a 15-fold purification with a 90% yield for chymase. Further one step purification on conventional sephadex, sephacryl and octyl sepharose columns was unsatisfactory because of further protein contamination of the fractions. Heparin affinity chromatography caused a high loss of tryptase and residual protein contamination. Gradient elution on a benzamidine sepharose 6B column resulted however in a single, low yield (17.9%) tryptase peak and a broader, high yield (>90%) chymase peak, and a 34% yield high purity fraction. The proteases thus purified exhibited their typical inhibitor profile. On Western blot analysis and on autoradiography in the presence of the serine protease inhibitor diisopropylfluorophosphate (DFP), only one 28 kD molecule with chymase activity was identified, whereas a broad 32-38 kD band of tryptase monomers was noted. Taken together, these data show that, after salt extraction and a single benzamidine affinity chromatography step, both mast cell chymase and tryptase can be separated and in case of chymase also highly purified, allowing thus for the study of biological activities of this molecule.

Evidence that non-caspase proteases are required for chromatin degradation during apoptosis.

Chromatin degradation into oligonucleosomal and approximately 30-50 Kb fragments is a hallmark of apoptosis. Crude nuclear extract from apoptotic rat thymocytes is able to recapitulate both types of DNA fragmentation in an assay using HeLa cell nuclei as an exogenous substrate. Using size exclusion chromatography we have identified a novel activity (approximately 260 Kd) that produces only approximately 30-50 Kb DNA fragments, and a 25 Kd activity that generates both approximately 30-50 Kb and oligonucleosomal fragments. Both activities produced DNA fragments with 3'-OH termini, are dependent on Ca2+ and Mg2+ and are inhibited by N-ethyl-maleimide, sodium tetrathionate, aurintricarboxylic acid and sodium chloride, similar to other nucleases implicated in apoptosis. These activities were inhibited by the serine protease inhibitors N-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone, but not by the serine protease inhibitor diisopropyl fluorophosphate, or by calpain inhibitors I or II, or the capsase inhibitors Ac-Asp-Glu-Val-Asp-aldehyde, Ac-Tyr-Val-Ala-Asp-aldehyde, or Z-Val-Ala-Asp-fluoromethyl ketone. Both activities were insensitive to protease inhibitors when extracts were incubated with naked linear DNA, indicating the presence of both nuclease and protease activities in the preparation. Together, these observations suggest the involvement of non-caspase proteases in apoptosis which perhaps function by altering chromatin substructure and exposing it to nucleolytic attack.

Characterization of membrane-bound serine protease related to degradation of oxidatively damaged erythrocyte membrane proteins

It has been shown that erythrocyte membrane proteins become susceptible to degradation by membrane-bound serine protease activity after oxidative modification of the membranes (M. Beppu, M. Inoue, T. Ishikawa, K. Kikugawa, Biochim. Biophys. Acta 1196 (1994) 81–87). The aim of the present study was to clarify the presence of the serine protease in oxidized erythrocyte membranes and to characterize the selectivity of the enzyme to oxidized proteins. Human erythrocytes were oxidized in vitro with xanthine/xanthine oxidase/Fe(III) and oxidized membranes isolated. Proteolytic activity of the membranes toward spectrin obtained from oxidized membranes and bovine serum albumin oxidized with H 2O 2/horseradish peroxidase was increased by membrane oxidation, and the degradability of the substrates was increased by substrate oxidation. The proteolytic activity was inhibited by the serine protease inhibitor diisopropyl fluorophosphate (DFP). The 72 kDa and 80 kDa proteins in the membranes were labeled by [ 3H]DFP when detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and subsequent fluorography. The 72 kDa protein was found to be a serine enzyme, acetylcholine esterase. The 80 kDa protein appeared to be responsible for the degradation of oxidatively damaged proteins. The 80 kDa protein was loosely bound to membranes and readily solubilized into a 0.1% NP-40 detergent solution. The presence of the same 80 kDa protease in intact erythrocyte cytosol was suggested. The increased serine protease activity in oxidized membranes can result from the increased adherence of the cytosolic 80 kDa serine protease to the membranes due to oxidation.

Cloning, Expression, and Catalytic Mechanism of Murine Lysophospholipase I

A lysophospholipase (LysoPLA I) has been purified and characterized from the mouse macrophage-like P388D1 cell line (Zhang, Y. Y, and Dennis, E. A. (1988) J. Biol. Chem. 263, 9965-9972). This enzyme has now been sequenced, cloned, and expressed in Escherichia coli cells. The enzyme contains 230 amino acid residues with a calculated molecular mass of 24.7 kDa. It has a high helical content in its predicated secondary structure, which is also indicated in its CD spectrum. The cloned LysoPLA I was purified to homogeneity from the transformed E. coli cells by a gel filtration column and an ion exchange column. The specific activity of the purified protein is 1. 47 micromol/min.mg toward 1-palmitoyl-sn-glycero-3-phosphorylcholine at pH 8.0 and 40 degrees C, corresponding to the reported value of 1.3-1.7 micromol/min.mg for the protein purified from the P388D1 cells. In addition, the cloned protein cross-reacted with an antibody raised against LysoPLA I also purified from the P388D1 cells. The deduced LysoPLA I sequence contains a well conserved GXSXG motif found in the active site of many serine enzymes, and the activity of the LysoPLA I was irreversibly inhibited by the classical serine protease inhibitor diisopropyl fluorophosphate. Furthermore, site-directed mutagenesis was employed to change Ser-119 in the GXSXG motif to an Ala. The resulting mutant protein lost all of its lysophospholipase activity, even though it had the same overall protein conformation as that of the wild-type LysoPLA I. Therefore, LysoPLA I has been demonstrated to be a serine enzyme with Ser-119 at the active site.

The serine protease inhibitor diisopropylfluorophosphate inhibits neutrophil NADPH-oxidase activity induced by the calcium ionophore ionomycin and serum opsonised yeast particles.

The effect on human neutrophil NADPH-oxidase activity of the serine protease inhibitor diisopropylfluorophosphate (DFP) was investigated. Pretreatment of neutrophils with the protease inhibitor did not affect the release of reactive oxygen species induced by fMLP. However, the intracellular production of reactive oxygen species induced by ionomycin and yeast particles was largely inhibited in DFP treated cells. Production of reactive oxygen species in subcellular fractions was not affected by the protease inhibitor, neither when the plasma membrane nor when the specific granules were used as source for the b cytochrome subunit of the oxidase. This shows that DFP does not affect the assembly of the oxidase or the activity of the assembled complex. We suggest that serine protease activity is of importance for the signal(s) induced by the Ca2+ ionophore and the yeast particles to reach the dormant NADPH oxidase present in the specific granules and phagolysosomes.

Protease Inhibitors Differentially Regulate Tumor Necrosis Factor-Induced Apoptosis, Nuclear Factor-κB Activation, Cytotoxicity, and Differentiation

We investigated the effect of various protease inhibitors on several tumor necrosis factor (TNF)-mediated cellular responses. Treatment of a human myelogenous leukemia cell line, ML-1a, with TNF in the presence of cycloheximide triggers endonucleolytic activity and apoptotic cell death within 90 minutes. The general serine protease inhibitor diisopropyl fluorophosphate (DFP) and the chymotrypsin-like protease inhibitor N-tosyl-L-lysyl chloromethyl ketone (TPCK) completely abrogated TNF-induced DNA fragmentation and the formation of apoptotic bodies. However, 13 other protease inhibitors, including serine protease inhibitors, did not. The addition of TPCK to cells 30 minutes after TNF treatment completely inhibited the cytokine action, indicating that TPCK-sensitive proteases are not involved in the early stages of signal transduction. TNF is cytotoxic and induces differentiation in ML-1a cells after a 3-day incubation. TPCK had no effect on the TNF-induced cytotoxicity and differentiation, indicating that TPCK-sensitive proteases are specific for DNA fragmentation. TPCK also blocked TNF-induced activation of nuclear factor (NF)-kappa B. The dose-response and the time-course of the inhibitor, however, indicated that the site of action of TPCK for NF-kappa B activation and for DNA fragmentation are quite distinct. Therefore, we conclude that TNF activates two distinct TPCK-sensitive pathways, one leading to apoptosis and the other to NF-kappa B activation.

Androgen Regulation of an Elastase-Like Protease Activity in the Seminal Vesicle1

The processing of secretory proteins in the guinea pig (GP) seminal vesicle epithelium (SVE) is altered by castration and restored by treatment of animals with androgens. To test the hypothesis that the changes in protein processing are due to changes in the activity of specific proteases, we examined the GPSVE for protease activities capable of cleaving a synthetic elastase substrate, succinyl-alanyl-alanyl-alanyl-p-nitroanilide (Suc(Ala)3pNA). We found that the GPSVE does contain a Suc(Ala)3pNA-cleaving activity that is sensitive to the serine protease inhibitor diisopropylfluorophosphate (DFP) and to the elastase inhibitor elastatinal. Furthermore, the amount of protease activity per milligram of SVE protein is reduced to about 50% of control levels by castration. The activity is completely restored within four days by treatment of castrated animals with androgens, but is not restored by treatment with estradiol, progesterone, or dexamethasone. Although the SVE enzyme did not yield a pattern of specific cleavage products when incubated with a secretory protein substrate in vitro, this enzyme activity was competitively inhibited by a peptide whose primary sequence included the cleavage site used by the processing machinery in vivo.

Downregulation of neutrophil CD43 by opsonized zymosan

CD43, a prevalent white blood cell molecule distinguished by its mucin- like surface region, has been proposed as a “functional barrier” that prevents or negatively regulates a variety of cell surface interactions. Implicit in this hypothesis is the expectation that CD43 will be altered or removed when white blood cells are activated. To investigate alterations of CD43 in a dramatic example of functional cell activation, suspension neutrophils were challenged with opsonized zymosan, a characterized stimulator of phagocytosis and respiratory burst oxidase. Flow cytometry showed decreased surface density of CD43 in opsonized zymosan-treated neutrophils, and immune precipitation showed decreased cellular CD43 content, indicating that opsonized zymosan downregulates CD43 by a proteolytic mechanism. Based on densitometry of immune precipitates, CD43 levels were decreased 42% +/- 6% in neutrophils treated for 10 minutes with opsonized zymosan and decreased 70% +/- 3% in neutrophils treated with phorbol 12-myristate 13-acetate (PMA). CD43 downregulation in response to opsonized zymosan, like PMA-induced CD43 downregulation, was insensitive to the serine protease inhibitor diisopropylfluorophosphate (DFP). In contrast, CD43 downregulation in response to opsonized zymosan or PMA was prevented by 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF) and 3′4′- dichloroisocoumarin (3,4-DCI), both of which are characterized serine protease inhibitors. Activation of the neutrophil respiratory burst oxidase by opsonized zymosan or PMA was also insensitive to DFP and prevented by AEBSF and 3,4-DCI. These findings indicate a requirement for a proteolytic step in activation of the respiratory burst of intact suspension neutrophils by opsonized zymosan and PMA and suggest that CD43 cleavage may be a required proteolytic event.

Downregulation of Neutrophil CD43 by Opsonized Zymosan

CD43, a prevalent white blood cell molecule distinguished by its mucin-like surface region, has been proposed as a "functional barrier" that prevents or negatively regulates a variety of cell surface interactions. Implicit in this hypothesis is the expectation that CD43 will be altered or removed when white blood cells are activated. To investigate alterations of CD43 in a dramatic example of functional cell activation, suspension neutrophils were challenged with opsonized zymosan, a characterized stimulator of phagocytosis and respiratory burst oxidase. Flow cytometry showed decreased surface density of CD43 in opsonized zymosan-treated neutrophils, and immune precipitation showed decreased cellular CD43 content, indicating that opsonized zymosan downregulates CD43 by a proteolytic mechanism. Based on densitometry of immune precipitates, CD43 levels were decreased 42% +/- 6% in neutrophils treated for 10 minutes with opsonized zymosan and decreased 70% +/- 3% in neutrophils treated with phorbol 12-myristate 13-acetate (PMA). CD43 downregulation in response to opsonized zymosan, like PMA-induced CD43 downregulation, was insensitive to the serine protease inhibitor diisopropylfluorophosphate (DFP). In contrast, CD43 downregulation in response to opsonized zymosan or PMA was prevented by 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF) and 3'4'-dichloroisocoumarin (3,4-DCI), both of which are characterized serine protease inhibitors. Activation of the neutrophil respiratory burst oxidase by opsonized zymosan or PMA was also insensitive to DFP and prevented by AEBSF and 3,4-DCI. These findings indicate a requirement for a proteolytic step in activation of the respiratory burst of intact suspension neutrophils by opsonized zymosan and PMA and suggest that CD43 cleavage may be a required proteolytic event.

Processing of human cathepsin G after transfection to the rat basophilic/mast cell tumor line RBL.

The azurophil granules of neutrophil granulocytes contain neutral proteases such as leukocyte elastase and cathepsin G. These are synthesized as inactive precursors, but following proteolytic processing, they are stored in granules as active enzymes. We describe the establishment of a transgenic cellular model for expression of the human myeloid serine protease cathepsin G. The cDNA for preprocathepsin G was stably expressed in the rat basophilic/mast cell line RBL-1 and the translation product was characterized by use of biosynthetic labeling followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography. Conversion into complex form of an asparagine-linked carbohydrate unit of approximately 3.5 kDa was shown, as judged by the products obtained upon treatment with endoglycosidase H and N-glycanase. Proteolytic processing of 32.5-kDa procathepsin G into a 31-kDa form, within 1-2 h after synthesis, was demonstrated by pulse-chase experiments. Further processing into a 30-kDa form also occurred to a minor extent. The processed forms were enzymatically active, as judged by affinity for the serine protease inhibitors diisopropylfluorophosphate and aprotinin. Translocation of processed forms of cathepsin G to high density fractions, indicating targeting of the protease to granules, was demonstrated by subcellular fractionation. The weak base NH4Cl was shown to delay the processing and enzymatic activation of cathepsin G, whereas the monovalent ionophore monensin completely inhibited both events. Our data demonstrate that human cathepsin G transfected to rat RBL-1 cells, is proteolytically processed into enzymatically active forms and that subcellular transfer to granular organelles occurs. As the processing of transgenic human cathepsin G corresponds to that of endogenous protease of myeloid cells, the model should provide new unique possibilities to further characterize the activation and granular targeting of myeloid serine proteases.

Human granzyme B degrades aggrecan proteoglycan in matrix synthesized by chondrocytes.

Cartilage degradation, a hallmark of rheumatoid arthritis, is attributed to serine and metalloproteases secreted by neutrophils, synovial lining cells, macrophages, and chondrocytes. A large proportion of synovial fluid lymphocytes contains the granule-associated serine proteases granzymes A and B. We report that lysates of IL-2-stimulated lymphocytes contain an enzymatic activity (ECMase; cartilage extracellular matrix 35S release assay; extracellular matrix degrading activity) that solubilizes matrix synthesized by chondrocyte monolayers. ECMase activity is inactivated by the serine protease inhibitor diisopropylfluorophosphate, is stored in dense granules and cleaves aggrecan proteoglycans but not free glycosaminoglycans, hyaluronic acid, or type II collagen. ECMase is mediated by a cationic protein with biochemical properties identical to granzyme B, inasmuch as it preferentially hydrolyzes the substrate Boc-Ala-Ala-Asp-SBzl, immunochemically cross-reacts with an antibody that binds to a conserved amino-terminal region of lymphoid-myeloid serine proteases, and has amino-terminal sequence identity with human Q31 granzyme B. Using an agarose gel electrophoresis technique to assess cleavage of the rat sarcoma aggrecan, the catalytic efficiency of granzyme B for the digestion of aggrecan (catalytic efficiency = 1.7 x 10(7) M-1 s-1) was 425-fold faster than the catalytic efficiency reported for human stromelysin-1 at pH 7.5 (catalytic efficiency 4000 M-1 s-1) and 3200-fold faster than granzyme A. Based on these observations, we propose that granzyme B, secreted from cytotoxic lymphocytes within the rheumatoid joint, may contribute to cartilage loss by degrading resident aggrecan.

Protease inhibitors selectively block T cell receptor-triggered programmed cell death in a murine T cell hybridoma and activated peripheral T cells.

The hypothesis that cytoplasmic proteases play a functional role in programmed cell death was tested by examining the effect of protease inhibitors on the T cell receptor-mediated death of the 2B4 murine T cell hybridoma and activated T cells. The cysteine protease inhibitors trans-epoxysuccininyl-L-leucylamido-(4-guanidino) butane (E-64) and leupeptin, the calpain selective inhibitor acetyl-leucyl-leucyl-normethional, and the serine protease inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, all showed dose-dependent blocking of the 2B4 death response triggered by the T cell receptor complex and by anti-Thy-1. These protease inhibitors enhanced rather than inhibited IL-2 secretion triggered by T cell receptor cross-linking, showing that they did not act by preventing signal transduction. Growth inhibition induced by cross-linking the 2B4 T cell receptor, measured by inhibition of thymidine incorporation, was not generally blocked by these protease inhibitors. All five of these protease inhibitors enhanced rather than blocked 2B4 cell death triggered by dexamethasone, an agent previously shown to have a death pathway antagonistic with that of the TCR. 2B4 cytolysis by the cytotoxic agents staphylococcal alpha-toxin and dodecyl imidazole, and that caused by hypotonic conditions, was not significantly affected by the five protease inhibitors tested. The selected protease inhibitors blocked both the apoptotic nuclear morphology changes and DNA fragmentation induced by T cell receptor cross-linking, and enhanced both these properties induced by dexamethasone in 2B4 cells. The T cell receptor-induced death of activated murine lymph node T cells and human peripheral blood CD4+ T cells was blocked by both cysteine and serine protease inhibitors, showing that the protease-dependent death pathway also operates in these systems.

Characterization and identification of a lipoprotein lipase from Manduca sexta flight muscle

Lipoprotein lipase (LpL) activity in Manduca sexta flight muscle tissue was measured using in vivo radiolabeled lipophorin as a substrate. LpL hydrolyses diacylglycerol in the low density lipophorin (that occurs during flight) at a higher rate than diacylglycerol in the high density lipophorin (present in the resting insect). LpL has a pH-optimum of 7.5 and is less sensitive to NaCl than mammalian LpL. LpL is inhibited by bovine albumin and chicken ovalbumin. LpL is inhibited by the serine protease inhibitors diisopropylfluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF), which indicates the presence of an active site serine similar to mammalin LpL. Flight muscle LpL shows affinity for immobilized copper as well as for immobilized heparin. Using radiolabeled DFP, a protein of 37 kDa was identified (after SDS-PAGE) as the DFP-binding protein in a partially purified preparation of LpL. This 37 kDa protein is proposed to be the LpL or a subunit thereof.