A functional proteomics screen of proteases in colorectal carcinoma

BACKGROUND: Proteases facilitate several steps in cancer progression. To identify proteases most suitable for drug targeting, actual enzyme activity and not messenger RNA levels or immunoassay of protein is the ideal assay readout. MATERIALS AND METHODS: An automated microtiter plate assay format was modified to allow detection of all four major classes of proteases in tissue samples. Fifteen sets of colorectal carcinoma biopsies representing primary tumor, adjacent normal colon, and liver metastases were screened for protease activity. RESULTS: The major proteases detected were matrix metalloproteases (MMP9, MMP2, and MMP1), cathepsin B, cathepsin D, and the mast cell serine proteases, tryptase and chymase. Matrix metalloproteases were expressed at higher levels in the primary tumor than in adjacent normal tissue. The mast cell proteases, in contrast, were at very high levels in adjacent normal tissue, and not detectable in the metastases. Cathepsin B activity was significantly higher in the primary tumor, and highest in the metastases. The major proteases detected by activity assays were then localized in biopsy sections by immunohistochemistry. Mast cell proteases were abundant in adjacent normal tissue, because of infiltration of the lamina propria by mast cells. Matrix metalloproteases were localized to the tumor cells themselves; whereas, cathepsin B was predominantly expressed by macrophages at the leading edge of invading tumors. Although only low levels of urinary plasminogen activator were detected by direct enzyme assay, immunohistochemistry showed abundant protein within the tumor. CONCLUSIONS: This analysis, surveying all major classes of proteases by assays of activity rather than immunolocalization or in situ hybridization alone, serves to identify proteases whose activity is not completely balanced by endogenous inhibitors and which may be essential for tumor progression. These proteases are logical targets for initial efforts to produce low molecular weight protease inhibitors as potential chemotherapy.     

Regulation of cyclic adenosine 3':5'-monophosphate phosphodiesterases: altered pattern in transformed myoblasts

Rat skeletal myoblasts, L6 and L8, have two major forms of phosphodiesterases, PDE II and PDE III. Only the former is activated by treatment with proteases. When the myoblasts are exposed to cAMP for 10-16 h, the activity of PDE III increases considerably. This increase is accompanied by a loss of activatability of PDE II by proteases. Leupeptin prevents the increase in the levels of PDE III suggesting that a protease in vivo may be responsible for the formation of PDE III from PDE II. Spontaneously or Rous sarcoma virus-transformed myoblasts, however, show altered regulation of the two forms of PDE. In the presence of cAMP in the medium, unlike the nontransformed cells, the levels of PDE III do not increase but the activity of PDE II rises. Simultaneously, PDE II becomes refractory to activation by proteases. The altered mode of PDE regulation in transformed cells is dominant in hybrids between normal and transformed myoblasts, which suggests that altered regulation is due to an "acquisition" of some new property by transformed cells.     

Inhibition of bovine factor IXa and factor Xabeta by antithrombin III.

Factor IXa and factor Xabeta are serine proteases which participate in the middle phase of blood coagulation. These two enzymes are inhibited by antithrombin III by the formation of an enzyme-inhibitor complex containing 1 mol of enzyme and 1 mol of antithrombin III. The complex was readily demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and loss of coagulant or esterase activity at increasing concentrations of inhibitor. The inactivation of factor IXa by antithrombin III was relatively slow, but the reaction was greatly accelerated by the addition of heparin.     

Proteases from human immunodeficiency virus and avian myeloblastosis virus show distinct specificities in hydrolysis of multidomain protein substrates.

The virally encoded proteases from human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) have been compared relative to their ability to hydrolyze a variant of the three-domain Pseudomonas exotoxin, PE66. This exotoxin derivative, missing domain I and referred to as LysPE40, is made up of a 13-kilodalton NH2-terminal translocation domain II connected by a segment of 40 amino acids to enzyme domain III of the toxin, a 23-kilodalton ADP-ribosyltransferase. HIV protease hydrolyzes two peptide bonds in LysPE40, a Leu-Leu bond in the interdomain region and a Leu-Ala bond in a nonstructured region three residues in from the NH2-terminus. Neither of these sites is cleaved by the AMV enzyme; hydrolysis occurs, instead, at an Asp-Val bond in another part of the interdomain segment and at a Leu-Thr bond in the NH2-terminal region of domain II. Synthetic peptides corresponding to these cleavage sites are hydrolyzed by the individual proteases with the same specificity displayed toward the protein substrate. Peptide substrates for one protease are neither substrates nor competitive inhibitors for the other. A potent inhibitor of HIV type 1 protease was more than 3 orders of magnitude less active toward the AMV enzyme. These results suggest that although the crystallographic models of Rous sarcoma virus protease (an enzyme nearly identical to the AMV enzyme) and HIV type 1 protease show a high degree of similarity, there exist structural differences between these retroviral proteases that are clearly reflected by their kinetic properties.     

Degradation of L-glutamate dehydrogenase from Escherichia coli: allosteric regulation of enzyme stability.

L-glutamate dehydrogenase (GDH) is stable in exponentially growing Escherichia coli cells but is degraded at a rate of 20-30% per hour in cells starved for either nitrogen or carbon. GDH degradation is energy-dependent, and mutations in ATP-dependent proteases, ClpAP or Lon lead to partial stabilization. Degradation is inhibited by chloramphenicol and is completely blocked in relA mutant cells, suggesting that ribosome-mediated signaling may facilitate GDH degradation. Purified GDH has a single tight site for NADPH binding. Binding of NADPH in the absence of other ligands leads to destabilization of the enzyme. NADPH-induced instability and sensitivity to proteolysis is reversed by tri- and dicarboxylic acids or nucleoside di- and triphosphates. GTP and ppGpp bind to GDH at an allosteric site and reverse the destabilizing effects of NADPH. Native GDH is resistant to degradation by several purified ATP-dependent proteases: ClpAP, ClpXP, Lon, and ClpYQ, but denatured GDH is degraded by ClpAP. Our results suggest that, in vivo, GDH is sensitized to proteases by loss of a stabilizing ligand or interaction with an destabilizing metabolite that accumulates in starving cells, and that any of several ATP-dependent proteases degrade the sensitized protein.     

Saturated Fatty Acid Mutant of Saccharomyces cerevisiae with an Intact Fatty Acid Synthetase

To determine directly the effects of streptomycin on translational fidelity in intact cells, we studied the synthesis of beta-galactosidase and of the coat protein of bacteriophage R17 in an Escherichia coli mutant in which the bactericidal effects of streptomycin are delayed. After the addition of streptomycin to exponentially growing mutant cells, protein synthesis continues at an undiminished rate for approximately an hour; however, as measured by enzyme assays, little functional protein is produced. Serological assays designed to detect beta-galactosidase and bacteriophage R17 coat protein show that substantial amounts of the protein synthesized can react with antisera prepared against active beta-galactosidase and phage R17, indicating the aberrance of the protein produced in the presence of the antibiotic. The polypeptides synthesized in the presence of streptomycin are degraded in the cell to a much greater extent than protein synthesized in the absence of the antibiotic. The proteolytic attack on this protein is not affected by inhibitors of serine proteases, suggesting that enzymes other than those involved in "normal turnover" of cellular protein are responsible. In this strain, certain of the multiple effects of streptomycin are separated in time and the production of abnormal protein (enzymatically inactive and susceptible to proteolytic attack) could be studied in the absence of the lethal effect of the drug.     

Differential influence of bacitracin on plant proteolytic enzyme activities

The effect of bacitracin on the activity of proteases extracted from pollen and sprouts of various plant species and compared to five commercially available proteases was studied. Bacitracin stimulates some pollen proteolytic enzyme activities, contrary to its inhibitory influence on proteases from the other sources. Proteases from maize pollen, inhibited by pepstatin and phenylmethylsulfonyl fluoride, immediately accelerate their activities after addition of bacitracin to the reaction mixture. The stimulating influence of peptide antibiotic on pollen proteases of some plants is unexpected and molecular mechanism of this phenomenon requires a further elucidation. The augmentation of allergenic response caused by pollen enzymes and drugs containing bacitracin is discussed.     

Sheep plasma protease inhibitors influencing protease activity and growth of Lucilia cuprina larvae in vitro

The enzyme inhibitors alpha 2-macroglobulin (alpha 2M), anti-thrombin III (AT III) and alpha 1-proteinase inhibitor (alpha 1PI) were isolated from sheep plasma and tested for their ability to affect L. cuprina larval proteases and larval growth in vitro. Casein radial diffusion gels indicated that both alpha 2M and alpha 1PI completely inhibited the protease activity of a larval excretory-secretory preparation, while AT III had a partial effect. Casein zymograms revealed that alpha 2M inhibited all of the larval proteases, while AT III was able to modify the normal plaque pattern; alpha 1PI inhibited all plaques except a doublet present at pI 8.5. Larval growth in vitro was significantly inhibited by alpha 2M and AT III (P less than 0.05) when compared to albumin controls but was not affected by alpha 1PI. The levels of alpha 2M in sheep serum were monitored over the course of a larval fly infection. A significant increase in alpha 2M (P less than 0.05) was recorded in the serum of flystruck sheep. It is suggested that, under certain circumstances, these inhibitors may be involved in influencing flystrike through reducing the activity of larval proteases necessary for wound formation and larval nutrition.     

Studies on the conversion of multiple forms of tyrosine aminotransferase in rat liver.

Studies from several laboratories have demonstrated the existence of at least three separable forms of the hepatic enzyme, tyrosine aminotransferase. The significance of these separable forms of the enzyme isolated in vitro for the nature and regulation of the enzyme in vivo has been the subject of some controversy. The studies reported in this paper demonstrate the existence of a heat-labile, pH- and temperature-dependent, nondialyzable component associated predominantly with the lysosomal and mitochondrial fraction of rat liver which catalyzes the conversion of form II to forms III and IV of the enzyme. The activity of this conversion factor is not significantly affected by F^?, molybdate ions, or two inhibitors of proteases. On the other hand, the cyanate ion completely inhibits the conversion of form II to forms III and IV of tyrosine aminotransferase, as do iodoacetate and oxidized glutathione. p-Chloromercuribenzoate also markedly inhibits the conversion. Kinetic studies suggest that the shift from one form to another follows the pathway: II to III to IV. Titration of the available sulfhydryl groups of the three forms of the enzyme demonstrates that form II possesses between 16 and 17 titratable SH groups per mole, while forms III and IV possess 15 and 13 or 14, respectively. The possible catalytic mechanism by which the conversion of the multiple forms of tyrosine aminotransferase is accomplished is discussed.     

秸秆纤维素分解菌的酶活力测定

目的:测定秸秆纤维素分解菌的酶活力。方法:从土壤中分离出具有分解纤维素能力的菌株,采用刚果红染色法进行粗选,得到7株透明圈较大的菌株。将这7株菌株液体发酵培养6d,再分别用滤纸分解度观察、羧甲基纤维素酶活法(CMC)、滤纸酶活法(FPA)和天然纤维素酶活法测定其酶活力。结果:在7株菌株中,F-1、F-2、F-3、F-5的酶活力测定结果与其溶解圈的测定结果、滤纸分解结果基本相同。且天然纤维素酶活力高的菌株,其CMC酶活、FPA酶活也高,滤纸分解效果也比较明显。结论:CMC法、FPA法和天然纤维素酶活法适于测定秸秆纤维素分解菌的酶活力。     

Identification of protease IV of E.coli: An outer membrane bound enzyme

The proteolytic activity of $\text{E.coli}$ measured using ¹²⁵I-labelled αS1 casein as substrate, is mainly localised in the outer membrane and is due to an intrinsic outer membrane protein which can be solubilized by deoxycholate. This enzyme exhibits maximum activity at pH 7,5 in Tris-HCl buffer, is resistant to thermal denaturation with a half-life of 28 min. at 90°C in deoxycholate-NaCl buffer and is inhibited by ethylene-diamine tetraacetate, high concentrations of p-aminobenzamidine, tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalaninechloromethyl ketone and by two inhibitors of the processing of the secreted protein precursors, procaine and phenehylalcohol. Whole cells do not exhibit proteolytic activity, nevertheless, some is unmasked when the outer membrane is permeabilized by Tris or ethylenediamine tetraacetate or when vesicles are sonicated. This suggests that the protease is on the inner side of the outer membrane. Because the protease is different from the soluble proteases described in $\text{E.coli}$, and especially from proteases I,II and III, it has been called protease IV.     

Proteolytic complex from Aspergillus terricola (review)

Results are presented of the studies on the proteolytic enzymes produced by the fungus Aspergillus terricola that comprise the antiinflammatory drug Terrilytin. Terrilytin was shown to be a complex of three proteolytic enzymes that contained amylase as well. Two of the proteases (proteases I and II) were of the group of serine proteases by their physico-chemical and enzymatic characteristics and might be considered as two types of the same enzyme. Protease III showed characteristics specific for metalloproteases of the microbial origin, its activity being exhibited in case of the zinc ion presence. Fibrinolytic properties of Terrilytin are associated with the ability of all the three proteases to hydrolyze fibrin and fibrinogen.     

Effect of cellulase size reduction on activity and accessibility

Summary Reducing the size of crude cellulase from Trichoderma reesei with various proteases showed that while the -glucosidase activity on CMC generally increased, the extent of hydrolysis of hardwood and pretreated hardwood decreased. The rank in the decrease in hydrolytic activity is the same as the rank in the decrease in enzyme adsorption on the hardwood or pretreated hardwood.     

Influence of the composition of the medium and the duration of cultivation on the growth of Actinomyces spheroides and their biosynthesis of intracellular proteases and novobiocin

The growth of Actinomyces spheroides 35 under different conditions and the biosynthesis of biomass, proteases and the antibiotic were studied with two types of growth medium: a control one and an optimized one. The rate of biomass accumulation was by 5--7 per cent higher on the latter medium than on the former one. The dynamics of accumulation was studied with novobiocin and proteases which hydrolyzed fibrin and casein. Fibrin hydrolyzing proteases were found to be synthesized under conditions that were unfavourable for the production of novobiocin. If the production of the antibiotic was supressed, the concentration of fibrinolytic proteases in the cultural broth increased almost proportionally. Such a relation has not been found for caseinolytic proteases.     

Characterization of the activation of rat liver beta-glucosidase by sialosylgangliotetraosylceramide

We show that sialosylgangliotetraosylceramide (GM1) is a potent activator of delipidated (sodium cholate- and 1-butanol-extracted) lysosomal rat liver glucocerebroside:beta-glucosidase. Stimulation of 4-methylumbelliferyl-beta-D-glucopyranoside hydrolysis by the beta-glucosidase was markedly dependent upon the concentration of GM1 in the assay medium. Estimations of critical micellar concentration (CMC) performed fluorometrically using the dye N-phenylnaphthylamine revealed two CMC values of GM1 above 18 degrees C; the CMC of the primary micelles (3.32 microM) was temperature-independent whereas that of the secondary micelles decreased with decreasing temperature (17.2 and 10.8 microM at 37 and 20 degrees C, respectively). In the temperature range of 18-39 degrees C, beta-glucosidase activity increased sharply when the GM1 concentration was above the CMC of the secondary micelles. Although a heat-stable factor, purified from the spleen of a patient with Gaucher's disease, had a profound effect on the activation of beta-glucosidase by GM1, it decreased the CMC only slightly (14.8 versus 17.2 microM at 37 degrees C). The heat-stable factor (8 micrograms/ml) changed the shape of the activation curve from sigmoidal to hyperbolic, suggesting that the heat-stable factor permits beta-glucosidase to be activated by primary micelles or monomers. The results of gel filtration chromatography and sucrose gradient centrifugation in H2O and D2O revealed that the activation of beta-glucosidase by GM1 was associated with an increase in the size of the enzyme from 45,800 to 178,500 daltons and an increase in the partial specific volume from 0.697 to 0.740 ml/g. The active, reconstituted beta-glucosidase appears to consist of 50% protein and 50% ganglioside (56 molecules/178,500 g). Concentrations of GM1 below the CMC of secondary micelles increased the rate of inactivation of the enzyme by the irreversible inhibitor conduritol B epoxide at 37 degrees C, indicating that GM1 monomers or primary micelles do interact with the enzyme, even though they do not increase the rate of hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside by the enzyme.     

Role of surfactant on the proteolysis of aqueous bovine serum albumin

Nonionic and ionic surfactants diminish the initial rate of proteolysis of aqueous bovine serum albumin (BSA) by subtilisin Carlsberg. Surfactants studied include: nonionic tetraethylene glycol monododecyl ether (C12E4); anionic sodium dodecyl sulfate (SDS), anionic sodium dodecylbenzenesulfonate (SDBS), and cationic dodecyltrimethylamonium bromide (DTAB). Kinetic data are obtained using fluorescence emission. Special attention is given to enzyme kinetic specificity determined by fitting initial-rate data to the Michaelis-Menten model. All surfactants reduce the rate of proteolysis, most strongly at concentrations near and above the critical micelle concentration (CMC). Circular dichroism (CD), tryptophan/tyrosine fluorescence spectra, and tryptophan fluorescence thermograms indicate that BSA partially unfolds at ionic surfactant concentrations near and above the CMC. Changes in BSA conformation are less apparent at ionic surfactant concentrations below the CMC and for the nonionic surfactant C12E4. Subtilisin Carlsberg activity against the polypeptide, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, decreased due to enzyme-surfactant interaction. At the concentrations and time frames studied, there was no enzyme autolysis. Importantly, aqueous proteolysis rates are significantly reduced at high surfactant concentrations where protein-micellar-surfactant aggregates occur. To explain the negative effect of surfactant on subtilisin Carlsberg proteolytic activity against BSA, we propose that micelle/protein complexes hinder enzyme access.     

Cleavage of bovine skin type III collagen by proteolytic enzymes. Relative resistance of the fibrillar form

We have studied the susceptibility of fibrils formed from fetal bovine skin type III collagen to proteolytic enzymes known to cleave within the helical portion of the molecule (vertebrate and microbial collagenase, polymorphonuclear elastase, trypsin, thermolysin) and to two general proteases of broad specificity (plasmin, Pronase). Fibrils reconstituted from neutral salt solutions, at 35 degrees C, were highly resistant to nonspecific proteolysis by general proteases such as polymorphonuclear elastase, trypsin, and thermolysin but were rapidly dissolved by bacterial and vertebrate collagenases at rates of 12-45 mol X mol-1 X h-1. In solution, type III collagen was readily cleaved by each of the proteases (with the exception of plasmin), as well as by the true collagenases, although at different rates. Turnover numbers determined by viscometry at 35 degrees C were: human collagenase, approximately equal to 1500 h-1; microbial (clostridial) collagenase, approximately equal to 100 h-1; and general proteases, 23-52 h-1. In addition it was shown that pronase cleaves type III collagen in solution at 22 degrees C by attacking the same Arg-Gly bond in the alpha 1(III) chain as trypsin. However, like other proteases, Pronase was rather ineffective against fibrillar forms of type III collagen. It was also shown that transition of type III collagen as well as type I collagen to the fibrillar form resulted in a significant gain of triple helical thermostability as evidenced by a 6.8 degrees C increase in denaturation temperature (Tm = 40.2 degrees C in solution; Tm = 47.0 degrees C in fibrils).     

Purification and some properties of the extracellular acid proteases from Mucor renninus

A complex of proteases was fractionated into three enzymes by chromatography of a crude enzyme preparation obtained from culture fluid of the fungus Mucor renninus on biospecific polystyrene adsorbent. Electrophoretically homogeneous proteases I-III were obtained by subsequent rechromatography on biospecific adsorbent and gel filtration on Sephadex G-75. Optimal proteolytic activities occurred at pH 4.25; 3.5 and 2.5 for enzymes I, II and III, respectively. Milk-clotting activity was exhibited only by protease II. All three proteases hydrolysed haemoglobin, Na caseinate and bovine serum albumin. Enzyme I hydrolysed Na caseinate the most effectively, while haemoglobin was the most effective substrate for proteases II and III. Trypsinogen was activated only by protease I. All three enzymes have a molecular weight ～35 000 as determined by gel chromatography on Sephadex G-75 column and by sodium dodecylsulphate disc electrophoresis. Isoelectric points, pH-stability range, amino acid composition, carbohydrate content were determined for each enzyme and the influence of metal ions (Ca²⁺, Mg²⁺, Cu²⁺, Co²⁺) on proteolytic activities of these enzymes studied.     

Localization of the type III isozyme of hexokinase at the nuclear periphery.

The distribution of the type III isozyme of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) in rat kidney, liver, spleen, lung, and brain was determined immunohistochemically, using a monoclonal antibody generated against the enzyme purified from rat Novikoff hepatoma.In all tissues, specific cell types exhibited intense staining at the nuclear periphery, as confirmed by analysis using confocal microscopy. Isolated nuclei from kidney or liver were devoid of detectable type III hexokinase, although the enzyme was found in the "soluble" fraction from kidney or liver homogenates; these results suggest that the type III isozyme is associated in a labile manner with the external surface of the nucleus, with this association being disrupted by conventional homogenization and nuclear isolation procedures. The nuclear localization of the type III isozyme contrasts with previously demonstrated association of the type I and II isozymes with mitochondria. The physiological significance of a nuclear localization for the type III isozyme remains unclear. However, it was noted that many of the cells exhibiting prominent nuclear staining for type III hexokinase are endothelial or epithelial cells, suggesting a possible relationship between nuclear type III hexokinase and transport functions which are prominent in such cells.     

Simultaneous expression of cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity to a syngeneic rat lymphoma: separation and partial characterization of two types of cytotoxic cells

The immune response of $\text{W}\text{Fu}$ rats to a syngeneic Gross virus-induced lymphoma (C58NT)D evokes the simultaneous generation of effector cells able specifically to destroy the tumor cells by two different cytotoxic pathways: cell-mediated cytotoxicity (CMC) and antibody-dependent cellular cytotoxicity (ADCC). The question of possible interdependence in the relationship between the effector cells mediating both cytotoxicities was approached in several ways: (a) Immunospecific competition of one form of cytotoxicity (CMC or ADCC) did not interfere with the full expression of the other cytotoxic effect (ADCC or CMC, respectively), (b) Elimination of T cells by anti-thymocyte serum and complement completely abrogated the CMC activity while not impairing the ADCC activity, (c) Specific depletion of cytotoxic (CMC) lymphoid cells on monolayers of target cells bearing the sensitizing antigens considerably diminished the CMC activity, but did not affect the ADCC activity, (d) Depletion of Fc receptor-bearing cells (non-T cells) markedly reduced the ADCC activity, but did not interfere with CMC activity. These findings indicate that, in this system, two forms of cell-mediated cytotoxicity to tumor-associated antigens exist concurrently in the immune host and are expressions of different lymphoid cell populations; CMC is mediated by T cells, whereas ADCC is a non-T cell function.