Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Three-dimensional structures of trypsin with the reversible inhibitor leupeptin have been determined in two different crystal forms. The first structure was determined at 1.7 A resolution with R-factor = 17.7% in the trigonal crystal space group P3(1)21, with unit cell dimensions of a = b = 55.62 A, c = 110.51 A. The second structure was determined at a resolution of 1.8 A with R-factor = 17.5% in the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions of a = 63.69 A, b = 69.37 A, c = 63.01 A. The overall protein structure is very similar in both crystal forms, with RMS difference for main-chain atoms of 0.27 A. The leupeptin backbone forms four hydrogen bonds with trypsin and a fifth hydrogen bond interaction is mediated by a water molecule. The aldehyde carbonyl of leupeptin forms a covalent bond of 1.42 A length with side-chain oxygen of Ser-195 in the active site. The reaction of trypsin with leupeptin proceeds through the formation of stable tetrahedral complex in which the hemiacetal oxygen atom is pointing out of the oxyanion hole and forming a hydrogen bond with His-57.
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PMID:Two crystal structures of the leupeptin-trypsin complex. 884 65

African trypanosomes are tsetse-transmitted protozoan parasites that cause sleeping sickness in humans and 'Nagana' in animals. A high relative molecular mass multicatalytic proteinase complex (MCP) was purified and biochemically characterized from the cytosolic fraction of Trypanosoma brucei brucei. The isolation procedure consisted of fractionation of the lysate by high speed centrifugation, chromatography on Q-sepharose molecular sieve filtration on Sephacryl S-300, chromatography on HA-Ultrogel and glycerol density gradient centrifugation (10-40%). The final enzyme preparation yielded a single protein band corresponding to a relative molecular mass of 630 kDa on a non-denaturing polyacrylamide gel. The enzyme hydrolyses a wide range of peptide substrates characteristic of chymotrypsin-like, trypsin-like, peptidylglutamylpeptide-hydrolysing activities determined by fluorogenic peptides, Z-Gly-Gly-Leu-NHMec, Z-Arg-Arg-NHMec and Z-Leu-Leu-Glu-beta NA, respectively. The enzyme was found to have a wide variation in pH optimal activity profile, with optimum activity against Z-Gly-Gly-Leu-NHMec at 7.8, Z-Arg-Arg-NHMec at pH 10.5 and Z-Leu-Leu-Glu-beta NA at pH 8.0, showing that the different activities are distinct. The enzyme hydrolysed oxidized proteins. In addition, the chymotryptic and trypsin-like activities were susceptible to inhibition by peptide aldehyde inhibitors with variable inhibition effects. The study demonstrates the presence of a non-lysosomal proteasome pathway of intracellular protein degradation in the bloodstream form of T. b. brucei. Further, the ability of the enzyme to hydrolyse most oxidized proteins, and the high immunogenicity exhibited suggests a possible involvement of the enzyme in pathogenesis of the disease.
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PMID:Characterization of a multicatalytic proteinase complex (20S proteasome) from Trypanosoma brucei brucei. 922 59

Mucosal mast cells (MCs) are normally found in the connective tissue stroma but are redistributed into the epithelium in conditions associated with immunoglobulin E responses, such as allergic inflammation and nematode infections, as well as in interstitial cystitis, a condition of unknown etiology. The potential role of epithelium-derived factors in this response prompted this inquiry into growth and differentiation signaling in normal tissue as well as in tissues from five different metaplastic conditions of the urothelium (cystitic cystica, cystitis glandularis, colonic metaplasia, squamous cell metaplasia, and nephrogenic metaplasia). Expression of the two major human MC growth factors, stem cell factor (or kit ligand) and interleukin 6, was detected using immunohistochemistry. In the case of interleukin 6, its mRNA expression was also detected using in situ reverse transcription-polymerase chain reaction. Among the different metaplastic lesions, nephrogenic metaplasia was the only one associated with an abundance of MCs, which were distributed within or in close relationship to the epithelium. Unlike in the other types of metaplasia, the epithelium strongly co-expressed interleukin 6 and stem cell factor. The MCs expressed the stem cell factor receptor CD117 and exhibited a variable tryptase immunoreactivity, but lacked chymase. They also displayed a relative deficiency of granular glycosaminoglycan, as indicated by a lack of metachromasia, and were sensitive to strong aldehyde fixation. The findings suggest that the MC response in nephrogenic metaplasia may be the result of local epithelial stem cell factor/interleukin 6 expression.
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PMID:Metaplastic transformation of urinary bladder epithelium: effect on mast cell recruitment, distribution, and phenotype expression. 966 75

Purified membrane fractions have been widely used for the study of the factors regulating the functions of Rho small GTP-binding proteins. Using brush border membranes from the rat kidney as a model, we observed that in vitro incubation of these membranes resulted in time- and temperature-dependent proteolytic degradation of Cdc42 and RhoA. Treatment of kidney brush border membranes with various nucleotides showed that GDP and GTP weakly protected Cdc42 but not RhoA and that their nonhydrolyzable counterparts, guanosine 5'-O-[beta-thio] diphosphate (GDP beta S) and guanosine 5'-O-[gamma-thio]triphosphate (GTP gamma S), were highly efficient in protecting both proteins from endogenous proteolytic activity whereas ADP and ATP were without effect. GTP gamma S also protected Cdc42 and RhoA from proteolytic degradation in crude cell membranes from several rat tissues including intestine, kidney, liver, and testis. In addition, Cdc42 and RhoA associated with brush border membranes were largely resistant to increased proteolytic degradation induced by membrane treatment with the denaturing reagent urea as well as to added trypsin when incubated in the presence of GTP gamma S. In brush border membranes, the resistance to endo- and exo-genous proteolytic activity conferred by GTP gamma S was usually lower for RhoA than for Cdc42. GTP gamma S also protected recombinant Cdc42 and RhoA from the action of proteases associated with brush border membranes. The only protease inhibitor protecting Cdc42 but not RhoA from proteolytic degradation in brush border membranes was the synthetic peptide acetyl-Tyr-Val-Ala-Asp-aldehyde, a selective inhibitor of interleukin-1 beta-converting enzyme. This latter result showed that different proteases cleaved the two Rho proteins. Taken together, these results suggest that the GTP gamma S-bound forms of Cdc42 and RhoA are maintained in a conformation that protects them from proteases found in many cell membranes.
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PMID:Guanine nucleotides protect Rho proteins from endogenous proteolytic degradation in renal membranes. 966 7

The crystal structures of three highly potent and selective low-molecular weight rigid peptidyl aldehyde inhibitors complexed with thrombin have been determined and refined to R values 0.152-0. 170 at 1.8-2.1 A resolution. Since the selectivity of two of the inhibitors was >1600 with respect to trypsin, the structures of trypsin-inhibited complexes of these inhibitors were also determined (R = 0.142-0.157 at 1.9-2.1 A resolution). The selectivity appears to reside in the inability of a benzenesulfonamide group to bind at the equivalent of the D-enantiomorphic S3 site of thrombin, which may be related to the lack of a 60-insertion loop in trypsin. All the inhibitors have a novel lactam moiety at the P3 position, while the two with greatest trypsin selectivity have a guanidinopiperidyl group at the P1 position that binds in the S1 specificity site. Differences in the binding constants of these inhibitors are correlated with their interactions with thrombin and trypsin. The kinetics of inhibition vary from slow to fast with thrombin and are fast in all cases with trypsin. The kinetics are examined in terms of the slow formation of a stable transition-state complex in a two-step mechanism. The structures of both thrombin and trypsin complexes show similar well-defined transition states in the S1 site and at the electrophilic carbon atom and Ser195OG. The trypsin structures, however, suggest that the first step in a two-step kinetic mechanism may involve formation of a weak transition-state complex, rather than binding dominated by the P2-P4 positions.
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PMID:Highly selective mechanism-based thrombin inhibitors: structures of thrombin and trypsin inhibited with rigid peptidyl aldehydes. 972 21

The specific inhibition of trypsin-like serine proteases has become an important therapeutic target. These proteases have been implicated in several physiological and pathophysiological processes, including blood coagulation, digestion, and inflammation. Proteases of this class cleave polypeptide chains C-terminal to a basic residue (arginine or lysine). It has been shown that selectivity for a particular serine protease can be conferred based upon the structural moiety incorporated in the P1 position. In this regard, the three isomers (ortho, meta, and para) of amidinophenylalanine represent modified arginine residues and are important synthetic targets. Herein, a convenient asymmetric synthesis of (S)-Nalpha-(tert-butyloxycarbonyl)-2-, (S)-Nalpha-(tert-butyloxycarbonyl)-3-, and (S)-Nalpha-(tert-butyloxycarbonyl)-4-amidinophenyl-alanine N,O-dimethylamides (Weinreb amides) will be described. These derivatives represent key synthetic intermediates for the synthesis of enzyme inhibitors because the amidine can be readily orthogonally protected, while the Weinreb amide is easily converted to a variety of electrophilic carbonyls via reduction to the corresponding aldehyde or by reaction with various lithiated heterocycles. Likewise, the Weinreb amide can be reduced to the aldehyde and subsequently oxidized to the corresponding carboxylate, which is suitable for solid- or solution-phase peptide synthetic strategies.
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PMID:The asymmetric synthesis of arginine mimetics: derivatives of (S)-2-, 3- and 4-amidinophenylalanine suitable for incorporation into enzyme inhibitors and/or peptides. 977 33

Inhibition of interleukin-1 beta converting enzyme (ICE), apopain, papain, thrombin and trypsin with substrate like peptidyl L- and D-alpha-aldehydes and their L-beta-homo-aldehyde analogues was investigated. The L-beta-homo-aspartals appear to be specific inhibitors for ICE and its homologues; the other enzymes were not inhibited with such L-beta-homo aldehydes. Papain shows tolerance for D-residues at P1 depending on their chiral stability.
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PMID:Peptidyl beta-homo-aspartals: specific inhibitors of interleukin-1 beta converting enzyme and its homologues (caspases). 987 73

Fixation and staining conditions for rat mast cell tryptase and its histochemical distribution in different rat tissues were investigated. Prostate, skin, lung, gut, stomach and salivary glands were fixed in either aldehyde or Carnoy fixatives and then frozen or embedded in paraffin wax. Preservation of tryptase enzymic activity against peptide substrates required aldehyde fixation and frozen sectioning. Of the peptide substrates examined, z-Ala-Ala-Lys-4-methoxy-2-naphthylamide and z-Gly-Pro-Arg-4-methoxy-2-naphthylamide proved the most effective for the demonstration of tryptase. Double staining by enzyme cytochemistry followed by immunological detection of tryptase showed that, in all tryptase-containing mast cells, the enzyme is at least in part active. Conventional dye-binding histochemistry was used to confirm the identity of mast cells. Aldehyde-fixed mucosal mast cells required a much shorter staining time with Toluidine Blue if tissue sections were washed directly in t-butyl alcohol. Double staining by enzyme cytochemistry and dye binding showed that tryptase is absent from mucosal and subepidermal mast cells, which are also smaller in size and appear to contain fewer granules than connective tissue mast cells. This study demonstrates that rat mast cell tryptase, unlike tryptases in other species, is a soluble enzyme. It is stored in an active form and is absent from some mast cell subpopulations in mucosa, skin and lung.
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PMID:Enzyme histochemistry of rat mast cell tryptase. 1019 50

We investigated the histochemical characteristics of mast cell tryptase in different mouse tissues. By use of peptide substrates, tryptase activity could be demonstrated in unfixed connective tissue mast cells in different tissues, including the stomach. Tryptase activity was better localized after aldehyde fixation and frozen sectioning, and under such conditions was also demonstrated in mucosal mast cells of the stomach but not in those of the gut mucosa. Double staining by enzyme histochemistry followed by toluidine blue indicated that the tryptase activity was present only in mast cells and that all mast cells in the stomach mucosa contained the enzyme. The peptide substrates z-Ala-Ala-Lys-4-methoxy-2-naphthylamide and z-Gly-Pro-Arg-4-methoxy-2-naphthlyamide, which are substrates of choice for demonstrating tryptase in other species, were most effective for demonstrating mouse tryptase. The use of protease inhibitors further indicated that activity present in all mast cells was tryptase. Safranin O did not stain stomach mucosal mast cells, suggesting that the tryptase present in these cells was active in the absence of heparin sulfate proteoglycan.
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PMID:Enzyme histochemistry of tryptase in stomach mucosal mast cells of the mouse. 1021 54

Interleukin-1 beta (IL-1 beta)-converting enzyme (ICE, caspase-1) processes the IL-1 beta precursor to mature inflammatory cytokine IL-1 beta. ICE has been identified as a unique cysteine protease, which cleaves Asp-X bonds, shows resistance to E-64 (an inhibitor of most cysteine proteases) and has a primary structure that is homologous to CED-3, a protein required for apoptosis (programmed cell death) in the nematode Caenorhabditis elegans, and to mammalian cysteine proteases that initiate and execute apoptosis, e.g., apopain/CPP32/caspase-3. The inhibitors of the ICE/CED-3 family or caspases, as they are called recently, may constitute therapeutic agents for amelioration of inflammatory and apoptosis-associated diseases. The most efficient ICE inhibitors are peptide aldehydes and peptidyl chloro or (acyloxy)methanes. A recent study revealed that both D- and L-Asp are accepted by ICE at the P1 of such inhibitors, and the peptidyl (acyloxy)methane analogues having the beta-homo-aspartyl residue [-NH-CH(CH2COOH)-CH2CO-] are inactive. These findings we reexamined in terms of two issues. (a) ICE's resistance to E-64. Since it was thought to be caused by the enzyme's unique substrate specificity, we prepared substrate-based analogues, which were not inhibitory suggesting significant structural difference between the active centers of ICE and papain-like enzymes. (b) Tolerance for D-stereochemistry at the P1 of these inhibitors. In view of the mechanism of cysteine protease inhibition by peptidyl X-methanes, we thought that this phenomenon should be a general characteristic of cysteine proteases and the hAsp-containing analogues should behave as reversible inhibitors. Here, we analyzed the inhibition of ICE and apopain in comparison with that of papain, thrombin, and trypsin by peptide L/D-alpha-aldehydes and their L-beta-homo-aldehyde [-NH-CH(R)-CH2-CHO] analogues. The following results were found. (1) The peptidyl L-beta-homo-aspartals are potent inhibitors for caspases. (2) The L-beta-homo analogues of peptide aldehyde inhibitors designed for other proteases are not inhibitory. (3) Unlike trypsin and thrombin (serine proteases), papain (cysteine protease) shows tolerance for D-stereochemistry at the P1 site of peptide aldehydes in proportion to the lability of the alpha-hydrogen of the P1-D-residue. The complete tolerance of ICE for P1-D-Asp may arise from this residue's high tendency to epimerization. (4) Reaction of cysteine proteases with peptide aldehyde or peptidyl X-methane inhibitors containing P1-D-residues may include alpha-proton abstraction followed by asymmetric induction leading to P1-L-residue-containing products.
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PMID:Peptidyl beta-homo-aspartals (3-amino-4-carboxybutyraldehydes): new specific inhibitors of caspases. 1038 Mar 58


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