Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The potent allergenicity of Der p 1, a major allergen of the house dust mite Dermatophagoides pteronyssinus, is thought to be related to its cysteine protease activity. Therefore, there is considerable interest in developing a sensitive assay for measuring Der p 1 activity to screen for specific inhibitors. This study demonstrates for the first time that the activity of Der p 1 can be measured conveniently in a continuous rate assay with the fluorogenic substrate Boc-Gln-Ala-Arg-AMC (K(m) = 280 microM and kcat/K(m) = 4.6 x 10(3)/M/s).
Mol Pathol 1998 Aug
PMID:A sensitive fluorescent assay for measuring the cysteine protease activity of Der p 1, a major allergen from the dust mite Dermatophagoides pteronyssinus. 989 50

Trypanosoma brucei contain a serine oligopeptidase (OP-Tb) that is released into (and remains active in) the blood of trypanosome-infected animals. Here a similar enzyme from Trypanosoma congolense is described. This oligopeptidase, called OP-Tc, was purified using three-phase partitioning, and ion-exchange and affinity chromatography. OP-Tc is inhibited by alkylating agents, by serine peptidase-specific inhibitors including 3,4-dichloroisocoumarin, 4-(2-aminoethyl)benzenesulfonylfluoride and diispropylfluoro-phosphate and by other peptidase inhibitors including leupeptin, antipain and peptidyl chloromethyl ketones. Reducing agents such as dithiothreitol enhanced activity as did heparin, spermine and spermidine. The enzyme has trypsin-like specificity since it cleaved fluorogenic peptides that have basic amino acid residues (Arg or Lys) in the P1 position. Potential substrates without a basic residue in P1 were not hydrolysed. Although OP-Tc has weak arginine aminopeptidase activity, the enzyme clearly preferred substrates that had amino acids in the P2 and P3 positions. Overall, OP-Tc appears to be less efficient than OP-Tb because it usually displayed lower k(cat)/Km values for the substrates tested. However, like OP-Tb, the best substrate for OP-Tc was Cbz-Arg-Arg-AMC (Km = 0.72 microM, k(cat) = 96 s(-1)). OP-Tc preference for amino acids in the P2 position was (Gly,Lys,Arg) > Phe > Leu > Pro. The results also suggest that the P3-binding site has hydrophobic characteristics. OP-Tc may not be a naturally immunodominant molecule because neither IgG nor IgM anti- OP-Tc antibodies were detected in the blood of experimentally infected cattle.
Mol Biochem Parasitol 1999 Jul 30
PMID:Purification and characterisation of a trypsin-like serine oligopeptidase from Trypanosoma congolense. 1047 83

Previous studies revealed that expression and activation of cyclooxygenase-2 (Cox-2) conveyed a protective principle in murine macrophages, thus attenuating pro-apoptotic actions of chemotherapeutic agents or programmed cell death as a result of massive nitric oxide (NO) generation. Expression of Cox-2 was achieved by treatment of cells with lipopolysaccharide/interferon-gamma or nontoxic doses of NO releasing agents. We reasoned E-type prostanoid formation, and in turn an intracellular cAMP increase as the underlying protective mechanism. To prove our hypothesis, we analyzed the effects of lipophilic cAMP-analogs on NO, cisplatin, or etoposide induced apoptosis in RAW 264.7 macrophages. Selected apoptotic parameters comprised DNA fragmentation (diphenylamine assay), annexin V staining of phosphatidylserine, caspase activity (quantitated by the cleavage of a fluorogenic caspase-3-like substrate Ac-DEVD-AMC), and mitochondrial membrane depolarisation (delta psi). Western blots detected accumulation of the tumor suppressor protein p53, relocation of cytochrome c to the cytosol, and expression of the anti-apoptotic protein Bcl-xL. Prestimulation with lipophilic cAMP-analogs attenuated apoptosis with the notion that cell death parameters were basically absent. To verify gene induction by cAMP in association with protection we established activation of cAMP response element binding protein (CREB) by gel-shift analysis and moreover, treated macrophages with oligonucleotides containing a cAMP-responsive element (CRE) in order to scavenge CREB. Decoy oligonucleotides, but not control oligonucleotides, attenuated cAMP-evoked protection and reestablished pro-apoptotic parameters. We conclude that gene induction by cAMP protects macrophages towards apoptosis that occurs as a result of excessive NO formation or addition of chemotherapeutica. Attenuating programmed cell death by the cAMP-signaling system may be found in association with Cox-2 expression and tumor formation.
Mol Cell Biochem 2000 Sep
PMID:Attenuation of macrophage apoptosis by the cAMP-signaling system. 1110 34

In order to design plasminogen activators with improved thrombolytic properties we sought to construct the bifunctional protein HLS-2 which combines both a plasminogen-activating and an anticoagulative activity. The chimeric protein comprises four elements: a derivative of thrombin inhibitor hirudin, a 6-amino acid spacer, the sequence of plasminogen-activator staphylokinase (Sak), and a 13-amino acid expression tag at the C-terminus. The gene of the fusion protein was obtained by SOE-PCR, cloned into pCANTAB5E, and expressed in E. coli BL21. HLS-2 was purified from periplasmatic extracts and characterized by Western blotting. Plasminogen-activation of HLS-2 and of Sak in equimolar mixtures with plasminogen showed near equivalence as measured by plasmin-mediated cleavage of chromogenic substrate S-2403. For catalytic amounts of plasminogen-activator, however, HLS-2 was less effective by a factor of 1.7. HLS-2 also inhibited both the amidolytic and the fibrinolytic activities of thrombin. Similar concentrations of either commercial HV1 (42 pmol/L) or HLS-2 (250 pmol/L) were required to halve the initial rate of thrombin reaction with fluorogenic substrate Tos-Gly-Pro-Arg-AMC, suggesting the retention of high-affinity inhibition of thrombin by the fusion protein sufficiently strong to substitute anticoagulative comedication during fibrinolytic treatment. The results provide a rationale for further testing the efficacy of HLS-2 for the lysis of platelet-rich arterial blood clots and for the prevention of reocclusion after thrombolysis.
Mol Genet Metab 2002 Mar
PMID:Modular design of a novel chimeric protein with combined thrombin inhibitory activity and plasminogen-activating potential. 1191 37

During post-germinative seedling growth, the major storage organ of the white spruce (Picea glauca) seed, the megagametophyte, undergoes programmed cell death (PCD). Protease activities in megagametophyte cells that arise post-germinatively were investigated. The accumulation of protease activities can be divided into two phases: the first phase correlated with degradation of storage proteins while the second phase was temporally associated with cell death, although some of the early proteases were also active during the later phase. Proteases induced during PCD were mainly serine and cysteine proteases. One of the PCD-associated cysteine proteases had homology to Cys-EP, a PCD-related cysteine protease of the castor bean endosperm. Transcripts encoding a Cys-EP-related protein were not present in megagametophytes when seeds were imbibed, nor were they present during germination and early post-germinative growth (radicle length ca. 2-5 mm). At a later post-germinative stage (i.e when the seed's radicle was ca. 15 mm), the Cys-EP-related transcripts (ca. 1.3 kb) became abundant and, at this time, the 48 kDa proform of the enzyme first appeared. The mature form of the Cys-EP (ca. 38 kDa) was predominant at a very late stage of post-germinative growth. Immunocytochemistry showed that the Cys-EP-related protein was localized to spherical organelles (ca. 2 microm) that may be equivalent to the 'ricinosomes' of castor bean endosperm cells. Caspase-like protease (CLP) activities were first detected 3 days after germination with the caspase-specific substrate Ac-DEVD-AMC; maximum activities occurred when the seed's radicle was ca. 20-25 mm. When germinated seeds were treated with a caspase-3 inhibitor, both the peak of CLP activities and the death of megagametophyte cells were delayed. We propose that the Cys-EP-related protein and CLP activity are involved in PCD of white spruce megagametophyte cells.
Plant Mol Biol 2003 Jul
PMID:Proteases associated with programmed cell death of megagametophyte cells after germination of white spruce (Picea glauca) seeds. 1367 63

The proteasome, a multienzymatic protease complex is present in human sperm. Here we present evidence indicating that the proteasome has an extracellular localization, on the plasma membrane of the sperm head. Motile sperm (>90%) in PBS were incubated with the proteasome inhibitors clasto-lactacystin beta-lactone or epoxomicin. Then, the substrate Suc-Leu-Leu-Val-Tyr-AMC (SLLVY-AMC) was added and the enzyme activity evaluated in a spectrofluorometer. Other aliquots were resuspended in Tyrode's medium and incubated at different concentrations for various times with or without inhibitors in the presence of 0.4% azocasein. Hydrolysis of azocasein was evaluated at 440 nm. In addition, sperm membrane proteins were obtained incubating the sperm with Triton X-114 or with 0.5 M KCl plus Triton X-100 and removing insoluble material by centrifugation at 5,000g for 40 min. Proteasomal activity was evaluated with SLLVY-AMC and its presence corroborated by Western blotting. Formaldehyde fixed, unpermeabilized sperm were incubated with anti-proteasome monoclonal antibodies and evaluated using indirect immunofluorescence. The effect of proteasome inhibitors upon the progesterone-induced acrosome reaction was also evaluated. Results indicated that (a) whole, intact sperm were able to hydrolyze the proteasome substrates SLLVY-AMC and azocasein; this activity was inhibited by proteasome inhibitors; (b) proteasomal activity was detected in soluble sperm membrane protein preparations and Western blotting revealed the presence of the proteasome in these fractions; (c) indirect immunofluorescence revealed staining of the head region, particularly of the post acrosomal region; and (d) the proteasome plays an important role during the acrosome reaction.
Mol Reprod Dev 2004 May
PMID:Extracellular localization of proteasomes in human sperm. 1503 55

Cysteine proteinases are the major class of enzymes responsible for digestive proteolysis in western corn rootworm (Diabrotica virgifera), a serious pest of maize. A larval gut extract hydrolysed typical cathepsin substrates, such as Z-phe-arg-AMC and Z-arg-arg-AMC, and hydrolysis was inhibited by Z-phe-tyr-DMK, specific for cathepsin L. A cDNA library representing larval gut tissue mRNA contained cysteine proteinase-encoding clones at high frequency. Sequence analysis of 11 cysteine proteinase cDNAs showed that 9 encoded cathepsin L-like enzymes, and 2 encoded cathepsin B-like enzymes. Three enzymes (two cathepsin L-like, DvRS5 and DvRS30, and one cathepsin B-like, DvRS40) were expressed as recombinant proteins in culture supernatants of the yeast Pichia pastoris. The cathepsin L-like enzymes were active proteinases, whereas the cathepsin B-like enzyme was inactive until treated with bovine trypsin. The amino acid residue in the S2 binding pocket, the major determinant of substrate specificity in cathepsin cysteine proteinases, predicted that the two cathepsin L-like enzymes, DvRS5 and DvRS30, should differ in substrate specificity, with the latter resembling cathepsin B in hydrolysing substrates with a positively charged residue at P2. This prediction was confirmed; DvRS5 only hydrolysed Z-phe-arg-AMC and not Z-arg-arg-AMC, whereas DvRS30 hydrolysed both substrates. The enzymes showed similar proteolytic activity towards peptide substrates.
Insect Biochem Mol Biol 2004 Apr
PMID:Characterisation of cysteine proteinases responsible for digestive proteolysis in guts of larval western corn rootworm (Diabrotica virgifera) by expression in the yeast Pichia pastoris. 1504 Oct 15

The proteasome is a multicatalytic cellular complex, which possess three different enzymatic activities, trypsin-like, chymotrypsin-like, and peptidylglutamyl peptidase. Its function is to remove abnormal or aged proteins. Recently, it has been suggested the participation of the sperm proteasome during mammalian fertilization. In this study, we present evidence that indicates that sperm extracts from several mammalian species, including hamster, mice, rats, bovine, rabbits, and humans all possess proteasome activity. We characterized the three specific activities of the proteasome using specific synthetic substrates and specific proteasome inhibitors. The results indicates that the highest specific activity detected was in mouse sperm toward the trypsin substrates and it was 1,114% of the activity of human sperm toward the chymotrypsin substrate Suc-Leu-Leu-Val-Tyr-AMC (SLLVY-AMC, which was considered as 100%). In all cases, the lowest activity was toward substrates for the peptidylglutamyl peptidase hydrolyzing activity, and it was lowest for rabbit sperm (1.7% of the activity of human sperm toward the chymotrypsin substrate SLLVY-AMC). In addition, specific proteasome inhibitors were able to block all proteasome activities almost 100%, with the exception of clasto-Lactacystin beta-lactone upon rat sperm. All sperm extracts tested evidenced bands of about 29-32 kDa by Western blots using a monoclonal antibody against proteasome subunits alpha 1, 2, 3, 5, 6, and 7. In conclusion, sperm from several mammals possess enzymatic activities that correspond to the proteasome. The proteasome from the different species hold similar but distinctive enzymatic characteristics.
Mol Reprod Dev 2004 Sep
PMID:Proteasomal activity in mammalian spermatozoa. 1527 8

1. A neurite outgrowth factor, neurocrescin (NC), which we previously identified from an extract of denervated skeletal muscle, was endoproteolytically processed in cell transfectants. In addition to the processing site identified in NC (DESD358/F) being similar to the optimal recognition sequence of group II caspases, DExD, cleavage site mutations confirmed the involvement of caspase(s) in NC processing. 2. However, both the recombinant NC and the synthetic octapeptide (YLDESDFG) were scarcely cleaved in vitro by caspase-3 or -7. Furthermore, transiently expressed NC was cleaved even in the caspase-3-deficient cell line, MCF-7 cells, and this efficiency was not altered by the transfectional expression of caspase-3. 3. Using the fluorescent substrate (Ac-DESD-AMC), the characteristic proteolytic activities, which cleaved it more effectively than caspase-3 and whose pH dependences were different from those of caspase-3, were endogenously identified in the muscle extract. These findings indicate the presence of proteolytic activities that are distinguishable from caspase-3.
Cell Mol Neurobiol 2004 Dec
PMID:Neurocrescin is specifically cleaved after the sequence DESD in a caspase-3-independent manner. 1567 75

Multicatalytic proteinase complex (MCP) was studied in skeletal muscle of the hibernating ground squirrel, Spermophilus tridecemlineatus. MCP was partially purified using a S-400 gel filtration column and Centricon concentrating devices and assayed fluorometrically using three AMC-labeled substrates. K(m) and V(max) values were determined for each substrate with no significant differences between the enzyme from euthermic versus hibernating animals when assayed at 23 degrees C. However, properties of MCP from euthermic and hibernating ground squirrels were differentially affected by low assay temperature (8-10 degrees C) and also differed from the mouse enzyme, the data indicating that ground squirrel MCP is better suited for low temperature function. MCP preferentially degrades oxidatively-damaged proteins and quantification of protein carbonyl content showed that the level of oxidatively-damaged protein in skeletal muscle decreased by > 75% during hibernation suggesting a continuing role for the MCP in the torpid state.
Mol Cell Biochem 2005 Mar
PMID:Effects of hibernation on multicatalytic proteinase complex in thirteen-lined ground squirrels, Spermophilus tridecemlineatus. 1588 72


<< Previous 1 2 3 4 5 6 Next >>