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:3.4.24.11 (CD10)
9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A recent report [Gil, Chaib-Oukadour, Pelliccioni and Aguilera (2000) FEBS Lett. 481, 177-182] describes activation of signal transduction pathways by tetanus toxin (TeTx), a Zn(2+)-dependent endopeptidase synthesized by the Clostridium tetani bacillus, which is responsible for tetanus disease. In the present work, specific activation of protein kinase C (PKC) isoforms and of intracellular signal-transduction pathways, which include nerve-growth-factor (NGF) receptor trkA, phospholipase C(PLC)gamma-1 and extracellular regulated kinases (ERKs) 1 and 2, by the recombinant C-terminal portion of the TeTx heavy chain (H(C)-TeTx) is reported. The activation of PKC isoforms was assessed through their translocation from the soluble (cytosolic) compartment to the membranous compartment, showing that clear translocation of PKC-alpha, -beta, -gamma and -delta isoforms exists, whereas PKC-epsilon showed a slight decrease in its soluble fraction immunoreactivity. The PKC-zeta isoform showed no consistent response. Using immunoprecipitation assays against phosphotyrosine residues, time- and dose-dependent increases in tyrosine phosphorylation were observed in the trkA receptor, PLCgamma-1 and ERK-1/2. The effects shown by the H(C)-TeTx fragment on tyrosine phosphorylation were compared with the effects produced by NGF. The trkA and ERK-1/2 activation were corroborated using phospho-specific antibodies against trkA phosphorylated on Tyr(490), and antibodies against Thr/Tyr phosphorylated ERK-1/2. Moreover, PLCgamma-1 phosphorylation was supported by its H(C)-TeTx-induced translocation to the membranous compartment, an event related to PLCgamma-1 activation. Since H(C)-TeTx is the domain responsible for membrane binding and lacks catalytic activity, the activations described here must be exclusively triggered by the interaction of TeTx with a membrane component.
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PMID:HC fragment (C-terminal portion of the heavy chain) of tetanus toxin activates protein kinase C isoforms and phosphoproteins involved in signal transduction. 1133 40

Streptococcus milleri NMSCC 061 produces an endopeptidase, millericin B, which hydrolyzes the peptide moiety of susceptible cell wall peptidoglycan. The nucleotide sequence of a 4.9-kb chromosomal region showed three open reading frames (ORFs) and a putative tRNA(Leu) sequence. The three ORFs encode a millericin B preprotein (MilB), a putative immunity protein (MilF), and a putative transporter protein (MilT). The milB gene encodes a 277-amino-acid preprotein with an 18-amino-acid signal peptide with a consensus IIGG cleavage motif. The predicted protein encoded by milT is homologous to ABC (ATP-binding cassette) transporters of several bacteriocin systems and to proteins implicated in the signal-sequence-independent export of Escherichia coli hemolysin A. These similarities strongly suggest that the milT gene product is involved in the translocation of millericin B. The gene milF encodes a protein of 302 amino acids that shows similarities to the FemA and FemB proteins of Staphylococcus aureus, which are involved in the addition of glycine to a pentapeptide peptidoglycan precursor. Comparisons of the cell wall mucopeptide of S. milleri NMSCC 061(resistant to lysis by millericin B) and S. milleri NMSCC 051(sensitive) showed a single amino acid difference. Serial growth of S. milleri NMSCC 051 in a cell wall minimal medium containing an increased concentration of leucine resulted in the in vivo substitution of leucine for threonine in the mucopeptide of the cell wall. A cell wall variant of S. milleri NMSCC 051 (sensitive) that contained an amino acid substitution (leucine for threonine) within its peptidoglycan cross bridge showed partial susceptibility to millericin B. The putative tRNA(Leu) sequence located upstream of milB may be a cell wall-specific tRNA and could together with the milF protein, play a potential role in the addition of leucine to the pentapeptide peptidoglycan precursor and thereby, contributing to self-protection to millericin B in the producer strain.
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PMID:Self-protection against cell wall hydrolysis in Streptococcus milleri NMSCC 061 and analysis of the millericin B operon. 1152 82

To evaluate the effect of peptidases on mu-opioid receptor (MOR) activation by endogenous opioids, we measured MOR-1 internalization in rat spinal cord slices. A mixture of inhibitors of aminopeptidases (amastatin), dipeptidyl carboxypeptidase (captopril), and neutral endopeptidase (phosphoramidon) dramatically increased the potencies of Leu-enkephalin and dynorphin A to produce MOR-1 internalization, and also enhanced the effects of Met-enkephalin and alpha-neoendorphin, but not endomorphins or beta-endorphin. The omission of any one inhibitor abolished Leu-enkephalin-induced internalization, indicating that all three peptidases degraded enkephalins. Amastatin preserved dynorphin A-induced internalization, and phosphoramidon, but not captopril, increased this effect, indicating that the effect of dynorphin A was prevented by aminopeptidases and neutral endopeptidase. Veratridine (30 microm) or 50 mm KCl produced MOR-1 internalization in the presence of peptidase inhibitors, but little or no internalization in their absence. These effects were attributed to opioid release, because they were abolished by the selective MOR antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2)) and were Ca(2+) dependent. The effect of veratridine was protected by phosphoramidon plus amastatin or captopril, but not by amastatin plus captopril or by phosphoramidon alone, indicating that released opioids are primarily cleaved by neutral endopeptidase, with a lesser involvement of aminopeptidases and dipeptidyl carboxypeptidase. Therefore, because the potencies of endomorphin-1 and endomorphin-2 to elicit internalization were unaffected by peptidase inhibitors, the opioids released by veratridine were not endomorphins. Confocal microscopy revealed that MOR-1-expressing neurons were in close proximity to terminals containing opioids with enkephalin-like sequences. These findings indicate that peptidases prevent the activation of extrasynaptic MOR-1 in dorsal horn neurons.
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PMID:Peptidases prevent mu-opioid receptor internalization in dorsal horn neurons by endogenously released opioids. 1262 89

The mini-chain of human cathepsin H has been identified as the major structural element determining the protease's substrate specificity. A genetically engineered mutant of human cathepsin H lacking the mini-chain, des[Glu(-18)-Thr(-11)]-cathepsin H, exhibits endopeptidase activity towards the synthetic substrate Z-Phe-Arg-NH-Mec (kcat = 0.4 s(-1), Km = 92 microM, kcat/Km = 4348 M(-1) s(-1)) which is not cleaved by r-wt cathepsin H. However, the mutant enzyme shows only minimal aminopeptidase activity for H-Arg-NH-Mec (kcat = 0.8 s(-1), Km = 3.6 mM, kcat/Km = 222 M(-1) s(-1)) which is one of the best known substrates for native human cathepsin H (kcat = 2.5 s(-1), Km = 150 microM, kcat/Km = 16666 M(-1) s(-1)). Inhibition studies with chicken egg white cystatin and E-64 suggest that the mini-chain normally restricts access of inhibitors to the active site. The kinetic data on substrates hydrolysis and enzyme inhibition point out the role of the mini-chain as a structural framework for transition state stabilization of free alpha-amino groups of substrates and as a structural barrier for endopeptidase-like substrate cleavage.
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PMID:Human cathepsin H: deletion of the mini-chain switches substrate specificity from aminopeptidase to endopeptidase. 1451 96

A gene encoding of glutamyl-specific endopeptidase precursor from Bacillus licheniformis has been cloned in Escherichia coli cells. The recombinant protein was expressed and accumulated as cytoplasmic insoluble inclusion bodies. Washed inclusion bodies were solubilized in 6 M guanidine-HCL in the presence of reducing agent. The following precursor renaturation was performed by fast frequent dilution method. The highest yield of the refolded protein was achieved at pH value of 8.5 and 4 degrees C. The renaturation process was accompanied by a gradual splitting of Glu(-48)/Thr(-47) and Glu(-13)/Lys(-12) peptide bonds. A 26-kDa protein proved to be an end product of in vitro renaturation. The mature glutamyl endopeptidase with a molecular mass of 25 kDa was obtained after a limited proteolysis of the 26-kDa protein was performed by subtilisin or trypsin. The 26-kDa protein was purified by gel filtration on a Superdex 75 column. Comparative characteristics of the thermal stability and catalytic properties of the 26-kDa and 25-kDa proteins showed that complete cleavage of the N-terminal pro-peptide by exogenous proteinase is necessary for a final packing and activation of the B. licheniformis glutamyl endopeptidase.
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PMID:In vitro maturation pathway of a glutamyl endopeptidase precursor from Bacillus licheniformis. 1593 78

Taspase1 was identified as the threonine endopeptidase that cleaves mixed-lineage leukemia (MLL) for proper Hox gene expression in vitro. To investigate its functions in vivo, we generated Taspase1(-/-) mice. Taspase1 deficiency results in noncleavage (nc) of MLL and MLL2 and homeotic transformations. Remarkably, our in vivo studies uncover an unexpected role of Taspase1 in the cell cycle. Taspase1(-/-) animals are smaller in size. Taspase1(-/-) mouse embryonic fibroblasts (MEFs) exhibit impaired proliferation, and acute deletion of Taspase1 leads to a marked reduction of thymocytes. Taspase1 deficiency incurs down-regulation of Cyclin Es, As, and Bs and up-regulation of p16(Ink4a) . We show that MLL and MLL2 directly target E2Fs for Cyclin expression. The uncleaved precursor MLL displays a reduced histone H3 methyl transferase activity in vitro. Accordingly, chromatin immunoprecipitation assays demonstrate a markedly decreased histone H3 K4 trimethylation at Cyclin E1 and E2 genes in Taspase1(-/-) cells. Furthermore, MLL(nc/nc;2nc/nc) MEFs are also impaired in proliferation. Our data are consistent with a model in which precursor MLLs, activated by Taspase1, target to Cyclins through E2Fs to methylate histone H3 at K4, leading to activation. Lastly, Taspase1(-/-) cells are resistant to oncogenic transformation, and Taspase1 is overexpressed in many cancer cell lines. Thus, Taspase1 may serve as a target for cancer therapeutics.
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PMID:Proteolysis of MLL family proteins is essential for taspase1-orchestrated cell cycle progression. 1695 Dec 54

Previous genome-wide screens identified over 100 host genes whose deletion/down-regulation affected tombusvirus replication and 32 host genes that affected tombusvirus RNA recombination in yeast, a model host for replication of Tomato bushy stunt virus (TBSV). Down-regulation of several of the identified host genes affected the accumulation levels of p33 and p92(pol) replication proteins, raising the possibility that these host factors could be involved in the regulation of the amount of viral replication proteins and, thus, they are indirectly involved in TBSV replication and recombination. To test this model, we developed a tightly regulated expression system for recombinant p33 and p92(pol) replication proteins in yeast. We demonstrate that high accumulation level of p33 facilitated efficient viral RNA replication, while the effect of p33 level on RNA recombination was less pronounced. On the other hand, high level of p92(pol) accumulation promoted TBSV RNA recombination more efficiently than RNA replication. As predicted, Rpb11p, which is part of the polII complex, affected the accumulation levels of p33 and p92(pol) as well as altered RNA replication and recombination. An in vitro assay with the tombusvirus replicase further supported that Rpb11p affects TBSV replication and recombination only indirectly, via regulating p33 and p92(pol) levels. In contrast, the mechanism by which Rpt4p endopeptidase/ATPase and Mps1p threonine/tyrosine kinase affect TBSV recombination is different from that proposed for Rpb11p. We propose a model that the concentration (molecular crowding) of replication proteins within the viral replicase is a factor affecting viral replication and recombination.
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PMID:Host transcription factor Rpb11p affects tombusvirus replication and recombination via regulating the accumulation of viral replication proteins. 1768 83

In the analytical approach called chemically targeted identification (CTID), peptides containing phosphorylated or glycosylated serine and threonine underwent beta-elimination to produce an unsaturated double bond. Nucleophilic addition of 2-aminoethanethiol to this bond occurred, yielding aminoethylcysteine. Thus, sites containing posttranslational modifications were made susceptible to lysine endopeptidase. Structural information could then be obtained by mass analysis of the proteolytic products. The method was demonstrated by the analysis of beta-casein tryptic digest peptides and an O-glycosylated peptide. Contrary to an earlier report, the glycopeptide was found to react with essentially the same kinetics as phosphopeptides. Conversion of all five phosphoserines in residues 15, 17, 18, 19, and 35 in N-terminal tryptic phosphopeptides from bovine beta-casein were followed by monitoring the time course of the addition reaction. The chemistry proceeded rapidly at room temperature with a half-reaction time of 15 min. No side reaction products were observed. However, care had to be taken to minimize all counterions, which either precipitate barium or neutralize the base. In the case of 2-aminoethanethiol, excess Ba(OH)2 was needed to offset the effect of the hydrochloride. Alternatively, pre-incubation with base followed by nucleophilic addition was found to work satisfactorily. The use of water-soluble thiol allowed the procedure to be carried out in the solid phase, with a micro pipet greatly facilitating sample cleanup.
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PMID:Determination of phosphorylated and O-glycosylated sites by chemical targeting (CTID) at ambient temperature. 1860 43

Two proteolytic enzymes capable of releasing the angiotensin I-converting enzyme (ACE) inhibitor Ile-Pro-Pro from casein were identified by purification of an Aspergillus oryzae extract by three-step column chromatography. First, proteins capable of producing Ile-Pro-Pro from beta-casein were eluted using a DEAE-sepharose FF column with a linear sodium chloride gradient. An endopeptidase capable of releasing Pro-Ile-Pro-Gln-Ser-Leu-Pro-Gln-Asn-Ile-Pro-Pro from Pro-Ile-Pro-Gln-Ser-Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu-Thr-Gln and an aminopeptidase producing Ile-Pro-Pro from Gln-Asn-Ile-Pro-Pro were separated from the resultant fraction using a hydroxyapatite column. Each active enzyme was then loaded onto a Develosil 300Diol gel filtration column for high performance liquid chromatography (HPLC) and purified to homogeneity. The endopeptidase had a molecular mass of approximately 46,000 Da and exhibited an N-terminal amino acid sequence identical to that of neutral protease I (NP I) of A. oryzae. Meanwhile, the aminopeptidase had a molecular mass of 36,000 Da and an N-terminal amino acid sequence similar to that of Leucine aminopeptidase (LAP), as reported in Aspergillus sojae and A. oryzae. The eluted endopeptidase and aminopeptidase were thus identified as NP I and LAP, respectively. Analysis of peptide production using synthetic proteins containing an Ile-Pro-Pro sequence showed that NP I processed the C-terminal end and LAP processed the N terminus to produce Ile-Pro-Pro. While Ile-Pro-Pro was successfully produced from casein by the addition of these two purified enzymes, it was not generated with the addition of only a single enzyme. Based on our experimental findings, we suggest that NP I and LAP are key proteolytic enzymes in the release of Ile-Pro-Pro from casein in A. oryzae.
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PMID:Purification and identification of proteolytic enzymes from Aspergillus oryzae capable of producing the antihypertensive peptide Ile-Pro-Pro. 1944 37

Resuscitation-promoting factor (Rpf) is a muralytic enzyme that increases the culturability of dormant bacteria. Recently, considerable progress has been made in understanding the structure, function and physiological role of Rpfs in different organisms, most notably the major human pathogen, Mycobacterium tuberculosis, which encodes multiple rpf-like genes. A key unresolved question, however, concerns the relationship between the predicted biochemical activity of Rpfs - cleavage of the beta-1,4 glycosidic bond in the glycan backbone of peptidoglycan - and their effect on culturability. In M. tuberculosis, the interaction between RpfB and the d,l-endopeptidase, Rpf interacting protein A (RipA), enables these proteins to synergistically degrade peptidoglycan to facilitate growth. Furthermore, the combined action of Rpfs with RipA and other peptidoglycan hydrolases might produce muropeptides that could exert diverse biological effects through host and/or bacterial signaling, the latter involving serine/threonine protein kinases. Here, we explore these possibilities in the context of the structure and composition of mycobacterial peptidoglycan. Clearly, a deeper understanding of the role of Rpfs and associated peptidoglycan remodeling enzymes in bacterial growth and culturability is necessary to establish the significance of dormancy and resuscitation in diseases such as tuberculosis, which are associated with long-term persistence of viable bacterial populations recalcitrant to antibiotic and immune clearance.
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PMID:Resuscitation-promoting factors as lytic enzymes for bacterial growth and signaling. 1979 29


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