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:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The nucleotide sequence of the helper component protease (HC-Pro) genes of three zucchini yellow mosaic virus (ZYMV) strains has been compared with that of a helper-deficient strain of ZYMV-HC. The comparisons revealed three unique deduced amino acid differences. Two of these mutations were located in regions which are conserved in other potyviruses. The role of these mutations in aphid transmissibility was examined by exchanging DNA fragments of part of the deficient HC-Pro gene with the respective section within the gene of the infectious full-length clone of the aphid-transmissible ZYMV. The first exchange included two of the three mutations, the first coding for a change from Asp to Gly (in a non-conserved region) and the second coding for a change from Arg to Ile [within the Phe-Arg-Asp-Lys (FRNK) conserved box]. This exchange resulted in a reduced transmission (20.6% for the mutated virus compared with 57.4% in the normal ZYMV when acquired from plants and 37.2% compared with 83.1%, respectively, when acquired from membranes). The second exchange incorporated a single mutation [conferring a change from Thr to Ala within the Pro-Thr-Lys (PTK) conserved box]. This single mutation resulted in almost total loss of HC activity in aphid transmission both from plants and from membranes. The Lys residue in the conserved Lys-Ile-Thr-Cys (KITC) box, which is related to loss of HC activity in potato virus Y, tobacco vein mottling virus and in the Michigan strain of ZYMV, is unchanged in the helper-deficient ZYMV. It is therefore proposed that more than one site in HC-Pro may be functionally related to aphid transmissibility. The possible reasons for the role of these mutations in helper activity in aphid transmission of ZYMV are discussed.
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PMID:Mutations in the helper component protease gene of zucchini yellow mosaic virus affect its ability to mediate aphid transmissibility. 820 4

The transmembrane PTPase HPTP beta differs from its related family members in having a single rather than a tandemly duplicated cytosolic catalytic domain. We have expressed the 354-amino acid, 41-kDa human PTP beta catalytic fragment in Escherichia coli, purified it, and assessed catalytic specificity with a series of pY peptides. HPTP beta shows distinctions from the related LAR PTPase and T cell CD45 PTPase domains: it recognizes phosphotyrosyl peptides of 9-11 residues from lck, src, and PLC gamma with Km values of 2, 4, and 1 microM, some 40-200-fold lower than the other two PTPases. With kcat values of 30-205 s-1, the catalytic efficiency, kcat/Km, of the HPTP beta 41-kDa catalytic domain is very high, up to 5.7 x 10(7) M-1 s-1. The peptides corresponding to PLC gamma (766-776) and EGFR (1,167-1,177) phosphorylation sites were used for structural variation to assess pY sequence context recognition by HPTP beta catalytic domain. While exchange of the alanine residue at the +2 position of the PLC gamma (Km of 1 microM) peptide to lysine or aspartic acid showed little or no effect on substrate affinity, replacement by arginine increased the Km 35-fold. Similarly, the high Km value of the EGFR pY peptide (Km of 104 microM) derives largely from the arginine residue at the +2 position of the peptide, since arginine to alanine single mutation at the -2 position of the EGFR peptide decreased the Km value 34-fold to 3 microM. Three thiophosphotyrosyl peptides have been prepared and act as substrates and competitive inhibitors of these PTPase catalytic domains.
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PMID:Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTP beta, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors. 831 1

The objectives of this investigation were to characterize neuropeptide-degrading enzymes on the surface of gastric muscle cells and to determine their physiological function. Neutral endopeptidase (NEP, EC 3.4.24.11) activity was measured using the fluorogenic substrate glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamine. The NEP inhibitors phosphoramidon and DL-thiorphan (1 microM) inhibited degradation of the substrate by gastric muscle membranes by 100% and by freshly dispersed gastric muscle cells by 55-60%. The phosphoramidon or DL-thiorphan-inhibitable activity, attributed to NEP, of membranes was 112 +/- 4.0 pmol h-1 (micrograms protein)-1 and of cells was 4.2 +/- 0.8 nmol h-1 (10(6) cells)-1. This activity was associated with membranes prepared from cells and was not detected in the cytoplasm or in the cell incubation solution. Gastric muscle membranes were fractionated by electrophoresis and analysed by Western blotting using two NEP antisera. Both antisera recognized a protein in membranes with an electrophoretic mobility identical to that of recombinant human NEP and an apparent molecular mass of approximately 95 kDa. Neuropeptides were degraded by membranes with specific activities in the order of [Leu5]enkephalin > [Met5]enkephalin > gastrin-releasing peptide-10 (GRP-10) > [D-Ala2][Leu5]enkephalin > somatostatin-14. Phosphoramidon and DL-thiorphan similarly inhibited the degradation of GRP-10 (mean of 35% inhibition), somatostatin-14 (57%) and the aminopeptidase-resistant analogue, [D-Ala2][Leu5]enkephalin (75%). When aminopeptidases were inhibited with amastatin (10 microM) phosphoramidon inhibited degradation of [Leu5]enkephalin (54%) and [Met5]enkephalin (100%). Phosphoramidon increased the potency of the contractile effects of neuropeptides on muscle cells by > 280-fold for somatostatin-14, 17-fold for GRP-10, 18-fold for [Met5]enkephalin and 14-fold for [Leu5]enkephalin. The results show that an NEP-like enzyme on the surface of gastric muscle cells degrades and inactivates enkephalins, GRP-10 and somatostatin-14 and acts in a manner analogous to that of acetylcholinesterase in the neuromuscular junction of skeletal muscle.
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PMID:Neutral endopeptidase (EC 3.4.24.11) modulates the contractile effects of neuropeptides on muscle cells from the guinea-pig stomach. 844 12

The distribution of neutral endopeptidase (NEP; EC 3.4.24.11) was examined in the alimentary tract of the rat. Immunoreactive NEP and NEP mRNA were localized to epithelial cells of the small intestine and to muscle cells in the stomach, small intestine, and colon by immunohistochemistry and in situ hybridization histochemistry. NEP antisera recognized a protein on Western blots of membranes from gastric, jejunal, and colonic mucosa and gastric muscle with an electrophoretic mobility identical to that of recombinant human NEP (approximately 95 kDa). An antisense cRNA probe to NEP hybridized to RNA of approximately 3.5 kb and approximately 6.5 kb, corresponding to the primary transcripts of rat NEP, on Northern blots of total RNA from the jejunal mucosa. NEP message was detected in mRNA from jejunal and colonic mucosa and gastric, jejunal, and colonic muscle using a ribonuclease protection assay. NEP enzymatic activity, assessed by DL-thiorphan-inhibitable degradation of glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamine, was highest in homogenates of jejunal mucosa (868 +/- 98 pmol.h-1 x micrograms protein-1) and was between 49- and 413-fold lower in other gastrointestinal tissues. The cellular origin of NEP in the gastric and colonic mucosa could not be determined.
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PMID:Distribution and abundance of neutral endopeptidase (EC 3.4.24.11) in the alimentary tract of the rat. 846 Jul 3

Lysosomal cathepsin B but not L degraded rAPP751 to yield C-terminal 19-25 kDa fragments containing beta A4, reinforcing the view that acidic proteases participate in endosomal-lysosomal processing to yield amyloidogenic fragments in situ. This mechanism is consistent with fragmentation of endogenous APPs within clathrin-coated vesicles (CVs) by vesicular hydrolases, with the appearance of C-terminal amyloidogenic fragments following incubation at pH 6.5. A neutral endopeptidase resembling NEP 24.11 (PS-NEP) purified from detergent extracts of human brain degraded rAPP751; however, breakdown was not blocked robustly by metal chelators or phosphoramidon, suggesting the presence of an alternative processing enzyme. Effects of other inhibitors showed that breakdown was mediated by serine-protease-like component(s). A phosphoramidon-insensitive metalloendopeptidase (PI-NEP) partially purified from rat brain P2 using detergents, and resembling NEP 24.15, showed no activity towards rAPP751. Peptides containing putative beta- or gamma-secretase sites were synthesized for purposes of examining their metabolism by the brain enzymes. Those containing beta-secretase sites were hydrolysed at one or more sites by the four enzymes, but only PI- and PS-NEP acted at the Met-Asp site of Ac-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg.NH2. In the case of substrates containing the gamma-site, these two categories of enzymes were the only ones degrading N-Ac-Ile-Ala.NH2. These data imply that the brain metalloendopeptidases, while inactive towards intact precursors, may be involved in turnover of intermediates containing beta- or gamma-sites.
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PMID:Brain cathepsin B but not metalloendopeptidases degrade rAPP751 with production of amyloidogenic fragments. Comparison with synthetic peptides emulating beta- and gamma-secretase sites. 853 84

Neutral endopeptidase (EC 3.4.24.11; NEP), originally isolated from renal tubular brush border, is a cell surface peptidase identical to the CD10 antigen (or CALLA; common acute lymphoblastic leukemia antigen) in lymphoid cells. We studied the serum NEP levels daily after transplantation (Tx) in 19 renal allograft recipients. The NEP activity was determined with a two-step enzymatic assay utilizing a fluorogenic substrate (Suc-Ala-Ala-Phe-AMC; see text) and related to clinical signs of graft rejection, to signs of immunoactivation in transplant fine-needle aspiration biopsy (FNAB) specimens, to renal function, and to serum levels of C-reactive protein. The serum NEP levels remained normal (peak level 10.3 +/- 1.8 micrograms/l on days 6-9 after Tx, initial level after Tx 7.3 +/- 1.4 micrograms/1 on day 2; mean values +/- SEM) in patients who neither showed clinical signs of rejection nor had findings of immunoactivation in FNAB samples. On the contrary, the serum NEP levels rose clearly in patients developing acute rejection verified clinically and in FNAB samples (peak value 90.4 +/- 18.7 micrograms/l on days 6-9 post-Tx; p < 0.001 compared with patients without sings of immunoactivation) and even in patients having immunoactivation in FNAB without clinical evidence of rejection (108.2 +/- 22.4 micrograms/l, p < 0.001). Serum NEP peak appeared 2-3 days before clinical diagnosis of rejection and a positive findings in FNAB samples. Serum NEP increments did not correlate with changes in serum creatinine, delayed onset of renal excretory function, blood leukocyte count, C-reactive protein level, or infections. Thus, the serum NEP activity was shown to increase after renal allotransplantation associated with early phases of immunoactivation and development of acute graft rejection. Because of the limited number of patients studied, the clinical implications of these preliminary observations for kidney transplant monitoring clearly need confirmation in larger studies.
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PMID:Increased serum neutral endopeptidase activity in acute renal allograft rejection. 873 78

1. The role of the metalloendopeptidase EC 3.4.24.15 (EP 24.15) in peptide metabolism in vivo is unknown, in part reflecting the lack of a stable enzyme inhibitor. The most commonly used inhibitor, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP-AAY-pAB, Ki = 16 nM), although selective in vitro, is rapidly degraded in the circulation to cFP-Ala-Ala, an angiotensin converting enzyme (ACE) inhibitor. This metabolite is thought to be generated by neutral endopeptidase (NEP; EC 3.4.24.11), as the Ala-Tyr bond of cFP-AAY-pAB is cleaved by NEP in vitro. In the present study, we have examined the role of NEP in the metabolism of cFP-AAY-pAB in vivo, and have tested a series of inhibitor analogues, substituted at the second alanine, for both potency and stability relative to the parent compound. 2. Analogues were screened for inhibition of fluorescent substrate cleavage by recombinant rat testes EP 24.15. D-Ala or Asp substitution abolished inhibitory activity, while Val-, Ser- and Leu-substituted analogues retained activity, albeit at a reduced potency. A relative potency order of Ala (1) > Val (0.3) > Ser (0.16) > Leu (0.06) was observed. Resistance to cleavage by NEP was assessed by incubation of the analogues with rabbit kidney membranes. The parent compound was readily degraded, but the analogues were twice (Ser) and greater than 10 fold (Leu and Val) more resistant to cleavage. 3. Metabolism of cFP-AAY-pAB and the Val-substituted analogue was also examined in conscious rabbits. A bolus injection of cFP-AAY-pAB (5 mg kg-1, i.v.) significantly reduced the blood pressure response to angiotensin I, indicating ACE inhibition. Pretreatment with NEP inhibitors, SCH 39370 or phosphoramidon, slowed the loss of cFP-AAY-pAB from the plasma, but did not prevent inhibition of ACE. Injection of 1 mg kg-1 inhibitor resulted in plasma concentrations at 10 s of 23.5 microM (cFP-AAY-pAB) and 18.0 microM (cFP-AVY-pAB), which fell 100 fold over 5 min. Co-injection of 125I-labelled inhibitor revealed that 80-85% of the radioactivity had disappeared from the circulation within 5 min, and h.p.l.c. analysis demonstrated that only 25-30% of the radiolabel remained as intact inhibitor at this time. Both analogues were cleared from the circulation at the same rate, and both inhibitors blunted the pressor response to angiotensin I, indicative of ACE inhibition. 4. These results suggest that both NEP and other clearance/degradation mechanisms severely limit the usefulness of peptide-based inhibitors such as cFP-AAY-pAB. To examine further EP 24.15 function in vivo, more stable inhibitors, preferably non-peptide, must be developed, for which these peptide-based inhibitors may serve as useful molecular templates.
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PMID:Synthetic inhibitors of endopeptidase EC 3.4.24.15: potency and stability in vitro and in vivo. 881 53

Selective and mixed inhibitors of the three zinc metallopeptidases that degrade neurotensin (NT), e.g. endopeptidase 24-16 (EC 3.4.24.16), endopeptidase 24-11 (EC 3.4.24.11 or neutral endopeptidase, NEP) and endopeptidase 24-15 (EC 3.4.24.15), and leucine-aminopeptidase (type IV-S), that degrades the NT-related peptides, Neuromedin N (NN), are of great interest. On the structural basis of compound JMV 390-1 (N-[3-[(hydroxyamino)carbonyl]-1-oxo-2(R)-benzylpropyl]-L- isoleucyl-L-leucine), which was a full inhibitor of the major NT degrading enzymes, several hydroxamate inhibitors corresponding to the general formula HONHCO-CH2-CH(CH2-C6H5)CO-X-Y-OH (with X-Y = dipeptide) have been synthesized. Compound 7a (X-Y = Ile-Ala) was nearly 40-times more potent in inhibiting EC 24-16 than NEP and more than 800-times more potent than EC 24-15, with an IC50 (12 nM) almost equivalent to that of compound JMV 390-1. Therefore, this compound is an interesting selective inhibitor of EC 24-16, and should be an interesting probe to explore the physiological involvement of EC 24-16 in the metabolism of neurotensin.
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PMID:New hydroxamate inhibitors of neurotensin-degrading enzymes. Synthesis and enzyme active-site recognition. 887 32

Human heat shock transcription factor 1 (HSF1) is responsible for stress-induced transcription of heat shock protein genes. The activity of the HSF1 transcriptional activation domains is modulated by a separate regulatory domain, which confers repression at control temperature and heat inducibility. We show here that two specific proline-directed serine motifs are important for function of the regulatory domain: Mutation of these serines to alanine derepresses HSF1 activity at control temperature, and mutation to glutamic acid, mimicking a phosphorylated serine, results in normal repression at control temperature and normal heat shock inducibility. Tryptic mapping shows that these serines are the major phosphorylation sites of HSF1 at control temperature in vivo. Stimulation of the Raf/ERK pathway in vivo results in an increased level of phosphorylation of these major sites and the regulatory domain is an excellent substrate in vitro for the mitogen-activated MAPK/ERK. We conclude that phosphorylation of the regulatory domain of HSF1 decreases the activity of HSF1 at control temperature, and propose a mechanism for modification of HSF1 activity by growth control signals.
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PMID:Repression of human heat shock factor 1 activity at control temperature by phosphorylation. 894 18

Intracellular reactive oxygen intermediate (ROI) levels play an important role in numerous physiological and pathophysiological conditions. Apart from causing oxidative stress and damage, ROI changes differentially activate gene expression. However the proto-oncogene encoding the AP-1 transcription factor subunit c-Fos is induced by both prooxidants and antioxidants. Here, the transcription factor Elk-1 is identified as being responsible for c-fos serum response element (SRE) induction in response to changes in the cellular redox status induced by treatment with either the oxidant H2O2 or various structurally unrelated antioxidants. A temporal correlation is observed between changes in the phosphorylation status of Elk-1 and the activation of the mitogen-activated protein kinase 2 (ERK2) in response to cellular redox changes. Correspondingly, the transcriptional response of the SRE to redox fluctuations is attenuated upon mutation of critical ERK2 target residues within the Elk-1 transactivation domain to alanine. Signals elicited by antagonistic intracellular redox changes converge at or above the level of Ras or an effector of Ras, leading to similar activation of c-fos transcription, since an [N17]Ras mutant interfered with redox signaling. Hence components of signaling pathways are revealed to be shared by mitogenic and redox-dependent stimuli.
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PMID:Antioxidants as well as oxidants activate c-fos via Ras-dependent activation of extracellular-signal-regulated kinase 2 and Elk-1. 906 44


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