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)

Aromatase from human placenta has been purified to homogeneity (MW 55,000). Enzymatic activity can be reconstituted with reductase from pig liver in an aqueous buffer or after incorporation of the enzyme into liposomes. In both cases the enzyme converts androstenedione to estrone and testosterone to estradiol. Aromatase shows a typical CO-spectrum when reduced with dithionite and a type I spectral shift with both substrates. The NH2 terminal amino acid sequence is hydrophobic but shows no homology to that of other cytochromes P-450. Five cysteine peptides have been isolated by HPLC following tryptic digestion of the [14C]-carboxymethylated protein. Amino acid sequences of these peptides reveal that histidine is the carboxy-terminal amino acid of the protein and that significant homology exists with corresponding peptides from other cytochromes P-450. Unique oligonucleotides (62 and 30 MER) synthesized on the basis of a 45 amino acid sequence near the center of the molecular have been used to clone the aromatase gene from a cDNA expression library from human placenta in lambda gt11.
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PMID:Purification and characterization of aromatase from human placenta. 350 67

cDNA clones encoding three classes of human actins have been isolated and characterized. The first two classes (gamma and beta, cytoplasmic actins) were obtained from a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA, and the third class (alpha, muscle actin) was obtained from a cDNA library constructed from adult human muscle mRNA. A new approach was developed to enrich for full-length cDNAs. The human fibroblast cDNA plasmid library was linearized with restriction enzymes that did not cut the inserts of interest; it was then size-fractionated on gels, and the chimeric molecules of optimal length were selected for retransformation of bacteria. When the resulting clones were screened for actin-coding sequences it was found that some full-length cDNAs were enriched as much as 50- to 100-fold relative to the original frequency of full-length clones in the total library. Two types of clones were distinguished. One of these clones encodes gamma actin and contains 100 base pairs of 5' untranslated region, the entire protein coding region, and the 3' untranslated region. The second class encodes beta actin, and the longest such clone contains 45 base pairs of 5' untranslated region plus the remainder of the mRNA extending to the polyadenylic acid tail. A third class, obtained from the human muscle cDNA library, encodes alpha actin and contains 100 base pairs of 5' untranslated region, the entire coding region, and the 3' untranslated region. Analysis of the DNA sequences of the 5' end of the clones demonstrated that although beta- and gamma-actin genes start with a methionine codon (MET-Asp-Asp-Asp and MET-Glu-Glu-Glu, respectively), the alpha-actin gene starts with a methionine codon followed by a cysteine codon (MET-CYS-Asp-Glu-Asp-Glu). Since no known actin proteins start with a cysteine, it is likely that post-translational removal of cysteine in addition to methionine accompanies alpha-actin synthesis but not beta- and gamma-actin synthesis. This observation has interesting implications both for actin function and actin gene regulation and evolution.
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PMID:Isolation and characterization of full-length cDNA clones for human alpha-, beta-, and gamma-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed. 686 42

alpha trans-inducing factor (alpha TIF, VP16, Vmw65) is an essential structural protein of herpes simplex virus, being required for virion assembly. The protein also forms complexes with host proteins and a response element and transactivates the alpha genes which carry this element. The protein contains an acidic carboxyl terminus required for transactivation and a much larger amino-terminal domain required for promoter recognition. We report the first set of temperature-sensitive (ts) mutations deliberately introduced into the protein by substitution of the cysteine codons with those specifying glycine at positions 78, 102, and 176, either singly or in combinations. We report the following results. (i) All mutated proteins synthesized in vitro formed complexes with the DNA response element at room temperature. However, the mutant with the triple substitution and two mutants with substitutions in two of the three cysteines exhibited a ts phenotype at 33 and 37 degrees C, and one exhibited a ts phenotype only at 37 degrees C. (ii) Replacement of wild-type alpha TIF with genes carrying substitutions in any two cysteines conferred a ts phenotype for replication at 39.5 degrees C. Shift-down experiments indicated that the 10(4)- to 10(5)-fold reduction in virus yield at the nonpermissive temperature was due to the disfunction of alpha TIF late in infection, presumably in virion maturation. (iii) The alpha TIF expressed in cells infected with mutant viruses exhibited the same ts phenotype in protein-DNA complex formation as those expressed in vitro from mutated plasmids. Although the virus carrying the alpha TIF substitutions at Cys-102 and Cys-176 failed to induce a reporter gene linked to the alpha 4 promoter at 39.5 degrees C, it replicated as well as the parent virus in cells maintained for the first 10 h of infection at 39.5 degrees C. We conclude the following. (i) Formation of DNA-protein complexes containing alpha TIF is a poor prognosticator of alpha TIF function. (ii) The data presented here and in the literature strongly support the hypothesis that the secondary structure of the alpha TIF is very sensitive to deletions or insertions which probably affect the interaction of alpha TIF with both viral proteins in the virion and cellular proteins during infection. As a consequence, deletion-insertion mutagenesis may not shed useful information on the role of transactivating function of alpha TIF in infection. (iii) Since cysteines may play a role in stabilizing the secondary structure of proteins, substitutions of cysteines may be a powerful technique for site-specific construction of ts mutants in essential viral proteins.
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PMID:The phenotype in vitro and in infected cells of herpes simplex virus 1 alpha trans-inducing factor (VP16) carrying temperature-sensitive mutations introduced by substitution of cysteines. 749 74

Oltipraz (5-pyrazinyl-4-methyl-1,2-dithiole-3-thione), which is undergoing clinical evaluation as an anticarcinogen, also inhibits HIV-1 replication (IC50 approximately equal to 10 microM). The inactivation of RT appears to be a relevant antiviral mechanism since oltipraz blocks viral replication in acutely infected T-cell lines, but is ineffective in chronically infected ACH-2 cells (H. J. Prochaska, W. G. Bornmann, P. Baron, and B. Polsky (1995) Mol. Pharmacol. 48, 15-20). Since a nucleophilic amino acid is a likely target for oltipraz, we assessed whether the conserved cysteine residues of HIV-1 RT (38Cys or 280Cys) were the target(s) for oltipraz, and we synthesized [Me 14C]oltipraz to determine if oltipraz forms a stable adduct with RT. Thus, HIV-2 RT as well as wild-type, 38Cys-->Ser, 280Cys-->Ser, and the Cys-->Ser double mutant of HIV-1 RT were purified from the lysates of transformed Escherichia coli strain DH5 alpha (A. Hizi, M. Shaharabany, R. Tal, and S. H. Hughes (1992) J. Biol. Chem. 267, 1293-1297) via a purification procedure that included (NH4)2SO4 fractionation followed by gel filtration, dye-ligand, and ion-exchange chromatographies. Procion yellow H4R was chosen as the dye-ligand chromatography since it was the most potent and selective inhibitor of RT among seventy reactive dyes that were screened. Mono Q anion-exchange chromatography with diethanolamine (pH 9) resulted in the generation of heterodimeric RT from a predominantly homodimeric enzyme preparation. Because the instability of dilute RT preparations at room temperature rendered the kinetic evaluation of inactivation difficult, we sought to identify conditions that prevent denaturation of these enzymes. High concentrations (25 mM) of MgCl2 had a stabilizing effect. Oltipraz behaved kinetically as an irreversible inhibitor of all RTs purified, and the kinetic constants for the inactivation of these enzymes were not significantly different from wild-type HIV-1 RT (Ki = 17.0 +/- 4.1 microM; k3 = 0.214 +/- 0.051 h-1). In stark contrast, oltipraz neither inhibited nor inactivated the Klenow fragment of DNA polymerase I, whose subdomain structure is similar to the p66 subunit of RT. Wild-type RT was incubated with 60 microM [Me 14C]oltipraz for 4 h and was then subjected to gel filtration chromatography. The [14C] label comigrated with RT with a stoichiometry of binding of 0.88 +/- 0.05 oltipraz per inactivated RT subunit (N = 3 experiments), and the [14C] label remained bound after treatment with 4 M urea.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Inactivation of human immunodeficiency virus type 1 reverse transcriptase by oltipraz: evidence for the formation of a stable adduct. 750 49

Medullary thyroid carcinoma (MTC) occurs sporadically or as part of the inherited cancer syndrome multiple endocrine neoplasia (MEN) type 2. In MEN 2A, germline missense mutations are found in one of five cysteine codons within exons 10 and 11 in the extracellular domain of the RET protooncogene. In MEN 2B, germline mutations occur in codon 918 (exon 16) within the catalytic core of the tyrosine kinase domain. To determine if RET mutations similar to those in MEN 2A and 2B play a role in the pathogenesis of sporadic MTC, we analysed 71 sporadic tumours comprising 68 primary tumours and three cell lines, for mutations in RET exons 10, 11, and 16. We found that 23% of sporadic MTC had RET codon 918 mutations, while only 3% had exon 10 mutations, and none had mutations in exon 11. We found no exon 16 mutations in MTC from 14 MEN 2A cases. Thus, exon 10 and 11 mutations, commonly found in familial MTC and MEN 2A, rarely occur in sporadic MTC; somatic mutation of RET codon 918 appears to play a role in the tumourigenesis of a significant minority of sporadic MTC but not MEN 2A tumours. In addition to their biological interest, these findings may have some clinical application in determining whether a patient presenting with isolated MTC is truly sporadic or is part of an inherited cancer syndrome.
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PMID:Mutation of the RET protooncogene in sporadic medullary thyroid carcinoma. 753 60

The hereditary multiple endocrine neoplasia syndromes types 2A and B (MEN 2A and B) were recently linked to germline mutations in the RET proto-oncogene, altering one of five cysteine residues in exon 10 or 11 (MEN 2A), or substituting a methionine for a threonine at codon 918 in exon 16 (MEN 2B). The latter mutation also occurs somatically in some sporadic medullary thyroid carcinomas (MTC), and has in a previous study been correlated with a less favorable clinical outcome. In the present study, 46 MTCs were selected for investigation of the codon 918 mutation. The mutation was found in 29 tumors (63%), and was significantly correlated with a poor outcome, with regard to distant metastasis or tumor recurrence (p < 10(-4)). Two tumors showed multifocal growth and C-cell hyperplasia, and these patients were therefore also investigated for germline mutations in exons 10, 11 and 16. The codon 918 mutation was found only in the tumors, thus of somatic origin. The RET codon 918 mutation may have prognostic impact, and therefore preoperative assessment may influence decision-making in the treatment of patients suffering from MTC.
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PMID:Mutations of codon 918 in the RET proto-oncogene correlate to poor prognosis in sporadic medullary thyroid carcinomas. 755 2

Multiple endocrine neoplasia (MEN) type 1 is an autosomal, dominantly inherited predisposition to develop neoplastic lesions of the parathyroid glands, the neuroendocrine pancreas-duodenum, and the anterior pituitary. The genetic defect was mapped to the centromeric part of the long arm of chromosome 11 based on studies of somatic deletions in MEN-1-associated tumors and linkage analysis in families in whom the disease is segregated. Combined family and tumor analysis has shown that tumorigenesis in MEN-1 involves loss of the wild-type chromosome, indicating that the putative MEN-1 gene is a tumor suppressor gene. Similar deletions are also seen in a proportion of sporadic parathyroid and pancreatic tumors, suggesting that tumorigenesis involves related mechanisms in both sporadic and familial cases. Based on results from linkage analysis in more than 40 MEN-1 families, predictive testing for MEN-1 using DNA polymorphisms can now be performed with high accuracy. Hence, biochemical screening programs can focus on individuals at risk to identify early signs of tumor development. MEN-2, an autosomal dominant cancer syndrome of variable expressivity, has previously been localized to chromosome 10q11.2 by positional cloning tactics. The RET protooncogene mapping to the MEN2 susceptibility locus has recently emerged as a candidate gene for MEN-2A. RET, a transmembrane receptor protein, has a large glycosylated extracellular domain containing clustered cysteine residues and calcium-binding motifs, a single hydrophobic transmembrane domain, and a cytoplasmic domain with tyrosine kinase catalytic activity. Several germline missense mutations in a codon specifying one of these highly conserved cysteine residues have been detected in patients affected with MEN-2A.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Genetic aspects of multiple endocrine neoplasia types 1 and 2. 758 11

The International RET Mutation Consortium was first convened as part of the Fifth International Workshop on Multiple Endocrine Neoplasia, Stockholm, Sweden, in an attempt to analyse the relationship of RET mutation and disease phenotype in the autosomal dominantly inherited multiple endocrine neoplasia type 2 (MEN 2) syndromes. Out of 361 families studied, 41% had MEN 2A, 17.7% MEN 2B, 6.4% FMTC and the remaining subjects were unclassified. RET mutations were detected in 87.3% of families overall. Over 93% of MEN 2B families had the RET 918 ATG-->ACG mutation, while the most frequent mutation detected in MEN 2A families was cysteine codon 634 (87% of all mutations).
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PMID:Genotype-phenotype correlation in multiple endocrine neoplasia type 2: report of the International RET Mutation Consortium. 759 70

To assess the pathophysiological role of the RET protooncogene in sporadic pheochromocytomas, we examined the 2 regions of the gene in which molecular defects are specifically associated with the multiple endocrine neoplasias (MEN) type 2A (the cysteine-rich domain encoded by exons 10 and 11), and type 2B (the tyrosine kinase domain encoded by exon 16). The sequences of both regions were amplified by reverse transcriptase-polymerase chain reaction (PCR) or PCR from tumor RNA and/or leukocyte DNA. The amplified fragments were analyzed by denaturing gradient gel electrophoresis using chemical clamps. In 28 patients with unilateral sporadic tumors, 6 RET mutations were found, 3 in the MEN 2A region, 3 in the MEN 2B region. Five patients had missense mutations: 2 in the MEN 2A region (C634W and D631Y), and 3 in the MEN 2B region (M918T). Analysis of leukocyte DNA in 3 of these patients confirmed that RET mutations were only present in tumor DNA. The sixth patient had lost exon 10 in the tumor complementary DNA as a result of the deletion of the dinucleotide -AG- at the 3'splice acceptor site of intron 9; this molecular defect was only found in the tumor DNA. Thus RET mutations of the MEN 2A and 2B regions are also found in about 20% of sporadic pheochromocytomas. We describe new types of molecular defects of the RET protooncogene in the MEN 2A region that involve noncysteine residues and loss of exon 10. Further studies should be extended to analyze the entire RET protooncogene. These findings have a profound clinical impact for the management of patients with supposedly sporadic pheochromocytomas.
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PMID:The RET protooncogene in sporadic pheochromocytomas: frequent MEN 2-like mutations and new molecular defects. 855 Jul 89

Medullary thyroid carcinoma occurs sporadically or as a part of the inherited cancer syndrome multiple endocrine neoplasia (MEN) type 2. The MEN 2 gene has been identified as the RET proto-oncogene on chromosome 10. In MEN 2A, RET mutations are detectable in one of five cysteine codons within exons 10 and 11 and in MEN 2B in codon 918 (exon 16). Direct DNA testing for RET proto-oncogene mutations is the method of first choice in presymptomatic screening of MEN 2 families. Gene carriers should be offered prophylactic thyroidectomy. The process of DNA analysis for RET proto-oncogene mutations is demonstrated in one family with hereditary medullary thyroid carcinoma. RET mutations were detectable in five of the nine family members at risk.
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PMID:Presymptomatic genetic screening in families with multiple endocrine neoplasia type 2. 767 Sep 26


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