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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Members of the protein kinase C (PKC) family of serine/threonine kinases play a key role in regulating the differentiation and growth of diverse cell types and, to date, the cloning of seven mammalian PKC genes encoding eight distinct isoforms has been reported. Here we describe the molecular cloning and deduced primary structure of a cDNA encoding a novel PKC isoform, termed PKC theta, which was isolated in the course of attempts to identify PKC genes that are expressed selectively in hematopoietic cells. Degenerate oligonucleotide primers corresponding to conserved sequence motifs, which distinguish the PKC family from other protein kinases, were employed in polymerase chain reactions (PCR) to amplify partial core sequences of putative PKC genes from a human peripheral blood lymphocyte-derived cDNA library. DNA sequencing of selected clones revealed several PKC-related sequences, including one that, on the basis of sequence comparison with known PKC isoforms, represented a novel PKC isoform. The complete cDNA sequence was determined by anchored PCR cloning and sequencing the entire coding sequence, using cDNA derived from a human leukemic T cell line (Jurkat). Included within this approximately 2.7-kilobase pair cDNA is an open reading frame of 2,118 nucleotides encoding a putative 82-kDa protein. The deduced primary structure contains consensus sequences characteristic of protein kinase catalytic domains and, based on its amino acid sequence and domain structure, is a member of the PKC family. PKC theta displays the highest homology to PKC delta, lacks the Ca(2+)-binding C2 domain and, thus, belongs to the subfamily of Ca(2+)-independent PKC enzymes which also includes the delta, epsilon, zeta, and eta isoforms. RNase protection assays and semiquantitative PCR analysis indicated that, although PKC theta transcripts are expressed ubiquitously, the highest levels are found in hematopoietic tissues and cell lines, including T cells and thymocytes. In contrast, the expression levels in the brain and testes are considerably lower, and no transcripts were detected in several human carcinoma cell lines. A rabbit antiserum raised against a unique (V3 domain) bacterially expressed PKC theta fragment immunoprecipitated specifically an 82-kDa protein from Jurkat cell lysates. Thus, PKC theta represents an additional member of the PKC family, and its predominant expression in hematopoietic cells suggests that it may play a role in signal transduction and growth regulatory pathways unique to these cells.
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PMID:Molecular cloning and characterization of PKC theta, a novel member of the protein kinase C (PKC) gene family expressed predominantly in hematopoietic cells. 844 77

Escherichia coli RNase T, which is responsible for the 3' processing and end-turnover of tRNA and the maturation of 5 S RNA, is extremely sensitive to sulfhydryl reagents and to oxidation, suggesting a role for cysteine residues in its activity. Titration of homogeneous RNase T with 5,5'-dithiobis-(2-nitrobenzoic acid) revealed that the 4 cysteine residues present in each of the two protein subunits are in a reduced form and that 1 or 2 of them are important for activity. To identify these residue(s), each of the cysteines in RNase T was changed individually to either serine or alanine. The serine mutant at position 168 is greatly reduced in RNase T activity both in vivo and in vitro; likewise, the serine mutant at position 112 and the alanine mutants at positions 112 and 168 also display decreased RNase T activity. Mutations at the other cysteine positions show little or no change. Kinetic analyses of the mutant enzymes showed that the Km values of C168S and C168A are increased considerably, whereas their Vmax values are reduced only slightly compared to the wild type enzyme. The other mutant enzymes are little changed. Additional amino acid replacements at position 168 showed that the in vivo and in vitro activities of RNase T are in the order Cys approximately Val > Ala >> Ser >> Asn approximately Asp, which closely follows the relative hydrophobicity of these amino acid residues. However, the affinity for tRNA, determined by fluorescence quenching, is not altered in C168S, suggesting that Cys-168 is not directly involved in substrate binding. Interestingly, proteins altered at position 168 showed increased temperature sensitivity as the residue at that position became less hydrophobic. These data indicate that Cys-168 contributes a hydrophobic group that influences the structure and ultimately the catalytic activity of RNase T.
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PMID:The role of individual cysteine residues in the activity of Escherichia coli RNase T. 855 40

It was shown that Cys-168 is required for RNase T function and thermostability and that its hydrophobic properties are important for this role (Li, Z., Zhan, L., and Deutscher, M. P. (1996) J. Biol Chem. 271, 1127-1132). To understand the molecular basis for these findings, further studies of Cys-168 and RNase T structure were carried out. Treatment of RNase T with the sulfhydryl-modifying agent 5,5'-dithiobis-(2-nitrobenzoic acid) leads not only to inactivation, but also to monomerization of the protein. Similarly, specifically converting Cys-168 to either serine or asparagine leads to loss of activity and to monomer formation at 37 degrees C. However, at 10 degrees C the serine mutant remains as a dimer and retains full RNase T activity, whereas the asparagine derivative shows only a low level of activity and of dimer formation. These data show a strong correlation between activity and the dimer form of RNase T. The importance of dimer formation was also shown in vivo using genetic studies. An inactive mutant of RNase T, termed HA2, which exists as a dimer at 37 degrees C in vitro, completely suppresses endogenous RNase T activity in vivo and in vitro when introduced into a RNase T+ cell on a multicopy phagemid, most likely as a consequence of inactive heterodimer formation. Introduction of the HA2 gene on a single-copy plasmid, as expected, leads to a proportionally smaller effect on endogenous activity. The dominant negative effect displayed by the HA2 protein can be relieved by an additional mutation in HA2 RNase T that abolishes its ability to dimerize. An inactive mutant asparagine derivative of Cys-168, which also does not dimerize, also shows little of the dominant negative phenotype. Thus, these data demonstrate that RNase T dimerizes in vivo, that the dimer form is required for RNase T activity, and that Cys-168 is needed for dimerization of the enzyme.
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PMID:Escherichia coli RNase T functions in vivo as a dimer dependent on cysteine 168. 855 41

The serine proteinase gene (sep) in Aspergillus flavus was disrupted by homologous recombination with a hygromycin resistance gene as the marker. The gene-disrupted mutant GR-2 contained a single-copy insertion of the marker gene and did not express the sep gene. Serine proteinase activity, 36-kDa protein labeled by 3H-diisopropylfluorophosphate, and immunologically detectable proteinase were not detected in the culture fluid of GR-2. Despite the absence of the serine proteinase, the total elastinolytic activity levels in the mutant and the wild-type A.flavus were comparable. Immunoblots revealed that the mutant secreted greater amounts of an elastinolytic metalloproteinase gene (mep20) product than did the wild type. Furthermore, mep20 mRNA levels, measured by RNase protection assay, in the mutant were higher than those in the wild type. Inhibition of the serine proteinase by Streptomyces subtilisin inhibitor (SSI) in the culture medium of wild-type A.flavus also resulted in an elevation of mep20 gene products. Although no serine proteinase activity could be detected, the level of elastinolytic activity of the SSI-treated culture was comparable to that of the control. Immunoblots revealed that the addition of SSI caused an elevation in the levels of metalloproteinase and its mRNA. These results suggest that the expression of the genes encoding serine and metalloproteinases are controlled by a common regulatory system and the fungus has a mechanism to sense the status of extracellular proteolytic activities.
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PMID:Disruption of the serine proteinase gene (sep) in Aspergillus flavus leads to a compensatory increase in the expression of a metalloproteinase gene (mep20). 868 96

CD36 is a multifunctional integral-membrane glycoprotein that acts as a receptor for thrombospondin, collagen, long-chain fatty acids, and oxidized LDL. Platelet CD36 deficiency can be divided into two groups. In type I, neither platelets nor monocytes/macrophages express CD36; in type II, monocytes/macrophages express CD36 but platelets do not. Two known mutations cause CD36 deficiency, ie, a 478C-->T substitution in codon 90 (proline90-->serine) and a dinucleotide deletion at nucleotide 539 in codon 110. In this study we investigated a type I Japanese subject (A.T.) and identified a new mutation, a single nucleotide insertion at nucleotide 1159 in codon 317. This mutation leads to a frameshift and the appearance of a premature stop codon. CD36 gene analysis indicated that A.T. was a compound heterozygote for a dinucleotide deletion at nucleotide 539 and the single nucleotide insertion at nucleotide 1159. RNase protection studies suggested that the new mutation as well as the dinucleotide deletion led to a marked reduction in the level of CD36 mRNA in her macrophages. However, the new mutation could be detected in macrophage but not platelet CD36 mRNA. These data suggest that the allele having the single nucleotide insertion in this subject has an additional abnormality that results in the absence of the mutated CD36 mRNA in platelets.
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PMID:A single nucleotide insertion in codon 317 of the CD36 gene leads to CD36 deficiency. 869 42

The invasive property of trophoblast cells is dependent on the activity of proteolytic enzymes of the metallo- and serine proteases family. Interleukin-1 (IL-1) was found to be involved in the regulation of these proteases in various systems, serving as an important modulator in trophoblast physiology (e.g. induction of hCG beta, cytokines, and others). Therefore, consideration is given in this report to the role of IL-1 in the regulation of metalloprotease activity in human trophoblasts. Human trophoblast cells were isolated from first trimester placentas by trypsin degradation and Percoll fractionation. Primary cell cultures of first trimester trophoblasts constitutively elaborated two species of collagenase type IV (92 and 72 kDa), as assessed in gelatin matrix. Treatment with IL-1 further augmented the 92-kDa type IV collagenase secretion in a dose-dependent manner. Furthermore, IL-1 significantly (P < 0.01) increased 92-kDa collagenase gene expression by trophoblast cells, as determined by solution hybridization/ribonuclease protection assay. Both the increase in gene expression and protein biosynthesis of the 92-kDa collagenase type IV were neutralized by the soluble IL-1 receptor, indirectly suggesting a receptor-mediated response. Interestingly, transforming growth factor-beta a putative modulator of IL-1 induced effects, was shown to induce the 92-kDa collagenase type IV secretion as well. These results provide indirect evidence supporting the idea that IL-1 and transforming growth factor-beta may play an intermediary role in trophoblast invasion at the feto-maternal interface by regulating trophoblast expression of 92-kDa type IV collagenase, a protease of prime importance in trophoblast invasion.
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PMID:Cytokine-mediated regulation of type IV collagenase expression and production in human trophoblast cells. 876 80

In previous studies using specific G alpha s antibodies we have identified several human myometrial G alpha s protein isoforms with molecular masses of 45, 46, 47, 54, and 58 kDa, respectively. During pregnancy, levels of the 46- and 54-kDa proteins are significantly increased compared to those in nonpregnant myometrium and then decreased at the onset of labor. In this study we investigated the expression of G alpha s messenger ribonucleic acid (mRNA) splice variants, which are generated as a result of alternative splicing of a single mRNA precursor, in term pregnancy and parturition to determine whether there was any correlation with the observed changes in G alpha s protein isoforms. A myometrial G alpha s complementary DNA was synthesized using RT-PCR and cloned into pCRtmII suitable for preparation of riboprobes for use in ribonuclease protection assays. Using this technique, we identified at least three myometrial G alpha s mRNAs, including two forms of G alpha s-Large (with or without the serine at amino acid 87) and one form of G alpha s-Small (with the serine at amino acid 72). G alpha s Small (with the serine) and G alpha s-Large (with the serine) mRNAs encode for the 46- and 54-kDa G alpha s protein isoforms, respectively, and were increased in term pregnancy and then subsequently decreased after the onset of labor. Our data suggest that posttranscriptional regulation of G alpha s mRNAs may be important in the differential expression of G alpha s protein isoforms during pregnancy and labor.
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PMID:Human myometrial G alpha s-small (with serine) and Gs-large (with serine) messenger ribonucleic acid splice variants promote the increased expression of 46- and 54-kilodalton G alpha s protein isoforms in pregnancy and their down-regulation during labor. 877 78

Receptor serine-threonine kinases (RSTK) mediate inhibitory as well as stimulatory signals for growth and differentiation by binding to members of the transforming growth factor-beta (TGF-beta) superfamily. Over 12 different RSTKs have been isolated so far, displaying wide expression in peripheral tissues and in the nervous system. Here we report the isolation and characterization of a novel type I RSTK termed activin receptor-like kinase-7 (ALK-7) that, unlike other members of this receptor family, is predominantly expressed in the adult central nervous system. The ALK-7 gene encodes a 55-kDa cell-surface protein that exhibits up to 78% amino acid sequence identity in the kinase domain to previously isolated type I receptors for TGF-beta and activin. In the extracellular domain, however, ALK-7 is more divergent, displaying comparable similarities with all members of the ALK subfamily. RNase protection and in situ hybridization studies demonstrated a highly specific mRNA distribution restricted to neurons in several regions of the adult rat central nervous system, including cerebellum, hippocampus, and nuclei of the brainstem. Receptor reconstitution and cross-linking experiments indicated that ALK-7 can form complexes with type II RSTKs for TGF-beta and activin in a ligand-dependent manner, although direct binding of ALK-7 to ligand in these complexes could not be demonstrated. The specific expression pattern of ALK-7, restricted to the postnatal central nervous system, indicates that this receptor may play an important role in the maturation and maintenance of several neuronal subpopulations.
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PMID:A novel type I receptor serine-threonine kinase predominantly expressed in the adult central nervous system. 894 33

We have shown previously that expression of 46 and 54 kDa human myometrial G alpha s protein isoforms is increased during gestation and then subsequently decreased during labour. These proteins appear to be coded for by G alpha s-Small (with a serine residue at position 72) and G alpha s-Large (with a serine residue at position 87) mRNA splice variants respectively. In the study presented here we have used a G alpha s cDNA template to generate [32P]cytidine cRNA riboprobes for use in RNase protection assays, so as to measure total myometrial G alpha s mRNA levels in relation to the pattern of expression of G alpha s mRNA splice variants during pregnancy and labour. We report that total levels of human myometrial G alpha s mRNA remain similar in non-pregnant and pregnant women but are substantially reduced during parturition. Our data also provide strong evidence that alternative splicing of G alpha s precursor mRNA has a primary role in regulating expression of G alpha s protein isoforms during pregnancy and labour. The inclusion of an additional serine codon in G alpha s mRNAs during pregnancy involves a switch in alternative splicing pathways. We speculate that this switch may be due to a change in specificity of splicing factors that are modulated during pregnancy and labour.
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PMID:Expression of human myometrial G alpha s messenger ribonucleic acid transcript during pregnancy and labour: involvement of alternative splicing pathways. 906 3

Granulosa cells are essential for follicular development and corpus luteum formation and their functions are regulated by gonadotrophins through G protein-coupled receptors. The dominant second messenger pathway involves the stimulation of cyclic AMP formation by G alpha s-linked receptors. In this paper we have investigated the expression of G alpha s mRNA splice variants in relation to expression of G alpha s protein isoforms in granulosa cells obtained from patients undergoing in vitro fertilization. We have carried out ribonuclease protection assays using cRNA riboprobes which are capable of detecting all G alpha s mRNA isoforms as well as quantifying total amounts of G alpha s mRNA. Granulosa cells express the message for G alpha s-Large and G alpha s-Small and the presence of two distinct protein products was confirmed by immunoblotting using the antibody RM/1. Moreover, the data show that a significant fraction of G alpha s-Large and G alpha s-Small mRNAs contain an extra CAG codon. This should generate proteins with an extra serine residue, resulting in G alpha s variants with the consensus sequence of a protein kinase C phosphorylation site. These results highlight the possible interaction between different signalling pathways in the control of cAMP production and the need to investigate the relationship between G alpha s variants and different adenylyl cyclase isozymes in patients with normal and abnormal ovarian function.
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PMID:Identification of G alpha s messenger ribonucleic acid splice variants in human granulosa cells. 906 4


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