Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.9 (ribonuclease)
 6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Seven hydrophobic residues ranging in size from glycine to phenylalanine have been substituted for the wild-type methionine residue at position 13 in a 15-residue truncated version (S15) of S-peptide, the small component of ribonuclease S. Complexes of both S-15 and the seven variants with S-protein yielded isomorphous crystals. The structures of all eight complexes have been refined to final R-factors in the range of 17-19%. [See Kim, E. E. Varadarajan, R., Wyckoff, H. W., and Richards, F. M. (1992) Biochemistry (preceding paper in this issue) for the description of the reference S-15 complex.] Multiple side-chain conformations were seen for six residues in all of the complexes and for two to three additional residues in at least some of the complexes. Three of the complexes, Gly, Ala, and alpha-amino-n-butyric acid (ANB), contained a single water molecule in the cavity near residue 13 that makes three hydrogen bonds to protein atoms. Although space is available, no evidence for additional water in this region, ordered or disordered, was found. The atoms in the cavity wall tend to shrink the cavity by moving in on the small residues and to swell the cavity by moving out for the larger Phe substitution. A swelling seen with leucine was attributed to a shape effect since Leu, Ile, and Met all have the same volume. A slight volume contraction of the collection of interior residues outside of the region of position 13 was also noted. (All changes noted are in the direction to maintain a constant packing density averaged over the whole protein.) Leu51, a surface hydrophobic residue, moved considerably in the G, A, and ANB complexes in directionswhich would tend to decrease the cavity volume. The only other major change in position, 1.5 A, was the 66-69 loop, which is about 25 A from position 13. His12, Phe120, and Asp121 appear to be involved in this movement, but the connection with position 13 is not clear at all. The thermodynamic data on the association reaction for all of these complexes have been previously reported [Connelly, P. R., Varadarajan, R., Sturtevant, J. M., & Richards, F. M. (1990) Biochemistry 29, 6108-6114; Varadarajan, R., Connelly, P. R., Sturtevant, J. M., & Richards, F. M. (1992) Biochemistry 31, 1421-1426]. Some comments are offered on our initial attempts to correlate the structural changes with the changes in the thermodynamic parameters.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Crystallographic structures of ribonuclease S variants with nonpolar substitution at position 13: packing and cavities. 146 20

We evaluated two independent models of eosinophil differentiation for their ability to synthesize the ribonuclease toxins eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP). Cells from the clone 15 subline of HL-60 (human promyelocytic leukemia) produced both EDN and ECP; production of EDN increased in response to butyric acid (BA). CD34+ peripheral blood progenitor cells (PBPCs) grown with cytokines promoting eosinophil differentiation also produced EDN. EDN from both the clone 15 and PBPCs was more heterogeneous and heavily glycosylated (approximately 22-45 kDa) than EDN from the mature peripheral blood eosinophils (18-25 kDa). The heterogeneity of EDN from the clone 15 cells was not altered by endoglycosidase Hf, whereas treatment with peptide-N-glycosidase F (PNGase F) produced a single-band immunoreactive band (approximately 15 kDa). In contrast, only the highest molecular weight forms of EDN from differentiated PBPCs were eliminated by PNTGase F (reduced to 22-35 kDa), suggesting the presence of uncharacteristic forms of posttranslational modification. Synthesis of hyperglycosylated proteins has not been previously reported in PBPCs and is a feature shared with tumor cells and cell lines.
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PMID:Hyperglycosylation of eosinophil ribonucleases in a promyelocytic leukemia cell line and in differentiated peripheral blood progenitor cells. 761 5

The eosinophil-derived neurotoxin (EDN/RNS2) is a member of the mammalian ribonuclease gene family and is one of four proteins found in the large specific granules of human eosinophilic leukocytes. The gene encoding EDN consists of two exons, including a noncoding exon 1, separated by a single intron from the coding sequence in exon 2. We have identified a functional promoter of the EDN gene and shown that optimal expression depends on interaction between the promoter and one or more sequence elements found in the single intron. Cells of the clone 15 eosinophilic variant of the human promyelocytic HL-60 cell line were transfected with constructs that included the promoter region of the EDN gene alone, promoter with exon 1, and promoter with both exon 1 and the intron positioned 5' to the chloramphenicol acetyltransferase (CAT) reporter gene (constructs referred to as PrCAT, PrExCAT, and PrExIn CAT, respectively). Although reporter gene activity from either PrCAT or PrExCAT was only 2-3 fold higher than baseline (CAT alone), inclusion of the single intron (PrExInCAT) resulted in a 28-fold increase in reporter gene activity in uninduced clone 15 cells, and an 80-fold in activity when clone 15 cells were induced to differentiate toward eosinophils with butyric acid. The intron-mediated enhancer activity was reproduced in other human hematopoietic cell lines (K562, Jurkat, U937, and HL-60), but was not found in human 293 kidney cells, suggesting that the function of the enhancer element(s) may be tissue-specific. A significant portion of the observed enhancer activity resides in the first 60 base pairs the the intron, which includes consensus binding sites for both AP-1, and NF-ATp transcription factors, and a 15-base pair segment that is identical to a sequence found in the promoter of the gene encoding the neutrophil granule protein, lactoferrin. The noncoding exon 1/single intron/coding exon 2 genomic structure is a common feature among the mammalian ribonucleases; this finding suggests the possibility of a conserved mechanism of regulation in this gene family.
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PMID:Enhanced expression of the eosinophil-derived neurotoxin ribonuclease (RNS2) gene requires interaction between the promoter and intron. 864 42

The present study tested the hypothesis that estradiol reduces tissue infarction after middle cerebral artery occlusion (MCAO) in estradiol-deficient females by augmenting glutamic acid decarboxylase (GAD) expression and thus activity, leading to increases in gamma-amino-butyric acid (GABA) tissue levels. Glutamic acid decarboxylase is the principal enzyme for GABA synthesis and has two isoforms, GAD65 and GAD67, which differ in size and cellular distribution. Rats were ovariectomized 7 to 8 days before receiving no hormone, placebo, or 25 microg estradiol via subcutaneous implant 7 to 10 days before harvesting tissue in either ischemic cohorts after 2 h of MCAO (end-ischemia) or in nonischemic cohorts. Selected cortical and striatal regions were microdissected from harvested brains. GAD65/67 mRNA levels were determined by microlysate ribonuclease protection assay. End-ischemic GABA concentrations were determined by HPLC. Steroid treatment selectively decreased ischemic cortical GAD67 mRNA levels. In most brain regions evaluated, regional GABA concentrations increased with ischemia regardless of treatment. Estradiol blocked MCAO-induced increases in GABA concentration only in dorsomedial cortex. These data suggest that estradiol repletion in ischemic rat brain selectively decreases GAD67 mRNA levels but does not alter steady-state GABA concentrations. It may be that estradiol under ischemic conditions is attenuating GABA metabolism rather than enhancing synthesis or is augmenting other aspects of GABAergic transmission such as GABA transporters and receptors.
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PMID:Estradiol alters only GAD67 mRNA levels in ischemic rat brain with no consequent effects on GABA. 1609 13

The transcription factors GATA-1 and GATA-2 have been implicated in promoting differentiation of eosinophilic leukocytes. In this study, we examined the roles of GATA-1 and GATA-2 in activating transcription of the secretory ribonuclease, the eosinophil-derived neurotoxin (EDN/RNase 2). Augmented expression of both GATA-1 and GATA-2 was detected in eosinophil promyelocyte HL-60 clone 15 cells in response to biochemical differentiation with butyric acid. Deletion or mutation of one or both of the two consensus GATA-binding sites in the extended 1000-bp 5' promoter of the EDN gene resulted in profound reduction in reporter gene activity. Antibody-augmented electrophoretic mobility shift and chromatin immunoprecipitation analyses indicate that GATA-1 and GATA-2 proteins bind to both functional GATA consensus sequences in the EDN promoter. Interestingly, RNA silencing of GATA-1 alone had no impact on EDN expression; silencing of GATA-2 resulted in diminished expression of EDN, and also diminished expression of GATA-1 in both butyric acid-induced HL-60 clone 15 cells and in differentiating human eosinophils derived from CD34(+) hematopoietic progenitors. Likewise, overexpression of GATA-2 in uninduced HL-60 clone 15 cells resulted in augmented transcription of both EDN and GATA-1. Taken together, our data suggest that GATA-2 functions directly via interactions with the EDN promoter and also indirectly, via its ability to regulate the expression of GATA-1 in differentiating eosinophils and eosinophil cell lines.
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PMID:GATA transcription factors regulate the expression of the human eosinophil-derived neurotoxin (RNase 2) gene. 1927 13