Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P01034 (cystatin C)
 3,397 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chloroacetaldehyde-modified poly(rC) or poly(dC) was prepared containing either 8-36% 3,N4-ethenocytidine (epsilon C) or 8-36% of a mixture of epsilon C and the hydrated epsilon C (epsilon C . H2O), with the hydrate greatly predominating (greater than 90%). These ribo- and deoxyribonucleotide templates were transcribed with DNA-dependent RNA polymerases from Escherichia coli and calf thymus, in the presence of either Mn2+ or Mg2+ and all four ribonucleoside triphosphates. All the polymers tested were transcribed with either cation present. In an earlier report from this laboratory [Spengler, S., & Singer, B. (1981) Nucleic Acids Res. 9. 365], transcriptional ambiguities resulting from epsilon C residues in enzymatically synthesized poly(rC, epsilon rC) were studied with E. coli DNA-dependent RNA polymerase in the presence of Mn2+. The misincorporations there reported were confirmed when poly(rC, epsilon rC) and poly(dC, epsilon dC), prepared by reaction of poly(rC) and poly(dC) with CAA, were transcribed in the presence of either Mn2+ or Mg2+. We now report that the presence of hydrated epsilon C in polymers also leads to misincorporations but with reproducible differences from those found with epsilon C alone. Nearest-neighbor analysis of the transcription products showed that the hydrate caused misincorporation of A greater than U much greater than C while epsilon C caused misincorporation of U greater than A much greater than C. The extent of misincorporation in transcription was less with Mg2+ than with Mn2+, but the pattern of ambiguity was the same with both cations and with both ribo- and deoxyribocytidylate polymers. Calf thymus DNA-dependent RNA polymerase IIB was also used to transcribe deoxyribocytidine polymers with Mn2+ as the cation. epsilon C and epsilon C . H2O both caused a high level of misincorporation of U , A, and C, but the preferred misincorporations differed slightly from those found with E. coli DNA-dependent RNA polymerase. For both prokaryotic and eukaryotic enzymes, the type of misincorporation resulting from the loss of hydrogen bonding by modification of the N-3 of C not only differed between epsilon C and the hydrated intermediate but also both differed from the transcriptional errors resulting from the presence of 3-methylcytidine in poly(dC) or poly(rC). We conclude that the errors made by these polymerases during transcription do not result primarily from the conditions used (cation, ribo- or deoxyribotemplate) but must be at least in part attributed to the enzyme recognizing some facet of the modified base other than the lack of normal hydrogen bonding.
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PMID:Chloroacetaldehyde-treated ribo- and deoxyribopolynucleotides. 2. Errors in transcription by different polymerases resulting from ethenocytosine and its hydrated intermediate. 675 74

The reading of glutamine and lysine codons during protein synthesis in vitro has been investigated using an MS2-RNA-programed system derived from Escherichia coli. Under conditions when either glutaminyl-tRNA1Gln (s2UUG) or glutaminyl-tRNA2Gln (CUG) was the only source of glutamine for protein synthesis both tRNAs were able to read the glutamine codons CAA and CAG as indicated by the incorporation of labeled glutamine into the pertinent coat protein tryptic peptides. On the other hand, when the two glutamine tRNAs competed for the codon CAA the reading efficiency of the anticodon s2UUG, which reads the codon according to the wobble rules, was almost 40 times higher than that of the competing anticodon CUG, which reads the codon by "two out of three," i.e. it cannot form a regular base pair with the third codon position. In reading the codon CAG the anticodon CUG was approximately eight times more efficient than the anticodon s2UUG. The lysyl-tRNA1Lys (CUU) could not alone sustain any detectable coat protein synthesis in the MS2 system indicating that there was no significant reading of the lysine codon AAA. This conclusion is supported by the outcome of experiments where lysyl-tRNA1Lys (CUU) and lysyl-tRNA2Lys (s2UUU) competed for the codon AAA. The reading efficiency of the anticodon CUU was less than 1% of that of the competing s2UUU which represents the limit of resolution of our experimental system. When the two lysine tRNAs competed for the codon AAG the anticodon CUU was about four times more efficient than s2UUU. These results are discussed in the context of the two out of three hypothesis, which attempts to relate the frequency of such reading to the hydrogen bonding properties of the codon nucleotides.
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PMID:Codon reading and translational error. Reading of the glutamine and lysine codons during protein synthesis in vitro. 678 93

The importance of the evolutionarily conserved Gly-4 residue for the affinity and kinetics of interaction of cystatin A with several cysteine proteinases was assessed by site-directed mutagenesis. Even the smallest replacement, by Ala, resulted in approximately 1000-, approximately 10- and approximately 6000-fold decreased affinities for papain, cathepsin L, and cathepsin B, respectively. Substitution by Ser gave further 3-8-fold reductions in affinity, whereas the largest decreases, >10(5)-fold, were observed for mutations to Arg and Glu. The kinetics of inhibition of papain by the mutants with small side chains, Ala and Ser, were compatible with a one-step bimolecular reaction similar to that with wild-type cystatin A. The decreased affinities of these mutants for papain and cathepsin L were due exclusively to increased dissociation rate constants, but the reduced affinities for cathepsin B were due also to decreased association rate constants. The latter finding indicates that the intact N-terminal region serves as a guide directing cystatin A to the active site of cathepsin B, as has been proposed for cystatin C. The kinetics of binding of the mutants with charged side chains, Arg and Glu, to papain were consistent with a two-step binding mechanism, in which the mutant side chains are accommodated in the complex by a conformational change. The NMR solution structure of the Ala and Trp mutants showed only minor changes compared with wild-type cystatin A, indicating that the large reductions in affinity for proteinases are not due to altered structures of the mutants. Instead, a side chain larger than a hydrogen atom at position 4 affects the interaction with the proteinase most likely by interfering with the binding of the N-terminal region.
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PMID:The role of Gly-4 of human cystatin A (stefin A) in the binding of target proteinases. Characterization by kinetic and equilibrium methods of the interactions of cystatin A Gly-4 mutants with papain, cathepsin B, and cathepsin L. 958 70

Ribonuclease A (RNase A) catalyzes the cleavage of RNA after pyrimidine nucleotides. When bound in the active site, the base of a pyrimidine nucleotide forms hydrogen bonds with the side chain of Thr45. Here, the role of Thr45 was probed by using the wild-type enzyme, its T45G variant, X-ray diffraction analysis, and synthetic oligonucleotides as ligands and substrates. Catalytic specificity was determined with the fluorogenic substrate: 6-carboxyfluorescein approximately dArXdAdA approximately 6-carboxytetramethylrhodamine (6-FAM approximately dArXdAdA approximately 6-TAMRA), where X = C, U, A, or G. Wild-type RNase A cleaves 10(6)-fold faster when X = C than when X = A. Likewise, its affinity for the non-hydrolyzable oligonucleotide 6-FAM approximately d(CAA) is 50-fold greater than for 6-FAM approximately d(AAA). T45G RNase A cleaves 6-FAM approximately dArAdAdA approximately 6-TAMRA 10(2)-fold faster than does the wild-type enzyme. The structure of crystalline T45G RNase A, determined at 1.8-A resolution by X-ray diffraction analysis, does not reveal new potential interactions with a nucleobase. Indeed, the two enzymes have a similar affinity for 6-FAM approximately d(AAA). The importance of pentofuranosyl ring conformation to nucleotide specificity was probed with 6-FAM approximately d(AU(F)AA), where U(F) is 2'-deoxy-2'-fluorouridine. The conformation of the pentofuranosyl ring in dU(F) is known to be more similar to that in rU than dU. The affinity of wild-type RNase A for 6-FAM approximately d(AU(F)AA) is 50-fold lower than for 6-FAM approximately d(AUAA). This discrimination is lost in the T45G enzyme. Together, these data indicate that the side chain of Thr45 plays multiple roles-interacting favorably with pyrimidine nucleobases but unfavorably with purine nucleobases. Moreover, a ribose-like ring disfavors the interaction of Thr45 with a pyrimidine nucleobase, suggesting that Thr45 enhances catalysis by ground-state destabilization.
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PMID:Excavating an active site: the nucleobase specificity of ribonuclease A. 1108 2

The structure of a 23 nt RNA sequence, rGGACCCGGGCUCAACCUGGGUCC, was elucidated using homonuclear NMR, distance geometry and restrained molecular dynamics. This RNA is analogous to residues 612-628 of the Escherichia coli 16S rRNA. The structure of the RNA reveals the presence of a pentaloop closed by a duplex stem in typical A-form conformation. The loop does not form a U-turn motif, as previously predicted. A non-planar A.C.A triple base interaction (hydrogen bonds A13 NH6-C10 O2 and C10 N3-A14 NH6) stabilizing the loop structure is inferred from structure calculations. The CUCAA loop structure is asymmetrical, characterized by a reversal of the phosphodiester backbone at the UC step (hydrogen bond C12 NH4-C10 O2') and 3'-stacking within the CAA segment. Loop base U11 is oriented towards the major groove and the consecutive adenosines on the 3'-end of the loop are well stacked, exposing their reactive functional groups in the minor groove defined by the duplex stem. The solution structure of the loop resembles that seen in the 3.3 A X-ray structure of the entire 30S subunit, where the analogous loop interacts with a ribosomal protein and a receptor RNA helix.
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PMID:NMR structure of a ribosomal RNA hairpin containing a conserved CUCAA pentaloop. 1181 46

Amyloid-beta protein (A beta) deposition in the cerebral vascular walls is one of the key features of Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). A beta(1-40) carrying the 'Dutch' mutation (HCHWA-D A beta(1-40)) induces pronounced degeneration of cultured human brain pericytes. In this study, we aimed to identify inhibitors of A beta-induced toxicity in human brain pericytes. The toxic effect of HCHWA-D A beta(1-40) on human brain pericytes was inhibited by co-incubation with catalase, but not with superoxide dismutase, glutathione or vitamin E analogue Trolox. Catalase interacts with A beta, both in cell cultures and in cell-free assays, and has a prominent effect on the amount and conformational state of A beta binding to the cell surface of human brain pericytes. This activity of catalase is likely based on its ability to bind and slowly degrade A beta and not by its usual capacity to convert hydrogen peroxide. Our data confirm that assembly of A beta at the cell surface of human brain pericytes is a crucial step in A beta-induced cellular degeneration of human brain pericytes. Inhibition of fibril formation at the cell surface could be an important factor in therapy aimed at reducing cerebral amyloid angiopathy.
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PMID:Inhibition of amyloid-beta-induced cell death in human brain pericytes in vitro. 1236 10

Oligomerization of human cystatin C (HCC) leads to amyloid deposits in brain arteries, and this process is greatly accelerated with a naturally occurring L68Q variant. The crystal structures of N-truncated and full-length HCC (cubic form) showed dimer formation via three-dimensional (3D) domain swapping, and this observation has led to the suggestion that an analogous domain-swapping mechanism, but propagated in an open-ended fashion, could be the basis of HCC fibril formation. Here we report that full-length HCC, when crystallized in a new, tetragonal form, dimerizes by swapping the same secondary structure elements but with a very different overall structure generated by the flexibility of the hinge linking the moveable elements. The beta-strands of the beta-cores of the two folding units of the present dimer are roughly parallel, while they formed an angle of about 100 degrees in the previous two structures. The dimers pack around a crystallographic dyad by extending their molecular beta-sheets in an intermolecular context. At the other edge of the molecular beta-sheet, side-chain-side-chain hydrogen bonds propagate the beta-structure in the same direction. In consequence, a supramolecular crystal structure is generated, with all the beta-strands of the domain-swapped dimers being perpendicular to one crystallographic direction. This observation is relevant to amyloid aggregation of HCC, as X-ray diffraction studies of amyloid fibrils show them to have ordered, repeating structure, consistent with the so-called cross-beta structure, in which extended polypeptide chains are perpendicular to the fiber axis and form infinite beta-sheets that are parallel to this axis.
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PMID:3D domain-swapped human cystatin C with amyloidlike intermolecular beta-sheets. 1617 Jul 82

We present the crystal structure of the DNA duplex formed by d(ATATATCT). The crystals contain seven stacked antiparallel duplexes in the asymmetric unit with A.T Hoogsteen base pairs. The terminal CT sequences bend over so that the thymines enter the minor groove and form a hydrogen bond with thymine 2 of the complementary strand in the Hoogsteen duplex. Cytosines occupy extra-helical positions; they contribute to the crystal lattice through various kinds of interactions, including a unique CAA triplet. The presence of thymine in the minor groove apparently contributes to the stability of the DNA duplex in the Hoogsteen conformation. These observations open the way toward finding under what conditions the Hoogsteen duplex may be stabilized in vivo. The present crystal structure also confirms the tendency of A.T-rich oligonucleotides to crystallize as long helical stacks of duplexes.
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PMID:Stabilization by extra-helical thymines of a DNA duplex with Hoogsteen base pairs. 1844 54

We here report on a surface plasmon resonance (SPR) sensor carrying small organic ligands for the detection of single-nucleotide polymorphisms (SNPs). Two kinds of ligands are prepared, both of which have a hydrogen-bond forming site suitable for nucleobase recognition, and have an active amino group for the immobilization to the sensor chip. While the sensor immobilized flavin does not show any useful responses, the sensor based on 3,5-diaminopyrazine shows a highly selective response to thymine over other nucleobases opposite an abasic site in DNA duplexes (5'-GTT GGA GCT GXG GGC GTA GGC-3'/3'-CAA CCT CGA CNC CCG CAT CCG-5', X = AP site, N = target; G, C, A, T). In PBS buffer (pH 6.4, 0.25 M NaCl, at 5 degrees C), the sensor can detect 10 nM of the sample solution, and the SPR signal for thymine is linear in the concentration range from 10 nM to 100 nM. These sensing functions of the present sensor are discussed for the development of SNPs detection chemistry based on DNA-binding small molecules.
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PMID:A surface plasmon resonance sensor based on 3,5-diaminopyrazine with a high selectivity for thymine in AP site-containing DNA duplex. 1877 84

The highly polymorphic microsatellite CAI described for Candida albicans genotyping was found to be located within the RLM1 gene which codes for a transcription factor from the MADS box family that, in Saccharomyces cerevisiae, is known to regulate the expression of genes involved in the cell wall integrity pathway. The aim of this work was to study CAI genetic variability in a wide group of C. albicans isolates and determine the response of genetic variants to cell wall damaging stress agents. One hundred twenty-three C. albicans isolates were genotyped with CAI microsatellite (CAA/G)(n), and 35 alleles were found with repeat units varying from 11 to 49. Alleles with less than 29 repetitions were the most frequent, while the longer ones were underrepresented and had a more complex internal structure. Combinations of RLM1 alleles generated 66 different genotypes. Significant differences (P < 0.05) in the susceptibility patterns to menadione, hydrogen peroxide, SDS, acetic acid, and CFW, stress agents affecting cell integrity, were found between strains harbouring alleles ranging from 17 to 28 repetitions and strains with longer alleles, suggesting that an increased number of repetitive units in the C. albicans RLM1 gene could be related to stress response.
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PMID:Increased number of glutamine repeats in the C-terminal of Candida albicans Rlm1p enhances the resistance to stress agents. 1948 3


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