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)

The gut-associated excretory antigen CAA (circulating anodic antigen) from adult Schistosoma mansoni worms was isolated by immunoaffinity chromatography. Amino acid analysis following alkaline borohydride treatment indicated that CAA is a glycoprotein, O-glycosylated at Thr. The primary structure of the released O-glycan moiety was investigated by one- and two-dimensional, homo- and heteronuclear 1H and 13C NMR spectroscopy. It was found that the major carbohydrate chains have a novel polysaccharide structure, consisting of a branched disaccharide repeating unit containing 2-acetamido-2-deoxy-beta-D- galactopyranose (beta-D-Galp-NAc) and beta-D-glucopyranuronic acid (beta-D-GlcpA). [formula: see text] The major antigenic character of CAA arises from this novel polysaccharide, which was shown to be an absolutely specific diagnostic marker in schistosomiasis. The cross-reactivity of CAA with anti-CCA (circulating cathodic antigen) monoclonal antibodies is caused by the presence of a small amount of O-linked CCA-poly-Lewis x carbohydrate chains on the CAA protein chain.
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PMID:The immunologically reactive part of immunopurified circulating anodic antigen from Schistosoma mansoni is a threonine-linked polysaccharide consisting of --> 6)-(beta-D-GlcpA-(1 --> 3))-beta-D-GalpNAc-(1 --> repeating units. 798 18

With the aim to improve our understanding of the structural basis for protein self-association and aggregation, in particular in relationship to protein refolding and amyloid formation, folding-related processes for human cystatin C have been studied. Using NMR spectroscopy together with chromatographic and electrophoretic methods, a self-association process resulting in dimer formation for protein samples treated with denaturing agents as well as for samples subjected to low pH or high temperature conditions could be studied with amino acid resolution. In all three cases, the dimerization involves properly folded molecules and proceeds via the reactive site of the inhibitor, which leads to complete loss of its biological activity. This dimerization process has potential relevance for amyloid formation by the brain hemorrhage-causing Leu58-Gln variant of cystatin C. The results also indicate that cystatin C dimerization and inactivation may occur in acidified compartments in vivo, which could be relevant for the physiological regulation of cysteine proteinase activity.
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PMID:Folding-related dimerization of human cystatin C. 857 18

The aim of the project has been to elucidate molecular events leading to amyloidosis in Hereditary Cystatin C Amyloid Angiopathy (HCCAA) patients, to enable simple diagnosis of the disease and with the ultimate goal to understand the amyloid formation process in detail, in order to develop inhibitors to the process. At the DNA level, a point mutation segregating with HCCAA was identified in the cystatin C gene on chromosome 20, after basic characterization of cDNA and gene for the wildtype protein. The mutation results in the amino acid substitution Leu-68-Gin (L68Q) and abolishes a recognition site for Alu I. This information was used to design a PCR based assay for simple and rapid mutation detection in DNA from blood samples to allow routine diagnosis of HCCAA. Studies at the protein level, allowed through E. coli expression of wildtype and L68Q mutated cystatin C genes, revealed that both protein variants effectively inhibit the cysteine proteinase cathepsin B (equilibrium constants for dissociation: 0.4 and 0.3 nM, respectively), but differ considerably in their tendency to dimerize and form aggregates. The initial dimerization of L68Q-cystatin C results in complete loss of biological activity and is highly temperature-dependent, with a rise in incubation temperature from 37 to 40 degrees C resulting in a 150% increase in dimerization rate. This result might be of clinical relevance, since medical intervention to abort febrile periods of carriers of the disease trait may reduce the in vivo formation of L68Q-cystatin C aggregates. The three-dimensional structure of normal cystatin C, crystallized in a complex with cathepsin B, was elucidated by X-ray analysis and subsequent refinement of the structure to 3.0 A resolution. Besides pinpointing the cystatin C structures resulting in efficient target enzyme inhibition, the results demonstrated that the Leu-68 residue is buried in the hydrophobic core of the protein. Studies of the three-dimensional solution structure of wildtype cystatin C by NMR spectroscopy revealed that cystatin C dimers can be formed as a result of slight, localized structural changes under conditions preceding complete defolding and denaturation of the protein. Dimers of L68Q-cystatin C are likely similar but are formed at temperatures nearly 30 degrees C lower than needed for the wildtype protein, indicating that the Leu-68-Gln substitution lowers the transition temperature for unfolding. Thus, the results presented suggest that cystatin C provides a system where decreased stability of a mutant protein correlates with its amyloidogenic nature. The NMR results furthermore imply that the hydrophobic proteinase-binding region of cystatin C is directly involved in dimer formation and that compounds designed to interact with this region could serve as inhibitors to the dimerization, and likely also the subsequent amyloid formation process, of cystatin C in HCCAA patients.
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PMID:Molecular basis for amyloidosis related to hereditary brain hemorrhage. 898 67

Human cystatin C undergoes dimerization before unfolding. Dimerization leads to a complete loss of its activity as a cysteine proteinase inhibitor. A similar process of dimerization has been observed in cells, and may be related to the amyloid formation seen for the L68Q variant of the protein. Dimerization is barrier controlled, and no dimer/monomer interconversion can be observed at physiological conditions. As a consequence, very stable, "trapped" dimers can be easily separated from monomers. A study of the structural aspects of cystatin C dimer formation was undertaken using NMR spectroscopy. The monomer/dimer model was verified by (pulse field gradient NMR) self-diffusion molecular mass measurements. Complete backbone resonance assignments and secondary structure determination were obtained for the monomer using data from triple resonance experiments performed on 13C/15N doubly labeled protein. A marked similarity of the cystatin C secondary structure to that of chicken cystatin was observed. Using uniformly and amino-acid-specific 15N-enriched protein, backbone NH signals were assigned for cystatin C in its dimeric state. Comparison of 1H -15N correlation NMR spectra of the monomer and dimer shows that the three-dimensional structure remains unchanged in the dimer and that only local perturbations occur. These are localized to the amino acid residues comprising the cysteine proteinase binding site. Such a mode of dimerization readily explains the complete loss of the inhibitory activity in the dimer. The NMR results also demonstrate that the dimer is symmetric.
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PMID:NMR structural studies of human cystatin C dimers and monomers. 926 58

Recombinant human cystatin C (cC), a cysteine protease inhibitor, contained methionine sulfoxide [Met(O)] residues when expressed in Escherichia coli under aerobic conditions or upon allowing osmotic shock solutions from anaerobically grown cultures to warm to room temperature. Oxidation occurred in the periplasmic space or intracellularly during aerobic expression. Both Met14 and Met41 were subject to oxidation, as determined by NMR spectroscopy and mass spectrometry. Oxidation of Met110 was not observed. Growth under anaerobic conditions and modified purification procedures prevented oxidation. Through the use of a new form of affinity purification, cC was purified to > 99% in one step on E-64-papain-Sepharose (E-64 is 1-[N-[(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]amino]-4-g uanidinobutane), with elution with sodium trichloroacetate. The dissociation equilibrium constants (Kd) for the interaction of unoxidized cC, (Met(O)14)cC, and (Met(O)41)cC with S-(N-ethylsuccinimidyl)papain were experimentally identical: 1.8 (+/-0.2) x 10(-7), 1.6 (+/-0.2) x 10(-7), and 1.4 (+/-0.5) x 10(-7) M, respectively. This implies that the structure of the protease-binding region of mono-oxidized cC's was unchanged. The NMR observation of small, localized conformational changes was consistent with this. (Met(O)14)cC and (Met(O)14,Met(O)41)cC eluted earlier upon analytical affinity chromatography.
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PMID:Affinity purification and elimination of methionine oxidation in recombinant human cystatin C. 932 46

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

In hereditary cystatin C amyloid angiopathy (HCCAA), presence of the Leu68 --> Gln substitution in cystatin C is coupled to a decreased concentration of this major cysteine proteinase inhibitor in cerebrospinal fluid and leads to its amyloid deposition in the brain. We established a high-yield expression system for L68Q cystatin C in Escherichia coli resulting in inclusion body accumulation at a level of 40% of the total cellular protein. Refolding of protein from purified inclusion bodies yielded a pure, almost completely monomeric and active inhibitor. CD and NMR spectroscopy demonstrated that so produced L68Q cystatin C is folded, conformationally homogeneous, and structurally very similar to wild-type cystatin C. Incubation at pH 7.0-5.5 caused the cystatin C variant to dimerize rapidly. The molecular form present at pH 6.0 displayed a slightly increased amount of hydrophobic parts on the surface as measured by 1-anilinonaphthalene-8-sulfonic acid (ANS) binding. NMR results showed that the dimer has a structure similar to that of the wild-type cystatin C dimer formed as a result of slight denaturation. Under more acidic conditions, at pH 4.5, another stable unfolding intermediate of L68Q cystatin C was identified. This molecular form exists in a monomeric state, is characterized by changes in secondary structure according to far UV CD spectroscopy, and shows an altered ANS binding resembling that of a molten globule state. The acidic pH also caused an almost complete monomerization of preformed dimers. The state of denaturation of L68Q cystatin C in vivo is thus a critical factor for the concentration of active cysteine proteinase inhibitor in cerebrospinal fluid and likely also for the development of amyloidosis, in HCCAA patients.
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PMID:Two stable unfolding intermediates of the disease-causing L68Q variant of human cystatin C. 986 Aug 45

In DNA duplexes, pyrimidine-purine steps are believed to be flexible or conformationally unstable. Indeed, several DNA crystal structures exhibit a multitude of conformations for CpA*TpG steps. The question arises of whether this structural flexibility is accompanied by dynamical flexibility, i.e., a question pertaining to the energy barrier between conformations. Except for TpA steps, slow motions on the microsecond-to-millisecond time scale have not been detected in duplexes until now. In the present study, such slow motion was investigated by 1H, 13C, and 15N NMR relaxation measurements on a DNA decamer d(CATTTGCATC)*d(GATGCAAATG). The DNA decamer was enriched with 15% 13C and 98% 15N isotopes for each adenosine and guanosine residue. Three lines of evidence support the notion of slow motion in the CAA*TTG moiety. Analysis of (15)N relaxation showed that the order parameter, S2, of guanosine imino NH groups was about 0.8, similar to that of CH groups for this oligomer. The strong temperature dependence of guanosine NH S2 in the CAA*TTG sequence indicated the presence of a large-amplitude motion. Signals of adenosine H8 protons in the CAA*TTG sequence were broadened in 2D 1H NOESY spectra, which also suggested the existence of slow motion. As well as being smaller than for other adenine residues, the 1H T2 values exhibited a magnetic field strength dependence for all adenosine H8 signals in the ATTTG*CAAAT region, suggesting slow motions more pronounced at the first adenosine in the CAA*TTG sequence but extending over the CAAAT*ATTTG region. This phenomenon was further examined by the pulse field strength dependence of the 1H, 13C, and 15N T1rho values. 1H and 13C T1rho values showed a pulse field strength dependence, but 15N T1rho did not. Assuming a two-site exchange process, an exchange time constant of 20-300 micros was estimated for the first adenosine in the CAA sequence. The exact nature of this motion remains unknown.
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PMID:Slow motion in the CAA*TTG sequence of a DNA decamer duplex studied by NMR. 1140 71

Benzo[a]pyrene (BP), a prototype polycyclic aromatic hydrocarbon (PAH), can be metabolically activated to the enantiomeric benzo[a]pyrene diol epoxides (BPDEs), (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and the (-)-(7S,8R,9R,10S) enantiomer. These can react with adenine residues in DNA, to produce the stereoisomeric 10S (+)- and 10R (-)-trans-anti-[BP]-N(6)-dA adducts. High-resolution NMR solution studies indicate that in DNA duplexes the 10R (-) adduct is intercalated on the 5'-side of the modified adenine, while the 10S (+) adduct is disordered, exhibits multiple adduct conformations, and is positioned on the 3'-side of the modified adenine. Duplexes containing the 10S (+) adduct positioned at A within codon 61 of the human N-ras sequence CAA are thermodynamically less stable and more easily excised by human DNA repair enzymes than those containing the 10R (-) adduct. However, the molecular origins of these differences are not understood and represent a fascinating opportunity for elucidating structure-function relationships. We have carried out a computational investigation to uncover the structural and thermodynamic origins of these effects in the 11-mer duplex sequence d(CGGACAAGAAG).d(CTTCTTGTCCG) by performing a 2-ns molecular dynamics simulation using NMR solution structures as the basis for the starting models. Then, we applied the MM-PBSA (molecular mechanics Poisson-Boltzmann surface area) method to compute free energy differences between the stereoisomeric adducts. The 10R (-) isomer is more stable by approximately 13 kcal/mol, of which approximately 10 kcal/mol is enthalpic, which agrees quite well with their observed differences in thermodynamic stability. The lower stability of the 10S (+) adduct is due to diminished stacking by the BP moiety in the intercalation pocket, more helix unwinding, and a diminished quality of Watson-Crick base pairing. The latter stems from conformational heterogeneity involving a syn-anti equilibrium of the glycosidic bond in the modified adenine residue. The lower stability and conformational heterogeneity of the 10S (+) adduct may play a role in its enhanced susceptibility to nucleotide excision repair.
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PMID:Stereochemical, structural, and thermodynamic origins of stability differences between stereoisomeric benzo[a]pyrene diol epoxide deoxyadenosine adducts in a DNA mutational hot spot sequence. 1145 84

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


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