35 results on '"Boteva, R"'
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2. Fluorescence analysis of the Hansenula polymorpha peroxisomal targeting signal-1 receptor, Pex5p
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Boteva, R., Koek, A., Visser, N.V., Krieger, E., Zlateva, T., Veenhuis, M., and Klei, I. van der
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Cellular energy metabolism [UMCN 5.3] - Abstract
Item does not contain fulltext
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- 2003
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3. Conformational Transitions Accompanying Oligomerization of Yeast Alcohol Oxidase, a Peroxisomal Flavoenzyme
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Boteva, R, Visser, AJWG, Filippi, B, Vriend, G, Veenhuis, M, van der Klei, IJ, Visser, Antonie J.W.G., Groningen Biomolecular Sciences and Biotechnology, and Molecular Cell Biology
- Subjects
LIPOAMIDE DEHYDROGENASE ,Models, Molecular ,FLAVIN FLUORESCENCE ,Protein Conformation ,Stereochemistry ,Biochemie ,Fluorescence Polarization ,MAXIMUM-ENTROPY METHOD ,Microbodies ,Biochemistry ,Pichia ,Protein Structure, Secondary ,Cofactor ,Pichia pastoris ,SACCHAROMYCES-CEREVISIAE ,chemistry.chemical_compound ,GLUTATHIONE-REDUCTASE ,Aromatic amino acids ,Life Science ,PRION PROTEIN ,Glucose oxidase ,Protein secondary structure ,chemistry.chemical_classification ,SECONDARY STRUCTURE ,Sequence Homology, Amino Acid ,biology ,IMPORT ,Circular Dichroism ,Tryptophan ,Peroxisome ,biology.organism_classification ,Alcohol oxidase ,Alcohol Oxidoreductases ,PICHIA-PASTORIS ,Spectrometry, Fluorescence ,Enzyme ,chemistry ,Flavin-Adenine Dinucleotide ,biology.protein ,HANSENULA-POLYMORPHA - Abstract
Alcohol oxidase (AO) is a homo-octameric flavoenzyme which catalyzes methanol oxidation in methylotrophic yeasts. AO protein is synthesized in the cytosol and subsequently sorted to peroxisomes where the active enzyme is formed. To gain further insight in the molecular mechanisms involved in AO activation, we studied spectroscopically native AO from Hansenula polymorpha and Pichia pastoris and three putative assembly intermediates. Fluorescence studies revealed that both Trp and FAD are suitable intramolecular markers of the conformation and oligomeric state of AO. A direct relationship between dissociation of AO octamers and increase in Trp fluorescence quantum yield and average fluorescence lifetime was found. The time-resolved fluorescence of the FAD cofactor showed a rapid decay component which reflects dynamic quenching due to the presence of aromatic amino acids in the FAD-binding pocket. The analysis of FAD fluorescence lifetime profiles showed a remarkable resemblance of pattern for purified AO and AO present in intact yeast cells. Native AO contains a high content of ordered secondary structure which was reduced upon FAD-removal. Dissociation of octamers into monomers resulted in a conversion of beta-sheets into alpha-helices. Our results are explained in relation to a 3D model of AO, which was built based on the crystallographic data of the homologous enzyme glucose oxidase from Aspergillus niger. The implications of our results for the current model of the in vivo AO assembly pathway are discussed.
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- 1999
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4. The Biliprotein C-Phycocyanin Modulates the DNA Damage Response in Lymphocytes from Nuclear Power Plant Workers
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Stankova, K., Ivanova, K., Nikolov, V., Minkova, K., Gigova, L., Georgieva, R., Boteva, R., Stankova, K., Ivanova, K., Nikolov, V., Minkova, K., Gigova, L., Georgieva, R., and Boteva, R.
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- 2011
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5. Dissociation equilibrium of human recombinant interferon gamma
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Boteva, R., Zlateva, T., Dorovska-Taran, V., Visser, A.J.W.G., Tsanev, R., and Salvato, B.
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Life Science ,Biochemie ,Biochemistry - Published
- 1996
6. Bystander responses in low dose irradiated cells treated with plasma from gamma irradiated blood
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Acheva, A, primary, Georgieva, R, additional, Rupova, I, additional, Boteva, R, additional, and Lyng, F, additional
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- 2008
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7. Fluorescence technique for comparative studies of substrate-binding subsites in serine proteinases. Application to subtilisins
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Genov, N C and Boteva, R N
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A fluorescence technique for comparative studies of substrate-binding subsites in serine proteinases is described. It consists of: selective labelling of the corresponding subsites with a fluorescent group by using N alpha-dansyl(5-dimethylaminonaphthalene-1-sulphonyl)ated peptide chloromethanes containing different numbers of amino acid residues, and probing the immediate environment of the subsites by quenching experiments using ionic and neutral quenchers. Intramolecular distances between the subsites and particular chromophores can be also determined. The technique is of general applicability to all serine proteinases. The above mentioned approach was applied to two proteinases: subtilisin Novo and mesentericopeptidase. It was concluded that the substrate-binding site of mesentericopeptidase is considerably more polar than that of subtilisin Novo. Intramolecular distances between the labelled subsites and tryptophan residues in the two proteinases were determined.
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- 1986
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8. Intramolecular distances between tryptophan residues and the active-site serine residue in alkaline bacterial proteinases as measured by fluorescence energy-transfer studies
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Genov, N C, Shopova, M, Boteva, R, Ricchelli, F, and Jori, G
- Abstract
Singlet-singlet energy transfer from the tryptophan residues to an active-site-serine-bound 5-dimethylaminonaphthalene-1-sulphonyl group was investigated in four subtilisins. The transfer distances for subtilisin Novo and mesentericopeptidase are 1.93 +/- 0.20 nm (19.3 +/- 2.0 A) and 1.81 +/- 0.20 nm (18.1 +/- 2.0 A) respectively. The positions of the indole groups in the three-dimensional structures of the two pairs of proteinases, namely subtilisin Novo and mesentericopeptidase on the one hand and subtilisins Carlsberg and DY on the other, are essentially identical.
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- 1983
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9. Chemical, photochemical and spectroscopic characterization of an alkaline proteinase from Bacillus subtilis variant DY
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Genov, N, Shopova, M, Boteva, R, Jori, G, and Ricchelli, F
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Circular-dichroism and fluorescence studies indicate that the 5-dimethylaminonaphthalene-1-sulphonyl and phenylmethanesulphonyl derivatives of subtilisin DY have three-dimensional structure closely similar to that of native enzyme. The single tryptophan residue is largely accessible to the aqueous solvent, and is not directly involved in the enzyme-substrate interactions, since its photochemical modification causes only a partial inhibition of the enzyme activity. It appears very likely that the location of the single tryptophan residue in the three-dimensional structure of subtilisin DY is similar to that of the single tryptophan residue in subtilisin Carlsberg. Fluorescence-quenching experiments further indicate that the 14 tyrosine residues are also largely accessible to the aqueous solvent, and probably interact with hydrated peptide carbonyl groups. The charge environment for tryptophan and tyrosine residues in subtilisin DY, as deduced by quenching experiments with ionic species, is also discussed. In general, subtilisin DY displays strong similarities to subtilisin Carlsberg, as suggested by a comparative analysis of the amino acid composition and fluorescence properties.
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- 1982
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10. Effects of pH and urea on the conformational properties of subtilisin DY
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Ricchelli, F, Jori, G, Filippi, B, Boteva, R, Shopova, M, and Genov, N
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Subtilisin DY is very resistant to the denaturing action of urea: the conformational properties are not affected up to 4.5 M-urea, and even in the presence of 8 M-urea there is only a slow loss of ordered structure and caseinolytic activity. C.d. and fluorescence-emission studies also show that this proteinase is stable in the 5.5-10.0 pH range, whereas below pH 5.5 a sharp denaturation occurs that is complete at pH 4.5. Protein denaturation leads to a change of the emission quantum yield; in particular, in the native protein, indole fluorescence is quenched by some amino groups. Moreover, subtilisin DY possesses two classes of tyrosine residues: one class of exposed residues titrates normally, with pKapp. = 10.24, whereas one class of partially buried or hydrogen-bonded residues ionizes with pKapp. = 11.58. In general, such conformational properties resemble those of other subtilisins. However, some differences occur: e.g., subtilisin DY is less stable at acidic pH values and its tyrosine residues are more accessible to the solvent. Such differences are probably due to small variations of the three-dimensional structure; e.g., subtilisin DY has a slightly lower alpha-helix content.
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- 1982
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11. THE NATURAL PROTEASOME INHIBITOR CELASTROL CAN ACCELERATE DNA REPAIR IN HUMAN LYMPHOCYTES
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Gergana Savova, Stankova, K., and Boteva, R.
12. Radiation can stimulate secretion of TNFα by human mononuclear cells treated with LPS
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Aneva, N., Zaharieva, E., Gergana Savova, Stankova, K., and Boteva, R.
13. Molecular markers for assessment of radiation-induced oxidative stress in occupationally exposed subjects
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Stankova, K., Zaharieva, E., Nevena Aneva, Katsarska, O., Savova, G., and Boteva, R.
14. Spectroscopic studies on the conformational stability of subtilisins in the presence of urea
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Shopova, M., primary, Boteva, R., additional, and Genov, N., additional
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- 1984
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15. Fluorescence titration of native and PMS-subtilisin Carlsberg
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Genov, N., primary, Boteva, R., additional, and Shopova, M., additional
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- 1984
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16. Labeling of tarantula hemocyanin (Eurypelma californicum) with dansyl-type fluorescent tags: identification of the dye-binding site by fluorescence spectroscopy
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Boteva, R., Ricchelli, F., Sartor, G., and Decker, H.
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- 1993
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17. Fluorescence properties of hemocyanin from tarantula (Eurypelma californicum): a comparison between the whole molecule and isolated subunits
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Boteva, R., Ricchelli, F., Sartor, G., and Decker, H.
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- 1993
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18. Inflammatory profile dysregulation in nuclear workers occupationally exposed to low-dose gamma radiation.
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Aneva N, Zaharieva E, Katsarska O, Savova G, Stankova K, Djounova J, and Boteva R
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- Adult, Antioxidants metabolism, Case-Control Studies, Cytokines blood, Dose-Response Relationship, Radiation, Humans, Hypertension blood, Linear Models, Male, Middle Aged, Gamma Rays, Inflammation pathology, Nuclear Power Plants, Occupational Exposure analysis, Radiation Exposure analysis
- Abstract
Chronic inflammation is a common denominator linking a wide range of health conditions, including tissue response to radiation exposure. This pilot study investigates whether inflammatory cytokines-interleukins IL-6, -8, -10, monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor α (TNFα)-can be used as early biomarkers of radiation-induced adverse health effects in occupationally exposed individuals. The study included 33 workers externally exposed to gamma radiation from the nuclear industry with cumulated doses from 0.11 to 190 mSv and 42 non-exposed controls of comparable age and socio-economic status. IL-6, IL-8, MCP-1, TNFα and IL-10 were analyzed by enzyme-linked assay (ELISA) in blood plasma samples. Total antioxidant status (TAS) of blood plasma was determined by a colorimetric assay. The radiation-exposed and control groups measured significantly different levels of MCP-1, TNFα and IL-10. Seventy-five percent of radiation workers had either high MCP-1 levels or low IL-10 levels and 30% had all three cytokines dysregulated. Approximately 50% of workers showed upregulated antioxidant status, which appeared to compensate the pro-inflammatory cytokine shift in these individuals. In contrast, only 2% of the control subjects were found to have three dysregulated cytokines, and all of them measured within the normal TAS range. The present study may represent an important step towards the establishment of a reliable set of biomarkers for health-risk estimation in population cohorts exposed to low radiation doses., (© The Author(s) 2019. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)
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- 2019
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19. The soluble receptor ST2 is positively associated with occupational exposure to radiation.
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Katsarska O, Zaharieva E, Aneva N, Savova G, Stankova K, and Boteva R
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- Absorption, Radiation, Adult, Biomarkers blood, Cells, Cultured, Dose-Response Relationship, Radiation, Female, Humans, Interleukin-1 Receptor-Like 1 Protein, Male, Middle Aged, Radiation Dosage, Lymphocytes metabolism, Lymphocytes radiation effects, Occupational Exposure analysis, Radiation Exposure analysis, Reactive Oxygen Species blood, Receptors, Cell Surface blood
- Abstract
Purpose Radiation exposure, besides the risk of cancer, may also increase the risk of non-cancer diseases, including cardiovascular disease (CVD). This study investigates whether the soluble form of the ST2 receptor (sST2), an emerging prognostic marker in patients with CVD, can be used to monitor the CVD risk in individuals occupationally exposed to radiation. Materials and methods sST2 in blood plasma from 69 individuals, 45 workers from the nuclear industry and 24 controls, was analyzed using enzyme-linked assay (ELISA). Total antioxidant status (TAS) of blood plasma and levels of reactive oxygen species (ROS) in lymphocytes were determined by colorimetric and fluorescence assays. Results The data suggest a 5-fold increase in the number of subjects with sST2 levels above the clinical threshold and a 10-fold increase in the number of subjects with TAS levels outside the reference range in the exposed group when compared to the group of non-exposed individuals. The strongest up-regulation of TAS was measured in the group of younger workers with cumulative doses not exceeding 50 mSv. Conclusion The present study may represent an initial step towards the establishment of sST2 as a biomarker for CVD risk estimation in the context of radiation exposure.
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- 2016
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20. HSP90 Inhibitor Geldanamycin as a Radiation Response Modificator in Human Blood Cells.
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Stankova K, Savova G, Nikolov V, and Boteva R
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Heat shock protein 90 (Hsp90) is a highly conserved molecular chaperone, involved in the folding, assembly, stabilization and activation of numerous proteins with unrelated amino acid sequences and functions. Geldanamycin (GA), a natural benzoquinone, can inhibit the chaperone activity of Hsp90. It has been shown that GA can produce superoxide anions and increase the intracellular oxidative stress, which, in addition to the direct inhibition of Hsp90, might also contribute to the modifying effects of the inhibitor on the early response in human mononuclear cells exposed to ionizing radiation. The present study shows that GA antagonizes the radiation-induced suppression on MnSOD and catalase, key enzymes of the radical scavenging systems. By significantly up-regulating catalase levels over the entire range of doses from 0.5 to 4 Gy, the inhibitor of Hsp90 exerted adaptive protection and modified the early radiation response of the human blood cells.
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- 2015
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21. Proteasome inhibition protects human peripheral blood mononuclear cells from radiation-induced oxidative stress.
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Stankova K, Ivanova K, Nikolov V, Aneva N, Georgieva R, and Boteva R
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- Cells, Cultured, Dose-Response Relationship, Drug, Dose-Response Relationship, Radiation, Humans, Leukocytes, Mononuclear radiation effects, Radiation Dosage, Radiation-Protective Agents pharmacology, Leukocytes, Mononuclear drug effects, Leukocytes, Mononuclear physiology, Oxidative Stress physiology, Oxidative Stress radiation effects, Proteasome Endopeptidase Complex metabolism, Proteasome Inhibitors pharmacology
- Abstract
Purpose: The study aimed to analyze the impact of the proteasome inhibitors MG132 (N-carbobenzyoxyl-L-leucyl-L-leucyl-L-leucinal), lactacystin and celastrol on manganese superoxide dismutase (MnSOD), catalase and glutathione-S-transferase-π (GST-π), and on the heat shock protein 70 (Hsp70) in human peripheral blood mononuclear cells (PBMC), exposed to ionizing radiation., Materials and Methods: Changes in protein levels were analyzed by Western blot. Cellular viability, proteasome activity, level of oxidative stress and apoptosis were determined by standard colorimetric and fluorescence assays., Results: MG132 and lactacystin induced an increase in the intracellular levels of Hsp70. MnSOD was up-regulated by MG132 and celastrol, and GST-π was up-regulated by MG132 and lactacystin. Notably, the proteasome inhibitors significantly modified the protein levels in the irradiated cells and dramatically reduced the intracellular pool of oxidative species. The combined effect of radiation and proteasome inhibition was a dose-dependent up-regulation of the antioxidant enzymes and Hsp70., Conclusions: All three proteasome inhibitors showed antioxidant effects in PBMC and up-regulated the antioxidant enzymes MnSOD, catalase and GST-π and the stress protein Hsp70, modifying the early radiation response, and conferring protection against the effects of ionizing radiation.
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- 2013
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22. Conformational transitions of proteins engaged in DNA double-strand break repair, analysed by tryptophan fluorescence emission and FRET.
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Stankova K, Ivanova K, Mladenov E, Rosidi B, Sharma A, Boteva R, and Iliakis G
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- Base Sequence, DNA Primers, Fluorescence Resonance Energy Transfer, Humans, Protein Binding, Protein Conformation, DNA Damage, DNA Repair, Proteins chemistry, Tryptophan chemistry
- Abstract
We analysed protein-DNA and protein-protein interactions relevant to the repair of DNA DSBs (double-strand breaks) by NHEJ (non-homologous end-joining). Conformational transitions in mammalian DNA ligases III (LigIII) and IV (LigIV), as well as in PARP-1 [poly(ADP-ribose) polymerase-1], were analysed upon binding to double-stranded DNA by changes in tryptophan emission and FRET (Förster resonance energy transfer) from tryptophan to DNA-conjugated Alexa Fluor® 532. For LigIII, two non-equivalent high- and low-affinity DNA-binding sites are detected interacting sequentially with DNA. PARP-1 displays a single high-affinity DNA-binding site and can displace bound DNA fragments from the low-affinity site of LigIII, consistent with its mediator role in LigIII-DNA interactions. For the LX [LigIV-XRCC4 (X-ray cross-complementation group 4)] complex, a single DNA-binding site is detected. Binding of Ku to DNA was accompanied by conformational changes in the protein and intermolecular FRET from dansyl chromophores of the labelled Ku to the Alexa Fluor® chromophores of Alexa Fluor® 532-conjugated DNA. The average distance of 5.7 nm calculated from FRET data is consistent with a location of Ku at the very end of the DNA molecule. Binding of LX to Ku-DNA complexes is associated with conformational changes in Ku, translocating the protein further towards the DNA ends. The protein-protein and protein-DNA interactions detected and analysed generate a framework for the characterization of molecular interactions fundamental to the function of NHEJ pathways in higher eukaryotes.
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- 2012
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23. Chaperonin TRiC promotes the assembly of polyQ expansion proteins into nontoxic oligomers.
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Behrends C, Langer CA, Boteva R, Böttcher UM, Stemp MJ, Schaffar G, Rao BV, Giese A, Kretzschmar H, Siegers K, and Hartl FU
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- Chaperonins metabolism, DNA Repeat Expansion, Green Fluorescent Proteins analysis, HSP40 Heat-Shock Proteins metabolism, HSP70 Heat-Shock Proteins metabolism, Peptides metabolism, Protein Folding, Recombinant Fusion Proteins analysis, Repetitive Sequences, Amino Acid, Saccharomyces cerevisiae cytology, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Chaperonins physiology, Peptides chemistry
- Abstract
Aberrant folding and fibrillar aggregation by polyglutamine (polyQ) expansion proteins are associated with cytotoxicity in Huntington's disease and other neurodegenerative disorders. Hsp70 chaperones have an inhibitory effect on fibril formation and can alleviate polyQ cytotoxicity. Here we show that the cytosolic chaperonin, TRiC, functions synergistically with Hsp70 in this process and is limiting in suppressing polyQ toxicity in a yeast model. In vitro reconstitution experiments revealed that TRiC, in cooperation with the Hsp70 system, promotes the assembly of polyQ-expanded fragments of huntingtin (Htt) into soluble oligomers of approximately 500 kDa. Similar oligomers were observed in yeast cells upon TRiC overexpression and were found to be benign, in contrast to conformationally distinct Htt oligomers of approximately 200 kDa, which accumulated at normal TRiC levels and correlated with inhibition of cell growth. We suggest that TRiC cooperates with the Hsp70 system as a key component in the cellular defense against amyloid-like protein misfolding.
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- 2006
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24. Proteolytic cleavage of polyglutamine-expanded ataxin-3 is critical for aggregation and sequestration of non-expanded ataxin-3.
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Haacke A, Broadley SA, Boteva R, Tzvetkov N, Hartl FU, and Breuer P
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- Amino Acid Sequence genetics, Animals, Ataxin-3, Cell Line, Cell Line, Tumor, Humans, Inclusion Bodies genetics, Inclusion Bodies pathology, Machado-Joseph Disease genetics, Machado-Joseph Disease pathology, Mice, Nerve Tissue Proteins genetics, Neuroblastoma genetics, Neuroblastoma metabolism, Neuroblastoma pathology, Neurons pathology, Nuclear Proteins genetics, Protein Structure, Tertiary genetics, Repressor Proteins genetics, Sequence Deletion genetics, Transcription Factors, Inclusion Bodies metabolism, Machado-Joseph Disease metabolism, Nerve Tissue Proteins metabolism, Neurons metabolism, Nuclear Proteins metabolism, Protein Processing, Post-Translational, Repressor Proteins metabolism
- Abstract
Spinocerebellar ataxia type 3 (SCA3), like other polyglutamine (polyQ) diseases, is characterized by the formation of intraneuronal inclusions, but the mechanism underlying their formation is poorly understood. Here, we tested the "toxic fragment hypothesis", which predicts that proteolytic production of polyQ-containing fragments from the full-length disease protein initiates the aggregation process associated with inclusion formation and cellular dysfunction. We demonstrate that the removal of the N-terminus of polyQ-expanded ataxin-3 (AT3) is required for aggregation in vitro and in vivo. Consistently, proteolytic cleavage of full-length, pathogenic AT3 initiates the formation of sodium dodecylsulfate-resistant aggregates in neuroblastoma cells. Although full-length AT3 does not readily aggregate on its own, it is susceptible to co-aggregation with polyQ-expanded AT3 fragments. Interestingly, interaction with soluble polyQ-elongated fragments causes a structural distortion of wild-type AT3 prior to the formation of stable co-aggregates. These results establish the critical role of C-terminal, proteolytic fragments of AT3 in the molecular pathomechanism of SCA3, in strong support of the toxic fragment hypothesis.
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- 2006
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25. Cysteine-free glutathione-S-tranferase as a tool for thiol-specific labeling of proteins.
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Tzvetkov N, Breuer P, and Boteva R
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- Amino Acid Substitution, Chromatography, Gel, Circular Dichroism, Escherichia coli genetics, Glutathione metabolism, Glutathione Transferase genetics, Glutathione Transferase isolation & purification, Plasmids, Point Mutation, Polymerase Chain Reaction, Sequence Analysis, DNA, Serine metabolism, Spectrometry, Fluorescence, Cysteine chemistry, Glutathione Transferase metabolism, Proteins metabolism, Sulfhydryl Compounds metabolism
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- 2006
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26. The highly abundant protein Ag-lbp55 from Ascaridia galli represents a novel type of lipid-binding proteins.
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Jordanova R, Radoslavov G, Fischer P, Torda A, Lottspeich F, Boteva R, Walter RD, Bankov I, and Liebau E
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- Amino Acid Sequence, Animals, Base Sequence, Blotting, Western, Caprylates chemistry, Carrier Proteins chemistry, Circular Dichroism, DNA Primers chemistry, DNA, Complementary metabolism, Electrophoresis, Gel, Two-Dimensional, Fatty Acid-Binding Proteins metabolism, Immunohistochemistry, Ligands, Lipid Metabolism, Models, Biological, Molecular Sequence Data, Oleic Acid chemistry, Oligonucleotides chemistry, Palmitic Acid chemistry, Peptides chemistry, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, Signal Transduction, Transcription, Genetic, Ascaridia physiology, Fatty Acid-Binding Proteins chemistry, Lipids chemistry
- Abstract
Lipid-binding proteins exhibit important functions in lipid transport, cellular signaling, gene transcription, and cytoprotection. Their functional analogues in nematodes are nematode polyprotein allergens/antigens and fatty acid and retinoid-binding proteins. This work describes a novel 55-kDa protein, Ag-lbp55, purified from the parasitic nematode Ascaridia galli. By direct N-terminal sequencing, a partial amino acid sequence was obtained that allowed the design of oligonucleotide primers to obtain the full-length cDNA sequence. Sequence analysis revealed the presence of an N-terminal signal peptide of 25 amino acid residues and a FAR domain at the C terminus. Data base searches showed almost no significant homologies to other described proteins. The secondary structure of Ag-lbp55 was predominantly alpha-helical (65%) as shown by CD spectroscopy. It was found to bind with high affinity fatty acids (caprylic, oleic, and palmitic acid) and their fluorescent analogue dansylaminoundecanic acid. Immunolocalization showed that Ag-lbp55 is a highly abundant protein, mainly distributed in the inner hypodermis and extracellularly in the pseudocoelomatic fluid. A similar staining pattern was observed in other pathogenic nematodes, indicating the existence of similar proteins in these species.
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- 2005
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27. Conformational and functional analysis of the lipid binding protein Ag-NPA-1 from the parasitic nematode Ascaridia galli.
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Jordanova R, Radoslavov G, Fischer P, Liebau E, Walter RD, Bankov I, and Boteva R
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- Animals, Circular Dichroism, Fluorescence Resonance Energy Transfer, Helminth Proteins metabolism, Immunohistochemistry, Protein Conformation, Spectrometry, Fluorescence, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Ascaridia chemistry, Helminth Proteins chemistry, Lipid Metabolism
- Abstract
Ag-NPA-1 (AgFABP), a 15 kDa lipid binding protein (LBP) from Ascaridia galli, is a member of the nematode polyprotein allergen/antigen (NPA) family. Spectroscopic analysis shows that Ag-NPA-1 is a highly ordered, alpha-helical protein and that ligand binding slightly increases the ordered secondary structure content. The conserved, single Trp residue (Trp17) and three Tyr residues determine the fluorescence properties of Ag-NPA-1. Analysis of the efficiency of the energy transfer between these chromophores shows a high degree of Tyr-Trp dipole-dipole coupling. Binding of fatty acids and retinol was accompanied by enhancement of the Trp emission, which allowed calculation of the affinity constants of the binary complexes. The distance between the single Trp of Ag-NPA-1 and the fluorescent fatty acid analogue 11-[(5-dimethylaminonaphthalene-1- sulfonyl)amino]undecanoic acid (DAUDA) from the protein binding site is 1.41 nm as estimated by fluorescence resonance energy transfer. A chemical modification of the Cys residues of Ag-NPA-1 (Cys66 and Cys122) with the thiol reactive probes 5-({[(2-iodoacetyl)amino]ethyl}amino) naphthalene-1-sulfonic acid (IAEDANS) and N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD), followed by MALDI-TOF analysis showed that only Cys66 was labeled. The observed similar affinities for fatty acids of the modified and native Ag-NPA-1 suggest that Cys66 is not a part of the protein binding pocket but is located close to it. Ag-NPA-1 is one of the most abundant proteins in A. galli and it is distributed extracellularly mainly as shown by immunohistology and immunogold electron microscopy. This suggests that Ag-NPA-1 plays an important role in the transport of fatty acids and retinoids.
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- 2005
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28. Cellular toxicity of polyglutamine expansion proteins: mechanism of transcription factor deactivation.
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Schaffar G, Breuer P, Boteva R, Behrends C, Tzvetkov N, Strippel N, Sakahira H, Siegers K, Hayer-Hartl M, and Hartl FU
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- Animals, CREB-Binding Protein, Cell Line, Tumor, Cell Nucleus metabolism, Exons, HSP70 Heat-Shock Proteins genetics, HSP70 Heat-Shock Proteins metabolism, Huntingtin Protein, Macromolecular Substances, Mice, Molecular Chaperones genetics, Molecular Chaperones metabolism, Mutation genetics, Nerve Tissue Proteins genetics, Nerve Tissue Proteins metabolism, Neurodegenerative Diseases etiology, Neurodegenerative Diseases genetics, Neurodegenerative Diseases metabolism, Nuclear Proteins antagonists & inhibitors, Nuclear Proteins genetics, Nuclear Proteins metabolism, Peptides metabolism, Protein Conformation, Protein Folding, Saccharomyces cerevisiae, TATA-Box Binding Protein antagonists & inhibitors, TATA-Box Binding Protein genetics, TATA-Box Binding Protein metabolism, Trans-Activators antagonists & inhibitors, Trans-Activators genetics, Trans-Activators metabolism, Transcription Factors genetics, Transcription Factors metabolism, Nerve Tissue Proteins toxicity, Nuclear Proteins toxicity, Peptides genetics, Transcription Factors antagonists & inhibitors, Trinucleotide Repeat Expansion genetics
- Abstract
The expression of polyglutamine-expanded mutant proteins in Huntington's disease and other neurodegenerative disorders is associated with the formation of intraneural inclusions. These aggregates could potentially cause cellular toxicity by sequestering essential proteins possessing normal polyQ repeats, including the transcription factors TBP and CBP. We show, in vitro and in cells, that monomers or small soluble oligomers of huntingtin exon1 accumulate in the nucleus and inhibit the function of TBP in a polyQ-dependent manner. FRET experiments indicate that these toxic forms are generated through a conformational rearrangement in huntingtin. Interaction of toxic huntingtin with the benign polyQ repeat of TBP structurally destabilizes the transcription factor, independent of the formation of insoluble coaggregates. Hsp70/Hsp40 chaperones interfere with the conformational change in mutant huntingtin and inhibit the deactivation of TBP. These results outline a molecular mechanism of cellular toxicity in polyQ disease and can explain the beneficial effects of molecular chaperones.
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- 2004
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29. Deflavination of flavo-oxidases by nucleophilic reagents.
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Zlateva T, Boteva R, Filippi B, Veenhuis M, and van der Klei IJ
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- Alcohol Oxidoreductases chemistry, Circular Dichroism, Cyanates, Cyanides, D-Amino-Acid Oxidase chemistry, Glucose Oxidase chemistry, Indicators and Reagents, Spectrometry, Fluorescence, Spectrophotometry, Thiocyanates, Flavin-Adenine Dinucleotide chemistry, Flavoproteins chemistry, Oxidoreductases chemistry
- Abstract
Using spectroscopic techniques we studied the effect of the nucleophilic reagents cyanide, cyanate and thiocyanate on three flavo-oxidases namely alcohol oxidase (AO), glucose oxidase (GOX) and D-amino acid oxidase (DAOX). All three ions, added at concentrations in the mM range, caused release of the flavin adenine dinucleotide (FAD) co-factors from the enzyme molecules. In the case of AO this was accompanied by significant conformational perturbations, which was not observed for GOX and DAOX. As suggested from fluorescence, absorption and circular dichroism spectral changes at least one phenolic hydroxyl group became ionized upon FAD release from AO and a new class of Trp residues, fluorescent only in apo-AO protein, was demasked.
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- 2001
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30. Dissociation equilibrium of human recombinant interferon gamma.
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Boteva R, Zlateva T, Dorovska-Taran V, Visser AJ, Tsanev R, and Salvato B
- Subjects
- Biopolymers chemistry, Fluorescence Polarization, Humans, Mathematics, Protein Conformation, Recombinant Proteins, Spectrometry, Fluorescence, Thermodynamics, Tryptophan chemistry, Tyrosine chemistry, Interferon-gamma chemistry
- Abstract
The biologically active form of interferon gamma is a dimer composed of two noncovalently bound identical polypeptide chains of 17 kDa each. In this study, it was found that dissociation of the dimer into monomers significantly reduced the fluorescence quantum yield and the efficiency of the intermolecular Tyr to Trp radiationless energy transfer. The same process caused significant changes in the fluorescence decay and in the fluorescence anisotropy decay. The kinetic and thermodynamic parameters of the dimer-monomer equilibrium were determined by fluorescence measurements at different temperatures and by a theoretical mathematical model. Dissociation of the dimers into monomers was an endothermic process and was favored by concentrations of the protein lower than 1 microM and by increasing the temperature. It was accompanied by formation of aggregates, a slow and partially reversible process leading to inactivation of the interferon. It is suggested that certain monomeric conformers are competent for aggregation.
- Published
- 1996
- Full Text
- View/download PDF
31. Catalytic mechanism of mitochondrial processing peptidase: fluorescence studies.
- Author
-
Boteva R and Salvato B
- Subjects
- Catalysis, Metalloendopeptidases chemistry, Metals metabolism, Mitochondria enzymology, Neurospora crassa enzymology, Protein Conformation, Protein Precursors metabolism, Protein Processing, Post-Translational, Spectrometry, Fluorescence, Substrate Specificity, Mitochondrial Processing Peptidase, Metalloendopeptidases metabolism
- Abstract
Processing of nuclear-encoded precursor proteins by mitochondrial processing peptidase (MPP) is an essential step for their sorting and function in mitochondria. We report spectroscopic studies on the catalytic mechanism of Neurospora crassa MPP. It is a complex enzyme consisting of two different subunits termed alpha-and beta-MPP. Following changes in the protein intrinsic fluorescence we register and characterize a complex formation between (i) the alpha- and the beta-subunit of MPP, (ii) the two subunits and a precursor protein, and (iii) the two subunits and some metal ions. The presequence of the precursor protein was absolutely necessary for its binding to MPP subunits. Mn2+ ions in concentrations enhancing the processing activity did not influence the substrate binding, whereas EDTA in concentrations inhibiting the enzyme completely abolished the binding of the substrate to the MPP subunits. Both MPP subunits bind metal ions such as Mn2+, Mg2+, and Zn2+. beta-MPP interacts stronger with these ions but alpha-MPP-Mn2+ conjugates seem to be important for the processing activity.
- Published
- 1996
- Full Text
- View/download PDF
32. Chaperonin-mediated protein folding at the surface of groEL through a 'molten globule'-like intermediate.
- Author
-
Martin J, Langer T, Boteva R, Schramel A, Horwich AL, and Hartl FU
- Subjects
- Adenosine Triphosphate metabolism, Chaperonin 60, Hydrolysis, Tetrahydrofolate Dehydrogenase chemistry, Thiosulfate Sulfurtransferase chemistry, Bacterial Proteins pharmacology, Heat-Shock Proteins pharmacology, Protein Conformation drug effects
- Abstract
Folding of two monomeric enzymes mediated by groE has been reconstituted in vitro. The groEL protein stabilizes the polypeptides in a conformation resembling the 'molten globule' state. Mg-ATP and groES then promote the acquisition of ordered tertiary structure at the surface of groEL. Folding requires the hydrolysis of about 100 ATP molecules per protein monomer. This active process of surface-mediated chain folding might represent a general mechanism for the formation of protein structure in vivo.
- Published
- 1991
- Full Text
- View/download PDF
33. Fluorescence properties of native and chemically modified mesentericopeptidase.
- Author
-
Ricchelli F, Jori G, Shopova M, Boteva R, and Genov N
- Subjects
- Dansyl Compounds, Energy Transfer, Fluorescence, Temperature, Tryptophan, Tyrosine, Endopeptidases, Serine Endopeptidases
- Abstract
Studies on the fluorescence properties of native mesentericopeptidase as a function of the temperature and/or in the presence of either neutral or ionic fluorescence quenchers demonstrate that the intrinsic emission f this protein is dominated by a partially exposed tryptophyl residue, which is probably located in a site of high dielectric constant containing positively charged amino acid side chains. One largely exposed tryptophan contributes about 14% of the total emission, whereas one deeply buried tryptophan is virtually non-fluorescent. The conversion of the active site serine to cysteine and the insertion of either one phenylmethanesulfonyl or one dansyl substituent into the active site induce only subtle differences in the conformational properties with respect to the native protein; in particular, the mutual distances and orientation between the 13 tyrosyl and 3 tryptophyl residues are unaffected, as shown by singlet-singlet energy transfer experiments.
- Published
- 1981
- Full Text
- View/download PDF
34. A novel extracellular subtilisin inhibitor produced by a Streptomyces sp.
- Author
-
Kourteva Y and Boteva R
- Subjects
- Amino Acids analysis, Caseins metabolism, Chromatography, High Pressure Liquid, Esters, Molecular Weight, Serine Proteinase Inhibitors, Protease Inhibitors pharmacology, Streptomyces metabolism, Subtilisins antagonists & inhibitors
- Abstract
The amino acid composition and inhibitory properties of a protein (SI-1-72) isolated from the culture medium of a Streptomyces sp. have been investigated. SI-1-72 appears to be a monomer protein of molecular mass about 13,100 Da and amino acid composition which differs from that of the inhibitors of the Streptomyces subtilisin inhibitor (SSI) family. Furthermore, it was found to exhibit novel specificity: strong inhibitory effect against microbial alkaline proteinases, moderate effect towards chymotrypsin and elastase, and no inhibition of the other serine proteinases, as well as of the cysteine, aspartate and metallo-proteinases.
- Published
- 1989
- Full Text
- View/download PDF
35. Comparative characterization of the substrate-binding subsites in subtilisins DY and Carlsberg by fluorescence and kinetic studies.
- Author
-
Boteva R, Dimov I, Genov N, Fittkau S, and Peters K
- Subjects
- Binding Sites, Dansyl Compounds pharmacology, Kinetics, Oligopeptides pharmacology, Spectrometry, Fluorescence, Structure-Activity Relationship, Subtilisins antagonists & inhibitors, Subtilisins metabolism
- Abstract
Peptide chloromethanes with the general formula dansyl-(Ala)n-Phe-CH2Cl where n = 0, 1, 2, 3 and dansyl fluoride were used to investigate the substrate-binding sites A and B in subtilisins DY and Carlsberg. Kinetic evidence for the introduction of the dansyl group at the subsites S2, S3, S4 and S5 were obtained. Fluorescence experiments showed that the micro-environment of these subsites is quite apolar. However, some differences in their accessibility to external reagents can be revealed in fluorescence quenching experiments. Efficient singlet-singlet radiationless energy transfer from the single Trp 113 to the dansyl group selectively bound at the respective subsites was observed and intramolecular distances between the chromophores were determined. The values calculated for the pairs Trp 113 plus Dns at S2, Trp 113 plus Dns at S4 and Trp 113 plus Dns at S5 are practically identical (1.7-2.0 nm) for the two enzymes. Conclusions on the shape of the substrate-binding sites in subtilisins DY and Carlsberg are drawn. The mutual spatial orientation of the donor (Trp 113) and acceptor (Dns at Sn) dipoles is also elucidated.
- Published
- 1988
- Full Text
- View/download PDF
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