Filtern
Erscheinungsjahr
- 2010 (45) (entfernen)
Dokumenttyp
- Wissenschaftlicher Artikel (45) (entfernen)
Volltext vorhanden
- ja (45)
Gehört zur Bibliographie
- nein (45)
Schlagworte
- Direct carbon (1)
- Ene Reaction (1)
- Evolution/Protein (1)
- Membrane/Proteins (1)
- NMR spectroscopy (1)
- Protein/Sorting (1)
- Protein/Targeting (1)
- Protein/Translocation (1)
- RNA (1)
- SiN Double Bond (1)
Institut
- Biochemie und Chemie (45) (entfernen)
Nep1 (Emg1) is a highly conserved nucleolar protein with an essential function in ribosome biogenesis. A mutation in the human Nep1 homolog causes Bowen–Conradi syndrome—a severe developmental disorder. Structures of Nep1 revealed a dimer with a fold similar to the SPOUT-class of RNA-methyltransferases suggesting that Nep1 acts as a methyltransferase in ribosome biogenesis. The target for this putative methyltransferase activity has not been identified yet. We characterized the RNA-binding specificity of Methanocaldococcus jannaschii Nep1 by fluorescence- and NMR-spectroscopy as well as by yeast three-hybrid screening. Nep1 binds with high affinity to short RNA oligonucleotides corresponding to nt 910–921 of M. jannaschii 16S rRNA through a highly conserved basic surface cleft along the dimer interface. Nep1 only methylates RNAs containing a pseudouridine at a position corresponding to a previously identified hypermodified N1-methyl-N3-(3-amino-3-carboxypropyl) pseudouridine (m1acp3-Psi) in eukaryotic 18S rRNAs. Analysis of the methylated nucleoside by MALDI-mass spectrometry, HPLC and NMR shows that the methyl group is transferred to the N1 of the pseudouridine. Thus, Nep1 is the first identified example of an N1-specific pseudouridine methyltransferase. This enzymatic activity is also conserved in human Nep1 suggesting that Nep1 is the methyltransferase in the biosynthesis of m1acp3-Psi in eukaryotic 18S rRNAs.
Experimental results are presented for 180 in silico designed octapeptide sequences and their stabilizing effects on the major histocompatibility class I molecule H-2Kb. Peptide sequence design was accomplished by a combination of an ant colony optimization algorithm with artificial neural network classifiers. Experimental tests yielded nine H-2Kb stabilizing and 171 nonstabilizing peptides. 28 among the nonstabilizing octapeptides contain canonical motif residues known to be favorable for MHC I stabilization. For characterization of the area covered by stabilizing and non-stabilizing octapeptides in sequence space, we visualized the distribution of 100,603 octapeptides using a self-organizing map. The experimental results present evidence that the canonical sequence motives of the SYFPEITHI database on their own are insufficient for predicting MHC I protein stabilization.
The title compound, [Li3(C4F9O)3(C3H6O)3], features an open Li/O cube with an Li ion missing at one corner. Three of the four bridging O atoms of the cube carry a fluorinated tert-butyl residue, whereas the fourth is part of an acetone molecule. Two of the Li atoms are further bonded to a non-bridging acetone molecule. Two of the lithium ion coordination geometries are very distorted LiO4 tetrahedra; the third could be described as a very distorted LiO3 T-shape with two distant F-atom neighbours. The Li[cdots, three dots, centered]Li contact distances for the three-coordinate Li+ ion [2.608 (14) and 2.631 (12) Å] are much shorter that the contact distance [2.940 (13) Å] between the tetrahedrally coordinated species.
The title compound, C8H11FN5 +·Cl-, crystallized with a monoprotonated 1-(4-fluorophenyl)biguanidinium cation and a chloride anion in the asymmetric unit. The biguanidium group is not planar [dihedral angle between the two CN3 groups = 52.0 (1)°] and is rotated with respect to the phenyl group [tau = 54.3 (3)°]. In the crystal, N—H ... N hydrogen-bonded centrosymmetric dimers are connected into ribbons, which are further stabilized by N—H ... Cl interactions, forming a three-dimensional hydrogen-bonded network.
In the title compound, C4H7N3O·C2H6OS, creatinine [2-amino-1-methyl-1H-imidazol-4(5H)one] exists in the amine form. The ring is planar (r.m.s. deviation for all non-H atoms = 0.017 Å). In the crystal, two creatinine molecules form centrosymmetric hydrogen-bonded dimers linked by pairs of N—H[cdots, three dots, centered]N hydrogen bonds. In addition, creatinine is linked to a dimethyl sulfoxide molecule by an N—H[cdots, three dots, centered]O interaction. The packing shows layers parallel to (120).
Human Transformer2-beta (hTra2-beta) is an important member of the serine/arginine-rich protein family, and contains one RNA recognition motif (RRM). It controls the alternative splicing of several pre-mRNAs, including those of the calcitonin/calcitonin gene-related peptide (CGRP), the survival motor neuron 1 (SMN1) protein and the tau protein. Accordingly, the RRM of hTra2-beta specifically binds to two types of RNA sequences [the CAA and (GAA)2 sequences]. We determined the solution structure of the hTra2-beta RRM (spanning residues Asn110–Thr201), which not only has a canonical RRM fold, but also an unusual alignment of the aromatic amino acids on the beta-sheet surface. We then solved the complex structure of the hTra2-beta RRM with the (GAA)2 sequence, and found that the AGAA tetra-nucleotide was specifically recognized through hydrogen-bond formation with several amino acids on the N- and C-terminal extensions, as well as stacking interactions mediated by the unusually aligned aromatic rings on the beta-sheet surface. Further NMR experiments revealed that the hTra2-beta RRM recognizes the CAA sequence when it is integrated in the stem-loop structure. This study indicates that the hTra2-beta RRM recognizes two types of RNA sequences in different RNA binding modes.
The transcription factor p63 is expressed as at least six different isoforms, of which two have been assigned critical biological roles within ectodermal development and skin stem cell biology on the one hand and supervision of the genetic stability of oocytes on the other hand. These two isoforms contain a C-terminal inhibitory domain that negatively regulates their transcriptional activity. This inhibitory domain contains two individual components: one that uses an internal binding mechanism to interact with and mask the transactivation domain and one that is based on sumoylation. We have carried out an extensive alanine scanning study to identify critical regions within the inhibitory domain. These experiments show that a stretch of ~13 amino acids is crucial for the binding function. Further, investigation of transcriptional activity and the intracellular level of mutants that cannot be sumoylated suggests that sumoylation reduces the concentration of p63. We therefore propose that the inhibitory function of the C-terminal domain is in part due to direct inhibition of the transcriptional activity of the protein and in part due to indirect inhibition by controlling the concentration of p63. Keywords: p63, transcriptional regulation, auto-inhibition, sumoylation
Leukotrienes constitute a group of bioactive lipids generated by the 5-lipoxygenase (5-LO) pathway. An increasing body of evidence supports an acute role for 5-LO products already during the earliest stages of pancreatic, prostate, and colorectal carcinogenesis. Several pieces of experimental data form the basis for this hypothesis and suggest a correlation between 5-LO expression and tumor cell viability. First, several independent studies documented an overexpression of 5-LO in primary tumor cells as well as in established cancer cell lines. Second, addition of 5-LO products to cultured tumor cells also led to increased cell proliferation and activation of anti-apoptotic signaling pathways. 5-LO antisense technology approaches demonstrated impaired tumor cell growth due to reduction of 5-LO expression. Lastly, pharmacological inhibition of 5-LO potently suppressed tumor cell growth by inducing cell cycle arrest and triggering cell death via the intrinsic apoptotic pathway. However, the documented strong cytotoxic off-target effects of 5-LO inhibitors, in combination with the relatively high concentrations of 5-LO products needed to achieve mitogenic effects in cell culture assays, raise concern over the assignment of the cause, and question the relationship between 5-LO products and tumorigenesis. Keywords: leukotriene, apoptosis, cell proliferation, mitogenic effects, cytotoxicity
Background: ClC-7 is a ubiquitous transporter which is broadly expressed in mammalian tissues. It is implied in the pathogenesis of lysosomal storage disease and osteopetrosis. Because of its endosomal/lysosomal localization it is still poorly characterized. Methodology/Principal Findings: An electrophysiological characterization of rat ClC-7 using solid-supported membrane-based electrophysiology is presented. The measured currents show the characteristics of ClC-7 and confirm its function as a Cl−/H+-antiporter. We have used rat ClC-7 in CHO cells as a model system to investigate the functionality and cellular localization of the wt transporter and its variant G213R ClC-7 which is the analogue of human G215R ClC-7 responsible for autosomal dominant osteopetrosis type II. Our study shows that rat G213R ClC-7 is functional but has a localization defect in CHO cells which prevents it from being correctly targeted to the lysosomal membrane. The electrophysiological assay is tested as a tool for drug discovery. The assay is validated with a number of drug candidates. It is shown that ClC-7 is inhibited by DIDS, NPPB and NS5818 at micromolar concentrations. Conclusions/Significance: It is suggested that the scenario found in the CHO model system also applies to the human transporter and that mislocalization rather than impaired functionality of G215R ClC-7 is the primary cause of the related autosomal dominant osteopetrosis type II. Furthermore, the robust solid-supported membrane-based electrophysiological assay is proposed for rapid screening for potential ClC-7 inhibitors which are discussed for treatment of osteoporosis.
The central structural element of the title compound, C24H29NO2, is a carbazole unit substituted with two acetyl residues and an octyl chain. The acetyl residues are nearly coplanar [dihedral angles = 5.37 (14) and 1.0 (3)°] with the carbazole unit which is essentially planar (r.m.s. deviation for all non-H atoms = 0.025 Å). The octyl chain adopts an all-trans conformation. The crystal packing is stabilized by C—H ... O hydrogen bonds.
The dihydropyrimidine ring of the title compound, C13H15ClN2S, adopts an envelope conformation with five almost coplanar atoms (r.m.s. deviation = 0.054 Å) and the C atom bearing the two methyl substituents deviating from this plane by 0.441 (2) Å. The best plane through the five almost coplanar atoms forms a dihedral angle of 89.56 (5)° with the benzene ring. The crystal packing is characterized by centrosymmetric dimers connected by pairs of N—H ... S hydrogen bonds.
The two rings in the title compound, C11H12N2O4S, are roughly coplanar [dihedral angle = 6.77 (8)°]. Whereas the two outer methyl groups of the three methoxy groups are almost coplanar with the aromatic ring to which they are attached [C—C—O—C torsion angles = 8.5 (3) and -8.3 (3)°], the methyl group of the central methoxy substituent is not [C—C—C—C = -78.4 (3)°]. The crystal packing is stabilized by N—H ... O hydrogen bonding.
Poster presentation at 5th German Conference on Cheminformatics: 23. CIC-Workshop Goslar, Germany. 8-10 November 2009 We demonstrate the theoretical and practical application of modern kernel-based machine learning methods to ligand-based virtual screening by successful prospective screening for novel agonists of the peroxisome proliferator-activated receptor gamma (PPARgamma) [1]. PPARgamma is a nuclear receptor involved in lipid and glucose metabolism, and related to type-2 diabetes and dyslipidemia. Applied methods included a graph kernel designed for molecular similarity analysis [2], kernel principle component analysis [3], multiple kernel learning [4], and, Gaussian process regression [5]. In the machine learning approach to ligand-based virtual screening, one uses the similarity principle [6] to identify potentially active compounds based on their similarity to known reference ligands. Kernel-based machine learning [7] uses the "kernel trick", a systematic approach to the derivation of non-linear versions of linear algorithms like separating hyperplanes and regression. Prerequisites for kernel learning are similarity measures with the mathematical property of positive semidefiniteness (kernels). The iterative similarity optimal assignment graph kernel (ISOAK) [2] is defined directly on the annotated structure graph, and was designed specifically for the comparison of small molecules. In our virtual screening study, its use improved results, e.g., in principle component analysis-based visualization and Gaussian process regression. Following a thorough retrospective validation using a data set of 176 published PPARgamma agonists [8], we screened a vendor library for novel agonists. Subsequent testing of 15 compounds in a cell-based transactivation assay [9] yielded four active compounds. The most interesting hit, a natural product derivative with cyclobutane scaffold, is a full selective PPARgamma agonist (EC50 = 10 ± 0.2 microM, inactive on PPARalpha and PPARbeta/delta at 10 microM). We demonstrate how the interplay of several modern kernel-based machine learning approaches can successfully improve ligand-based virtual screening results.
In prokaryotes, RNA thermometers regulate a number of heat shock and virulence genes. These temperature sensitive RNA elements are usually located in the 5'-untranslated regions of the regulated genes. They repress translation initiation by base pairing to the Shine–Dalgarno sequence at low temperatures. We investigated the thermodynamic stability of the temperature labile hairpin 2 of the Salmonella fourU RNA thermometer over a broad temperature range and determined free energy, enthalpy and entropy values for the base-pair opening of individual nucleobases by measuring the temperature dependence of the imino proton exchange rates via NMR spectroscopy. Exchange rates were analyzed for the wild-type (wt) RNA and the A8C mutant. The wt RNA was found to be stabilized by the extraordinarily stable G14–C25 base pair. The mismatch base pair in the wt RNA thermometer (A8–G31) is responsible for the smaller cooperativity of the unfolding transition in the wt RNA. Enthalpy and entropy values for the base-pair opening events exhibit linear correlation for both RNAs. The slopes of these correlations coincide with the melting points of the RNAs determined by CD spectroscopy. RNA unfolding occurs at a temperature where all nucleobases have equal thermodynamic stabilities. Our results are in agreement with a consecutive zipper-type unfolding mechanism in which the stacking interaction is responsible for the observed cooperativity. Furthermore, remote effects of the A8C mutation affecting the stability of nucleobase G14 could be identified. According to our analysis we deduce that this effect is most probably transduced via the hydration shell of the RNA.
We present here a set of 13C-direct detected NMR experiments to facilitate the resonance assignment of RNA oligonucleotides. Three experiments have been developed: (1) the (H)CC-TOCSY-experiment utilizing a virtual decoupling scheme to assign the intraresidual ribose 13C-spins, (2) the (H)CPC-experiment that correlates each phosphorus with the C40 nuclei of adjacent nucleotides via J(C,P) couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C10,H10 ribose signals. The experiments were applied to two RNA hairpin structures. The current set of 13C-direct detected experiments allows direct and unambiguous assignment of the majority of the hetero nuclei and the identification of the individual ribose moieties following their sequential assignment. Thus, 13C-direct detected NMR methods constitute useful complements to the conventional 1H-detected approach for the resonance assignment of oligonucleotides that is often hindered by the limited chemical shift dispersion. The developed methods can also be applied to large deuterated RNAs. Keywords: NMR spectroscopy , Direct carbon , detection , RNA
The asymmetric unit of the title compound, [K(C5HF6N2)(H2O)2]n, is composed of two 3,5-bis(trifluoromethyl)pyrazolide anions, two potassium cations and four water molecules. The water molecules and 3,5-bis(trifluoromethyl)pyrazolide anions act as bridges between the potassium cations. Each potassium cation is surrounded by four O atoms [K—O = 2.705 (3)–2.767 (3) Å] and four F atoms [K—F = 2.870 (7)–3.215 (13) Å]. The water molecules and the 3,5-bis(trifluoromethyl)pyrazolide anions are connected by O—H ... N hydrogen bonds, forming layers in the ab plane. All –CF3 groups show rotational disorder between two orientations each.
The title compound, [Na(CF3O3S)(C12H24O6)], features a sodium cation that is coordinated by eight O atoms in an irregular hexagonal bipyramidal environment. The equatorial positions are occupied by the six O atoms of an 18-crown-6 ether ring. In the axial positions, there is one O atom of a trifluoromethanesulfonate anion and an ether O atom of a symmetry-equivalent crown ether ring. In this way, centrosymmetric dimers are formed.