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To facilitate the measurement of intramolecular distances in solvated RNA systems, a combination of spin-labeling, electron paramagnetic resonance (EPR), and molecular dynamics (MD) simulation is presented. The fairly rigid spin label 2,2,5,5-tetramethyl-pyrrolin-1-yloxyl-3-acetylene (TPA) was base and site specifically introduced into RNA through a Sonogashira palladium catalyzed crosscoupling on column. For this purpose 5-iodouridine, 5-iodo-cytidine and 2-iodo-adenosine phosphoramidites were synthesized and incorporated into RNA-sequences. Application of the recently developed ACE (R) chemistry presented the main advantage to limit the reduction of the nitroxide to an amine during the oligonucleotide automated synthesis and thus to increase substantially the reliability of the synthesis and the yield of labeled oligonucleotides. 4-Pulse Electron Double Resonance (PELDOR) was then successfully used to measure the intramolecular spin–spin distances in six doubly labeled RNA-duplexes. Comparison of these results with our previous work on DNA showed that A- and B-Form can be differentiated. Using an all-atom force field with explicit solvent, MD simulations gave results in good agreement with the measured distances and indicated that the RNA A-Form was conserved despite a local destabilization effect of the nitroxide label. The applicability of the method to more complex biological systems is discussed.
Background Olfactory receptors work at the interface between the chemical world of volatile molecules and the perception of scent in the brain. Their main purpose is to translate chemical space into information that can be processed by neural circuits. Assuming that these receptors have evolved to cope with this task, the analysis of their coding strategy promises to yield valuable insight in how to encode chemical information in an efficient way. Results We mimicked olfactory coding by modeling responses of primary olfactory neurons to small molecules using a large set of physicochemical molecular descriptors and artificial neural networks. We then tested these models by recording in vivo receptor neuron responses to a new set of odorants and successfully predicted the responses of five out of seven receptor neurons. Correlation coefficients ranged from 0.66 to 0.85, demonstrating the applicability of our approach for the analysis of olfactory receptor activation data. The molecular descriptors that are best-suited for response prediction vary for different receptor neurons, implying that each receptor neuron detects a different aspect of chemical space. Finally, we demonstrate that receptor responses themselves can be used as descriptors in a predictive model of neuron activation. Conclusions The chemical meaning of molecular descriptors helps understand structure-response relationships for olfactory receptors and their 'receptive fields'. Moreover, it is possible to predict receptor neuron activation from chemical structure using machine-learning techniques, although this is still complicated by a lack of training data.
Wirkungen von Heilpflanzen, Gewürzen, Tees und Lebensmitteln werden in der Naturheilkunde seit der Antike genutzt. Pharmakologisch wirksam sind in der Regel nur die sekundären Pflanzeninhaltsstoffe. Diese in den oft aus vielen Bestandteilen zusammengesetzten Naturstoffen aufzuspüren und ihren molekularbiologischen Wirkungsmechanismus im Körper aufzuklären, ist das Ziel eines Forschungsnetzwerks am Frankfurter ZAFES (Zentrum für Arzneimittelforschung, -Entwicklung und -Sicherheit). So konnten Pharmazeuten und Kliniker gemeinsam herausfinden, wie ein Bestandteil des Rotweins, das Resveratrol, vor Darmkrebs schützt. Die Inhaltsstoffe von Salbei und Rosmarin bieten vielversprechende Ausgangspunkte für neue Medikamente gegen Altersdiabetes. Weihrauch, Myrte und Johanniskraut enthalten Wirkstoffe, die Schlüsselenzyme für Entzündungsreaktionen – etwa bei rheumatischen Beschwerden – hemmen.
Host cells infected with obligate intracellular bacteria Chlamydia trachomatis are profoundly resistant to diverse apoptotic stimuli. The molecular mechanisms underlying the block in apoptotic signaling of infected cells is not well understood. Here we investigated the molecular mechanism by which apoptosis induced via the tumor necrosis factor (TNF) receptor is prevented in infected epithelial cells. Infection with C. trachomatis leads to the up-regulation of cellular inhibitor of apoptosis (cIAP)-2, and interfering with cIAP-2 up-regulation sensitized infected cells for TNF-induced apoptosis. Interestingly, besides cIAP-2, cIAP-1 and X-linked IAP, although not differentially regulated by infection, are required to maintain apoptosis resistance in infected cells. We detected that IAPs are constitutively organized in heteromeric complexes and small interfering RNA-mediated silencing of one of these IAPs affects the stability of another IAP. In particular, the stability of cIAP-2 is modulated by the presence of X-linked IAP and their interaction is stabilized in infected cells. Our observations suggest that IAPs are functional and stable as heteromers, a thus far undiscovered mechanism of IAP regulation and its role in modulation of apoptosis.
Riboswitches are highly structured elements in the 50-untranslated regions (50-UTRs) of messenger RNA that control gene expression by specifically binding to small metabolite molecules. They consist of an aptamer domain responsible for ligand binding and an expression platform. Ligand binding in the aptamer domain leads to conformational changes in the expression platform that result in transcription termination or abolish ribosome binding. The guanine riboswitch binds with high-specificity to guanine and hypoxanthine and is among the smallest riboswitches described so far. The X-ray-structure of its aptamer domain in complex with guanine/ hypoxanthine reveals an intricate RNA-fold consisting of a three-helix junction stabilized by longrange base pairing interactions. We analyzed the conformational transitions of the aptamer domain induced by binding of hypoxanthine using highresolution NMR-spectroscopy in solution. We found that the long-range base pairing interactions are already present in the free RNA and preorganize its global fold. The ligand binding core region is lacking hydrogen bonding interactions and therefore likely to be unstructured in the absence of ligand. Mg2+-ions are not essential for ligand binding and do not change the structure of the RNA-ligand complex but stabilize the structure at elevated temperatures. We identified a mutant RNA where the long-range base pairing interactions are disrupted in the free form of the RNA but form upon ligand binding in an Mg2+-dependent fashion. The tertiary interaction motif is stable outside the riboswitch context.
Infektionen mit Herpesviren sind bereits seit der Antike bekannt. So beschrieb zum Beispiel schon Hippokrates in seinem »Corpus Hippocraticum« die sich auf der Haut ausbreitenden Herpes Simplex Läsionen und gab der Krankheit ihren bis heute gültigen Namen. Verbürgt ist auch, dass der römische Kaiser Tiberius vor etwa 2000 Jahren während einer auftretenden Herpes labialis-Epidemie das Küssen bei öffentlichen Zeremonien per Dekret verbat. Shakespeare war ebenfalls bestens vertraut mit den periodisch auftretenden Herpes-Bläschen; in seinem Werk »Romeo & Julia« spricht Mercutio zu Romeo: »O’er ladies lips, who straight on kisses dream, which oft the angry Mab with blisters plagues, ….« Doch erst in den 1960er Jahren erkannte man die virale Herkunft der Erkrankung.
Ribosomal proteins are assumed to stabilize specific RNA structures and promote compact folding of the large rRNA. The conformational dynamics of the protein between the bound and unbound state play an important role in the binding process. We have studied those dynamical changes in detail for the highly conserved complex between the ribosomal protein L11 and the GTPase region of 23S rRNA. The RNA domain is compactly folded into a well defined tertiary structure, which is further stabilized by the association with the C-terminal domain of the L11 protein (L11ctd). In addition, the N-terminal domain of L11 (L11ntd) is implicated in the binding of the natural thiazole antibiotic thiostrepton, which disrupts the elongation factor function. We have studied the conformation of the ribosomal protein and its dynamics by NMR in the unbound state, the RNA bound state and in the ternary complex with the RNA and thiostrepton. Our data reveal a rearrangement of the L11ntd, placing it closer to the RNA after binding of thiostrepton, which may prevent binding of elongation factors. We propose a model for the ternary L11–RNA–thiostrepton complex that is additionally based on interaction data and conformational information of the L11 protein. The model is consistent with earlier findings and provides an explanation for the role of L11ntd in elongation factor binding.
Oral presentations Background: We selected peptide ligands for the HIV-1 packaging signal PSI by screening phage displayed peptide libraries. Peptide ligands were optimized by screening spot synthesis peptide membranes. The aim of this study is the functional characterization of these peptide ligands with respect to inhibition of HIV-1 replication. Methods: Phage displayed peptide libraries were screened with PSI-RNA structures. The Trp-rich peptide motifs were optimized for specific binding on spot synthesis peptide membranes. The best binding peptide was expressed intracellularly in fusion with RFP or linked to a protein transduction domain (PTD) for intracellular delivery. The effects on virion production were analyzed using pseudotyped lentiviral particles. Results: After positive and negative selection rounds, phages binding specifically to PSI-RNA were identified by ELISA. Peptide inserts contained conserved motifs of aromatic amino acids known to be implicated in binding of PSI-RNA by the natural Gag ligand. The filter assay identified HKWPWW as the best binding ligand for PSI-RNA, which is delivered into several cell lines by addition of a PTD. Compared to a control peptide, the HKWPWW peptide inhibited HIV-1 replication as deduced from reduced titers of culture supernatants. As HKWPWW also binds to the TAR-RNA like the natural nucleocapsid PSI-RNA ligand, the effect on Tat-TAR inhibition will also be analyzed. Currently T-cell lines are established which stably express HKWPWW as well as a control peptide, which will be infected with HIV-1 to monitor the ability of HKWPWW to inhibit wild type HIV-1 replication. Conclusion: The selection of a peptide ligand for PSI-RNA able to inhibit HIV-1 replication proves the suitability of the phage display technology for the selection of peptides binding to RNA-structures. This enables the indentification of peptides serving as leads to interfere with additional targets in the HIV-1 replication cycle.
Background: Particle Swarm Optimization (PSO) is an established method for parameter optimization. It represents a population-based adaptive optimization technique that is influenced by several "strategy parameters". Choosing reasonable parameter values for the PSO is crucial for its convergence behavior, and depends on the optimization task. We present a method for parameter meta-optimization based on PSO and its application to neural network training. The concept of the Optimized Particle Swarm Optimization (OPSO) is to optimize the free parameters of the PSO by having swarms within a swarm. We assessed the performance of the OPSO method on a set of five artificial fitness functions and compared it to the performance of two popular PSO implementations. Results: Our results indicate that PSO performance can be improved if meta-optimized parameter sets are applied. In addition, we could improve optimization speed and quality on the other PSO methods in the majority of our experiments. We applied the OPSO method to neural network training with the aim to build a quantitative model for predicting blood-brain barrier permeation of small organic molecules. On average, training time decreased by a factor of four and two in comparison to the other PSO methods, respectively. By applying the OPSO method, a prediction model showing good correlation with training-, test- and validation data was obtained. Conclusion: Optimizing the free parameters of the PSO method can result in performance gain. The OPSO approach yields parameter combinations improving overall optimization performance. Its conceptual simplicity makes implementing the method a straightforward task.
DCD – a novel plant specific domain in proteins involved in development and programmed cell death
(2005)
Background: Recognition of microbial pathogens by plants triggers the hypersensitive reaction, a common form of programmed cell death in plants. These dying cells generate signals that activate the plant immune system and alarm the neighboring cells as well as the whole plant to activate defense responses to limit the spread of the pathogen. The molecular mechanisms behind the hypersensitive reaction are largely unknown except for the recognition process of pathogens. We delineate the NRP-gene in soybean, which is specifically induced during this programmed cell death and contains a novel protein domain, which is commonly found in different plant proteins.
Results: The sequence analysis of the protein, encoded by the NRP-gene from soybean, led to the identification of a novel domain, which we named DCD, because it is found in plant proteins involved in d evelopment and c ell d eath. The domain is shared by several proteins in the Arabidopsis and the rice genomes, which otherwise show a different protein architecture. Biological studies indicate a role of these proteins in phytohormone response, embryo development and programmed cell by pathogens or ozone.
Conclusion: It is tempting to speculate, that the DCD domain mediates signaling in plant development and programmed cell death and could thus be used to identify interacting proteins to gain further molecular insights into these processes.
Poster presentation: NO-sensitive guanylyl cyclases (GC) are the principal receptors for nitric oxide (NO) and convert GTP into the second messenger cGMP. We showed that GC is prone to tyrosine phosphorylation in COS1 cells overexpressing the human holoenzyme. Similar results were obtained in PC12 cells and in rat aortic tissue slices. The major phosphorylation site was mapped to position 192 in the regulatory domain of the beta1 subunit. Tyrosine phosphorylation of GC was reduced in the presence of the inhibitors PP1 and PP2 indicating that Src-like kinases are critically involved in phosphorylation. Moreover, co-immunoprecipitation experiments revealed an interaction between Src and GC. To further analyse the relevance of this posttranslational modification we generated a phospho-specific antibody raised against pTyr192. This antibody clearly distinguishes between phosphorylated and non-phosphorylated GC and may be a powerful tool to analyse the subcellular localisation of the phosphorylated enzyme.
Poster presentation: NO-sensitive guanylyl cyclases (sGCs) are cytosolic receptors for nitric oxide (NO) catalyzing the conversion of GTP to cGMP. sGCs are obligate heterodimers composed of one alpha and beta subunit each. The allosteric mechanism of sGC activation via NO is well understood, however, our knowledge about alternative mechanisms such as protein-protein interactions regulating activity, availability, translocation and expression of sGC is rather limited. In a search by the yeast two-hybrid system using the catalytic domain of the alpha1 subunit as the bait, we have identified two structurally related proteins AGAP1 [1] and MRIP2 as novel sGC interacting proteins. MRIP2 is a multi-domain protein of 75 kDa comprising a single PH and ArfGAP domain each and two ankyrin repeats. Co-immunoprecipitation experiments using COS1 cells overexpressing both proteins demonstrated the interaction of MRIP2 with both subunits of the sGC alpha1beta1. Confocal microscopical analysis showed a prominent plasma membrane staining of MRIP2. This membrane association is mediated through an N-terminal myristoylation site and through binding of its PH domain to phospholipids such as phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2). We hypothesize that MRIP2 may represent an acceptor protein for sGC that mediates recruitment of cytosolic sGC to the plasma membrane or other subcellular compartments.
Poster presentation NO-sensitive guanylyl cyclases (soluble guanylyl cyclase, sGC) are among the key regulators of intracellular cGMP concentration. The mechanisms underlying NO-mediated activation of sGC are quite well understood, however, little is known about the fine-tuning of sGC activity through alternative mechanisms such as protein phosphorylation. Several reports have demonstrated the reversible phosphorylation of sGC on serine/threonine residues, and it has been speculated, though not experimentally proven, that sGC might also be phosphorylated on tyrosine residues. Using broad-spectrum phosphatase inhibitors we were able to demonstrate tyrosine phosphorylation at Tyr192 of the beta 1 subunit of human sGC in COS1 cells. This residue forms part of a sequence segment (YEDL) representing a preferential binding site for SH2 domains of Src-like kinases. Pull-down assays and co-immunoprecipitation experiments showed that Src can indeed bind via its SH2 domain to pTyr192 of beta 1 indicating that tyrosine phosphorylation of sGC may be followed by recruitment of Src-like kinases to the phosphorylated beta 1 subunit. In support of this hypothesis, immunofluorescence studies showed a colocalization of overexpressed sGC and Src at the plasma membrane of COS1 and Hela cells. Together, our results point to an unexpected crosstalk between tyrosine kinase pathway(s) and the NO/cGMP signalling cascade which may result in translocation of the predominantly cytosolic sGC to the cytosolic face of the plasma membrane.
The NO/cGMP pathway inhibits Rap1 activation in human platelets via cGMP-dependent protein kinase I
(2005)
The NO/cGMP signalling pathway strongly inhibits agonist-induced platelet aggregation. However, the molecular mechanisms involved are not completely defined.We have studied NO/cGMP effects on the activity of Rap1, an abundant guanine-nucleotidebinding protein in platelets. Rap1-GTP levels were reduced by NO-donors and activators of NO-sensitive soluble guanylyl cyclase. Four lines of evidence suggest that NO/cGMP effects are mediated by cGMP-dependent protein kinase (cGKI): (i) Rap1 inhibition correlated with cGKI activity as measured by the phosphorylation state ofVASP, an established substrate of cGKI, (ii) 8-pCPT-cGMP, a membrane permeable cGMP-analog and activator of cGKI, completely blocked Rap1 activation, (iii) Rp- 8pCPT-cGMPS, a cGKI inhibitor, reversed NO effects and (iv) expression of cGKI in cGKI-deficient megakaryocytes inhibited Rap1 activation. NO/cGMP/cGKI effects were independent of the type of stimulus used for Rap1 activation.Thrombin-,ADPand collagen-induced formation of Rap1-GTP in platelets as well as turbulence-induced Rap1 activation in megakaryocytes were inhibited. Furthermore, cGKI inhibited ADP-induced Rap1 activation induced by the G a i -coupled P2Y12 receptor alone, i.e. independently of effects on Ca2+-signalling. From these studies we conclude that NO/cGMP inhibit Rap1 activation in human platelets and that this effect is mediated by cGKI. Since Rap1 controls the function of integrin a IIbß 3 , we propose that Rap1 inhibition might play a central role in the anti-aggregatory actions of NO/cGMP.
Background: The flavin in its FMN and FAD forms is a versatile cofactor that is involved in catalysis of most disparate types of biological reactions. These include redox reactions such as dehydrogenations, activation of dioxygen, electron transfer, bioluminescence, blue light reception, photobiochemistry (as in photolyases), redox signaling etc. Recently, hitherto unrecognized types of biological reactions have been uncovered that do not involve redox shuffles, and might involve the reduced form of the flavin as a catalyst. The present work addresses properties of reduced flavin relevant in this context. Results: N(5)-H exchange reactions of the flavin reduced form and its pH dependence were studied using the 15N-NMR-signals of 15N-enriched, reduced flavin in the pH range from 5 to 12. The chemical shifts of the N(3) and N(5) resonances are not affected to a relevant extent in this pH range. This contrasts with the multiplicity of the N(5)-resonance, which strongly depends on pH. It is a doublet between pH 8.45 and 10.25 that coalesces into a singlet at lower and higher pH values. From the line width of the 15N(5) signal the pH-dependent rate of hydrogen exchange was deduced. The multiplicity of the 15N(5) signal and the proton exchange rates are little dependent on the buffer system used. Conclusion: The exchange rates allow an estimation of the pKa value of N(5)-H deprotonation in reduced flavin to be ≥ 20. This value imposes specific constraints for mechanisms of flavoprotein catalysis based on this process. On the other hand the pK ≈ 4 for N(5)-H protonation (to form N(5)+-H2) would be consistent with a role of N(5)-H as a base.
In order to further understand how DNA polymerases discriminate against incorrect dNTPs, we synthesized two sets of dNTP analogues and tested them as substrates for DNA polymerase a (pol alpha) and Klenow fragment (exo-) of DNA polymerase I (Escherichia coli ). One set of analogues was designed to test the importance of the electronic nature of the base. The bases consisted of a benzimidazole ring with one or two exocyclic substituent(s) that are either electron-donating (methyl and methoxy) or electronwithdrawing (trifluoromethyl and dinitro). Both pol a and Klenow fragment exhibit a remarkable inability to discriminate against these analogues as compared to their ability to discriminate against incorrect natural dNTPs. Neither polymerase shows any distinct electronic or steric preferences for analogue incorporation. The other set of analogues, designed to examine the importance of hydrophobicity in dNTP incorporation, consists of a set of four regioisomers of trifluoromethyl benzimidazole. Whereas pol a and Klenow fragment exhibited minimal discrimination against the 5- and 6-regioisomers, they discriminated much more effectively against the 4- and 7-regioisomers. Since all four of these analogues will have similar hydrophobicity and stacking ability, these data indicate that hydrophobicity and stacking ability alone cannot account for the inability of pol a and Klenow fragment to discriminate against unnatural bases. After incorporation, however, both sets of analogues were not efficiently elongated. These results suggest that factors other than hydrophobicity, sterics and electronics govern the incorporation of dNTPs into DNA by pol {alpha} and Klenow fragment.
Im Zuge der steigenden Bedeutung der Proteomforschung und der »Molekularisierung« der Medizin werden neue, effizientere Plattformen zur Untersuchung von Proteinen und deren Wechselwirkungen notwendig. Hier bietet die Nanotechnologie, eine Wissenschaft mit Ursprüngen in der Physik und der Halbleiterindustrie, attraktive Lösungsperspektiven. Ein Bereich der Forschung am Institut für Biochemie der Universität Frankfurt um Prof. Dr. Robert Tampé widmet sich den Aspekten der Nanotechnologie zur Entwicklung von Protein-Chips für die Proteomforschung und Erzeugung von Mustern im Kleinstformat.
Chemically modified bases are frequently used to stabilize nucleic acids, to study the driving forces for nucleic acid structure formation and to tune DNA and RNA hybridization conditions. In particular, fluorobenzene and fluorobenzimidazole base analogues can act as universal bases able to pair with any natural base and to stabilize RNA duplex formation. Although these base analogues are compatible with an A-form RNA geometry, little is known about the influence on the fine structure and conformational dynamics of RNA. In the present study, nano-second molecular dynamics (MD) simulations have been performed to characterize the dynamics of RNA duplexes containing a central 1'-deoxy-1'-(2,4-difluorophenyl)-ß-D-ribofuranose base pair or opposite to an adenine base. For comparison, RNA with a central uridine:adenine pair and a 1'-deoxy-1'-(phenyl)-ß-D-ribofuranose opposite to an adenine was also investigated. The MD simulations indicate a stable overall A-form geometry for the RNAs with base analogues. However, the presence of the base analogues caused a locally enhanced mobility of the central bases inducing mainly base pair shear and opening motions. No stable ‘base-paired’ geometry was found for the base analogue pair or the base analogue:adenine pairs, which explains in part the universal base character of these analogues. Instead, the conformational fluctuations of the base analogues lead to an enhanced accessibility of the bases in the major and minor grooves of the helix compared with a regular base pair.
The shortage of functional information compared to the abundance of sequence information characterizes today’s situation in functional genomics. For many years the knock-down of a gene’s product has been the most powerful way of analysing its function. In addition to the complete knock-out by homologous recombination, several different techniques have been developed to temporarily knock down gene expression through methods based on specific sequence recognition, such as knockdown by antisense oligonucleotides, ribozymes, aptamers or RNAi.
The ESF workshop on ‘Impact of Nucleic Acid Chemistry on Gene Function Analysis’ brought together researchers who use techniques that are different but highly related. It offered an opportunity for an in-depth discussion of recent progress and common problems. Antisense oligonucleotides aptamers and ribozymes are techniques that have been used successfully for many years to validate targets. However, recent developments, such as increased tightness of binding (e.g. locked nucleic acids) or the combination of different methods (e.g. using aptamers to design ribozymes), have continued to improve the existing techniques. RNA interference (RNAi) is a defence mechanism of the cell against viruses. Since the exact mechanism of action within the cell is still unclear, RNAi was a particularly exciting topic at the workshop and was addressed in the largest number of presentations. Predictability of positional effects (accessibility of RNA) is a problem shared by all techniques using sequence-specific recognition and was the subject of quite controversial debates.
The meeting comprised over 50 people from 14 countries (13 European countries and the USA).
The conditionally-lethal pso4-1 mutant allele of the spliceosomal-associated PRP19 gene allowed us to study this gene’s influence on pre-mRNA processing, DNA repair and sporulation. Phenotypes related to intron-containing genes were correlated to temperature. Splicing reporter systems and RT–PCR showed splicing efficiency in pso4-1 to be inversely correlated to growth temperature. A single amino acid substitution, replacing leucine with serine, was identified within the N-terminal region of the pso4-1 allele and was shown to affect the interacting properties of Pso4-1p. Amongst 24 interacting clones isolated in a two-hybrid screening, seven could be identified as parts of the RAD2, RLF2 and DBR1 genes. RAD2 encodes an endonuclease indispensable for nucleotide excision repair (NER), RLF2 encodes the major subunit of the chromatin assembly factor I, whose deletion results in sensitivity to UVC radiation, while DBR1 encodes the lariat RNA splicing debranching enzyme, which degrades intron lariat structures during splicing. Characterization of mutagen-sensitive phenotypes of rad2{Delta}, rlf2{Delta} and pso4-1 single and double mutant strains showed enhanced sensitivity for the rad2{Delta} pso4-1 and rlf2{Delta} pso4-1 double mutants, suggesting a functional interference of these proteins in DNA repair processes in Saccharomyces cerevisiae.