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Folding of RNA molecules into their functional three-dimensional structures is often supported by RNA chaperones, some of which can catalyse the two elementary reactions helix disruption and helix formation. Hfq is one such RNA chaperone, but its strand displacement activity is controversial. Whereas some groups found Hfq to destabilize secondary structures, others did not observe such an activity with their RNA substrates. We studied Hfq’s activities using a set of short RNAs of different thermodynamic stabilities (GC-contents from 4.8% to 61.9%), but constant length. We show that Hfq’s strand displacement as well as its annealing activity are strongly dependent on the substrate’s GC-content. However, this is due to Hfq’s preferred binding of AU-rich sequences and not to the substrate’s thermodynamic stability. Importantly, Hfq catalyses both annealing and strand displacement with comparable rates for different substrates, hinting at RNA strand diffusion and annealing nucleation being rate-limiting for both reactions. Hfq’s strand displacement activity is a result of the thermodynamic destabilization of the RNA through preferred single-strand binding whereas annealing acceleration is independent from Hfq’s thermodynamic influence. Therefore, the two apparently disparate activities annealing acceleration and duplex destabilization are not in energetic conflict with each other.
The loading of antigenic peptides onto major histocompatibility complex class I (MHC I) molecules is an essential step in the adaptive immune response against virally or malignantly transformed cells. The ER-resident peptide-loading complex (PLC) consists of the transporter associated with antigen processing (TAP1 and TAP2), assembled with the auxiliary factors tapasin and MHC I. Here, we demonstrated that the N-terminal extension of each TAP subunit represents an autonomous domain, named TMD0, which is correctly targeted to and inserted into the ER membrane. In the absence of coreTAP, each TMD0 recruits tapasin in a 1:1 stoichiometry. Although the TMD0s lack known ER retention/retrieval signals, they are localized to the ER membrane even in tapasin-deficient cells. We conclude that the TMD0s of TAP form autonomous interaction hubs linking antigen translocation into the ER with peptide loading onto MHC I, hence ensuring a major function in the integrity of the antigen-processing machinery.
High-throughput protein localization studies require multiple strategies. Mass spectrometric analysis of defined cellular fractions is one of the complementary approaches to a diverse array of cell biological methods. In recent years, the protein content of different cellular (sub-)compartments was approached. Despite of all the efforts made, the analysis of membrane fractions remains difficult, in that the dissection of the proteomes of the envelope membranes of chloroplasts or mitochondria is often not reliable because sample purity is not always warranted. Moreover, proteomic studies are often restricted to single (model) species, and therefore limited in respect to differential individual evolution. In this study we analyzed the chloroplast envelope proteomes of different plant species, namely, the individual proteomes of inner and outer envelope (OE) membrane of Pisum sativum and the mixed envelope proteomes of Arabidopsis thaliana and Medicago sativa. The analysis of all three species yielded 341 identified proteins in total, 247 of them being unique. 39 proteins were genuine envelope proteins found in at least two species. Based on this and previous envelope studies we defined the core envelope proteome of chloroplasts. Comparing the general overlap of the available six independent studies (including ours) revealed only a number of 27 envelope proteins. Depending on the stringency of applied selection criteria we found 231 envelope proteins, while less stringent criteria increases this number to 649 putative envelope proteins. Based on the latter we provide a map of the outer and inner envelope core proteome, which includes many yet uncharacterized proteins predicted to be involved in transport, signaling, and response. Furthermore, a foundation for the functional characterization of yet unidentified functions of the inner and OE for further analyses is provided.
Both the genomes of the epsilonproteobacteria Wolinella succinogenes and Campylobacter jejuni contain operons (sdhABE) that encode for so far uncharacterized enzyme complexes annotated as ‘non-classical’ succinate:quinone reductases (SQRs). However, the role of such an enzyme ostensibly involved in aerobic respiration in an anaerobic organism such as W. succinogenes has hitherto been unknown. We have established the first genetic system for the manipulation and production of a member of the non-classical succinate:quinone oxidoreductase family. Biochemical characterization of the W. succinogenes enzyme reveals that the putative SQR is in fact a novel methylmenaquinol:fumarate reductase (MFR) with no detectable succinate oxidation activity, clearly indicative of its involvement in anaerobic metabolism. We demonstrate that the hydrophilic subunits of the MFR complex are, in contrast to all other previously characterized members of the superfamily, exported into the periplasm via the twin-arginine translocation (tat)-pathway. Furthermore we show that a single amino acid exchange (Ala86→His) in the flavoprotein of that enzyme complex is the only additional requirement for the covalent binding of the otherwise non-covalently bound FAD. Our results provide an explanation for the previously published puzzling observation that the C. jejuni sdhABE operon is upregulated in an oxygen-limited environment as compared with microaerophilic laboratory conditions.
Background: In macrophages Toll-like receptor 4 (TLR4) is activated in response to lipopolysaccharide (LPS) and induces proinflammatory cytokine expression. Therefore, mechanisms terminating proinflammatory gene expression are important. Autophagy plays a central role in controlling innate immune responses by lysosomal degradation of signaling proteins, thus contributing to the resolution of inflammation. Autophagic proteins like p62 directly interact with molecules involved in the TLR4-signaling pathway, but a correlation with the IRAK E3 ligase and scaffold protein Pellino3 remains obscure. Hence, we are interested in elucidating the function of Pellino3 to prove our hypothesis that it is a key regulator in the TLR4-signaling cascade.
Methods: We used the cecal ligation and puncture (CLP) mouse model causing polymicrobial sepsis to analyze Pellino3 protein and mRNA expression. Furthermore, we induced endotoxemia in RAW264.7 mouse macrophages by LPS treatment to verify in vivo experiments. Lentiviral Pellino3 knockdown in RAW264.7 macrophages was used for cytokine measurements at mRNA level. To analyze potential Pellino3 binding partners in TLR4-signaling by mass spectrometry (MS), we overexpressed FLAG-tagged Pellino3 in RAW264.7 macrophages, treated cells for 3, 6 and 24 hours with LPS and immunoprecipitated Pellino3 via its FLAG-tag. To consider Pellino3 degradation as a result of p62-mediated autophagy, we transiently knocked down p62 by siRNA in RAW264.7 macrophages and also pharmacologically blocked LPS-induced autophagy by Bafilomycin A1.
Results: We demonstrated Pellino3 protein degradation in primary CD11b+ splenocytes after 24 hours following CLP operation and confirmed this in RAW264.7 macrophages after 24-hour LPS stimulation. Knockdown of Pellino3 attenuates proinflammatory cytokines, for example IL-6 mRNA, after 6 hours of LPS. Furthermore, we found by MS and verifying immunoprecipitation experiments that p62 is a Pellino3 binding partner, thus targeting Pellino3 for degradation. In line, both p62 knockdown and Bafilomycin A1 treatment prevent Pellino3 degradation, supporting an autophagic mechanism.
Conclusion: Our observations highlight a regulatory role of Pellino3 on TLR4 signaling. Thus, antagonism of Pellino3 in the hyperinflammatory phase of sepsis may counteract the cytokine storm. Furthermore, stabilization of Pellino3 by inhibition of autophagy in the hypoinflammatory phase of sepsis may improve immunity. In consideration of these two conflictive sepsis phases, modulation of Pellino3 may provide a new strategy for the development of a therapy approach in sepsis.
Attenuated NOX2 expression impairs ROS production during the hypoinflammatory phase of sepsis
(2012)
Background: The multicomponent phagocytic NADPH oxidase produces reactive oxygen species (ROS) after activation by microorganisms or inflammatory mediators. In the hypoinflammatory phase of sepsis, macrophages are alternatively activated by contact with apoptotic cells or their secretion products. This inhibits NADPH oxidase and leads to attenuated ROS production and furthermore contributes among others to a hyporeactive host defense. Due to this immune paralysis, sepsis patients suffer from recurrent and secondary infections. We focused on the catalytic subunit of NADPH oxidase, the transmembrane protein NOX2. We assume that after induction of sepsis the expression of NOX2 is reduced and hence ROS production is decreased.
Methods: We induced polymicrobial sepsis in mice by cecal ligation and puncture. The ability of peritoneal macrophages (PMs) to produce ROS was determined by FACS via hydroethidine assay. NOX2 expression of PMs was determined by western blot and qPCR. To elucidate the mechanism causing mRNA destabilization, we performed in vitro experiments using J774 macrophages. To obtain an alternatively activated phenotype, macrophages were stimulated with conditioned medium from apoptotic T cells (CM). By luciferase assays we figured out a 3'UTR-dependent regulation of NOX2 mRNA stability. Assuming that a protein is involved in the mRNA degradation, we performed a RNA pulldown with biotinylated NOX2-3'UTR constructs followed by mass spectrometry. We verified the role of SYNCRIP by siRNA approach. Additionally, we overexpressed NOX2 in J774 cells and analyzed the ROS production (w/wo CM treatment) by FACS.
Results: We found an impaired expression of NOX2 at RNA and protein level along with decreased ROS production after induction of sepsis in mice as well as stimulating J774 macrophages with CM of apoptotic T cells. This is due to a time-dependent NOX2 mRNA degradation depending on SYNCRIP, a RNA-binding protein, which stabilizes NOX2 mRNA through binding to its 3'UTR under normal conditions. In line, knockdown of SYNCRIP also decreases NOX2 mRNA expression. We assume that a CM-dependent modification or degradation of SYNCRIP prevents its stabilizing function. As the overexpression of NOX2 restores ROS production of CM-treated J774 cells, we assume that NOX2 expression is crucial for maintaining NADPH activity during the hypoinflammatory phase of sepsis.
Conclusion: Our data imply a regulatory impact of SYNCRIP on NOX2 stability during the late phase of sepsis. Therefore, further understanding of the regulation of NADPH oxidase could lead to the design of a therapy to reconstitute NADPH oxidase function, finally improving immune function in sepsis patients.
Background: The ligand-activated transcription factor, peroxisome-proliferator-activated receptor gamma (PPARγ), has been shown to play an essential role in immunosuppression during sepsis. PPARγ is upregulated in T cells of septic patients, sensitizing these cells to PPARγ-dependent apoptosis and thus contributing to T-cell depletion. In the polymicrobial cecum ligation and puncture (CLP) sepsis model in mice, both T-cell-specific gene knockout (Lck-Cre PPARγfl/fl) and systemic pharmacological PPARγ antagonism by GW9662 improved survival. Because GW9662 was only effective when applied 3 hours after CLP, we were interested to extend this time frame. For this reason we characterized the kinetics of SPPARγMs when administered before or in combination with the agonist thiazolidinedione, rosiglitazone.
Methods: A PPARγ-dependent transactivation assay was used in HEK293T cells. It is based on the vector pFA-PPARγ-LBD-GAL4-DBD encoding the hybrid protein PPARγ-LBD-GAL4-DBD and the reporter vector pFR-Luc, carrying a GAL4-responsive element in front of the Firefly luciferase gene. These two vectors were co-transfected, in combination with a control vector encoding Renilla luciferase (pRL-CMV) to normalize Firefly luciferase activity for transfection efficiency. Following transfection, cells were incubated with the SPPARγMs F-MOC and MCC-555 and the PPARγ antagonist GW9662 for different times (2 to 48 hours) and at increasing doses (0.01 to 10 μM), with or without rosiglitazone (0.01 to 10 μM). Transactivation was analyzed using a 96-well plate format.
Results: Rosiglitazone transactivated PPARγ in a time-dependent and dose-dependent manner, the response gradually increasing to a maximum at 48 hours with 10 μM. Low concentrations (0.01 to 0.1 μM) of SPPARγMs F-MOC and MCC-555 and the PPARγ antagonist GW9662 all exerted dose-independent antagonistic effects at an early incubation time point (2 hours). From 10 hours onwards, MCC-555 and GW9662, given alone, both exerted PPARγ agonistic effects, MCC-555 in parallel to responses to rosiglitazone, but GW9662 with characteristics of partial antagonism. F-MOC showed no dose-dependent effect at any concentration at later time points. Only GW9662 (1 to 10 μM) was able to inhibit rosiglitazone (0.1 to 1 μM)-induced PPARγ transactivation after 10 hours.
Conclusion: Our kinetic analysis reveals clear differences in the modulatory characteristics of PPARγ inhibitors, with previously unreported early inhibitory effects and late agonistic or partial agonistic activity. New SPPARγMs with extended inhibitory activity may prove useful in the therapy of sepsis.
Ubiquitination relies on a subtle balance between selectivity and promiscuity achieved through specific interactions between ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). Here, we report how a single aspartic to glutamic acid substitution acts as a dynamic switch to tip the selectivity balance of human E2s for interaction toward E3 RING-finger domains. By combining molecular dynamic simulations, experimental yeast-two-hybrid screen of E2-E3 (RING) interactions and mutagenesis, we reveal how the dynamics of an internal salt-bridge network at the rim of the E2-E3 interaction surface controls the balance between an “open”, binding competent, and a “closed”, binding incompetent state. The molecular dynamic simulations shed light on the fine mechanism of this molecular switch and allowed us to identify its components, namely an aspartate/glutamate pair, a lysine acting as the central switch and a remote aspartate. Perturbations of single residues in this network, both inside and outside the interaction surface, are sufficient to switch the global E2 interaction selectivity as demonstrated experimentally. Taken together, our results indicate a new mechanism to control E2-E3 interaction selectivity at an atomic level, highlighting how minimal changes in amino acid side-chain affecting the dynamics of intramolecular salt-bridges can be crucial for protein-protein interactions. These findings indicate that the widely accepted sequence-structure-function paradigm should be extended to sequence-structure-dynamics-function relationship and open new possibilities for control and fine-tuning of protein interaction selectivity.
Measles virus glycoprotein-based lentiviral targeting vectors that avoid neutralizing antibodies
(2012)
Lentiviral vectors (LVs) are potent gene transfer vehicles frequently applied in research and recently also in clinical trials. Retargeting LV entry to cell types of interest is a key issue to improve gene transfer safety and efficacy. Recently, we have developed a targeting method for LVs by incorporating engineered measles virus (MV) glycoproteins, the hemagglutinin (H), responsible for receptor recognition, and the fusion protein into their envelope. The H protein displays a single-chain antibody (scFv) specific for the target receptor and is ablated for recognition of the MV receptors CD46 and SLAM by point mutations in its ectodomain. A potential hindrance to systemic administration in humans is pre-existing MV-specific immunity due to vaccination or natural infection. We compared transduction of targeting vectors and non-targeting vectors pseudotyped with MV glycoproteins unmodified in their ectodomains (MV-LV) in presence of α-MV antibody-positive human plasma. At plasma dilution 1:160 MV-LV was almost completely neutralized, whereas targeting vectors showed relative transduction efficiencies from 60% to 90%. Furthermore, at plasma dilution 1:80 an at least 4-times higher multiplicity of infection (MOI) of MV-LV had to be applied to obtain similar transduction efficiencies as with targeting vectors. Also when the vectors were normalized to their p24 values, targeting vectors showed partial protection against α-MV antibodies in human plasma. Furthermore, the monoclonal neutralizing antibody K71 with a putative epitope close to the receptor binding sites of H, did not neutralize the targeting vectors, but did neutralize MV-LV. The observed escape from neutralization may be due to the point mutations in the H ectodomain that might have destroyed antibody binding sites. Furthermore, scFv mediated cell entry via the target receptor may proceed in presence of α-MV antibodies interfering with entry via the natural MV receptors. These results are promising for in vivo applications of targeting vectors in humans.
The C. elegans nervous system is particularly well suited for optogenetic analyses of circuit function: Essentially all connections have been mapped, and light can be directed at the neuron of interest in the freely moving, transparent animals, while behavior is observed. Thus, different nodes of a neuronal network can be probed for their role in controlling a particular behavior, using different optogenetic tools for photo-activation or –inhibition, which respond to different colors of light. As neurons may act in concert or in opposing ways to affect a behavior, one would further like to excite these neurons concomitantly, yet independent of each other. In addition to the blue-light activated Channelrhodopsin-2 (ChR2), spectrally red-shifted ChR variants have been explored recently. Here, we establish the green-light activated ChR chimera C1V1 (from Chlamydomonas and Volvox ChR1′s) for use in C. elegans. We surveyed a number of red-shifted ChRs, and found that C1V1-ET/ET (E122T; E162T) works most reliable in C. elegans, with 540–580 nm excitation, which leaves ChR2 silent. However, as C1V1-ET/ET is very light sensitive, it still becomes activated when ChR2 is stimulated, even at 400 nm. Thus, we generated a highly efficient blue ChR2, the H134R; T159C double mutant (ChR2-HR/TC). Both proteins can be used in the same animal, in different neurons, to independently control each cell type with light, enabling a further level of complexity in circuit analyses.
Loss of the tumor suppressor Pdcd4 was reported for various tumor entities and proposed as a prognostic marker in tumorigenesis. We previously characterized decreased Pdcd4 protein stability in response to mitogenic stimuli, which resulted from p70S6K1-dependent protein phosphorylation, β-TrCP1-mediated ubiquitination, and proteasomal destruction. Following high-throughput screening of natural product extract libraries using a luciferase-based reporter assay to monitor phosphorylation-dependent proteasomal degradation of the tumor suppressor Pdcd4, we succeeded in showing that a crude extract from Eriophyllum lanatum stabilized Pdcd4 from TPA-induced degradation. Erioflorin was identified as the active component and inhibited not only degradation of the Pdcd4-luciferase-based reporter but also of endogenous Pdcd4 at low micromolar concentrations. Mechanistically, erioflorin interfered with the interaction between the E3-ubiquitin ligase β-TrCP1 and Pdcd4 in cell culture and in in vitro binding assays, consequently decreasing ubiquitination and degradation of Pdcd4. Interestingly, while erioflorin stabilized additional β-TrCP-targets (such as IκBα and β-catenin), it did not prevent the degradation of targets of other E3-ubiquitin ligases such as p21 (a Skp2-target) and HIF-1α (a pVHL-target), implying selectivity for β-TrCP. Moreover, erioflorin inhibited the tumor-associated activity of known Pdcd4- and IκBα-regulated αtranscription factors, that is, AP-1 and NF-κB, altered cell cycle progression and suppressed proliferation of various cancer cell lines. Our studies succeeded in identifying erioflorin as a novel Pdcd4 stabilizer that inhibits the interaction of Pdcd4 with the E3-ubiquitin ligase β-TrCP1. Inhibition of E3-ligase/target-protein interactions may offer the possibility to target degradation of specific proteins only as compared to general proteasome inhibition.
Riboswitch RNAs fold into complex tertiary structures upon binding to their cognate ligand. Ligand recognition is accomplished by key residues in the binding pocket. In addition, it often crucially depends on the stability of peripheral structural elements. The ligand-bound complex of the guanine-sensing riboswitch from Bacillus subtilis, for example, is stabilized by extensive interactions between apical loop regions of the aptamer domain. Previously, we have shown that destabilization of this tertiary loop–loop interaction abrogates ligand binding of the G37A/C61U-mutant aptamer domain (Gswloop) in the absence of Mg2+. However, if Mg2+ is available, ligand-binding capability is restored by a population shift of the ground-state RNA ensemble toward RNA conformations with pre-formed loop–loop interactions. Here, we characterize the striking influence of long-range tertiary structure on RNA folding kinetics and on ligand-bound complex structure, both by X-ray crystallography and time-resolved NMR. The X-ray structure of the ligand-bound complex reveals that the global architecture is almost identical to the wild-type aptamer domain. The population of ligand-binding competent conformations in the ground-state ensemble of Gswloop is tunable through variation of the Mg2+ concentration. We quantitatively describe the influence of distinct Mg2+ concentrations on ligand-induced folding trajectories both by equilibrium and time-resolved NMR spectroscopy at single-residue resolution.
Optogenetic approaches using light-activated proteins like Channelrhodopsin-2 (ChR2) enable investigating the function of populations of neurons in live Caenorhabditis elegans (and other) animals, as ChR2 expression can be targeted to these cells using specific promoters. Sub-populations of these neurons, or even single cells, can be further addressed by restricting the illumination to the cell of interest. However, this is technically demanding, particularly in free moving animals. Thus, it would be helpful if expression of ChR2 could be restricted to single neurons or neuron pairs, as even wide-field illumination would photostimulate only this particular cell. To this end we adopted the use of Cre or FLP recombinases and conditional ChR2 expression at the intersection of two promoter expression domains, i.e. in the cell of interest only. Success of this method depends on precise knowledge of the individual promoters' expression patterns and on relative expression levels of recombinase and ChR2. A bicistronic expression cassette with GFP helps to identify the correct expression pattern. Here we show specific expression in the AVA reverse command neurons and the aversive polymodal sensory ASH neurons. This approach shall enable to generate strains for optogenetic manipulation of each of the 302 C. elegans neurons. This may eventually allow to model the C. elegans nervous system in its entirety, based on functional data for each neuron.
Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1
(2011)
The respiratory chain in the inner mitochondrial membrane contains three large multi-enzyme complexes that together establish the proton gradient for ATP synthesis, and assemble into a supercomplex. A 19-Å 3D map of the 1.7-MDa amphipol-solubilized supercomplex I1III2IV1 from bovine heart obtained by single-particle electron cryo-microscopy reveals an amphipol belt replacing the membrane lipid bilayer. A precise fit of the X-ray structures of complex I, the complex III dimer, and monomeric complex IV indicates distances of 13 nm between the ubiquinol-binding sites of complexes I and III, and of 10–11 nm between the cytochrome c binding sites of complexes III and IV. The arrangement of respiratory chain complexes suggests two possible pathways for efficient electron transfer through the supercomplex, of which the shorter branch through the complex III monomer proximal to complex I may be preferred.
Atomic-level analyses of non-native protein ensembles constitute an important aspect of protein folding studies to reach a more complete understanding of how proteins attain their native form exhibiting biological activity. Previously, formation of hydrophobic clusters in the 6 M urea-denatured state of an ultrafast folding mini-protein known as TC5b from both photo-CIDNP NOE transfer studies and FCS measurements was observed. Here, we elucidate the structural properties of this mini-protein denatured in 6 M urea performing 15N NMR relaxation studies together with a thorough NOE analysis. Even though our results demonstrate that no elements of secondary structure persist in the denatured state, the heterogeneous distribution of R2 rate constants together with observing pronounced heteronuclear NOEs along the peptide backbone reveals specific regions of urea-denatured TC5b exhibiting a high degree of structural rigidity more frequently observed for native proteins. The data are complemented with studies on two TC5b point mutants to verify the importance of hydrophobic interactions for fast folding. Our results corroborate earlier findings of a hydrophobic cluster present in urea-denatured TC5b comprising both native and non-native contacts underscoring their importance for ultra rapid folding. The data assist in finding ways of interpreting the effects of pre-existing native and/or non-native interactions on the ultrafast folding of proteins; a fact, which might have to be considered when defining the starting conditions for molecular dynamics simulation studies of protein folding.
Essentially any behavior in simple and complex animals depends on neuronal network function. Currently, the best-defined system to study neuronal circuits is the nematode Caenorhabditis elegans, as the connectivity of its 302 neurons is exactly known. Individual neurons can be activated by photostimulation of Channelrhodopsin-2 (ChR2) using blue light, allowing to directly probe the importance of a particular neuron for the respective behavioral output of the network under study. In analogy, other excitable cells can be inhibited by expressing Halorhodopsin from Natronomonas pharaonis (NpHR) and subsequent illumination with yellow light. However, inhibiting C. elegans neurons using NpHR is difficult. Recently, proton pumps from various sources were established as valuable alternative hyperpolarizers. Here we show that archaerhodopsin-3 (Arch) from Halorubrum sodomense and a proton pump from the fungus Leptosphaeria maculans (Mac) can be utilized to effectively inhibit excitable cells in C. elegans. Arch is the most powerful hyperpolarizer when illuminated with yellow or green light while the action spectrum of Mac is more blue-shifted, as analyzed by light-evoked behaviors and electrophysiology. This allows these tools to be combined in various ways with ChR2 to analyze different subsets of neurons within a circuit. We exemplify this by means of the polymodal aversive sensory ASH neurons, and the downstream command interneurons to which ASH neurons signal to trigger a reversal followed by a directional turn. Photostimulating ASH and subsequently inhibiting command interneurons using two-color illumination of different body segments, allows investigating temporal aspects of signaling downstream of ASH.
Using an electrophysiological assay the activity of NhaA was tested in a wide pH range from pH 5.0 to 9.5. Forward and reverse transport directions were investigated at zero membrane potential using preparations with inside-out and right side-out-oriented transporters with Na+ or H+ gradients as the driving force. Under symmetrical pH conditions with a Na+ gradient for activation, both the wt and the pH-shifted G338S variant exhibit highly symmetrical transport activity with bell-shaped pH dependences, but the optimal pH was shifted 1.8 pH units to the acidic range in the variant. In both strains the pH dependence was associated with a systematic increase of the Km for Na+ at acidic pH. Under symmetrical Na+ concentration with a pH gradient for NhaA activation, an unexpected novel characteristic of the antiporter was revealed; rather than being down-regulated, it remained active even at pH as low as 5. These data allowed a transport mechanism to advance based on competing Na+ and H+ binding to a common transport site and a kinetic model to develop quantitatively explaining the experimental results. In support of these results, both alkaline pH and Na+ induced the conformational change of NhaA associated with NhaA cation translocation as demonstrated here by trypsin digestion. Furthermore, Na+ translocation was found to be associated with the displacement of a negative charge. In conclusion, the electrophysiological assay allows the revelation of the mechanism of NhaA antiport and sheds new light on the concept of NhaA pH regulation.
The conformational dynamics induced by ligand binding to the tetracycline-binding aptamer is monitored via stopped-flow fluorescence spectroscopy and time-correlated single photon counting experiments. The fluorescence of the ligand is sensitive to changes within the tertiary structure of the aptamer during and after the binding process. In addition to the wild-type aptamer, the mutants A9G, A13U and A50U are examined, where bases important for regulation are changed to inhibit the aptamer’s function. Our results suggest a very fast two-step-mechanism for the binding of the ligand to the aptamer that can be interpreted as a binding step followed by a reorganization of the aptamer to accommodate the ligand. Binding to the two direct contact points A13 and A50 was found to occur in the first binding step. The exchange of the structurally important base A9 for guanine induces an enormous deceleration of the overall binding process, which is mainly rooted in an enhancement of the back reaction of the first binding step by several orders of magnitude. This indicates a significant loss of tertiary structure of the aptamer in the absence of the base A9, and underlines the importance of pre-organization on the overall binding process of the tetracycline-binding aptamer.
Sucrose- and H+-dependent charge movements associated with the gating of sucrose transporter ZmSUT1
(2010)
Background: In contrast to man the majority of higher plants use sucrose as mobile carbohydrate. Accordingly proton-driven sucrose transporters are crucial for cell-to-cell and long-distance distribution within the plant body. Generally very negative plant membrane potentials and the ability to accumulate sucrose quantities of more than 1 M document that plants must have evolved transporters with unique structural and functional features.
Methodology/Principal Findings: To unravel the functional properties of one specific high capacity plasma membrane sucrose transporter in detail, we expressed the sucrose/H+ co-transporter from maize ZmSUT1 in Xenopus oocytes. Application of sucrose in an acidic pH environment elicited inward proton currents. Interestingly the sucrose-dependent H+ transport was associated with a decrease in membrane capacitance (Cm). In addition to sucrose Cm was modulated by the membrane potential and external protons. In order to explore the molecular mechanism underlying these Cm changes, presteady-state currents (Ipre) of ZmSUT1 transport were analyzed. Decay of Ipre could be best fitted by double exponentials. When plotted against the voltage the charge Q, associated to Ipre, was dependent on sucrose and protons. The mathematical derivative of the charge Q versus voltage was well in line with the observed Cm changes. Based on these parameters a turnover rate of 500 molecules sucrose/s was calculated. In contrast to gating currents of voltage dependent-potassium channels the analysis of ZmSUT1-derived presteady-state currents in the absence of sucrose (I = Q/τ) was sufficient to predict ZmSUT1 transport-associated currents.
Conclusions: Taken together our results indicate that in the absence of sucrose, ‘trapped’ protons move back and forth between an outer and an inner site within the transmembrane domains of ZmSUT1. This movement of protons in the electric field of the membrane gives rise to the presteady-state currents and in turn to Cm changes. Upon application of external sucrose, protons can pass the membrane turning presteady-state into transport currents.
5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes (LT), a group of inflammatory lipid mediators derived from arachidonic acid. Here, we investigated the regulation of 5-LO mRNA expression by alternative splicing and nonsense-mediated mRNA decay (NMD). In the present study, we report the identification of 2 truncated transcripts and 4 novel 5-LO splice variants containing premature termination codons (PTC). The characterization of one of the splice variants, 5-LOΔ3, revealed that it is a target for NMD since knockdown of the NMD factors UPF1, UPF2 and UPF3b in the human monocytic cell line Mono Mac 6 (MM6) altered the expression of 5-LOΔ3 mRNA up to 2-fold in a cell differentiation-dependent manner suggesting that cell differentiation alters the composition or function of the NMD complex. In contrast, the mature 5-LO mRNA transcript was not affected by UPF knockdown. Thus, the data suggest that the coupling of alternative splicing and NMD is involved in the regulation of 5-LO gene expression.
We present a computational method for the reaction-based de novo design of drug-like molecules. The software DOGS (Design of Genuine Structures) features a ligand-based strategy for automated ‘in silico’ assembly of potentially novel bioactive compounds. The quality of the designed compounds is assessed by a graph kernel method measuring their similarity to known bioactive reference ligands in terms of structural and pharmacophoric features. We implemented a deterministic compound construction procedure that explicitly considers compound synthesizability, based on a compilation of 25'144 readily available synthetic building blocks and 58 established reaction principles. This enables the software to suggest a synthesis route for each designed compound. Two prospective case studies are presented together with details on the algorithm and its implementation. De novo designed ligand candidates for the human histamine H4 receptor and γ-secretase were synthesized as suggested by the software. The computational approach proved to be suitable for scaffold-hopping from known ligands to novel chemotypes, and for generating bioactive molecules with drug-like properties.
Ubiquitin ligases and beyond
(2012)
First paragraph (this article has no abstract): In a review published in 2004 [1] and that still repays reading today, Cecile Pickart traced the evolution of research on ubiquitination from its origins in the proteasomal degradation of proteins through the revelation that it has a central role in cell cycle regulation and the recognition of regulatory roles for ubiquitin in intracellular membrane transport, cell signalling, transcription, translation, and DNA repair.
Fibroblast growth factor receptor substrate 2 (FRS2α) is a signaling adaptor protein that regulates downstream signaling of many receptor tyrosine kinases. During signal transduction, FRS2 can be both tyrosine and threonine phosphorylated and forms signaling complexes with other adaptor proteins and tyrosine phosphatases. We have here identified flotillin-1 and the cbl-associated protein/ponsin (CAP) as novel interaction partners of FRS2. Flotillin-1 binds to the phosphotyrosine binding domain (PTB) of FRS2 and competes for the binding with the fibroblast growth factor receptor. Flotillin-1 knockdown results in increased Tyr phosphorylation of FRS2, in line with the inhibition of ERK activity in the absence of flotillin-1. CAP directly interacts with FRS2 by means of its sorbin homology (SoHo) domain, which has previously been shown to interact with flotillin-1. In addition, the third SH3 domain in CAP binds to FRS2. Due to the overlapping binding domains, CAP and flotillin-1 appear to compete for the binding to FRS2. Thus, our results reveal a novel signaling network containing FRS2, CAP and flotillin-1, whose successive interactions are most likely required to regulate receptor tyrosine kinase signaling, especially the mitogen activated protein kinase pathway.
Bacterial porin disrupts mitochondrial membrane potential and sensitizes host cells to apoptosis
(2009)
The bacterial PorB porin, an ATP-binding beta-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (delta psi m). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of beta-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of delta psi m. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce delta psi m loss and apoptosis, demonstrating that dissipation of delta psi m is a requirement for cell death caused by neisserial infection.
Background: Threonine Aspartase 1 (Taspase1) mediates cleavage of the mixed lineage leukemia (MLL) protein and leukemia provoking MLL-fusions. In contrast to other proteases, the understanding of Taspase1's (patho)biological relevance and function is limited, since neither small molecule inhibitors nor cell based functional assays for Taspase1 are currently available. Methodology/Findings: Efficient cell-based assays to probe Taspase1 function in vivo are presented here. These are composed of glutathione S-transferase, autofluorescent protein variants, Taspase1 cleavage sites and rational combinations of nuclear import and export signals. The biosensors localize predominantly to the cytoplasm, whereas expression of biologically active Taspase1 but not of inactive Taspase1 mutants or of the protease Caspase3 triggers their proteolytic cleavage and nuclear accumulation. Compared to in vitro assays using recombinant components the in vivo assay was highly efficient. Employing an optimized nuclear translocation algorithm, the triple-color assay could be adapted to a high-throughput microscopy platform (Z'factor = 0.63). Automated high-content data analysis was used to screen a focused compound library, selected by an in silico pharmacophor screening approach, as well as a collection of fungal extracts. Screening identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamideand 2-benzyltriazole-4,5-dicarboxylic acid, which partially inhibited Taspase1 cleavage in living cells. Additionally, the assay was exploited to probe endogenous Taspase1 in solid tumor cell models and to identify an improved consensus sequence for efficient Taspase1 cleavage. This allowed the in silico identification of novel putative Taspase1 targets. Those include the FERM Domain-Containing Protein 4B, the Tyrosine-Protein Phosphatase Zeta, and DNA Polymerase Zeta. Cleavage site recognition and proteolytic processing of these substrates were verified in the context of the biosensor. Conclusions: The assay not only allows to genetically probe Taspase1 structure function in vivo, but is also applicable for high-content screening to identify Taspase1 inhibitors. Such tools will provide novel insights into Taspase1's function and its potential therapeutic relevance.
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.
Reciprocal t(9;22) ABL/BCR fusion proteins: leukemogenic potential and effects on B cell commitment
(2009)
Background: t(9;22) is a balanced translocation, and the chromosome 22 breakpoints (Philadelphia chromosome – Ph+) determine formation of different fusion genes that are associated with either Ph+ acute lymphatic leukemia (Ph+ ALL) or chronic myeloid leukemia (CML). The "minor" breakpoint in Ph+ ALL encodes p185BCR/ABL from der22 and p96ABL/BCR from der9. The "major" breakpoint in CML encodes p210BCR/ABL and p40ABL/BCR. Herein, we investigated the leukemogenic potential of the der9-associated p96ABL/BCR and p40ABL/BCR fusion proteins and their roles in the lineage commitment of hematopoietic stem cells in comparison to BCR/ABL. Methodology: All t(9;22) derived proteins were retrovirally expressed in murine hematopoietic stem cells (SL cells) and human umbilical cord blood cells (UCBC). Stem cell potential was determined by replating efficiency, colony forming - spleen and competitive repopulating assays. The leukemic potential of the ABL/BCR fusion proteins was assessed by in a transduction/transplantation model. Effects on the lineage commitment and differentiation were investigated by culturing the cells under conditions driving either myeloid or lymphoid commitment. Expression of key factors of the B-cell differentiation and components of the preB-cell receptor were determined by qRT-PCR. Principal Findings: Both p96ABL/BCR and p40ABL/BCR increased proliferation of early progenitors and the short term stem cell capacity of SL-cells and exhibited own leukemogenic potential. Interestingly, BCR/ABL gave origin exclusively to a myeloid phenotype independently from the culture conditions whereas p96ABL/BCR and to a minor extent p40ABL/BCR forced the B-cell commitment of SL-cells and UCBC. Conclusions/Significance: Our here presented data establish the reciprocal ABL/BCR fusion proteins as second oncogenes encoded by the t(9;22) in addition to BCR/ABL and suggest that ABL/BCR contribute to the determination of the leukemic phenotype through their influence on the lineage commitment.
Background: The human pathogen Helicobacter pylori (H. pylori) is a main cause for gastric inflammation and cancer. Increasing bacterial resistance against antibiotics demands for innovative strategies for therapeutic intervention. Methodology/Principal Findings: We present a method for structure-based virtual screening that is based on the comprehensive prediction of ligand binding sites on a protein model and automated construction of a ligand-receptor interaction map. Pharmacophoric features of the map are clustered and transformed in a correlation vector (‘virtual ligand’) for rapid virtual screening of compound databases. This computer-based technique was validated for 18 different targets of pharmaceutical interest in a retrospective screening experiment. Prospective screening for inhibitory agents was performed for the protease HtrA from the human pathogen H. pylori using a homology model of the target protein. Among 22 tested compounds six block E-cadherin cleavage by HtrA in vitro and result in reduced scattering and wound healing of gastric epithelial cells, thereby preventing bacterial infiltration of the epithelium. Conclusions/Significance: This study demonstrates that receptor-based virtual screening with a permissive (‘fuzzy’) pharmacophore model can help identify small bioactive agents for combating bacterial infection.
Chlamydia are obligate intracellular bacteria that cause variety of human diseases. Host cells infected with Chlamydia are protected against many different apoptotic stimuli. The induction of apoptosis resistance is thought to be an important immune escape mechanism allowing Chlamydia to replicate inside the host cell. Infection with C. trachomatis activates the Raf/MEK/ERK pathway and the PI3K/AKT pathway. Here we show that inhibition of these two pathways by chemical inhibitors sensitized C. trachomatis infected cells to granzyme B-mediated cell death. Infection leads to the Raf/MEK/ERK-mediated up-regulation and PI3K-dependent stabilization of the anti-apoptotic Bcl-2 family member Mcl-1. Consistently, interfering with Mcl-1 up-regulation sensitized infected cells for apoptosis induced via the TNF receptor, DNA damage, granzyme B and stress. Our data suggest that Mcl-1 up-regulation is primarily required to maintain apoptosis resistance in C. trachomatis-infected cells.
The continuous progress in the structural and functional characterization of aquaporins increasingly attracts attention to study their roles in certain mammalian diseases. Although several structures of aquaporins have already been solved by crystallization, the challenge of producing sufficient amounts of functional proteins still remains. CF (cell free) expression has emerged in recent times as a promising alternative option in order to synthesize large quantities of membrane proteins, and the focus of this report was to evaluate the potential of this technique for the production of eukaryotic aquaporins. We have selected the mouse aquaporin 4 as a representative of mammalian aquaporins. The protein was synthesized in an E. coli extract based cell-free system with two different expression modes, and the efficiencies of two modes were compared. In both, the P-CF (cell-free membrane protein expression as precipitate) mode generating initial aquaporin precipitates as well as in the D-CF (cell-free membrane protein expression in presence of detergent) mode, generating directly detergent solubilized samples, we were able to obtain mg amounts of protein per ml of cell-free reaction. Purified aquaporin samples solubilized in different detergents were reconstituted into liposomes, and analyzed for the water channel activity. The calculated Pf value of proteoliposome samples isolated from the D-CF mode was 133 µm/s at 10°C, which was 5 times higher as that of the control. A reversible inhibitory effect of mercury chloride was observed, which is consistent with previous observations of in vitro reconstituted aquaporin 4. In this study, a fast and convenient protocol was established for functional expression of aquaporins, which could serve as basis for further applications such as water filtration.
Ataxia represents a pathological coordination failure that often involves functional disturbances in cerebellar circuits. Purkinje cells (PCs) characterize the only output neurons of the cerebellar cortex and critically participate in regulating motor coordination. Although different genetic mutations are known that cause ataxia, little is known about the underlying cellular mechanisms. Here we show that a mutated axJ gene locus, encoding the ubiquitin-specific protease 14 (Usp14), negatively influences synaptic receptor turnover. AxJ mouse mutants, characterized by cerebellar ataxia, display both increased GABAA receptor (GABAAR) levels at PC surface membranes accompanied by enlarged IPSCs. Accordingly, we identify physical interaction of Usp14 and the GABAAR alpha 1 subunit. Although other currently unknown changes might be involved, our data show that ubiquitin-dependent GABAAR turnover at cerebellar synapses contributes to axJ-mediated behavioural impairment.
Mitochondrial complex I, the largest and most complicated proton pump of the respiratory chain, links the electron transfer from NADH to ubiquinone to the pumping of four protons from the matrix into the intermembrane space. In humans, defects in complex I are involved in a wide range of degenerative disorders. Recent progress in the X-ray structural analysis of prokaryotic and eukaryotic complex I confirmed that the redox reactions are confined entirely to the hydrophilic peripheral arm of the L-shaped molecule and take place at a remarkable distance from the membrane domain. While this clearly implies that the proton pumping within the membrane arm of complex I is driven indirectly via long-range conformational coupling, the molecular mechanism and the number, identity, and localization of the pump-sites remains unclear. Here, we report that upon deletion of the gene for a small accessory subunit of the Yarrowia complex I, a stable subcomplex (nb8m delta) is formed that lacks the distal part of the membrane domain as revealed by single particle analysis. The analysis of the subunit composition of holo and subcomplex by three complementary proteomic approaches revealed that two (ND4 and ND5) of the three subunits with homology to bacterial Mrp-type Na+/H+ antiporters that have been discussed as prime candidates for harbouring the proton pumps were missing in nb8m delta. Nevertheless, nb8m delta still pumps protons at half the stoichiometry of the complete enzyme. Our results provide evidence that the membrane arm of complex I harbours two functionally distinct pump modules that are connected in series by the long helical transmission element recently identified by X-ray structural analysis.
Channelrhodopsin-2 (ChR2) is widely used for rapid photodepolarization of neurons, yet, as it requires high-intensity blue light for activation, it is not suited for long-term in vivo applications, e.g. for manipulations of behavior, or photoactivation of neurons during development. We used “slow” ChR2 variants with mutations in the C128 residue, that exhibit delayed off-kinetics and increased light sensitivity in Caenorhabditis elegans. Following a 1 s light pulse, we could photodepolarize neurons and muscles for minutes (and with repeated brief stimulation, up to days) with low-intensity light. Photoactivation of ChR2(C128S) in command interneurons elicited long-lasting alterations in locomotion. Finally, we could optically induce profound changes in animal development: Long-term photoactivation of ASJ neurons, which regulate larval growth, bypassed the constitutive entry into the “dauer” larval state in daf-11 mutants. These lack a guanylyl cyclase, which possibly renders ASJ neurons hyperpolarized. Furthermore, photostimulated ASJ neurons could acutely trigger dauer-exit. Thus, slow ChR2s can be employed to long-term photoactivate behavior and to trigger alternative animal development.
Structured RNA regions are important gene control elements in prokaryotes and eukaryotes. Here, we show that the mRNA of a cyanobacterial heat shock gene contains a built-in thermosensor critical for photosynthetic activity under stress conditions. The exceptionally short 5´-untranslated region is comprised of a single hairpin with an internal asymmetric loop. It inhibits translation of the Synechocystis hsp17 transcript at normal growth conditions, permits translation initiation under stress conditions and shuts down Hsp17 production in the recovery phase. Point mutations that stabilized or destabilized the RNA structure deregulated reporter gene expression in vivo and ribosome binding in vitro. Introduction of such point mutations into the Synechocystis genome produced severe phenotypic defects. Reversible formation of the open and closed structure was beneficial for viability, integrity of the photosystem and oxygen evolution. Continuous production of Hsp17 was detrimental when the stress declined indicating that shutting-off heat shock protein production is an important, previously unrecognized function of RNA thermometers. We discovered a simple biosensor that strictly adjusts the cellular level of a molecular chaperone to the physiological need.
The mfl-riboswitch regulates expression of ribonucleotide reductase subunit in Mesoplasma florum by binding to 2´-deoxyguanosine and thereby promoting transcription termination. We characterized the structure of the ligand-bound aptamer domain by NMR spectroscopy and compared the mfl-aptamer to the aptamer domain of the closely related purine-sensing riboswitches. We show that the mfl-aptamer accommodates the extra 2´-deoxyribose unit of the ligand by forming a more relaxed binding pocket than these found in the purine-sensing riboswitches. Tertiary structures of the xpt-aptamer bound to guanine and of the mfl-aptamer bound to 2´-deoxyguanosine exhibit very similar features, although the sequence of the mfl-aptamer contains several alterations compared to the purine-aptamer consensus sequence. These alterations include the truncation of a hairpin loop which is crucial for complex formation in all purine-sensing riboswitches characterized to date. We further defined structural features and ligand binding requirements of the free mfl-aptamer and found that the presence of Mg2+ is not essential for complex formation, but facilitates ligand binding by promoting pre-organization of key structural motifs in the free aptamer.
Background: The automation of objectively selecting amino acid residue ranges for structure superpositions is important for meaningful and consistent protein structure analyses. So far there is no widely-used standard for choosing these residue ranges for experimentally determined protein structures, where the manual selection of residue ranges or the use of suboptimal criteria remain commonplace. Results: We present an automated and objective method for finding amino acid residue ranges for the superposition and analysis of protein structures, in particular for structure bundles resulting from NMR structure calculations. The method is implemented in an algorithm, CYRANGE, that yields, without protein-specific parameter adjustment, appropriate residue ranges in most commonly occurring situations, including low-precision structure bundles, multi-domain proteins, symmetric multimers, and protein complexes. Residue ranges are chosen to comprise as many residues of a protein domain that increasing their number would lead to a steep rise in the RMSD value. Residue ranges are determined by first clustering residues into domains based on the distance variance matrix, and then refining for each domain the initial choice of residues by excluding residues one by one until the relative decrease of the RMSD value becomes insignificant. A penalty for the opening of gaps favours contiguous residue ranges in order to obtain a result that is as simple as possible, but not simpler. Results are given for a set of 37 proteins and compared with those of commonly used protein structure validation packages. We also provide residue ranges for 6351 NMR structures in the Protein Data Bank. Conclusions: The CYRANGE method is capable of automatically determining residue ranges for the superposition of protein structure bundles for a large variety of protein structures. The method correctly identifies ordered regions. Global structure superpositions based on the CYRANGE residue ranges allow a clear presentation of the structure, and unnecessary small gaps within the selected ranges are absent. In the majority of cases, the residue ranges from CYRANGE contain fewer gaps and cover considerably larger parts of the sequence than those from other methods without significantly increasing the RMSD values. CYRANGE thus provides an objective and automatic method for standardizing the choice of residue ranges for the superposition of protein structures. Additional files Additional file 1: Dependence of Q on the order parameter rank. The quantity Qi is plotted against the order parameter rank i for 9 different protein structure bundles. Additional file 2: Dependence of P on the clustering stage. The quantity Pi is plotted against the clustering stage i for 9 different protein structure bundles. Additional file 3: Dependence of CYRANGE results on the minimal cluster size parameter my. The sequence coverage (red) and RMSD (blue) of the residue ranges determined by CYRANGE were plotted as a function of my for 9 different protein structure bundles. The dotted vertical line indicates the default value, my = 8. Where CYRANGE found two domains, the RMSD values of the individual domains are shown in light and dark blue. Additional file 4: Dependence of CYRANGE results on the domain boundary extension parameter m. See Additional File 3 for details. Additional file 5: Dependence of CYRANGE results on the minimal gap width g. See Additional File 3 for details. Additional file 6: Dependence of CYRANGE results on the relative RMSD decrease parameter delta. See Additional File 3 for details. Additional file 7: Dependence of CYRANGE results on the absolute RMSD decrease parameter delta abs. See Additional File 3 for details. Additional file 8: Dependence of CYRANGE results on the gap penalty parameter gamma. See Additional File 3 for details. Additional file 9: Correlation between the sequence coverage from CYRANGE, FindCore and PSVS, and the GDT total score, GDT_TS. Each data point represents a protein shown in Figures 3 and 4. The coverage is the percentage of amino acid residues included in the residue ranges found by the different methods. The GDT_TS value is defined by GDT_TS = (P1 + P2 + P4 + P8)/4, where Pd is the fraction of residues that can be superimposed under a distance cutoff of d Å. Additional file 10: Correlation between the RMSD value for the residue ranges from CYRANGE, FindCore and PSVS, and the GDT total score, GDT_TS. Each data point represents one protein domain. See Additional File 9 for details.
PERIOD proteins are central components of the Drosophila and mammalian circadian clocks. The crystal structure of a Drosophila PERIOD (dPER) fragment comprising two PER-ARNT-SIM (PAS) domains (PAS-A and PAS-B) and two additional C-terminal alpha-helices (alphaE and alphaF) has revealed a homodimer mediated by intermolecular interactions of PAS-A with tryptophane 482 in PAS-B and helix alphaF. Here we present the crystal structure of a monomeric PAS domain fragment of dPER lacking the alphaF helix. Moreover, we have solved the crystal structure of a PAS domain fragment of the mouse PERIOD homologue mPER2. The mPER2 structure shows a different dimer interface than dPER, which is stabilized by interactions of the PAS-B beta-sheet surface including tryptophane 419 (equivalent to Trp482dPER). We have validated and quantitatively analysed the homodimer interactions of dPER and mPER2 by site-directed mutagenesis using analytical gel filtration, analytical ultracentrifugation, and co-immunoprecipitation experiments. Furthermore we show, by yeast-two-hybrid experiments, that the PAS-B beta-sheet surface of dPER mediates interactions with TIMELESS (dTIM). Our study reveals quantitative and qualitative differences between the homodimeric PAS domain interactions of dPER and its mammalian homologue mPER2. In addition, we identify the PAS-B beta-sheet surface as a versatile interaction site mediating mPER2 homodimerization in the mammalian system and dPER-dTIM heterodimer formation in the Drosophila system.
Aptamers that can be regulated with light allow precise control of protein activity in space and time and hence of biological function in general. In a previous study, we showed that the activity of the thrombin-binding aptamer HD1 can be turned off by irradiation using a light activatable "caged" intramolecular antisense-domain. However, the activity of the presented aptamer in its ON state was only mediocre. Here we studied the nature of this loss in activity in detail and found that switching from 5'- to 3'-extensions affords aptamers that are even more potent than the unmodified HD1. In particular we arrived at derivatives that are now more active than the aptamer NU172 that is currently in phase 2 clinical trials as an anticoagulant. As a result, we present light-regulatable aptamers with a superior activity in their ON state and an almost digital ON/OFF behavior upon irradiation.
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.
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
Metal-ion binding and metal-ion induced folding of the adenine-sensing riboswitch aptamer domain
(2007)
Divalent cations are important in the folding and stabilization of complex RNA structures. The adenine-sensing riboswitch controls the expression of mRNAs for proteins involved in purine metabolism by directly sensing intracellular adenine levels. Adenine binds with high affinity and specificity to the ligand binding or aptamer domain of the adenine-sensing riboswitch. The X-ray structure of this domain in complex with adenine revealed an intricate RNA-fold consisting of a three-helix junction stabilized by long-range base-pairing interactions and identified five binding sites for hexahydrated Mg2+-ions. Furthermore, a role for Mg2+-ions in the ligand-induced folding of this RNA was suggested. Here, we describe the interaction of divalent cations with the RNA–adenine complex in solution as studied by high-resolution NMR spectroscopy. Paramagnetic line broadening, chemical shift mapping and intermolecular nuclear Overhauser effects (NOEs) indicate the presence of at least three binding sites for divalent cations. Two of them are similar to those in the X-ray structure. The third site, which is important for the folding of this RNA, has not been observed previously. The ligand-free state of the RNA is conformationally heterogeneous and contains base-pairing patterns detrimental to ligand binding in the absence of Mg2+, but becomes partially pre-organized for ligand binding in the presence of Mg2+. Compared to the highly similar guanine-sensing riboswitch, the folding pathway for the adenine-sensing riboswitch aptamer domain is more complex and the influence of Mg2+ is more pronounced.
High-resolution NMR structure of an RNA model system : the 14-mer cUUCGg tetraloop hairpin RNA
(2009)
We present a high-resolution nuclear magnetic resonance (NMR) solution structure of a 14-mer RNA hairpin capped by cUUCGg tetraloop. This short and very stable RNA presents an important model system for the study of RNA structure and dynamics using NMR spectroscopy, molecular dynamics (MD) simulations and RNA force-field development. The extraordinary high precision of the structure (root mean square deviation of 0.3 Å) could be achieved by measuring and incorporating all currently accessible NMR parameters, including distances derived from nuclear Overhauser effect (NOE) intensities, torsion-angle dependent homonuclear and heteronuclear scalar coupling constants, projection-angle-dependent cross-correlated relaxation rates and residual dipolar couplings. The structure calculations were performed with the program CNS using the ARIA setup and protocols. The structure quality was further improved by a final refinement in explicit water using OPLS force field parameters for non-bonded interactions and charges. In addition, the 2'-hydroxyl groups have been assigned and their conformation has been analyzed based on NOE contacts. The structure currently defines a benchmark for the precision and accuracy amenable to RNA structure determination by NMR spectroscopy. Here, we discuss the impact of various NMR restraints on structure quality and discuss in detail the dynamics of this system as previously determined.
Long-range tertiary interactions determine the three-dimensional structure of a number of metabolite-binding riboswitch RNA elements and were found to be important for their regulatory function. For the guanine-sensing riboswitch of the Bacillus subtilis xpt-pbuX operon, our previous NMR-spectroscopic studies indicated pre-formation of long-range tertiary contacts in the ligand-free state of its aptamer domain. Loss of the structural pre-organization in a mutant of this RNA (G37A/C61U) resulted in the requirement of Mg2+ for ligand binding. Here, we investigate structural and stability aspects of the wild-type aptamer domain (Gsw) and the G37A/C61U-mutant (Gswloop) of the guanine-sensing riboswitch and their Mg2+-induced folding characteristics to dissect the role of long-range tertiary interactions, the link between pre-formation of structural elements and ligand-binding properties and the functional stability. Destabilization of the long-range interactions as a result of the introduced mutations for Gswloop or the increase in temperature for both Gsw and Gswloop involves pronounced alterations of the conformational ensemble characteristics of the ligand-free state of the riboswitch. The increased flexibility of the conformational ensemble can, however, be compensated by Mg2+. We propose that reduction of conformational dynamics in remote regions of the riboswitch aptamer domain is the minimal pre-requisite to pre-organize the core region for specific ligand binding.
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.
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.
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.
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.
Poster presentation at 5th German Conference on Cheminformatics: 23. CIC-Workshop Goslar, Germany. 8-10 November 2009 Protein kinases are important targets for drug development. The almost identical protein folding of kinases and the common co-substrate ATP leads to the problem of inhibitor selectivity. Type II inhibitors, targeting the inactive conformation of kinases, occupy a hydrophobic pocket with less conserved surrounding amino acids. Human polo-like kinase 1 (Plk1) represents a promising target for approaches to identify new therapeutic agents. Plk1 belongs to a family of highly conserved serine/threonine kinases, and is a key player in mitosis, where it modulates the spindle checkpoint at metaphase/anaphase transition. Plk1 is over-expressed in all today analyzed human tumors of different origin and serves as a negative prognostic marker in cancer patients. The newly identified inhibitor, SBE13, a vanillin derivative, targets Plk1 in its inactive conformation. This leads to selectivity within the Plk family and towards Aurora A. This selectivity can be explained by docking studies of SBE13 into the binding pocket of homology models of Plk1, Plk2 and Plk3 in their inactive conformation. SBE13 showed anti-proliferative effects in cancer cell lines of different origins with EC50 values between 5 microM and 39 microM and induced apoptosis. Increasing concentrations of SBE13 result in increasing amounts of cells in G2/M phase 13 hours after double thymidin block of HeLa cells. The kinase activity of Plk1 was inhibited with an IC50 of 200 pM. Taken together, we could show that carefully designed structure-based virtual screening is well-suited to identify selective type II kinase inhibitors targeting Plk1 as potential anti-cancer therapeutics.
The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity.
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