Humaira Masood Siddiqi
- The aromatic rings in the title compound, C13H8ClNO4, enclose a dihedral angle of 39.53 (3)°. The nitro group is almost coplanar with the ring to which it is attached [dihedral angle = 4.31 (1)°]. In the crystal, molecules are connected by C-H...O hydrogen bonds into chains running along . Key indicators: single-crystal X-ray study; T = 173 K; mean σ(C–C) = 0.002 A°; R factor = 0.044; wR factor = 0.105; data-to-parameter ratio = 18.9.
Jens Michael Breunig
- The crystal structure of the title salt, [Li(CH3CN)4][B(NCS)4], is composed of discrete cations and anions. Both the Li and B atoms show a tetrahedral coordination by four equal ligands. The acetonitrile and isothiocyanate ligands are linear. The bond angles at the B atom are close to the ideal tetrahedral value [108.92 (18)–109.94 (16)°], but the bond angles at the Li atom show larger deviations [106.15 (17)–113.70 (17)°].
WeNMR: the tale of virtual research community in NMR and structural biology
EGI user forum 2011 : book of abstracts
Simple Method for Sub-Diffraction Resolution Imaging of Cellular Structures on Standard Confocal Microscopes by Three-Photon Absorption of Quantum Dots
- This study describes a simple technique that improves a recently developed 3D sub-diffraction imaging method based on three-photon absorption of commercially available quantum dots. The method combines imaging of biological samples via tri-exciton generation in quantum dots with deconvolution and spectral multiplexing, resulting in a novel approach for multi-color imaging of even thick biological samples at a 1.4 to 1.9-fold better spatial resolution. This approach is realized on a conventional confocal microscope equipped with standard continuous-wave lasers. We demonstrate the potential of multi-color tri-exciton imaging of quantum dots combined with deconvolution on viral vesicles in lentivirally transduced cells as well as intermediate filaments in three-dimensional clusters of mouse-derived neural stem cells (neurospheres) and dense microtubuli arrays in myotubes formed by stacks of differentiated C2C12 myoblasts.
CD69 Is a TGF-β/1α,25-dihydroxyvitamin D3 Target Gene in Monocytes
Thea K. Wöbke
Andreas von Knethen
Bernd L. Sorg
- CD69 is a transmembrane lectin that can be expressed on most hematopoietic cells. In monocytes, it has been functionally linked to the 5-lipoxygenase pathway in which the leukotrienes, a class of highly potent inflammatory mediators, are produced. However, regarding CD69 gene expression and its regulatory mechanisms in monocytes, only scarce data are available. Here, we report that CD69 mRNA expression, analogous to that of 5-lipoxygenase, is induced by the physiologic stimuli transforming growth factor-β (TGF-β) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in monocytic cells. Comparison with T- and B-cell lines showed that the effect was specific for monocytes. CD69 expression levels were increased in a concentration-dependent manner, and kinetic analysis revealed a rapid onset of mRNA expression, indicating that CD69 is a primary TGF-β/1α,25(OH)2D3 target gene. PCR analysis of different regions of the CD69 mRNA revealed that de novo transcription was initiated and proximal and distal parts were induced concomitantly. In common with 5-lipoxygenase, no activation of 0.7 kb or ~2.3 kb promoter fragments by TGF-β and 1α,25(OH)2D3 could be observed in transient reporter assays for CD69. Analysis of mRNA stability using a transcription inhibitor and a 3′UTR reporter construct showed that TGF-β and 1α,25(OH)2D3 do not influence CD69 mRNA stability. Functional knockdown of Smad3 clearly demonstrated that upregulation of CD69 mRNA, in contrast to 5-LO, depends on Smad3. Comparative studies with different inhibitors for mitogen activated protein kinases (MAPKs) revealed that MAPK signalling is involved in CD69 gene regulation, whereas 5-lipoxygenase gene expression was only partly affected. Mechanistically, we found evidence that CD69 gene upregulation depends on TAK1-mediated p38 activation. In summary, our data indicate that CD69 gene expression, conforming with 5-lipoxygenase, is regulated monocyte-specifically by the physiologic stimuli TGF-β and 1α,25(OH)2D3 on mRNA level, although different mechanisms account for the upregulation of each gene.
Real-Time Analysis and Visualization for Single-Molecule Based Super-Resolution Microscopy
- Accurate multidimensional localization of isolated fluorescent emitters is a time consuming process in single-molecule based super-resolution microscopy. We demonstrate a functional method for real-time reconstruction with automatic feedback control, without compromising the localization accuracy. Compatible with high frame rates of EM-CCD cameras, it relies on a wavelet segmentation algorithm, together with a mix of CPU/GPU implementation. A combination with Gaussian fitting allows direct access to 3D localization. Automatic feedback control ensures optimal molecule density throughout the acquisition process. With this method, we significantly improve the efficiency and feasibility of localization-based super-resolution microscopy.
Genome-wide multi-parametric analysis of H2AX or γH2AX distributions during ionizing radiation-induced DNA damage response
Maria Cristina Cardoso
- Background: After induction of DNA double strand breaks (DSBs), the DNA damage response (DDR) is activated. One of the earliest events in DDR is the phosphorylation of serine 139 on the histone variant H2AX (gH2AX) catalyzed by phosphatidylinositol 3-kinases-related kinases. Despite being extensively studied, H2AX distribution across the genome and gH2AX spreading around DSBs sites in the context of different chromatin compaction states or transcription are yet to be fully elucidated.
Materials and methods: gH2AX was induced in human hepatocellular carcinoma cells (HepG2) by exposure to 10 Gy X-rays (250 kV, 16 mA). Samples were incubated 0.5, 3 or 24 hours post irradiation to investigate early, intermediate and late stages of DDR, respectively. Chromatin immunoprecipitation was performed to select H2AX, H3 and gH2AX-enriched chromatin fractions. Chromatin-associated DNA was then sequenced by Illumina ChIP-Seq platform. HepG2 gene expression and histone modification (H3K36me3, H3K9me3) ChIP-Seq profiles were retrieved from Gene Expression Omnibus (accession numbers GSE30240 and GSE26386, respectively).
Results: First, we combined G/C usage, gene content, gene expression or histone modification profiles (H3K36me3, H3K9me3) to define genomic compartments characterized by different chromatin compaction states or transcriptional activity. Next, we investigated H3, H2AX and gH2AX distributions in such defined compartments before and after exposure to ionizing radiation (IR) to study DNA repair kinetics during DDR. Our sequencing results indicate that H2AX distribution followed H3 occupancy and, thus, the nucleosome pattern. The highest H2AX and H3 enrichment was observed in transcriptionally active compartments (euchromatin) while the lowest was found in low G/C and gene-poor compartments (heterochromatin). Under physiological conditions, the body of highly and moderately transcribed genes was devoid of gH2AX, despite presenting high H2AX levels. gH2AX accumulation was observed in 5’ or 3’ flanking regions, instead. The same genes showed a prompt gH2AX accumulation during the early stage of DDR which then decreased over time as DDR proceeded.
Finally, during the late stage of DDR the residual gH2AX signal was entirely retained in heterochromatic compartments. At this stage, euchromatic compartments were completely devoid of gH2AX despite presenting high levels of non-phosphorylated H2AX.
Conclusions: We show that gH2AX distribution ultimately depends on H2AX occupancy, the latter following H3 occupancy and, thus, nucleosome pattern. Both H2AX and H3 levels were higher in actively transcribed compartments. However, gH2AX levels were remarkably low over the body of actively transcribed genes suggesting that transcription levels antagonize gH2AX spreading. Moreover, repair processes did not take place uniformly across the genome; rather, DNA repair was affected by genomic location and transcriptional activity. We propose that higher H2AX density in euchromaticcompartments results in high relative gH2AXconcentration soon after the activation of DDR, thus favoring the recruitment of the DNA repair machinery to those compartments. When the damage is repaired and gH2AX is removed, its residual fraction is retained in the heterochromatic compartments which are then targeted and repaired at later times.
Pulsed EPR characterization of membrane transport protein complexes
- Pulsed electron–electron double resonance (PELDOR) spectroscopy is a powerful tool for measuring nanometer distances in spin-labeled systems and recently is increasingly applied to membrane proteins. However, after reconstitution of labeled proteins into liposomes, spin labels often exhibit a much faster transversal relaxation (Tm) than in detergent micelles, thus limiting application of the method in lipid bilayers. In the first part of the thesis, optimization of transversal relaxation in phospholipid membranes was systematically investigated by use of spin-labeled derivatives of stearic acid and phosphatidylcholine as well as spin-labeled derivatives of the channel-forming peptide gramicidin A under the conditions typically employed for PELDOR distance measurements. Our results clearly show that dephasing due to instantaneous diffusion that depends on dipolar interaction among electron spins is an important contributor to the fast echo decay in cases of high local concentrations of spin labels in membranes. The main difference between spin labels in detergent micelles and membranes is their local concentration. Consequently, avoiding spin aggregation and suppressing instantaneous diffusion is the key step for maximizing PELDOR sensitivity in lipid membranes. Even though proton spin diffusion is an important relaxation mechanism, only in samples with low local concentrations does deuteration of acyl chains and buffer significantly prolong Tm. In these cases, values of up to 7 μs have been achieved. Furthermore, our study revealed that membrane composition and labeling position in the membrane can also affect Tm, either by promoting the segregation of spin-labeled species or by altering their exposure to matrix protons. Effects of other experimental parameters including temperature (<50 K), presence of oxygen, and cryoprotectant type are negligible under our experimental conditions.
In the second part of the thesis, inhomogeneous distribution of spin-labels in detergent micelles has been studied. A common approach in PELDOR is measuring the distance between two covalently attached spin labels in a macromolecule or singly-labeled components of an oligomer. This situation has been described as a spin-cluster. The PELDOR signal, however, does not only contain the desired dipolar coupling between the spin-labels of the molecule or cluster under study. In samples of finite concentration the dipolar coupling between the spin-labels of the randomly distributed molecules or spin-clusters also contributes significantly. In homogeneous frozen solutions or lipid vesicle membranes this second contribution can be considered to be an exponential or stretched exponential decay, respectively. In this study, it is shown that this assumption is not valid in detergent micelles. Spin-labeled fatty acids that are randomly partitioned into different detergent micelles give rise to PELDOR time traces which clearly deviate from stretched exponential decays. As a main conclusion a PELDOR signal deviating from a stretched exponential decay does not necessarily prove the observation of specific distance information on the molecule or cluster. These results are important for the interpretation of PELDOR experiments on membrane proteins or lipophilic peptides solubilized in detergent micelles or small vesicles, which often do not show pronounced dipolar oscillations in their time traces.
In the third part, PELDOR has been utilized to study the structural flexibility of the Toc34 GTPase homodimer, a preprotein receptor of the translocon of the outer envelope of chloroplasts (TOC). Toc34 belongs to GAD subfamily of G-proteins that are regulated and activated by nucleotide-dependent dimerization. However, the function of Toc34 dimerization is not yet fully understood. Previous structural investigations of the Toc34 dimer yielded only marginal structural changes in response to different nucleotide loads. PELDOR revealed a nucleotide-dependent transition of the dimer flexibility from a tight GDP to a flexible GTP-loaded state. Substrate-binding stabilizes the dimer in the transition state mimicked by GDP-AlFx, but induces an opening in the GDP or GTP-loaded state. Thus, the structural dynamics of bona fide GTPases induced by GTP hydrolysis is replaced by substrate-dependent dimer flexibility, which represents the regulatory mode for dimerizing GTPases.
In the fourth part of the thesis, conformational flexibility and relative orientation of the N-terminal POTRA domains of a cyanobacterial Omp85 from Anabaena sp. PCC 7120, a key component of the outer membrane protein assembly machinery, were investigated by PELDOR spectroscopy. Membrane proteins of the Omp85-TpsB superfamily are composed of a C-terminal β-barrel and a different number of N-terminal POTRA domains, three in the case of cyanobacterial Omp85. It has been suggested that the N-terminal POTRA domains (P1 and P2) might have functions in substrate recognition. Molecular dynamics (MD) simulations predicted a fixed orientation for P2 and P3 and a flexible hinge between P1 and P2. The PELDOR distances measured between the P2 and P3 POTRA domains are in good agreement with the structure determined by X-ray, and compatible with the MD simulations suggesting a fixed orientation between these domains. PELDOR constraints between the P1 and P2 POTRA domains imply a rather rigid structure with a slightly different relative orientation of these domains compared with the X-ray structure. Moreover, the large mobility predicted from MD is not observed in the frozen solution. The PELDOR results further highlight the restricted relative orientation of the POTRA domains of the Omp85-TpsB proteins as a conserved characteristic feature that might be important for the processive sliding of the unfolded substrate towards the membrane.
TIP47 plays a crucial role in HCV morphogenesis and release by its interaction with viral nonstructural protein 5A and host protein Rab9
- Hepatitis C virus (HCV) assembly and production is closely linked to lipid metabolism. Indeed, lipid droplets (LD) have been shown to serve as a platform for HCV assembly. To investigate the effect of HCV on the host cell proteome, 2D-gelelectrophoresis with subsequent MALDI-TOF mass spectrometry of HCV replicating and the corresponding control cells were done. Based on this analysis, it was found out that HCV-replicating Huh7.5 cells revealed lower amounts of TIP47 (tail interacting protein of 47kD) compared to HCV-negative cells. TIP47, a cytoplasmic sorting factor, has been shown to be associated with lipid droplets. As it is known that HCV-replication and assembly takes place at the so called ”membranous web” that is composed of LDs and rearranged ER-derived membranes, it was tempting to investigate the role of TIP47 in HCV life-cycle. Western blot analysis did reveal that overexpression of TIP47 in HCV replicating Huh7.5 cells leads to decreased amounts of the HCV core protein while the levels of non-structural protein (NS)5A and intracellular HCVgenomes are increased. Moreover, in TIP47 overproducing cells higher amounts of infectious HCV particles are secreted. Vice versa, inhibition of TIP47 expression by siRNA results in a decreased level of intracellular NS5A, increased amounts of intracellular core and less infectious viral particles in the supernatant. In addition, complete silencing of TIP47 by lentiviral transduction abolishes HCV replication that can be restored by transfection of these cells with a TIP47 expression construct. It has been shown recently that apoE binds to NS5A and that this interaction plays an important role for the HCV life cycle (Benga et al., 2010). The C-terminal part of TIP47 harbours a 4 helix bundle motif and displays high homology to the N-terminus of apoE. Therefore, we investigated the interaction of NS5A and TIP47. Confocal double immunofluorescence microscopy revealed that a fraction of NS5A colocalizes with TIP47. Coimmunoprecipitation experiments and a yeast-two-hybrid screening confirmed the interaction between NS5A and TIP47 and deletion of the N-terminal-TIP47-PAT domain abolishes this interaction. From this we conclude that the TIP47-NS5A interaction is required for virus morphogenesis. Moreover, TIP47 can bind to Rab9 and this is relevant for targeting the viral particle out of the cell. In accordance to this, TIP47 was identified to be associated to the viral particle. Mutants of TIP47 that fail to bind Rab9 reveal lower amounts and a changed distribution of the HCV core protein. Furthermore, we could see that the core staining colocalizes with subcellular structures that were identified as autophagosomes using a p62-specific antibody which is a specific autophagosome-marker. Based on this, we hypothized that destruction of the Rab9 binding domain misdirects the viral particle towards the lysosomal compartment.
For the first time it could be shown that TIP47 interacts with NS5A and is associated to the viral particle, therefore plays a crucial role for the virus morphogenesis and secretion of the viral article.
Taken together, these results indicate that TIP47 is an essential cellular factor for the life cycle of HCV Abstract and might be used as target for antiviral treatment, e.g. by targeting the NS5A-TIP47 interaction, based on small molecules that mimic the NS5A-specific sequence that binds to TIP47 which might result in a competition of the TIP47/NS5A interaction.