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The widespread application of human stem-cell-derived neurons for functional studies is impeded by complicated differentiation protocols, immaturity, and deficient optogene expression as stem cells frequently lose transgene expression over time. Here we report a simple but precise Cre-loxP-based strategy for generating conditional, and thereby stable, optogenetic human stem-cell lines. These cells can be easily and efficiently differentiated into functional neurons, and optogene expression can be triggered by administering Cre protein to the cultures. This conditional expression system may be applied to stem-cell-derived neurons whenever timed transgene expression could help to overcome silencing at the stem-cell level.
The order-disorder phase transition which occurs at Tc = 296.9 K in anilinium bromide, C6H5NH3Br, is studied by inelastic neutron time-of-flight measurements. Data are taken from hydrogenated and partially deuterated samples (C6D5NH3Br and C6H5ND3Br) below and above the transition temperature. From the inelastic scattering data the phonon frequency distribution in the range of the intermolecular vibrations is calculated. The assignment of bands is given and changes in the spectra with temperature are discussed. The analysis of the quasielastic line revealed substantial broadening due to rotational diffusion of the -NH3⊕ group. These jump diffusion processes were observed in the orthorhombic high temperature and - with markedly diminished rate - in the monoclinic phase below Tc. From the correlation times at different temperatures the activation energies are estimated for both phases. Comparison with calculated elastic incoherent structure factors suggests jumps of 60° for hindered rotation.
The crystal structure of the high temperature phase of anilinium bromide, C6H5NH3⊕Br⊖ , was studied by X-ray and neutron diffraction at T = 343 K. The refinement supports disordered positions of the -NH3⊕ group. A split-atom model is proposed which includes disorder of the benzene ring. The thermal parameters, hydrogen bond distances, and other experimental data (NMR, NQR, inelastic neutron scattering) are in accordance with this model.
The crystal structure of the low temperature phase of anilinium bromide, C6H5NH3⊕Br⊖, was studied by neutron diffraction at T = 100 K. The refinement supports an ordered structure. The structures of the low and high temperature phases are compared and the mechanism of the phase transformation is discussed.
The interactions between human haptoglobin, Hp II (genetic types 2 - 1 and 2-2), and bovine hemoglobin, Hb, were investigated taking inhibition of complex formation and complex dissociation in various solvent media as criteria.
As shown by relative peroxidase activity and gel chromatography, complex dissociation occurs at high concentrations of guanidine HCl, urea, sodium chloride, dioxane, and formaldehyde, while in case of sodium dodecylsulfate a low molar ratio (SDS/Hb -Hp<5) is sufficient to split the complex. In general the formation of the complex stabilizes the structure of its constituents.
Excluding solvent conditions which lead to denaturation (as measured by optical rotation), ionpairs and H-bonds seem to prevail in the stabilization of the complex, while hydrophobic interactions should be of minor importance. Chemical modification of histidine and tyrosine with diazonium-1-H-tetrazole and N-acetylimidazole, respectively, prove histidyl-groups in Hb and tyrosylgroups in Hp to participate in the Hb-Hp contact, thus confirming earlier results.
The photodynamic deactivation of lysozyme in presence of acridine orange is caused by a reaction between singlet oxygen formed via the dye triplet state and the protein. In order to identify the region where the singlet oxygen reacts with the protein we have investigated the kinetics of the deactivation in presence ofthe inhibitor of the enzymatic reaction N-acetylglucosamine (GlcNAc). The overall experimental rate constant becomes slower with increasing saccharide concentrations. As we can exclude experimentally that this kinetical effect is caused in presence of the saccharide by a physical quenching of singlet oxygen or of the dye triplet state it has to be assumed that GlcNAc protects the surrounding of its bindings place at subsite C of the enzymatic center sterically against an attack of singlet oxygen. In this region three tryptophan residues are located, which could be sensitive against singlet oxygen. Surprisingly, however, it has been found that only those species are protected, in which a second saccharide molecule is bound to the protein, probably at subsite E at the enzymatic center, where no sensitive amino acid side chains are located.
The kinetics of the photodynamic desactivation of lysozyme in presence of acridine orange as the sensitizer have been investigated in detail varying oxygen, protein, dye concentration, ionic strength and pH value. The kinetics can be approximately described as an over all pseudo-first- order rate process. Changing the solvent from water to D2O or by quenching experiments in presence of azide ions it could be shown that the desactivation of lysozyme is caused exclusively by singlet oxygen. The excited oxygen occurs via the triplet state of the dye with a rate constant considerably lower than that to be expected for a diffusionally controlled reaction. Singlet oxygen reacts chemically (desactivation, k=2.9 × 107 ᴍ-1 sec-1) and physically (quenching process, k = 4.1 × 108 ᴍ-1sec-1) with the enzyme. The kinetical analysis shows that additional chemical reactions between singlet oxygen and lysozyme would have only little influence on the kinetics of the desactivation as long as their products would be enzymatically active and their kinetical constants would be less than about 1 × 108 ᴍ-1 sec-1.
Spectrophotometric investigation of the kinetics of the spontaneous reduction of the central metal ion in K2[Mn (IV)-2-α-hydroxyethyl-isochlorine e4] acetate in aqueous alkaline solution in the absence of any reducing agent reveals that it is a pseudo-first order reaction which is specifically hydroxide ion catalyzed. The pKα-value of the acid-base equilibrium has been estimated to be 14.4.
Electron transfer to the central metal ion is the rate limiting step. The measurements of its temperature dependence yields an activation enthalpy of ∆H‡ = 12 kcal/mol and an entropy of activation ∆S‡ = - 30 e.u. thus indicating that the electron transfer step is a bimolecular reaction. The most likely reactant is water. The reduction reaction does not take place with appreciable reaction rates at physiological pH. Thus, when bound to a suitable ligand of the chlorin type, Mn (IV)-compounds are sufficiently stable with respect to autoxidation to play some role in biological redox reactions as postulated recently for the photoreactivation process of the water splitting system in photosynthesis.
Inositol, 1,2,3,4,5,6-hexahydroxycyclohexane, exists in nine stereoisomers with different crystal structures and melting points. In a previous paper on the relationship between the melting points of the inositols and the hydrogen-bonding patterns in their crystal structures [Simperler et al. (2006[Simperler, A., Watt, S. W., Bonnet, P. A., Jones, W. & Motherwell, W. D. S. (2006). CrystEngComm, 8, 589-600.]). CrystEngComm 8, 589], it was noted that although all inositol crystal structures known at that time contained 12 hydrogen bonds per molecule, their melting points span a large range of about 170 °C. Our preliminary investigations suggested that the highest melting point must be corrected for the effect of molecular symmetry, and that the three lowest melting points may need to be revised. This prompted a full investigation, with additional experiments on six of the nine inositols. Thirteen new phases were discovered; for all of these their crystal structures were examined. The crystal structures of eight ordered phases could be determined, of which seven were obtained from laboratory X-ray powder diffraction data. Five additional phases turned out to be rotator phases and only their unit cells could be determined. Two previously unknown melting points were measured, as well as most enthalpies of melting. Several previously reported melting points were shown to be solid-to-solid phase transitions or decomposition points. Our experiments have revealed a complex picture of phases, rotator phases and phase transitions, in which a simple correlation between melting points and hydrogen-bonding patterns is not feasible.
The carnitine transporter CaiT from Escherichia coli belongs to the betaine, choline, and carnitine transporter family of secondary transporters. It acts as an L-carnitine/gamma-butyrobetaine exchanger and is predicted to span the membrane 12 times. Unlike the other members of this transporter family, it does not require an ion gradient and does not respond to osmotic stress (Jung, H., Buchholz, M., Clausen, J., Nietschke, M., Revermann, A., Schmid, R., and Jung, K. (2002) J. Biol. Chem. 277, 39251-39258). The structure and oligomeric state of the protein was examined in detergent and in lipid bilayers. Blue native gel electrophoresis indicated that CaiT was a trimer in detergent solution. This result was further supported by gel filtration and cross-linking studies. Electron microscopy and single particle analysis of the protein showed a triangular structure of three masses or two parallel elongated densities. Reconstitution of CaiT into lipid bilayers yielded two-dimensional crystals that indicated that CaiT was a trimer in the membrane, similar to its homologue BetP. The implications of the trimeric structure on the function of CaiT are discussed.
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.
Levels of the purine nucleoside triphosphates are de creasing towards the end of log phase growth of Streptomyces hydrogenans. Induction of 20β-hydroxysteroid dehy-drogenase by addition of 11β,21-dihydroxy-4,17 (20) -pregna-dien-3-one to the growth medium leads to a pronounced drop in purine nucleoside triphosphate levels with is irreversible in contrast to the initial loss and later accumulation of RNA.
Bacteria are highly organized organisms which are able to adapt to and propagate under a multitude of environmental conditions. Propagation hereby requires reliable chromosome replication and segregation which has to occur cooperatively with other cellular processes such as transcription, translation or signaling. Several mechanisms were proposed for segregation of the Escherichia coli (E. coli) chromosome, for example a mitotic-like active segregation model or entropy-based passive chromosome segregation. Another segregation model suggests coupled transcription, translation and insertion of membrane proteins (termed "transertion"), which links the replicating chromosome (nucleoid) to the growing cell cylinder.
Fluorescence microscopy was widely used to provide evidence for a distinct segregation model. However, the dynamic nature of bacterial chromosomes, the small bacterial size and the optical resolution limit of ~ 200-300 nm impair unveiling the underlying mechanisms. With the emergence of super-resolution fluorescence microscopy techniques and advanced labeling methods, a new toolbox became available enabling scientists to visualize biomolecules and cellular processes in unprecedented detail. Single-molecule localization microscopy (SMLM) represents a set of super-resolution microscopy techniques which relies on the temporal separation of the fluorescence signal and detection of single fluorophores. Separation can be achieved using photoactivatable or -convertible fluorescent proteins (FPs) in photoactivated localization microscopy (PALM), photoswitchable organic dyes in direct stochastic optical reconstruction microscopy (dSTORM) or dynamically binding fluorescent probes in point accumulation for imaging in nanoscale topography (PAINT). In all these techniques, the fluorescence emission pattern of single fluorophores is spatially localized with nanometer-precision. An artificial image is finally reconstructed from the coordinates of all single fluorophores detected. This provides a spatial resolution of ~ 20 nm, which is perfectly suited to investigate cellular processes in bacteria. In this thesis, different SMLM techniques were applied to study fundamental processes in E. coli. This includes determination of protein copy numbers and distributions as well as the nanoscale organization of nucleic acids and lipids.
A novel labeling approach was applied and used for super-resolution imaging of the E. coli nucleoid. It is based on the incorporation of the modified thymidine analogue 5-ethynyl-2’- deoxyuridine (EdU) into the replicating chromosome. Azide-functionalized organic fluorophores can be covalently attached to the ethynyl group of incorporated EdU bases using a copper-catalyzed "click chemistry" reaction. Under the investigated growth condition, E. coli cells exhibited overlapping replication cycles, which is commonly referred to as multi-fork replication and enables cells to divide faster than they can replicate the entire chromosome. dSTORM imaging of such labeled nucleoids revealed chromosome features with diameters of 50 - 200 nm, representing highly condensed DNA filaments. Sorting single E. coli cells by length allowed visualizing structural changes of the nucleoid throughout the cell cycle. Replicating nucleoids segregated and expanded along the bacterial long axis, while constantly covering the entire width of the cell. Measuring cell and nucleoid length revealed a relative nucleoid expansion rate of 78 ± 6 %. At the same time, nucleoids populated 63 ± 8 % of the cell length, almost exclusively being localized to the cylindrical part of the cell. This value was hence normalized to the cylindrical fraction of the cell, yielding a value of 79 ± 10 % (nucleoid-populated fraction of the cell cylinder), which is in good agreement with the observed relative nucleoid expansion rate. These results therefore support a growth-mediated segregation model, in which the chromosome is anchored to the inner membrane and passively segregated into the prospective daughter cells upon cell growth. 3-dimensional dSTORM imaging of labeled nucleoids confirmed that compacted nucleoids helically wrap along the inner membrane. Similar results were obtained by imaging orthogonally aligned E. coli cells using a holographic optical tweezer approach.
In order to visualize particular proteins together with the nucleoid, several correlative imaging workflows were established, facilitating multi-color SMLM imaging in single E. coli cells. These workflows bypass prior limitations of SMLM, including destruction of FPs by reactive oxygen species in copper-catalyzed click reactions or incompatibility of PALM imaging with dSTORM imaging buffers. A sequential SMLM imaging routine was developed which is based on postlabeling and retrieval of previously imaged cells. Optimal imaging conditions can be maintained for each fluorophore, enabling to extract quantitative information from PALM measurements while correlating the protein distribution to the nucleoid ultrastructure within the highly resolved cell envelope. Applying this workflow to an E. coli strain carrying a chromosomal rpoC - photoactivatable mCherry (PAmCh) fusion, transcribing RNA polymerase (RNAP) was found to be localized on the surface of nucleoids, where active genes are exposed towards the cytosol. During growth in nutrient-rich medium, the majority of RNAP molecules was bound to the chromosome, thus ensuring that the RNAP pool is equally distributed to the daughter cells upon cell division. This work represented the first triple-color SMLM study performed in E. coli cells. ...
Nuclear pore complexes (NPCs) constitute giant channels within the nuclear envelope that mediate nucleocytoplasmic exchange. NPC diameter is thought to be regulated by nuclear envelope tension, but how such diameter changes are physiologically linked to cell differentiation, where mechanical properties of nuclei are remodeled and nuclear mechanosensing occurs, remains unstudied. Here we used cryo-electron tomography to show that NPCs dilate during differentiation of mouse embryonic stem cells into neural progenitors. In Nup133-deficient cells, which are known to display impaired neural differentiation, NPCs however fail to dilate. By analyzing the architectures of individual NPCs with template matching, we revealed that the Nup133-deficient NPCs are structurally heterogeneous and frequently disintegrate, resulting in the formation of large nuclear envelope openings. We propose that the elasticity of the NPC scaffold mechanically safeguards the nuclear envelope. Our studies provide a molecular explanation for how genetic perturbation of scaffolding components of macromolecular complexes causes tissue-specific phenotypes.
The RAF family of kinases constitutes the members A, B and CRAF. They mediate RAS signaling by linking it to the MEK/ERK transduction module, which regulates cellular processes such as cell proliferation, migration, survival and cell death. As the RAS/RAF/MEK/ERK (MAPK) pathway is found to be activated in human cancers, the RAF kinases have been exploited as valuable therapeutic targets and RAF inhibitors show promising results in the clinic, esp. with tumors harboring an activating BRAFV600E mutation. However, RAF inhibitors paradoxically accelerate metastasis in RAS mutant and BRAF wildtype tumors. They also become ineffective over time in BRAFV600E tumors because of reactivation of downstream mitogen-activated protein kinase (MAPK) signaling by promoting RAF dimerization. Aims of the present work were 1) to investigate the role of ARAF kinase in the paradoxical activation of the enzymatic cascade by RAF inhibitors downstream of mutated RAS and 2) to study the consequences of the loss of ARAF function on signal transduction in vitro and in vivo (nude mice). We have engineered several cell lines that would allow the study of basal and RAF inhibitor induced effects on MAPK activation, tumor cell migration and invasion.
In summary, we were able to show that the RAF isoform ARAF has an obligatory role in promoting MAPK activity and tumor cell invasion in a cell type dependent manner. In these cell types, ARAF depletion prevented the activation of MAPK kinase 1 (MEK1) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) and led to a significant decrease of protrusions growing out of tumor cell spheroids in a three-dimensional (3D) culture that were otherwise induced by BRAFV600E-specific or BRAF/CRAF inhibitors (GDC-0879 and sorafenib, respectively). RAF inhibitors stimulated homodimerization of ARAF and heteromerization of BRAF with CRAF and the scaffolding protein KSR1. However, induced oligomerization was not sufficient to activate MAPK signaling if ARAF was depleted. By employing full length recombinant kinases, we were able to show for the first time that the three RAF isoforms competed for the binding to MEK1. In cell culture models, the overexpression of dimer-deficient ARAF mutants impaired the interaction between ARAF and endogenous MEK1 and thus prevented the subsequent phosphorylation of MEK1 and ERK1/2. Our findings reveal a new role for ARAF in directly activating the MAPK cascade through homodimerization and thereby promoting tumor cell invasion, suggesting the conserved RAF-dimer interface as a target for RAS- and RAF mediated cancer therapy.
Collectively, we provide evidence for the dual role ARAF plays in controlling MAPK signaling and cancer as loss of ARAF promoted strong lung metastasis formation in nude mice. Preliminary data describing the underlying mechanisms behind ARAF-regulated metastases have been presented and discussed.
In Philadelphia Chromosome (Ph) positive ALL and CML the fusion between BCR and ABL leads to the BCR/ABL fusion proteins, which induces the leukemic phenotype because of the constitutive activation of multiple signaling pathways down-stream to the aberrant BCR/ABL fusion tyrosine kinase. Targeted inhibition of BCR/ABL by ABL-kinase inhibitors induces apoptosis in BCR/ABL transformed cells and leads to complete remission in Ph positive leukemia patients. However, a large portion of patients with advanced Ph+ leukemia relapse and acquire resistance. Kinase domain (KD) mutations interfering with inhibitor binding represent the major mechanism of acquired resistance in patients with Ph+ leukemia. Tetramerization of BCR/ABL through the N-terminal coiled-coil region (CC) of BCR is essential for the ABL-kinase activation. Targeting the CC-domain forces BCR/ABL into a monomeric conformation, reduces its kinase activity and increases the sensitivity for Imatinib. Here we show that i.) targeting the tetramerization by a peptide representing the Helix-2 of the CC efficiently reduced the autophosphorylation of both WT BCR/ABL and its mutants; ii.) Helix-2 inhibited the transformation potential of BCR/ABL independently of the presence of mutations; iii.) Helix-2 efficiently cooperated with Imatinib as revealed by their effects on the transformation potential and the factor-independence related to BCR/ABL with the exception of mutant T315I. These findings suggest that BCR/ABL harboring the T315I mutation have a transformation potential which is at least partially independent from its kinase activity. Targeted inhibition of BCR/ABL by small molecule inhibitors reverses the transformation potential of BCR/ABL. We definitively proved that targeting the tetramerization of BCR/ABL mediated by the N-terminal coiled-coil domain (CC) using competitive peptides, representing the Helix-2 of the CC, represents a valid therapeutic approach for treating Ph+ leukemia. To further develop competitive peptides for targeting BCR/ABL, we created a membrane permeable Helix-2 peptide (MPH-2) by fusing the Helix-2 peptide with a peptide transduction tag. In this study, we report that the MPH-2: (i) interacted with BCR/ABL in vivo; (ii) efficiently inhibited the autophosphorylation of BCR/ABL; (iii) suppressed the growth and viability of Ph+ leukemic cells; and (iv) was efficiently transduced into mononuclear cells (MNC) in an in vivo mouse model. The T315I mutation confers resistance against all actually approved ABL-kinase inhibitors and competitive peptides. It seems not only to decrease affinity for kinase inhibitors but to confer additional features to the leukemogenic potential of BCR/ABL. To determine the role of T315I in resistance to the inhibition of oligomerization and in the leukemogenic potential of BCR/ABL, we investigated its influence on loss-of-function mutants with regard to the capacity to mediate factor-independence. Thus we studied the effects of T315I on BCR/ABL mutants lacking functional domains in the BCR portion indispensable for the oncogenic activity of BCR/ABL such as the N-terminal coiled coil (CC), the tyrosine phosphorylation site Y177 and the serine/threonine kinase domain (ST), as well as on the ABL portion of BCR/ABL (#ABL-T315I) with or without the inhibitory SH3 (delta SH3-ABL) domain. Here we report that i.) T315I restored the capacity to mediate factor independence of oligomerization_deficient p185BCR/ABL; ii.) resistance of p185-T315I against inhibition of the oligomerization depends on the phosphorylation at Y177; iii.) autophosphorylation at Y177 is not affected by the oligomerization inhibition, but phosphorylation at Y177 of endogenous BCR parallels the effects of T315I; iv.) the effects of T315I are associated with an intact ABL_kinase activity; v.) the presence of T315I is associated with an increased ABL_kinase activity also in mutants unable to induce Y177 phosphorylation of endogenous BCR; vi.) there is no direct relationship between the ABL-kinase activity and the capacity to mediate factor_independence induced by T315I as revealed by the #ABL-T315I mutant, which was unable to induce Y177 phosphorylation of BCR only in the presence of the SH3 domain. In contrast to its physiological counterpart c-ABL, the BCR/ABL kinase is constitutively activated, inducing the leukemic phenotype. The N-terminus of c-ABL (Cap region) contributes to the regulation of its kinase function. It is myristoylated, and the myristate residue binds to a hydrophobic pocket in the kinase domain known as the myristoyl binding pocket in a process called “capping”, which results in an auto-inhibited conformation. Because the cap region is replaced by the N-terminus of BCR, BCR/ABL “escapes” this auto-inhibition. Allosteric inhibition by myristate “mimics”, such as GNF-2, is able to inhibit unmutated BCR/ABL, but not the BCR/ABL that harbors the “gatekeeper” mutation T315I. Here we investigated the possibility of increasing the efficacy of allosteric inhibition by blocking BCR/ABL oligomerization. We demonstrate that inhibition of oligomerization was able not only to increase the efficacy of GNF-2 on unmutated BCR/ABL, but also to overcome the resistance of BCR/ABL-T315I to allosteric inhibition. These results strongly suggest that the response to allosteric inhibition by GNF-2 is inversely related to the degree of oligomerization of BCR/ABL. Taken together these data suggest that the inhibition of tetramerization inhibits BCR/ABL-mediated transformation and can contribute to overcome Imatinib-resistance. The study provides the first evidence that an efficient peptide transduction system facilitates the employ-ment of competitive peptides to target the oligomerization interface of BCR/ABL in vivo. Further the data show that T315I confers additional leukemogenic activity to BCR/ABL, which might explain the clinical behavior of patients with BCR/ABL -T315I-positive blasts. In summary, our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants represented by the combination of oligomerization and allosteric inhibitors.
The p53 family of transcription factors (p53, p63 and p73) covers a wide range of functions critical for development, homeostasis and health of mammals across their lifespan. Beside the well-established tumor suppressor role, recent evidence has highlighted novel non-oncogenic functions exerted by p73. In particular, p73 is required for multiciliated cell (MCC) differentiation; MCCs have critical roles in brain and airways to move fluids across epithelial surfaces and to transport germ cells in the reproductive tract. This novel function of p73 provides a unifying cellular mechanism for the disparate inflammatory and immunological phenotypes of p73-deficient mice. Indeed, mice with Trp73 deficiency suffer from hydrocephalus, sterility and chronic respiratory tract infections due to profound defects in ciliogenesis and complete loss of mucociliary clearance since MCCs are essential for cleaning airways from inhaled pollutants, pathogens and allergens. Cross-species genomic analyses and functional rescue experiments identify TAp73 as the master transcriptional integrator of ciliogenesis, upstream of previously known central nodes. In addition, TAp73 shows a significant ability to regulate cellular metabolism and energy production through direct transcriptional regulation of several metabolic enzymes, such as glutaminase-2 and glucose-6 phosphate dehydrogenase. This recently uncovered role of TAp73 in the regulation of cellular metabolism strongly affects oxidative balance, thus potentially influencing all the biological aspects associated with p73 function, including development, homeostasis and cancer. Although through different mechanisms, p63 isoforms also contribute to regulation of cellular metabolism, thus indicating a common route used by all family members to control cell fate. At the structural level, the complexity of p73's function is further enhanced by its ability to form heterotetramers with some p63 isoforms, thus indicating the existence of an intrafamily crosstalk that determines the global outcome of p53 family function. In this review, we have tried to summarize all the recent evidence that have emerged on the novel non-oncogenic roles of p73, in an attempt to provide a unified view of the complex function of this gene within its family.
NMR spectroscopy is a potent method for the structural and biophysical characterization of RNAs. The application of NMR spectroscopy is restricted in RNA size and most often requires isotope‐labeled or even selectively labeled RNAs. Additionally, new NMR pulse sequences, such as the heteronuclear‐detected NMR experiments, are introduced. We herein provide detailed protocols for the preparation of isotope‐labeled RNA for NMR spectroscopy via in vitro transcription. This protocol covers all steps, from the preparation of DNA template to the transcription of milligram RNA quantities. Moreover, we present a protocol for a chemo‐enzymatic approach to introduce a single modified nucleotide at any position of any RNA. Regarding NMR methodology, we share protocols for the implementation of a suite of heteronuclear‐detected NMR experiments including 13C‐detected experiments for ribose assignment and amino groups, the CN‐spin filter heteronuclear single quantum coherence (HSQC) for imino groups and the 15N‐detected band‐selective excitation short transient transverse‐relaxation‐optimized spectroscopy (BEST‐TROSY) experiment.
Basic Protocol 1: Preparation of isotope‐labeled RNA samples with in vitro transcription using T7 RNAP, DEAE chromatography, and RP‐HPLC purification
Alternate Protocol 1: Purification of isotope‐labeled RNA from in vitro transcription with preparative PAGE
Alternate Protocol 2: Purification of isotope‐labeled RNA samples from in vitro transcription via centrifugal concentration
Support Protocol 1: Preparation of DNA template from plasmid
Support Protocol 2: Preparation of PCR DNA as template
Support Protocol 3: Preparation of T7 RNA Polymerase (T7 RNAP)
Support Protocol 4: Preparation of yeast inorganic pyrophosphatase (YIPP)
Basic Protocol 2: Preparation of site‐specific labeled RNAs using a chemo‐enzymatic synthesis
Support Protocol 5: Synthesis of modified nucleoside 3′,5′‐bisphosphates
Support Protocol 6: Preparation of T4 RNA Ligase 2
Support Protocol 7: Setup of NMR spectrometer for heteronuclear‐detected NMR experiments
Support Protocol 8: IPAP and DIPAP for homonuclear decoupling
Basic Protocol 3: 13C‐detected 3D (H)CC‐TOCSY, (H)CPC, and (H)CPC‐CCH‐TOCSY experiments for ribose assignment
Basic Protocol 4: 13C‐detected 2D CN‐spin filter HSQC experiment
Basic Protocol 5: 13C‐detected C(N)H‐HDQC experiment for the detection of amino groups
Support Protocol 9: 13C‐detected CN‐HSQC experiment for amino groups
Basic Protocol 6: 13C‐detected “amino”‐NOESY experiment
Basic Protocol 7: 15N‐detected BEST‐TROSY experiment
Despite the great interest in glycoproteins, structural information reporting on conformation and dynamics of the sugar moieties are limited. We present a new biochemical method to express proteins with glycans that are selectively labeled with NMR‐active nuclei. We report on the incorporation of 13C‐labeled mannose in the C‐mannosylated UNC‐5 thrombospondin repeat. The conformational landscape of the C‐mannose sugar puckers attached to tryptophan residues of UNC‐5 is characterized by interconversion between the canonical 1C4 state and the B03 / 1S3 state. This flexibility may be essential for protein folding and stabilization. We foresee that this versatile tool to produce proteins with selectively labeled C‐mannose can be applied and adjusted to other systems and modifications and potentially paves a way to advance glycoprotein research by unravelling the dynamical and conformational properties of glycan structures and their interactions.
The Taiwan cobra (Naja naja atra) chymotrypsin inhibitor (NACI) consists of 57 amino acids and is related to other Kunitz-type inhibitors such as bovine pancreatic trypsin inhibitor (BPTI) and Bungarus fasciatus fraction IX (BF9), another chymotrypsin inhibitor. Here we present the solution structure of NACI. We determined the NMR structure of NACI with a root-mean-square deviation of 0.37 Å for the backbone atoms and 0.73 Å for the heavy atoms on the basis of 1,075 upper distance limits derived from NOE peaks measured in its NOESY spectra. To investigate the structural characteristics of NACI, we compared the three-dimensional structure of NACI with BPTI and BF9. The structure of the NACI protein comprises one 310-helix, one α-helix and one double-stranded antiparallel β-sheet, which is comparable with the secondary structures in BPTI and BF9. The RMSD value between the mean structures is 1.09 Å between NACI and BPTI and 1.27 Å between NACI and BF9. In addition to similar secondary and tertiary structure, NACI might possess similar types of protein conformational fluctuations as reported in BPTI, such as Cys14–Cys38 disulfide bond isomerization, based on line broadening of resonances from residues which are mainly confined to a region around the Cys14–Cys38 disulfide bond.
This thesis deals with the NMR characterization of the structure and the folding dynamics of DNA G quadruplexes as potential therapeutic target in cancer therapy and building block for DNA based nanotechnology.
The first part of this thesis (Chapters 1-5) introduces the reader to the world of G quadruplexes.
The main features of the classic Watson Crick double helix and alternative non B DNA structures are illustrated in Chapter 1. Many different base pairing schemes are possible, besides the canonical Watson Crick motif, thereby expanding the structural complexity of DNA. Non canonical base pairing, such as Hoogsteen hydrogen bonding, enables the assembly of triplets and quartets, which are the building blocks of triplex and quadruplex structures, respectively.
The structural characteristics of DNA G quadruplexes are delineated in detail in Chapter 2.
G quadruplex structures are extremely polymorphic, in terms of strands orientation, loops geometry, grooves width and arrangement of the glycosidic torsion angles. The various structural elements as well as the different cation coordination geometries are here presented, with a special emphasis on the diversity of conformations reported for the telomeric DNA G quadruplexes.
Chapter 3 describes the biological roles of G quadruplex structures in the genome. After introducing the architecture of the telomeric DNA and its interacting proteins, the mechanism of the telomeres elongation catalysed by the telomerase enzyme and its implications for cancer are discussed. The occurrence of G quadruplex structures in functional regions of the genome, such as promoter regions of oncogenes, and their possible roles in regulating the gene transcription are then outlined in the second part of the chapter.
The potential of G quadruplex as a novel anti cancer target is examined in Chapter 4 and the proposed anti cancer mechanisms for a ligand stabilizing G quadruplex structures are discussed.
RNA G quadruplexes and their putative role in gene regulation at the level of translation are briefly illustrated at the end of the chapter.
A general overview on the NMR methods to investigate the G quadruplex structures is presented in Chapter 5. The experimental set up used for the real time NMR studies of the G quadruplex folding is also described.
The second part of the thesis (Chapters 6-8), which is the cumulative part, comprises the original publications grouped in three Chapters according to the topic.
The state of the art on small molecules targeting G quadruplex structures is given at the beginning of Chapter 6, including a summary of the experimental structures of G quadruplexes in complex with ligands available up to date. The publications presented in Chapters 6.1-6.3 are concerned with the elucidation of the interaction modes between DNA G quadruplexes and selected ligands with potential therapeutic applications.
The binding ability of two natural alkaloids (berberine and sanguinarine) to telomeric G quadruplexes is examined in Chapter 6.1. The ability of carbazole and diguanosine derivatives (synthetized in the group of Prof. Dash, IISER, Kolkata) to interact with c-MYC G quadruplex and down regulate c-MYC expression is explored in Chapter 6.2 and Chapter 6.3, respectively.
The energy landscape of human telomeric G quadruplex structures is discussed in Chapter 7, in light of the experimental kinetic studies as well as molecular dynamics simulations reported in literature until now. Up to date there is no general consensus regarding the folding pathway of unimolecular human telomeric G quadruplex, in particular due to the lack of atomic resolution data on the species involved in the folding. Chapter 7.1 presents the first real time NMR study of the human telomeric G quadruplex folding kinetics.
The final chapter of this thesis (Chapter 8) outlines the potential of G-quadruplex structures as building blocks in nanotechnology. After illustrating briefly the additional possibilities offered by alternative non B DNA structures to programme nanomaterials, a number of applications employing G quadruplex structures in different fields of nanotechnology are described. The article presented in Chapter 8.1 investigates the structural and photoswitching properties of a novel intermolecular azobenzene containing G quadruplex synthetized in the group of Prof. Heckel (Goethe University, Frankfurt).
Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot, J. L., and Engelman, D. M. (1990) Biochemistry 29, 4031–4037) is postulated to proceed in 2 steps: partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two, and the first three transmembrane (TM) segments, as well as a construct comprising TM1–TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin, A., Cohen, L. S., Arshava, B., Tantry, S., Becker, J. M., Zerbe, O., and Naider, F. (2009) Biophys. J. 96, 3187–3196), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123, and TM127 adopt predominantly native-like topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior.
In this thesis, the structure of the C-terminal domain of presenilin-1, the catalytic component of the y-secretase complex, is investigated by NMR spectroscopy. The ysecretase complex has a definitive role in the pathogenic development of Alzheimer's disease, in that it mediates the cleavage of aprecursor to create the amyloid ß peptide. Aggregates of amyloid ß which form amyloid plaques are the most overt clinieal feature observed in the post-mortem brains of Alzheimer's patient. In addition, many of the mutations found in the aggressive early onset familial Alzheimer's disease have been linked to presenilin-1, highlighting its importance in disease progression and deeming it an important target for investigation. One of the greatest challenges for the structural investigation of the y-secretase components is their low expression yields in cell-based systems. We therefore applied continuous-exchange cell-free expression to obtain sufficient amounts of protein for our structural studies. An added benefit of the cell-free expression system is the freedom to incorporate any desired combination of stable-isotope labels directly into sampies. We were therefore able to develop a labeling scheme which targets the amino acid composition of transmembrane a-helices, allowing us to simplify an assignment procedure whieh tends to be cumbersome and diffieult for most a-helical transmembrane proteins. The y-secretase complex is a member of the intramembrane cleaving proteases which, as their name implies, cleave their transmembrane substrates within the bilayer. Single particle analysis of the y-secretase (1) as weil as crystal structures of rhomboid (2) and S2P (3) have revealed the presence of hydrophilie po res within the membrane where catalysis occurs. In light of evidence that certain elements of CTF reside in close proximity or even contribute to the formation of the hydrophilic pore, we chose to study the structure of CTF in mieelles, whieh may be better suited to accommodate CTF in isolation as compared with solid membranes in the absence of the other y-secretase components. The structure of CTF was solved to 1.7 A (backbone r.m.s.d) and revealed the presence of unusual features, including a partially membrane-spanning helix which situates the catalytic asparte at its N-terminus in what would be the center of the membrane where catalysis is proposed to occur, as weil as a severely kinked helix which is partially embedded beneath the surface of the membrane (P6). Interestingly, similar features have been observed in the crystal structure of the GlpG rhomboid. In addition, a soluble helix was found in the long N-terminal loop of CTF which until now has been described as unstructured. The first part of the thesis is designed to provide an introduction to Alzheimer's disease, the role of y-secretase and its presenilin-l catalytic component in disease progression, as weil as cell-free expression and liquid-state NMR techniques involved in the structural investigation of membrane proteins. In chapter 2, the reader is familiarized with the history, the clinical manifestation, and biochemical features of Alzheimer's disease. The chapter goes further to describe the role of the y-secretase complex and its individual components in disease progression and substrate processing. Chapter 3 focuses more specifically on presenilin-l in the context of the newly emerging class of intramembrane proteases. In chapter 4, attention is shifted to the cell-free expression system with special focus on the expression of membrane proteins, and chapter 5 explores the various liquid-state NMR techniques that were required for the characterization of CTF. The second part of the thesis is cumulative and contains original research, method, and review articles that were produced during the course of study. Chapter 6 explores the various techniques and innovations used to study membrane proteins using continuous exchange cell-free expression coupled with NMR spectroscopy. In chapter 7, a new technique, transmembrane segment targeted labeling, is described as a tool that facilitates the backbone assignment of transmembrane proteins which display severe overlap in NMR spectra. Chapter 8 presents the novel NMR structure of the C-terminal fragment of presenilin-l solved in SOS micelles.
NHC supersilyl silver complex [Ag(IPr)SitBu3] as a promising agent for substitution reactions
(2020)
The NHC supersilyl silver complex [Ag(IPr)SitBu3] (IPr = NHCIPr) was prepared by treatment of Ag(IPr)Cl with Na(thf)2[SitBu3] in benzene/thf at room temperature. X‐ray quality crystals of the NHC supersilyl silver complex [Ag(IPr)SitBu3] (monoclinic, space group P21/m) were grown from heptane at room temperature. The 29Si NMR spectrum of a solution of [Ag(IPr)SitBu3] in C6D6 revealed two doublets caused by coupling to 107Ag and 109Ag nuclei. We further investigated the possibility of a conversion of triel halides EX3 by treatment with [Ag(IPr)SitBu3]. At ambient temperature the reaction of [Ag(IPr)SitBu3] with an excess of EX3 yielded tBu3SiEX2 (E = B, Al; X = Cl, Br; E = Ga; X = Cl) and IPr·EX3 (EX3 = BCl3, BBr3, AlCl3, AlBr3, GaCl3). The identity of tBu3SiEX2 and IPr·EX3 was confirmed by comparison with authentic samples.
pH and Na+ homeostasis in all cells requires Na+/H+ antiporters. The crystal structure, obtained at pH 4, of NhaA, the main antiporter of Escherichia coli, has provided general insights into an antiporter mechanism and its unique pH regulation. Here, we describe a general method to select various NhaA mutants from a library of randomly mutagenized NhaA. The selected mutants, A167P and F267C are described in detail. Both mutants are expressed in Escherichia coli EP432 cells at 70–95% of the wild type but grow on selective medium only at neutral pH, A167P on Li+ (0.1 M) and F267C on Na+ (0.6 M). Surprising for an electrogenic secondary transporter, and opposed to wild type NhaA, the rates of A167P and F267C are almost indifferent to membrane potential. Detailed kinetic analysis reveals that in both mutants the rate limiting step of the cation exchange cycle is changed from an electrogenic to an electroneutral reaction.
New reactive coenzyme analogues for affinity labeling of NAD+ and NADP+ dependent dehydrogenases
(1995)
Reactive coenzyme analogues ω-(3-diazoniumpyridinium)alkyl adenosine diphosphate were prepared by reaction of ω-(3-aminopyridinium)alkyl adenosine diphosphate with nitrous acid. In these compounds the nicotinamide ribose is substituted by hydrocarbon chains of varied lengths (n-ethyl to n-pentyl). The diazonium compounds are very unstable and decompose rapidly at room temperature. They show a better stability at 0 °C. L actate and alcohol dehydrogenase do not react with any of the analogues. Glyceraldehyde-3-phosphate dehydrogenase reacts rapidly with the diazonium pentyl compound. Decreasing the length of the alkyl chain significantly decreases the inactivation velocity. 3α,20β-Hydroxysteroid dehydrogenase reacts at 0 °C with the ethyl homologue and slowly with the propyl compound. The butyl-and pentyl analogues do not inactivate at 0 °C. Tests with 14C -labeled 2-(3-diazoniumpyridinium)ethyl adenosine diphosphate show that complete loss of enzyme activity results after incorporation of 2 moles of inactivator into 1 mole of tetrameric enzyme. 4-(3-Acetylpyridinium)butyl 2 ′-phospho-adenosine diphosphate, a structural analogue of NADP +, was prepared by condensation of adenosine-2,3-cyclophospho-5′-phosphomorpholidate with (3-acetylpyridinium)butyl phosphate, followed by hydrolysis of the cyclic phosphoric acid ester with 2 ′:3′-cyclonucleotide-3′-phosphodiesterase. Because of the redox potential (-315 mV) and the distance between the pyridinium and phosphate groups, this analogue is a hydrogen acceptor and its reduced form a hydrogen donor in tests with alcohol dehyd rogenase from Thermoanaerobium brockii. The reduced form of the coenzyme analogue also is a hydrogen donor with glutathione reductase. With other NADP +-dependent dehydrogenases the com pound has been show n to be a competitive inhibitor against the natural coenzyme. The acetyl group reacts with bromine to form the bromoacetyl group. This reactive bromoacetyl analogue is a specific active-site directed irreversible inhibitor of isocitrate dehydrogenase.
Enantioselective carbon-carbon bond-forming reactions, particularly, using organocatalysts represent one of the most important areas in modern synthetic chemistry. New concepts and methods in organocatalysis are emerging continuously, allowing more selective, economically more appealing and environmentally friendlier transformations. Chiral Brønsted-acid catalysts have recently emerged as a new class of organocatalysts for a number of enantioselective carbon-carbon bond-forming reactions. The first part of this thesis focused on the new development of new Brønsted acid-catalyzed enantioselective Nazarov cyclizations. The Nazarov reaction belongs to the group of electrocyclic reactions and is one of the most versatile methods for the synthesis of five-membered rings, which are the key structural elements of numerous natural products. In general, the Nazarov cyclization can be catalyzed by Brønsted or Lewis acids. However, only a few asymmetric variations have been described, of which most require the use of large amounts of chiral metal complexes. The reactivities of Nazarov cyclizations are also depending on the substituents of the divinyl ketone substrates as described in the first chapter. The substrates to study Brønsted acid-catalyzed enantioselective Nazarov cyclization were prepared following the known procedures. The dihydropyran was treated with tBuLi in THF at –78 oC and then the α,β-unsaturated aldehydes 1 were added to the reaction mixture to afford the corresponding alcohols 2 in moderate to good yields. The alcohols 2 were oxidized to divinyl ketones 3 employing Dess-Martin periodinane/pyridine (DMP/py) in CH2Cl2 at room temperature to obtain the divinyl ketones 3 in moderate to good yields (Scheme 1). Scheme 1. Preparation of substrates in order to study Brønsted acid-catalyzed enantioselective Nazarov cyclization and subsequent transformations. At the starting point, an evaluation of suitable Brønsted acid catalysts for the enantioselective Nazarov cyclization of divinyl ketone 3a was performed. The initial reactions conducted with various BINOL-phosphoric acids 4a-4e in toluene at 60 oC provided the mixture of cis and trans cyclopentenones 5a with enantioselectivities of up to 82% ee (Table 1, entries 1-5). Eventually, improved reactivity could be achieved by using the corresponding N-triflylphosphoramides 4f and 4g, which even at 0 oC gave complete conversion after ten minutes. Additionally, it was shown that the use of these catalysts significantly enhanced both the diastereoselectivity (cis/trans ratio up to 7:1) and the enantioselectivity (up to 96% ee; Table 1, entries 6 and 7). Table 1. Evaluation of Brønsted acids 4a-4g in the enantioselective Nazarov cyclization. The scope of the Brønsted acid-catalyzed enantioselective Nazarov cyclization of various divinyl ketones 3 was explored under an optimized reaction condition (Scheme 2). Treatment of divinyl ketones 3 in CHCl3 in the presence of 2 mol% chiral BINOL-Ntriflylphosphoramide 4g at 0 oC for 1-6 h provided the corresponding cyclopentenone 5 in good yields (45-92%) with excellent enantioselectivities (up to 93% ee) (Scheme 2). Furthermore, the isomerization of cis-cyclopentenone under basic condition led to the corresponding trans-cyclopentenone without loss of enantiomeric purity. This efficient method introduced here was not only the first example of an organocatalytic electrocyclic reaction but also represented the first enantioselective activation of a carbonyl group catalyzed by a chiral BINOL phosphoric acid. Compared to the metal-catalyzed reaction, special features of this new Brønsted acid-catalyzed electrocyclization are the lower catalyst loadings (2 mol%), higher enantioselectivities, accessibility to all possible stereoisomers, as well as the mild conditions. ....
Cytochrome c oxidase (COX), the last enzyme of the respiratory chain of aerobic organisms, catalyzes the reduction of molecular oxygen to water. It is a redox-linked proton pump, whose mechanism of proton pumping has been controversially discussed, and the coupling of proton and electron transfer is still not understood. Here, we investigated the kinetics of proton transfer reactions following the injection of a single electron into the fully oxidized enzyme and its transfer to the hemes using time-resolved absorption spectroscopy and pH indicator dyes. By comparison of proton uptake and release kinetics observed for solubilized COX and COX-containing liposomes, we conclude that the 1-μs electron injection into CuA, close to the positive membrane side (P-side) of the enzyme, already results in proton uptake from both the P-side and the N (negative)-side (1.5 H+/COX and 1 H+/COX, respectively). The subsequent 10-μs transfer of the electron to heme a is accompanied by the release of 1 proton from the P-side to the aqueous bulk phase, leaving ∼0.5 H+/COX at this side to electrostatically compensate the charge of the electron. With ∼200 μs, all but 0.4 H+ at the N-side are released to the bulk phase, and the remaining proton is transferred toward the hemes to a so-called “pump site.” Thus, this proton may already be taken up by the enzyme as early as during the first electron transfer to CuA. These results support the idea of a proton-collecting antenna, switched on by electron injection.
NEK family kinases are serine/threonine kinases that have been functionally implicated in the regulation of the disjunction of the centrosome, the assembly of the mitotic spindle, the function of the primary cilium and the DNA damage response. NEK1 shows pleiotropic functions and has been found to be mutated in cancer cells, ciliopathies such as the polycystic kidney disease, as well as in the genetic diseases short-rib thoracic dysplasia, Mohr-syndrome and amyotrophic lateral sclerosis. NEK1 is essential for the ionizing radiation DNA damage response and priming of the ATR kinase and of Rad54 through phosphorylation. Here we report on the structure of the kinase domain of human NEK1 in its apo- and ATP-mimetic inhibitor bound forms. The inhibitor bound structure may allow the design of NEK specific chemo-sensitizing agents to act in conjunction with chemo- or radiation therapy of cancer cells. Furthermore, we characterized the dynamic protein interactome of NEK1 after DNA damage challenge with cisplatin. Our data suggest that NEK1 and its interaction partners trigger the DNA damage pathways responsible for correcting DNA crosslinks.
Imaging non-adherent cells by super-resolution far-field fluorescence microscopy is currently not possible because of their rapid movement while in suspension. Holographic optical tweezers (HOTs) enable the ability to freely control the number and position of optical traps, thus facilitating the unrestricted manipulation of cells in a volume around the focal plane. Here we show that immobilizing non-adherent cells by optical tweezers is sufficient to achieve optical resolution well below the diffraction limit using localization microscopy. Individual cells can be oriented arbitrarily but preferably either horizontally or vertically relative to the microscope’s image plane, enabling access to sample sections that are impossible to achieve with conventional sample preparation and immobilization. This opens up new opportunities to super-resolve the nanoscale organization of chromosomal DNA in individual bacterial cells.
The 3,5-methoxy groups in the title compound, C16H23NO4, are almost coplanar with the aromatic ring, whereas the 4-methoxy group is bent out of this plane. The three CH3—O—C—C torsion angles are -1.51 (18), 0.73 (19) and 75.33 (15)°. The cyclohexane ring adopts a chair conformation. In the crystal, molecules are connected by intermolecular N—H ... O hydrogen bonds into chains running along the b axis.
The crystal packing of the title compound, C13H19NO·0.33C7H8, shows a channel at [001], which contains grossly disordered toluene solvent molecules. The angle between the benzene ring and the mean plane of the formamide group is 71.1 (1)°. The amide groups of neighbouring molecules are connected by N—H(...)O hydrogen bonds, forming 21 helical chains propagating along [001]. Molecules are also connected by weak intermolecular C—H(...)O hydrogen bonds, forming 61 helices.
The title compound, [Li3(C4F9O)3(C3H6O)3], features an open Li/O cube with an Li ion missing at one corner. Three of the four bridging O atoms of the cube carry a fluorinated tert-butyl residue, whereas the fourth is part of an acetone molecule. Two of the Li atoms are further bonded to a non-bridging acetone molecule. Two of the lithium ion coordination geometries are very distorted LiO4 tetrahedra; the third could be described as a very distorted LiO3 T-shape with two distant F-atom neighbours. The Li[cdots, three dots, centered]Li contact distances for the three-coordinate Li+ ion [2.608 (14) and 2.631 (12) Å] are much shorter that the contact distance [2.940 (13) Å] between the tetrahedrally coordinated species.
The von Willebrand factor (VWF) is a glycoprotein in the blood that plays a central role in hemostasis. Among other functions, VWF is responsible for platelet adhesion at sites of injury via its A1 domain. Its adjacent VWF domain A2 exposes a cleavage site under shear to degrade long VWF fibers in order to prevent thrombosis. Recently, it has been shown that VWF A1/A2 interactions inhibit the binding of platelets to VWF domain A1 in a force-dependent manner prior to A2 cleavage. However, whether and how this interaction also takes place in longer VWF fragments as well as the strength of this interaction in the light of typical elongation forces imposed by the shear flow of blood remained elusive. Here, we addressed these questions by using single molecule force spectroscopy (SMFS), Brownian dynamics (BD), and molecular dynamics (MD) simulations. Our SMFS measurements demonstrate that the A2 domain has the ability to bind not only to single A1 domains but also to VWF A1A2 fragments. SMFS experiments of a mutant [A2] domain, containing a disulfide bond which stabilizes the domain against unfolding, enhanced A1 binding. This observation suggests that the mutant adopts a more stable conformation for binding to A1. We found intermolecular A1/A2 interactions to be preferred over intramolecular A1/A2 interactions. Our data are also consistent with the existence of two cooperatively acting binding sites for A2 in the A1 domain. Our SMFS measurements revealed a slip-bond behavior for the A1/A2 interaction and their lifetimes were estimated for forces acting on VWF multimers at physiological shear rates using BD simulations. Complementary fitting of AFM rupture forces in the MD simulation range adequately reproduced the force response of the A1/A2 complex spanning a wide range of loading rates. In conclusion, we here characterized the auto-inhibitory mechanism of the intramolecular A1/A2 bond as a shear dependent safeguard of VWF, which prevents the interaction of VWF with platelets.
The core of photosystem I (PS1) is composed of the two related integral membrane polypeptides, PsaA and PsaB, which bind two symmetrical branches of cofactors, each consisting of two chlorophylls and a phylloquinone, that potentially link the primary electron donor and the tertiary acceptor. In an effort to identify amino acid residues near the phylloquinone binding sites, all tryptophans and histidines that are conserved between PsaA and PsaB in the region of the 10th and 11th transmembrane alpha-helices were mutated in Chlamydomonas reinhardtii. The mutant PS1 reaction centers appear to assemble normally and possess photochemical activity. An electron paramagnetic resonance (EPR) signal attributed to the phylloquinone anion radical (A(1)(-)) can be observed either transiently or after illumination of reaction centers with pre-reduced iron-sulfur clusters. Mutation of PsaA-Trp(693) to Phe resulted in an inability to photo-accumulate A(1)(-), whereas mutation of the analogous tryptophan in PsaB (PsaB-Trp(673)) did not produce this effect. The PsaA-W693F mutation also produced spectral changes in the time-resolved EPR spectrum of the P(700)(+) A(1)(-) radical pair, whereas the analogous mutation in PsaB had no observable effect. These observations indicate that the A(1)(-) phylloquinone radical observed by EPR occupies the phylloquinone-binding site containing PsaA-Trp(693). However, mutation of either tryptophan accelerated charge recombination from the terminal Fe-S clusters.
The cytochrome bc1 complex is a dimeric enzyme of the inner mitochondrial membrane that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is rereduced at a second center, referred to as center N. To better understand the mechanism of ubiquinol oxidation, we have examined catalytic activities and pre-steady-state reduction kinetics of yeast cytochrome bc1 complexes with mutations in cytochrome b that we expected would affect oxidation of ubiquinol. We mutated two residues thought to be involved in proton conduction linked to ubiquinol oxidation, Tyr132 and Glu272, and two residues proposed to be involved in docking ubiquinol into the center P pocket, Phe129 and Tyr279. Substitution of Phe129 by lysine or arginine yielded a respiration-deficient phenotype and lipid-dependent catalytic activity. Increased bypass reactions were detectable for both variants, with F129K showing the more severe effects. Substitution with lysine leads to a disturbed coordination of a b heme as deduced from changes in the midpoint potential and the EPR signature. Removal of the aromatic side chain in position Tyr279 lowers the catalytic activity accompanied by a low level of bypass reactions. Pre-steady-state kinetics of the enzymes modified at Glu272 and Tyr132 confirmed the importance of their functional groups for electron transfer. Altered center N kinetics and activation of ubiquinol oxidation by binding of cytochrome c in the Y132F and E272D enzymes indicate long range effects of these mutations.
The effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochrome c oxidase was studied using a combination of optical spectroscopy, electrochemical and rapid kinetics techniques, and time-resolved measurements of electrical membrane potential. The mutation resulted in a blue-shift of the heme a alpha-band by 15 nm and partial occupation of the low-spin heme site by heme O. Additionally, there was a marked decrease in the midpoint potential of the low-spin heme, resulting in slow reduction of this heme species. A stopped-flow investigation of the reaction with ferrocytochrome c yielded a kinetic difference spectrum resembling that of heme a(3). This observation, and the absence of transient absorbance changes at the corresponding wavelength of the low-spin heme, suggests that, in the mutant enzyme, electron transfer from Cu(A) to the binuclear center may not occur via heme a but that instead direct electron transfer to the high-spin heme is the dominating process. This was supported by charge translocation measurements where Deltapsi generation was completely inhibited in the presence of KCN. Our results thus provide an example for how the interplay between protein and cofactors can modulate the functional properties of the enzyme complex.
Many interesting and important membrane proteins are hetero-oligomeric. However, besides naturally abundant examples, the structures of relatively few such complexes are known. Partly, this is due to difficulties in expression, stoichiometric assembly, and in the evaluation of their stability prior to crystallization trials. Here we describe a new approach, which allows rapid assessment of protein complex quality, assembly and stoichiometry, simplifying the search for conditions conducive to long-term stability and crystallization. Multicolour fluorescence size-exclusion chromatography (MC-FSEC) is used to enable tracking of individual subunits through expression, solubilization and purification steps. We show how the method has been applied to the heterodimeric transporter associated with antigen processing (TAP) and demonstrate how it may be extended in order to analyse membrane multisubunit assemblies.
Prostaglandin E2 (PGE2) favors multiple aspects of tumor development and immune evasion. Therefore, microsomal prostaglandin E synthase (mPGES-1/-2), is a potential target for cancer therapy. We explored whether inhibiting mPGES-1 in human and mouse models of breast cancer affects tumor-associated immunity. A new model of breast tumor spheroid killing by human PBMCs was developed. In this model, tumor killing required CD80 expression by tumor-associated phagocytes to trigger cytotoxic T cell activation. Pharmacological mPGES-1 inhibition increased CD80 expression, whereas addition of PGE2, a prostaglandin E2 receptor 2 (EP2) agonist, or activation of signaling downstream of EP2 reduced CD80 expression. Genetic ablation of mPGES-1 resulted in markedly reduced tumor growth in PyMT mice. Macrophages of mPGES-1-/- PyMT mice indeed expressed elevated levels of CD80 compared to their wildtype counterparts. CD80 expression in tumor-spheroid infiltrating mPGES-1-/- macrophages translated into antigen-specific cytotoxic T cell activation. In conclusion, mPGES-1 inhibition elevates CD80 expression by tumor-associated phagocytes to restrict tumor growth. We propose that mPGES-1 inhibition in combination with immune cell activation might be part of a therapeutic strategy to overcome the immunosuppressive tumor microenvironment.
Molecular recognition of M1-linked ubiquitin chains by native and phosphorylated UBAN domains
(2019)
Although the Ub-binding domain in ABIN proteins and NEMO (UBAN) is highly conserved, UBAN-containing proteins exhibit different Ub-binding properties, resulting in their diverse biological roles. Post-translational modifications further control UBAN domain specificity for poly-Ub chains. However, precisely, how the UBAN domain structurally confers such functional diversity remains poorly understood. Here we report crystal structures of ABIN-1 alone and in complex with one or two M1-linked di-Ub chains. ABIN-1 UBAN forms a homo-dimer that provides two symmetrical Ub-binding sites on either side of the coiled-coil structure. Moreover, crystal structures of ABIN1 UBAN in complex with di-Ub chains reveal a concentration-dependency of UBAN/di-Ub binding stoichiometry. Analysis of UBAN/M1-linked di-Ub binding characteristics indicates that phosphorylated S473 in OPTN and its corresponding phospho-mimetic residue in ABIN-1 (E484) are essential for high affinity interactions with M1-linked Ub chains. Also, a phospho-mimetic mutation of A303 in NEMO, corresponding to S473 of OPTN, increases binding affinity for M1-linked Ub chains. These findings are in line with the diverse physiological roles of UBAN domains, as phosphorylation of OPTN UBAN is required to enhance its binding to Ub during mitophagy.
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.
Transcription factor IIS (TFIIS) is a protein known for catalyzing the cleavage reaction of the 3′-end of backtracked RNA transcript, allowing RNA polymerase II (Pol II) to reactivate the transcription process from the arrested state. Recent structural studies have provided a molecular basis of protein-protein interaction between TFIIS and Pol II. However, the detailed dynamic conformational changes of TFIIS upon binding to Pol II and the related thermodynamic information are largely unknown. Here we use computational approaches to investigate the conformational space of TFIIS in the Pol II-bound and Pol II-free (unbound) states. Our results reveal two distinct conformations of TFIIS: the closed and the open forms. The closed form is dominant in the Pol II-free (unbound) state of TFIIS, whereas the open form is favorable in the Pol II-bound state. Furthermore, we discuss the free energy difference involved in the conformational changes between the two forms in the presence or absence of Pol II. Additionally, our analysis indicates that hydrophobic interactions and the protein-protein interactions between TFIIS and Pol II are crucial for inducing the conformational changes of TFIIS. Our results provide novel insights into the functional interplay between Pol II and TFIIS as well as mechanism of reactivation of Pol II transcription by TFIIS.
Na+/H+ antiporters are integral membrane proteins that are present in almost every cell and in every kingdom of life. They are essential for the regulation of intracellular pH-value, Na+-concentration and cell volume. These secondary active transporters exchange sodium ions against protons via an alternating access mechanism, which is not understood in full detail. Na+/H+ antiporters show distinct species-specific transport characteristics and regulatory properties that correlate with respective physiological functions. Here we present the characterization of the Na+/H+ antiporter NhaA from Salmonella enterica serovar Thyphimurium LT2, the causing agent of food-born human gastroenteritis and typhoid like infections. The recombinant antiporter was functional in vivo and in vitro. Expression of its gene complemented the Na+-sensitive phenotype of an E. coli strain that lacks the main Na+/H+ antiporters. Purified to homogeneity, the antiporter was a dimer in solution as accurately determined by size-exclusion chromatography combined with multi-angle laser-light scattering and refractive index monitoring. The purified antiporter was fully capable of electrogenic Na+(Li+)/H+-antiport when reconstituted in proteoliposomes and assayed by solid-supported membrane-based electrophysiological measurements. Transport activity was inhibited by 2-aminoperimidine. The recorded negative currents were in agreement with a 1Na+(Li+)/2H+ stoichiometry. Transport activity was low at pH 7 and up-regulation above this pH value was accompanied by a nearly 10-fold decrease of KmNa (16 mM at pH 8.5) supporting a competitive substrate binding mechanism. K+ does not affect Na+ affinity or transport of substrate cations, indicating that selectivity of the antiport arises from the substrate binding step. In contrast to homologous E. coli NhaA, transport activity remains high at pH values above 8.5. The antiporter from S. Typhimurium is a promising candidate for combined structural and functional studies to contribute to the elucidation of the mechanism of pH-dependent Na+/H+ antiporters and to provide insights in the molecular basis of species-specific growth and survival strategies.
Methanogenic archaea share one ion gradient forming reaction in their energy metabolism catalyzed by the membrane-spanning multisubunit complex N5-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH or simply Mtr). In this reaction the methyl group transfer from methyl-tetrahydromethanopterin to coenzyme M mediated by cobalamin is coupled with the vectorial translocation of Na+ across the cytoplasmic membrane. No detailed structural and mechanistic data are reported about this process. In the present work we describe a procedure to provide a highly pure and homogenous Mtr complex on the basis of a selective removal of the only soluble subunit MtrH with the membrane perturbing agent dimethyl maleic anhydride and a subsequent two-step chromatographic purification. A molecular mass determination of the Mtr complex by laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) and size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) resulted in a (MtrABCDEFG)3 heterotrimeric complex of ca. 430 kDa with both techniques. Taking into account that the membrane protein complex contains various firmly bound small molecules, predominantly detergent molecules, the stoichiometry of the subunits is most likely 1:1. A schematic model for the subunit arrangement within the MtrABCDEFG protomer was deduced from the mass of Mtr subcomplexes obtained by harsh IR-laser LILBID-MS.
Some anaerobic bacteria use biotin-dependent Na+-translocating decarboxylases (Bdc) of β-keto acids or their thioester analogs as key enzymes in their energy metabolism. Glutaconyl-CoA decarboxylase (Gcd), a member of this protein family, drives the endergonic translocation of Na+ across the membrane with the exergonic decarboxylation of glutaconyl-CoA (ΔG0’ ≈−30 kJ/mol) to crotonyl-CoA. Here, we report on the molecular characterization of Gcd from Clostridium symbiosum based on native PAGE, size exclusion chromatography (SEC) and laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS). The obtained molecular mass of ca. 400 kDa fits to the DNA sequence-derived mass of 379 kDa with a subunit composition of 4 GcdA (65 kDa), 2 GcdB (35 kDa), GcdC1 (15 kDa), GcdC2 (14 kDa), and 2 GcdD (10 kDa). Low-resolution structural information was achieved from preliminary electron microscopic (EM) measurements, which resulted in a 3D reconstruction model based on negative-stained particles. The Gcd structure is built up of a membrane-spanning base primarily composed of the GcdB dimer and a solvent-exposed head with the GcdA tetramer as major component. Both globular parts are bridged by a linker presumably built up of segments of GcdC1, GcdC2 and the 2 GcdDs. The structure of the highly mobile Gcd complex represents a template for the global architecture of the Bdc family.
Das Enzym 5-Lipoxygenase (5-LO) spielt eine entscheidende Rolle in der Generierung von Leukotrienen. Diese fungieren als wichtige proinflammatorische Mediatoren. Darüber hinaus ist die 5-LO anhand ihrer N-terminalen Domäne in der Lage mit verschiedenen Proteinen zu interagieren. Unter den Interaktionspartnern befindet sich Dicer, ein Enzym welches für den finalen Schritt der microRNA (miRNA)-Biosynthese verantwortlich ist. MiRNA sind kurze, nicht kodierende RNA Stränge mit einer typischen Länge von etwa 23 Nukleotiden, die an der posttranskriptionalen Regulierung der Proteinbiosynthese beteiligt sind.
Ziel dieser Arbeit war es den Einfluss der 5-LO auf die miRNA-Prozessierung im zellulären Kontext zu untersuchen. Als Modellsystem wurde die MonoMac6 (MM6) Zelllinie ausgewählt. MM6-Zellen exprimieren im undifferenzierten Grundzustand nur geringe Mengen an 5-LO. Erst nach Differenzierung mittels transformierenden Wachstumsfaktors ß (TGFß) und Calcitriol kommt es zur Induktion der 5-LO Proteinbiosynthese. Darüber hinaus war es Basavarajappa et al. möglich die 5-LO-Expression in diesen Zellen mittels RNA-Interferenz stark herunter zu regulieren (Δ5-LO).
Um die Frage der Auswirkungen des 5-LO knockdowns auf die miRNA-Expression analysieren zu können, wurde ein Microarray in differenzierten Kontroll-und Δ5-LO-Zellen durchgeführt.Es wurden 37 miRNAs identifiziert deren Expression 5-LO abhängig ist. Dabei war das Niveau von 30 Vertretern in Abwesenheit der 5-LO erhöht, wohingegen die Expression von sieben miRNAs reduziert war. Unter diesen sieben herunter regulierten miRNAs befanden sich miR-99b-5p und miR-125a-5p, die einem gemeinsamen Cluster entstammen. Als Cluster wird eine Gruppe von miRNAs bezeichnet, die aus einem gemeinsamen primären Transkript (pri-miRNA) hervorgeht. Diese Eigenschaft führte zur Vermutung, dass bereits die Expression dieser pri-miRNA durch die 5-LO reguliert wird. Allerdings zeigte sichim Verlauf dieser Arbeit, dass die Expression der pri-miRNA 5-LO unabhängig verläuft. Im Gegensatz dazu wies die Zwischenstufe zwischen pri-miRNA und reifer miRNA eine reduzierte Expression in Δ5-LO Zellen auf. Für die Prozessierung dieser sogenannten precursor miRNAs (pre-miRNA) ist die Ribonuklease III Drosha verantwortlich, welche die pre-miRNA aus der jeweiligen pri-miRNAs chneidet. Das verringerte pre-miR-99b-und pre-miR-125a-Niveau ist daher ein Hinweis darauf, dass überDicerhinausmöglicherweise ebenfalls die Drosha Aktivität mittels 5-LO reguliert wird.
Des Weiteren wurde untersucht iniefern Leukotriene beziehungsweise 5-LO-Inhibitoren die Expression von miR-99b-5p und miR-125a-5p beeinflussen. Dabei stellte sich heraus, dass das miRNA-Niveau unabhängig von der vorhandenen Leukotrien-Menge ist. Das 5-LO aktivierende Protein (FLAP) besitzt dahingegen einen mit der 5-LO vergleichbaren Einfluss auf die reife miRNA. FLAP ist ein weiterer Interaktionspartner der 5-LO und essentiell für die Leukotrien-Biosynthese in vivo. Anhand von Protein-Lokalisationsstudien mittels Immunofluoreszenz konnte gezeigt werden, dass FLAP außerdem in der Lage zu sein scheint die Relokalisation der 5-LO aus dem Zytoplasma in den Nukleus einzuschränken. Im Zytoplasma ist die 5-LO in der Lage mit Dicer zu interagieren. Daten bezüglich einer Interaktion zwischen Drosha und 5-LO im Zellkern liegen bisher nicht vor. Eine etwaige Interaktion könnte allerdings helfen die reduzierten pre-miRNA Spiegel in Abwesenheit der 5-LO zu erklären.
Im Laufe dieser Arbeit wurden weiterhin die Auswirkungen von proinflammatorischen Lipopolysacchariden (LPS) auf die Prozessierung von miR-99b-5p und miR-125a-5p analysiert. Ausschließlich in Anwesenheit von 5-LO zeigte sich eine differenzierungsunabhängig gesteigerte Biosynthese der pri-und der reifen miRNA. Allerdings konnte kein Einfluss von LPS auf die 5-LO-Lokalisation beziehungsweise Expression festgestellt werden. Aufgrund dessen sind weiterführende Studien, die den Zusammenhang zwischen LPS induzierter miR-99b-5p- beziehungsweise miR-125a-5p-Biosynthese und 5-LO herstellen, nötig.
Abschließend hat sich diese Arbeit mit den Zielgenen der durch 5-LO regulierten miRNAs auseinandergesetzt. Es konnte gezeigt werden, dass in Abwesenheit von miR-99b-5p und miR-125a-5p die Freisetzung der beiden durch LPS stimulierten Zytokine Interleukin 6 (IL-6) und Tumornekrosefaktor α (TNFα) gesteigert ist. Interessanterweise besitzt TNFα einen stimulierenden Effekt auf die Leukotrien-Biosynthese. Allerdings konnte kein direkter Zusammenhang zwischen miR-99b-5p/miR-125a-5p Expression, TNFα und der 5-LO Aktivität hergestellt werden. Der Einsatz von miR-99b-5p-und miR-125a-5p-Inhibitoren zeigte keine Auswirkungen auf die Leukotrien-Biosynthese nach LPS Stimulation. Im Gegensatz dazu konnte in unstimulierten Zellen eine signifikante Aktivitätssteigerung in Abwesenheit von miR-125a-5p festgestellt werden. Diese Beobachtungen legen nahe, dass miR-125a-5p einen TNFα unabhängigen Einfluss auf die 5-LO Aktivität besitzt. In LPS stimulierten Zellen kommt es möglicherweise zu Überlagerungen dieses Effektes.
Zusammenfassend konnte in dieser Arbeit gezeigt werden, dass 5-LO eine regulierende Funktion auf die Reifung der beiden miRNAs miR-99b-5p und miR-125a-5p aufweist. Dieser Effekt könnte einer direkten Interaktion zwischen 5-LO und Dicer zuzuschreiben sein. Des Weiteren konnte gezeigt werden, dass die Regulierung der Expression bestimmter miRNAs mittels 5-LO nicht auf deren kanonischer enzymatischer Aktivität beruht. Diese Ergebnisse schlagen eine neue Richtung der 5-LO-Forschung ein und können in Zukunft dazu beitragen 5-LO vermittelte Effekte besser charakterisieren zu können.
The miRNA biogenesis is tightly regulated to avoid dysfunction and consequent disease development. Here, we describe modulation of miRNA processing as a novel noncanonical function of the 5-lipoxygenase (5-LO) enzyme in monocytic cells. In differentiated Mono Mac 6 (MM6) cells, we found an in situ interaction of 5-LO with Dicer, a key enzyme in miRNA biogenesis. RNA sequencing of small noncoding RNAs revealed a functional impact, knockout of 5-LO altered the expression profile of several miRNAs. Effects of 5-LO could be observed at two levels. qPCR analyses thus indicated that (a) 5-LO promotes the transcription of the evolutionarily conserved miR-99b/let-7e/miR-125a cluster and (b) the 5-LO-Dicer interaction downregulates the processing of pre-let-7e, resulting in an increase in miR-125a and miR-99b levels by 5-LO without concomitant changes in let-7e levels in differentiated MM6 cells. Our observations suggest that 5-LO regulates the miRNA profile by modulating the Dicer-mediated processing of distinct pre-miRNAs. 5-LO inhibits the formation of let-7e which is a well-known inducer of cell differentiation, but promotes the generation of miR-99b and miR-125a known to induce cell proliferation and the maintenance of leukemic stem cell functions.
Drug-induced liver injury (DILI) has become a major problem for patients and for clinicians, academics and the pharmaceutical industry. To date, existing hepatotoxicity test systems are only poorly predictive and the underlying mechanisms are still unclear. One of the factors known to amplify hepatotoxicity is the tumor necrosis factor alpha (TNFα), especially due to its synergy with commonly used drugs such as diclofenac. However, the exact mechanism of how diclofenac in combination with TNFα induces liver injury remains elusive. Here, we combined time-resolved immunoblotting and live-cell imaging data of HepG2 cells and primary human hepatocytes (PHH) with dynamic pathway modeling using ordinary differential equations (ODEs) to describe the complex structure of TNFα-induced NFκB signal transduction and integrated the perturbations of the pathway caused by diclofenac. The resulting mathematical model was used to systematically identify parameters affected by diclofenac. These analyses showed that more than one regulatory module of TNFα-induced NFκB signal transduction is affected by diclofenac, suggesting that hepatotoxicity is the integrated consequence of multiple changes in hepatocytes and that multiple factors define toxicity thresholds. Applying our mathematical modeling approach to other DILI-causing compounds representing different putative DILI mechanism classes enabled us to quantify their impact on pathway activation, highlighting the potential of the dynamic pathway model as a quantitative tool for the analysis of DILI compounds.
Air pollution of particulate matter (PM) from traffic emissions has a significant impact on human health. Risk assessments for different traffic participants are often performed on the basis of data from local air quality monitoring stations. Numerous studies demonstrated the limitation of this approach. To assess the risk of PM exposure to a car driver more realistically, we measure the exposure to PM in a car cabin with a mobile aerosol spectrometer in Frankfurt am Main under different settings (local variations, opened versus a closed window) and compare it with data from stationary measurement. A video camera monitored the surroundings for potential PM source detection. In-cabin concentrations peaked at 508 µg m−3 for PM10, 133.9 µg m−3 for PM2.5, and 401.3 µg m−3 for coarse particles, and strongly depended on PM size and PM concentration in ambient air. The concentration of smaller particles showed low fluctuations, but the concentration of coarse particles showed high fluctuations with maximum values on busy roads. Several of these concentration peaks were assigned to the corresponding sources with characteristic particle size distribution profiles. The closure of the car window reduced the exposure to PM, and in particular to coarse particles. The mobile measured PM values differed significantly from stationary PM measures, although good correlations were computed for finer particles. Mobile rather than stationary measurements are essential to assess the risk of PM exposure for car passengers.
Mitochondria are important for cellular health and their dysfunction is linked to a variety of diseases, especially neurodegeneration. Thus, the renewal and degradation of dysfunctional mitochondria is crucial for the well-being of organisms. The selective digestion of damaged mitochondria via the lysosome (mitophagy), is the main pathway to do so.
In my dissertational work, I investigated the connection between protein misfolding, protein import into mitochondria and the degradation of mitochondria via mitophagy. Here, I present a new model for the initiation of mitophagy without collapse of the membrane potential. This model provides the link between protein import into mitochondria, stress signal transduction to the cytosol and the mitochondrial stress sensor PINK1. To comprehensively examine how mitophagy can be triggered, I performed a genome-wide CRISPR knockout screen utilizing the mitophagy reporter mitochondrial mKEIMA. Thereby, I observed numerous novel gene deletions that induce mitophagy. Prominently, I identified an accumulation of gene deletions of the protein import and of protein quality control factors. I validated several of those and examined HSPA9 (mitochondrial HSP70) and LONP1 (a mitochondrial matrix AAA protease) in more detail, regarding their effect on mitophagy and protein import. For this, I used an established fluorescence-based, mitochondrial-targeted EGFP, as well as a newly-developed pulsed-SILAC mass spectrometry approach (mePRODmt). Depletions of both genes resulted in reduced protein import and PINK1-dependent mitophagy. Strikingly, I did not observe any loss of mitochondrial membrane potential, which was hitherto believed to be essential for activation of PINK1-mediated mitophagy. Literature shows that certain mitochondrial stressors can also induce mitophagy without mitochondrial membrane depolarization, which I confirmed with my assays. Next, I characterized the impact of LONP1 and HSPA9 depletion, which are involved in proteostasis maintenance, and the mtHSP90 inhibitor GTPP on mitochondrial protein folding in more detail. GTPP treatment and LONP1 depletion both resulted in the accumulation of an insoluble protein fraction, as judged by proteomic analysis. This insoluble protein fraction enriched several components of the presequence translocase-associated motor PAM, including TIMM44. TIMM44 acts as a link between the translocon, the import pore of the inner mitochondrial membrane (TIM) complex and the PAM complex. Thus, I hypothesized that TIMM44 dissociates from the TIM complex upon protein folding stress, when it becomes part of the insoluble protein fraction. To validate this model, I measured the TIMM44 interactome upon proteostasis disturbance using proximity labeling. Indeed, interaction of TIMM44 with the import pore was almost completely abolished, explaining the loss of matrix-targeted import upon protein folding stress. From these findings, I reasoned that an import reduction mediated by the PAM complex would likely also inhibit the degradation of PINK1. Consistent with this hypothesis, I observed that mitophagy induced by HSPA9 or LONP1 deletion was prevented when PINK1 was genetically deleted. In comparison, non-processed PINK1 was stabilized on mitochondria in wild type cells when mitochondrial protein import was impaired. On this basis, I drew the conclusion that the loss of mitochondrial import was the stress signal, which leads to the stabilization of PINK1, as it could not be processed anymore via the inner mitochondrial membrane protease PARL. PINK1 auto-activates itself upon accumulation and signals to the cytosol that this mitochondrion is damaged. Mitophagy is subsequently initiated by the ubiquitin kinase activity of PINK1. As a result, the autophagy apparatus gets activated, damaged mitochondria are engulfed by a double membrane and removed via lysosomal digestion. This proposed model is, to the best of my knowledge, the first to provide an explanation for protein folding stress-induced and protein import inhibition-triggered mitophagy without mitochondrial depolarization. The model thus extends the PINK1/PARKIN-dependent mitophagy pathway to milder stresses and clears some of the open questions in the field. Furthermore, this work is also important, because protein misfolding stress and dysfunctional mitochondria are two hallmarks of neurodegeneration. In particular, mitochondrial protein import inhibition during Parkinson’s and Huntington disease might be driver of mitochondrial dysfunction. Hence, I hope and anticipate that the newly developed protein import method, mePRODmt, and the proposed model will be beneficial to further characterize underlying processes and to establish which factors prevent or drive these disorders on molecular level.
Mistakes in translation of messenger RNA into protein are clearly a detriment to the recombinant production of pure proteins for biophysical study or the biopharmaceutical market. However, they may also provide insight into mechanistic details of the translation process. Mistakes often involve the substitution of an amino acid having an abundant codon for one having a rare codon, differing by substitution of a G base by an A base, as in the case of substitution of a lysine (AAA) for arginine (AGA). In these cases one expects the substitution frequency to depend on the relative abundances of the respective tRNAs, and thus, one might expect frequencies to be similar for all sites having the same rare codon. Here we demonstrate that, for the ADP-ribosylation factor from yeast expressed in E. coli, lysine for arginine substitutions frequencies are not the same at the 9 sites containing a rare arginine codon; mis-incorporation frequencies instead vary from less than 1 to 16%. We suggest that the context in which the codons occur (clustering of rare sites) may be responsible for the variation. The method employed to determine the frequency of mis-incorporation involves a novel mass spectrometric analysis of the products from the parallel expression of wild type and codon-optimized genes in 15N and 14N enriched media, respectively. The high sensitivity and low material requirements of the method make this a promising technology for the collection of data relevant to other mis-incorporations. The additional data could be of value in refining models for the ribosomal translation elongation process.
A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.
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.
Experimental results are presented for 180 in silico designed octapeptide sequences and their stabilizing effects on the major histocompatibility class I molecule H-2Kb. Peptide sequence design was accomplished by a combination of an ant colony optimization algorithm with artificial neural network classifiers. Experimental tests yielded nine H-2Kb stabilizing and 171 nonstabilizing peptides. 28 among the nonstabilizing octapeptides contain canonical motif residues known to be favorable for MHC I stabilization. For characterization of the area covered by stabilizing and non-stabilizing octapeptides in sequence space, we visualized the distribution of 100,603 octapeptides using a self-organizing map. The experimental results present evidence that the canonical sequence motives of the SYFPEITHI database on their own are insufficient for predicting MHC I protein stabilization.
The knowledge of three-dimensional structures of biomolecules is fundamental for the understanding of their function. Nuclear magnetic resonance (NMR) spectroscopy represents besides X-ray crystallography one of the two most widely used techniques to study macromolecules at atomic resolution. Its application has long been a laborious task that could take months and required the expertise of an experienced scientist, however, owing to the tremendous effort that has been put into the development of respective computer algorithms, structure determination by NMR spectroscopy of small- to medium sized proteins is nowadays routinely performed. CYANA is one widely used software package, which combines the majority of individual steps towards a three-dimensional structure. The most common application of the program, however, restricts to the combined automated NOE assignment and structure calculation based on NOESY peak lists and an existing chemical shift assignment. Completely automated structure determination starting from NMR spectra is to date technically possible with CYANA, however, not yet routinely applied. In order to achieve this long-term goal, the individual steps need to become more robust with regard to data imperfections such as peak overlap, spectral artifacts or a limited amount of NMR data. The work presented in this thesis should be placed within the context of increasing the reliability and improving the accuracy of structures determined by CYANA on the basis of solution- as well as solid-state NMR data.
The chapter “Systematic evaluation of combined automated NOE assignment and structure calculation with CYANA” comprises an extensive study on the robustness of the combined automated NOE assignment and structure calculation algorithm based on experimental solution NMR data sets that were modified in multiple ways to mimic different kinds of data imperfections. The results show that the algorithm is remarkably robust with regard to imperfections of the NOESY peak lists and the chemical shift tolerances but susceptible to lacking or erroneous resonance assignments, in particular for nuclei that are involved in many NOESY cross peaks.
In the chapter “Peakmatch – A simple and robust tool for peaklist matching” a method to achieve self-consistency of the chemical shift referencing among a set of peak lists is presented. The Peakmatch algorithm matches a set of peak lists to a specified reference peak list, neither of which have to be assigned, by optimizing an assignment-free match-score function. The algorithm has been extensively tested on the basis of experimental NMR data sets of five different proteins. The results show that peak lists from many different types of spectra can be matched reliably as long as they contain at least two corresponding dimensions.
NMR structures are represented by bundles of conformers whose spread indicates the precision of the atomic coordinates. However, there is as yet no reliable measure of structural accuracy, i.e. how close NMR conformers are to the “true” structure. Instead, the precision of structure bundles is widely (mis)interpreted as a measure of structural quality. Attempts to increase the precision thus often yield tight structure bundles where the precision overestimates the accuracy. To overcome this problem, the chapter “Increased reliability of NMR protein structures by consensus structure bundles” introduces a new protocol for NMR structure determination with the software package CYANA that produces bundles of conformers with a realistic precision that is throughout a large number of test data sets a much better estimate of the structural accuracy than the precision of conventional structure bundles.
Solid-state NMR is a powerful technique to study molecules which are not amenable to either solution NMR or X-ray crystallography. Despite the reporting of individual atomic resolution structures of membrane proteins and amyloid fibrils based on solid-state NMR data, the application is far from routine. One major obstacle that hinders structure determination by solid-state NMR is the overall lower quality of the solid-state NMR spectra. It is therefore necessary to increase the robustness of the computer algorithms in order to improve the results when using lower quality solid-state NMR spectra. The chapter “Structure calculations of the model protein GB1 from solid-state NMR data” presents structure calculations on the basis of a set of two-dimensional solid-state NMR experiments of the model protein GB1. The most important result obtained from these test calculations is that the limitation of structural accuracy can be attributed to inaccurate distance information resulting from the limited correlation between peak intensities and distance, which is especially severe in spin diffusion-based solid-state NMR experiments.
The chapter “Full relaxation matrix-based correction of relayed polarization transfer for solid-state NMR structure calculation” therefore introduces a method which corrects experimental peak intensities for spin diffusion in order to improve the distance information from solid-state NMR spectra. The results show that the structural accuracy can be significantly improved when using the corrected distance information, however, strongly dependent on the preliminary structural model that is required as input for the method.
Proteomic analysis is the large-scale identification and characterization of proteins including post translational modifications. Proteomics encompasses a number of approaches including bottom-up and top-down workflows which are widely used independently and complementary as tools for the successful study of protein species. However, up to the present day these techniques have not been able to overcome every analytical limitation. Mass spectrometry has played a vital role alongside proteomics in providing the required analytical means of detecting protein amounts down to the atomole range. Soft ionization methods such as matrix assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) have permitted the transfer of peptides and intact proteins into the gas phase without extensive degradation. The introduction of recent developments in MALDI technology such as the highly sensitive 4-chloro-alpha-cyanocinnamic acid matrix (Cl-CCA) as well as the commercial availability of a MALDI-LTQ-Orbitrap which boosts peptide mass accuracy below 3 parts per million (ppm), have offered new prospective in protein analysis. The aim of the current study is to incorporate these new aspects and provide further advancements in gel-based as well as gel-free proteomic workflows.
Peptides of proteolytically digested proteins are routinely analyzed by means of peptide mass fingerprinting (PMF) often combined with MS/MS analyses to complement and substantiate PMF results by peptide sequence information. The most widely used protease for enzymatic digestion is trypsin, since it exhibits a very specific cleavage behavior limited to C-terminal hydrolyses after basic amino acids. However, less specific enzymes such as chymotrypsin, elastase and pepsin have emerged as useful tools in the analysis of particular protein classes e.g. membrane, cereal, and phosphorylated proteins. In this work a comprehensive bottom-up proteomic investigation including in-solution and in-gel protein digestions of analytes covering small to large, acidic to basic, and hydrophobic to hydrophilic proteins in combination with a series of less specific enzymes are presented in order to show the superiority of the novel MALDI matrix Cl-CCA. The Cl-CCA matrix proved to be highly superior compared to standard α-cyano-4-hydroxycinnamic acid (CHCA) since an average detection of more than 2- to 3-fold peptide amount was possible depending on the used protease and, therefore, resulting in strongly increased sequence coverage. Additionally, protein identification of chymotrypsin and elastase in-gel digested protein standards was evaluated. The MALDI-LTQ-Orbitrap providing peptide mass accuracy below and up to 3 ppm in combination with Cl-CCA as matrix and newly optimized digestion conditions led to unambiguous protein identifications of all chymotryptic digests outperforming its tryptic counterparts in the case of hydrophobic bacteriorhodopsin and α-globin from hemoglobin A (α-HgbA). In addition, significantly higher sequence coverage and increased number of detected peptides was acquired. Moreover, a proposed workaround for elastase digestions was capable of providing a solution for successful identification results.
Apart from digestions of singly separated proteins, solution isoelectic focusing (sIEF) was evaluated. OFFGEL fractionation is an efficient means of fractionating peptides and proteins according to their isoelectric point (pI) values through immobilized pH gel (IPG) strips after which samples are recovered in solution. Consequently, an issue of peptide recovery arises as a category of peptides relatively insoluble to the recovery solution should be present. A method was developed including the scraping of gel matrix from the IPG strips and peptide extraction using acetonitrile as organic solvent in combination with analytical techniques such as nLC-MALDI-MS/MS for peptide identification. The nature of the peptide species remaining in-gel was analysed and attributed to peptide solubility. A general trend in which a high percentage of neutral and hydrophobic peptides remaining entrapped in the IPG gel strip was observed.
The present work also examines a new top-down proteomic workflow involving protein elution from cleavable gels containing the labile crosslinker ethylene-glycol-diacrylate (EDA). Protein amounts of as low as 100 ng loaded onto EDA gels were detected using MALDI-TOF MS in the linear acquisition mode. Proteins from 8.5 up to 78 kDa were successfully measured including a hydrophobic 15 kDa core protein attaining a GRAVY score of +0.079. Additionally, the method was compatible with one dimensional protein separation as well as for 2-D IEF/SDS-PAGE. Lastly, two methods for protein identification were tested and found to be compatible to the proposed technique.
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.
Tyrosine kinase inhibitors (TKIs) are currently the standard chemotherapeutic agents for the treatment of chronic myeloid leukemia (CML). However, due to TKI resistance acquisition in CML patients, identification of new vulnerabilities is urgently required for a sustained response to therapy. In this study, we have investigated metabolic reprogramming induced by TKIs independent of BCR-ABL1 alterations. Proteomics and metabolomics profiling of imatinib-resistant CML cells (ImaR) was performed. KU812 ImaR cells enhanced pentose phosphate pathway, glycogen synthesis, serine-glycine-one-carbon metabolism, proline synthesis and mitochondrial respiration compared with their respective syngeneic parental counterparts. Moreover, the fact that only 36% of the main carbon sources were utilized for mitochondrial respiration pointed to glycerol-phosphate shuttle as mainly contributors to mitochondrial respiration. In conclusion, CML cells that acquire TKIs resistance present a severe metabolic reprogramming associated with an increase in metabolic plasticity needed to overcome TKI-induced cell death. Moreover, this study unveils that KU812 Parental and ImaR cells viability can be targeted with metabolic inhibitors paving the way to propose novel and promising therapeutic opportunities to overcome TKI resistance in CML.
Metabolic differences between symbiont subpopulations in the deep-sea tubeworm Riftia pachyptila
(2020)
The hydrothermal vent tube worm Riftia pachyptila lives in intimate symbiosis with intracellular sulfur-oxidizing gammaproteobacteria. Although the symbiont population consists of a single 16S rRNA phylotype, bacteria in the same host animal exhibit a remarkable degree of metabolic diversity: They simultaneously utilize two carbon fixation pathways and various energy sources and electron acceptors. Whether these multiple metabolic routes are employed in the same symbiont cells, or rather in distinct symbiont subpopulations, was unclear. As Riftia symbionts vary considerably in cell size and shape, we enriched individual symbiont cell sizes by density gradient centrifugation in order to test whether symbiont cells of different sizes show different metabolic profiles. Metaproteomic analysis and statistical evaluation using clustering and random forests, supported by microscopy and flow cytometry, strongly suggest that Riftia symbiont cells of different sizes represent metabolically dissimilar stages of a physiological differentiation process: Small symbionts actively divide and may establish cellular symbiont-host interaction, as indicated by highest abundance of the cell division key protein FtsZ and highly abundant chaperones and porins in this initial phase. Large symbionts, on the other hand, apparently do not divide, but still replicate DNA, leading to DNA endoreduplication. Highest abundance of enzymes for CO2 fixation, carbon storage and biosynthesis in large symbionts indicates that in this late differentiation stage the symbiont’s metabolism is efficiently geared towards the production of organic material. We propose that this division of labor between smaller and larger symbionts benefits the productivity of the symbiosis as a whole.
Membrane-suspended nanopores in microchip arrays for stochastic transport recording and sensing
(2021)
The transport of nutrients, xenobiotics, and signaling molecules across biological membranes is essential for life. As gatekeepers of cells, membrane proteins and nanopores are key targets in pharmaceutical research and industry. Multiple techniques help in elucidating, utilizing, or mimicking the function of biological membrane-embedded nanodevices. In particular, the use of DNA origami to construct simple nanopores based on the predictable folding of nucleotides provides a promising direction for innovative sensing and sequencing approaches. Knowledge of translocation characteristics is crucial to link structural design with function. Here, we summarize recent developments and compare features of membrane-embedded nanopores with solid-state analogues. We also describe how their translocation properties are characterized by microchip systems. The recently developed silicon chips, comprising solid-state nanopores of 80 nm connecting femtoliter cavities in combination with vesicle spreading and formation of nanopore-suspended membranes, will pave the way to characterize translocation properties of nanopores and membrane proteins in high-throughput and at single-transporter resolution.
The human MET receptor tyrosine kinase contributes to vertebrate development and cell proliferation. As a proto‐oncogene, it is a target in cancer therapies. MET is also relevant for bacterial infection by Listeria monocytogenes and is activated by the bacterial protein internalin B. The processes of ligand binding, receptor activation, and the diffusion behavior of MET within the plasma membrane as well as its interconnections with various cell components are not fully understood. We investigated the receptor diffusion dynamics using single‐particle tracking and imaging fluorescence correlation spectroscopy and elucidated mobility states of resting and internalin B‐bound MET. We show that internalin B‐bound MET exhibits lower diffusion coefficients and diffuses in a more confined area in the membrane. We report that the fraction of immobile receptors is larger for internalin B‐bound receptors than for resting MET. Results of single‐particle tracking in cells treated with various cytotoxins depleting cholesterol from the membrane and disrupting the actin cytoskeleton and microtubules suggest that cholesterol and actin influence MET diffusion dynamics, while microtubules do not have any effect.
The synthesis and single crystal structure of a new cocrystal, which is composed of OHphenolic∙∙∙OHphenolic∙∙∙Naminalic supramolecular heterosynthons assembled from 4-tert-butylphenol and the macrocyclic aminal TATU, is presented. This cocrystal was prepared by solvent-free assisted grinding, which is a commonly used mechanochemical method. Crystal structure, supramolecular assembly through hydrogen bonding interactions as well as the physical and spectroscopic properties of the title cocrystal are presented in this paper.
Solvent-free treatment of 1,3,6,8-tetraazatricyclo[4.3.1.13,8]undecano (TATU) with 4-chloro-3,5-dimethylphenol led to the formation of the title co-crystal, C7H14N4·2C8H9ClO. The asymmetric unit contains one aminal cage molecule and two phenol molecules linked via two O-H...N hydrogen bonds. In the aminal cage, the N-CH2-CH2-N unit is slightly distorted from a syn periplanar geometry. Aromatic [pi]-[pi] stacking between the benzene rings from two different neighbouring phenol molecules [centroid-centroid distance = 4.0570 (11) Å] consolidates the crystal packing.
FUSE Binding Protein 1 (FUBP1) is a transcriptional regulator, which is overexpressed in various cancer entities, including hepatocellular carcinoma (HCC) and colorectal cancer (CRC). It fulfills pro-proliferative and anti-apoptotic functions in cancer cells, resulting in increased proliferation and reduced sensitivity towards apoptotic stimuli.
Previously, camptothecin (CPT) and its clinically used analog 7-ethyl 10hydroxycamptothecin (SN-38) were shown to inhibit FUBP1 in biophysical interaction displacement assays (AlphaScreen; surface plasmon resonance, SPR), and first insights into the cellular effects of FUBP1 inhibition were obtained. CPT and SN-38 are known to potently inhibit topoisomerase 1 (TOP 1), and until today, these inhibitors were thought to be specific for this target. This could be disproved by our FUBP1 binding studies. An open issue, which is addressed in this thesis, was the contribution of FUBP1 inhibition to SN-38-mediated apoptosis apoptosis.
During this thesis, a low micromolar efficacy of CPT/SN-38-induced inhibition of FUBP1 binding to the Far Upstream Sequence Element (FUSE) oligonucleotide of p21 was determined. Furthermore, FUBP1 was for the first time shown to directly interact with a potential FUSE sequence upstream of the transcription start in pro-apoptotic gene BIK. In proof of-principle experiments, an effective inhibition of the binding of FUBP1 to the FUSE BIK DNA by CPT/SN-38 was verified.
One of the main goals of this thesis was to further elucidate the contribution of cellular FUBP1-inhibition by CPT/SN-38 to the anti-cancer potential of these substances. For this purpose, the TOP 1 mutant and TOP 1 wild type colorectal cancer sub-cell lines HCT116 G7 and HCT116 S were used. CPT/SN-38 was shown to induce apoptosis in single and combinatorial treatments with mitomycin c (MMC), independently of the TOP 1 mutation status of the cells. Furthermore, a prominent induction of a FUBP1 target gene signature was observed upon treatment of both cell lines with CPT/SN-38. Consequently, CPT/SN-38 was able to fulfill its anticancer effects in these cells, although TOP 1 could not be the main target in the mutant cell line.
In a second approach to gain indirect evidence for FUBP1 dependent effects of CPT/SN-38, the TOP 1-specific inhibitors topotecan (TTN) and β lapachone (BL) were used for the treatment of HCC and CRC cell lines. Interestingly, the TOP 1 inhibitors TTN and BL exhibited a reduced potency in apoptosis induction compared to the dual (FUBP1 and TOP 1) inhibitor SN-38.
Finally, two independent screens for a specific FUBP1 inhibitor were performed. In the first approach, a small number of structural and functional CPT-derivatives that exhibited a reduced inhibitory potential against TOP 1, were tested for their ability to interfere with the FUBP1/FUSE binding. Two particular indenoisoquinoline derivatives revealed potent in vitro inhibition of FUBP1 with low micromolar IC50 values.
In a second approach, previously identified candidate FUBP1 inhibitors that had been isolated from the Maybridge Hit Finder library served as lead structures for a structure activity relationship (SAR) study of the inhibition of FUBP1 binding to the FUSE oligonucleotide. After two cycles of optimization, a medium-potent FUBP1 inhibitor was obtained that induced effective deregulation of FUBP1 target genes in cell culture experiments.
Proton-pumping complex I of the mitochondrial respiratory chain is among the largest and most complex membrane protein complexes. The enzyme contributes substantially to oxidative energy-conversion in eukaryotic cells. Its malfunctions are implicated in many hereditary and degenerative disorders. Here, we report the X-ray structure of mitochondrial complex I at 3.6- 3.9 Å resolution describing in detail the central subunits that execute the bioenergetic function. A continuous axis of basic and acidic residues running centrally through the membrane arm connects the ubiquinone reduction site in the hydrophilic arm to four putative proton-pumping units. The binding position for a substrate analogous inhibitor and blockage of the predicted ubiquinone binding site provide a model for the ‘deactive’ form of the enzyme. The proposed transition into the active form is based on a concerted structural rearrangement at the ubiquinone reduction site rendering support for a two-state stabilization-change mechanism of protonpumping.
The light-harvesting complex of photosystem II (LHC-II) is the major antenna complex in plant photosynthesis. It accounts for roughly 30% of the total protein in plant chloroplasts, which makes it arguably the most abundant membrane protein on Earth, and binds about half of plant chlorophyll (Chl). The complex assembles as a trimer in the thylakoid membrane and binds a total of 54 pigment molecules, including 24 Chl a, 18 Chl b, 6 lutein (Lut), 3 neoxanthin (Neo) and 3 violaxanthin (Vio). LHC-II has five key roles in plant photosynthesis. It: (1) harvests sunlight and transmits excitation energy to the reaction centres of photosystems II and I, (2) regulates the amount of excitation energy reaching each of the two photosystems, (3) has a structural role in the architecture of the photosynthetic supercomplexes, (4) contributes to the tight appression of thylakoid membranes in chloroplast grana, and (5) protects the photosynthetic apparatus from photo damage by non photochemical quenching (NPQ). A major fraction of NPQ is accounted for its energy-dependent component qE. Despite being critical for plant survival and having been studied for decades, the exact details of how excess absorbed light energy is dissipated under qE conditions remain enigmatic. Today it is accepted that qE is regulated by the magnitude of the pH gradient (ΔpH) across the thylakoid membrane. It is also well documented that the drop in pH in the thylakoid lumen during high-light conditions activates the enzyme violaxanthin de-epoxidase (VDE), which converts the carotenoid Vio into zeaxanthin (Zea) as part of the xanthophyll cycle. Additionally, studies with Arabidopsis mutants revealed that the photosystem II subunit PsbS is necessary for qE. How these physiological responses switch LHC-II from the active, energy transmitting to the quenched, energy-dissipating state, in which the solar energy is not transmitted to the photosystems but instead dissipated as heat, remains unclear and is the subject of this thesis. From the results obtained during this doctoral work, five main conclusions can be drawn concerning the mechanism of qE: 1. Substitution of Vio by Zea in LHC-II is not sufficient for efficient dissipation of excess excitation energy. 2. Aggregation quenching of LHC-II does not require Vio, Neo nor a specific Chl pair. 3. With one exception, the pigment structure in LHC-II is rigid. 4. The two X-ray structures of LHC-II show the same energy transmitting state of the complex. 5. Crystalline LHC-II resembles the complex in the thylakoid membrane. Models of the aggregation quenching mechanism in vitro and the qE mechanism in vivo are presented as a corollary of this doctoral work. LHC-II aggregation quenching in vitro is attributed to the formation of energy sinks on the periphery of LHC-II through random interaction with other trimers, free pigments or impurities. A similar but unrelated process is proposed to occur in the thylakoid membrane, by which excess excitation energy is dissipated upon specific interaction between LHC-II and a PsbS monomer carrying Zea. At the end of this thesis, an innovative experimental model for the analysis of all key aspects of qE is proposed in order to finally solve the qE enigma, one of the last unresolved problems in photosynthesis research.
Mechanism of Na+-dependent citrate transport from the structure of an asymmetrical CitS dimer
(2015)
The common human pathogen Salmonella enterica takes up citrate as a nutrient via the sodium symporter SeCitS. Uniquely, our 2.5 Å x-ray structure of the SeCitS dimer shows three different conformations of the active protomer. One protomer is in the outside-facing state. Two are in different inside-facing states. All three states resolve the substrates in their respective binding environments. Together with comprehensive functional studies on reconstituted proteoliposomes, the structures explain the transport mechanism in detail. Our results indicate a six-step process, with a rigid-body 31° rotation of a helix bundle that translocates the bound substrates by 16 Å across the membrane. Similar transport mechanisms may apply to a wide variety of related and unrelated secondary transporters, including important drug targets.
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.
Potential energy, dipole moment, and electronic transition moment functions for the A 3Πand X3Σ- states of PH have been calculated from highly correlated electronic wavefunctions. The electric dipole moments in the vibrational ground state of PH are calculated to be 0.637 Debye (A 3Π) and 0.403 Debye (X3Σ-). The predicted rates of spontaneous emission between low lying vibrational states of the X state lie in the range of 46 to 109 sec-1 (PH) and 12 to 30 sec-1 (PD). The calculated radiative lifetime of the v' = 0 level in the A 3Π state of 400 ns is lower by about 10 percent than the most recent experimental value. The classical intersection of the 5Σ- and the A 3Πstate has been calculated to lie between v' = 2 and 3 with an expected uncertainty of about 500 cm−1, whereas the onset of the rotationally dependent predissociation lies at v' = 0, J' = 11.
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.
In der vorliegenden Arbeit werden Verfahren der Mathematik und Informatik entwickelt und eingesetzt, um Struktur, Dynamik und biologische Aktivität aus NMR spektroskopischen und empirischen Parametern zu bestimmen. Dolastatin 10 und Epothilon A sind potentielle Wirkstoffe gegen Krebs, da sie durch Wechselwirkung mit Tubulin die Zellteilung unterbinden. Die 3D Struktur beider Wirkstoffe in Lösung und die Struktur von an Tubulin gebundenem Epothilon A wird aus NMR spektroskopischen Parametern bestimmt. Dolastatin 10 liegt in einem konformationellen Gleichgewicht zwischen der cis -- und trans -- Konformation in der ungewöhnlichen Aminosäure DAP vor. Beide Konformationen des flexiblen Pentapeptids können bestimmt werden mit RMSD = 1.423 Å für das cis -- Konformer und RMSD = 1.488 Å für das trans -- Konformer. Während das trans -- Konformer gestreckt vorliegt, faltet das cis -- Konformer am DAP zurück. Epothilone A ist durch einen Makrozyklus weniger flexibel und sowohl die an Tubulin gebundene Struktur (RMSD = 0.537 Å) als auch freie Form (RMSD = 0.497 Å) kann mit geringen RMSD -- Werten bestimmt werden. Die Struktur der freien Form, welche in Lösung hauptsächlich vorliegt, ist mit der Röntgenstruktur weitgehend identisch. In der an Tubulin gebundenen Form wird eine essentielle Umorientierung der Seitenkette beobachtet, die für die Wechselwirkung mit Tubulin entscheidend ist. Dipolare Kopplungen eines Proteins sind geeignet, eine 3D Homologiesuche in der PDB durchzuführen, da die relative Orientierung von Sekundärstrukturelementen und Domänen durch sie beschrieben wird 85 . Die frühe Erkennung 3D homologer Proteinfaltungen eröffnet die Möglichkeit, die Bestimmung von Proteinstrukturen zu beschleunigen. Eine Homolgiesuche unter Nutzung dipolarer Kopplungen ist in der Lage, Proteine oder zumindest Fragmente mit ähnlicher 3D Struktur zu finden, auch wenn die Primärsequenzhomologie gering ist. Darüber hinaus wird eine Transformation für experimentelle dipolare Kopplungen entwickelt, die die indirekte Orientierungsinformation eines Vektors relativ zu einem externen Tensor in den möglichen Bereich für den Projektionswinkel zwischen zwei Vektoren und somit in eine intramolekulare Strukturinformation übersetzt. Diese Einschränkungen können in der Strukturbestimmung von Proteinen mittels Molekulardynamik genutzt werden 92 . Im Gegensatz zu allen existierenden Implementierungen wird die Konvergenz der Rechnung durch die auf diese Weise eingeführten dipolare Kopplungsinformation kaum beeinflusst. Die dipolaren Kopplungen werden trotzdem von den errechneten Strukturen erfüllt. Auch ohne die Nutzung bereits bekannter Protein oder Fragmentstrukturen kann so ein erheblicher Teil der NOE -- Information substituiert werden. Die Dynamik des Vektors, der die beiden wechselwirkenden Dipole verbindet, beeinflusst den Messwert der dipolaren Kopplung. Dadurch wird Information über die Dynamik von Molekülen auf der µsZeitskala zugänglich, die bisher nur schwer untersucht werden konnte. Die Messung dipolarer Kopplungen für einen Vektor in verschiedenen Orientierungen erlaubt die Analyse seiner Bewegung 89 . Im besonderen ist die Ableitung eines modellfreien Ordnungsparameters 2 S möglich. Weiterhin lassen sich ebenso modellfrei eine mittlere Orientierung des Vektors, axialsymmetrische Anteile und nichtaxialsymmetrische Anteile der Dynamik ableiten und auswerten. Die Anwendung der so entwickelten Protokolle auf experimentelle Daten 90 lässt Proteine deutlich dynamischer erscheinen als auf der Zeitskala der Relaxationsexperimente zu erkennen ist. Der mittlere Ordnungsparameter sinkt von 0.8 auf 0.6. Dies entspricht einer Erhöhung des Öffnungswinkels der Bewegung von ca. 22 ° auf ca. 33°. Die Bewegungen weichen teilweise bis zu 40% und im Mittel 15% von der Axialsymmetrie ab. Neuronale Netze erlauben eine schnelle (ca. 5000 chemische Verschiebungen pro Sekunde) und exakte (mittleren Abweichung von 1.6 ppm) Berechnung der 13 C NMR chemischen Verschiebung 115 . Dabei kombinieren sie die Vorteile bisher bekannter Datenbankabschätzungen (hohe Genauigkeit) und Inkrementverfahren (hohe Geschwindigkeit). Das 13 C NMR Spektrum einer organischen Verbindung stellt eine detaillierte Beschreibung seiner Struktur dar. Resultate des Strukturgenerators COCON können durch den Vergleich des experimentellen mit den berechneten 13 C NMR Spektren auf ca. 1 o/oo der vorgeschlagenen Strukturen eingeschränkt werden, die eine geringe Abweichung zum experimentellen Spektrum haben 122 . Die Kombination mit einer Substrukturanalyse erlaubt weiterhin die Erkennung wahrscheinlicher, geschlossener Ringsysteme und gibt einen Überblick über die Struktur des generierten Konstitutionssubraumes. Genetische Algorithmen können die Struktur organischer Moleküle ausgehend von derer Summenformel auf eine Übereinstimmung mit dem experimentellen 13 C NMR Spektrum optimieren. Die Konstitution von Molekülen wird dafür durch einen Vektor der Bindungszustände zwischen allen Atom -- Atom Paaren beschrieben. Selbige Vektoren sind geeignet, in einem genetischen Algorithmus als genetischer Code von Konstitutionen betrachtet zu werden. Diese Methode erlaubt die automatisierte Bestimmung der Konstitution von Molekülen mit 10 bis 20 Nichtwasserstoffatomen 123 . Symmetrische neuronale Netze können fünf bzw. sieben dimensionale, heterogene Parameterrepräsentationen der 20 proteinogenen Aminosäuren unter Erhalt der wesentlichen Information in den dreidimensionalen Raum projizieren 134 . Die niederdimensionalen Projektionen ermöglichen eine Visualisierung der Beziehungen der Aminosäuren untereinander. Die reduzierten Parameterrepräsentationen sind geeignet, als Eingabe für ein neuronales Netz zu dienen, welches die Sekundärstruktur eines Proteins mit einer Genauigkeit von 66 % im Q 3 -- Wert berechnet. Neuronale Netzte sind aufgrund ihrer flexiblen Struktur besonders geeignet, quantitative Beziehungen zwischen Struktur und Aktivität zu beschreiben, da hier hochgradig nichtlineare, komplexe Zusammenhänge vorliegen. Eine numerische Codierung der über 200 in der Literatur beschriebenen Epothilonderivate erlaubt es, Modelle zur Berechnung der Induktion der Tubulin Polymerisation (R = 0.73) und der Inhibierung des Krebszellenwachstums (R = 0.94) zu erstellen 136 . Die trainierten neuronalen Netze können in einer Sensitivitätsanalyse genutzt werden, um die Bindungsstellen des Moleküls zu identifizieren. Aus der Berechnung der Aktivität für alle Moleküle des durch die Parameter definierten Strukturraums ergeben sich Vorschläge für Epothilonderivate, die bis zu 1 000 mal aktiver als die bisher synthetisierten sein könnten.
The gas phase ion chemistry of the simplest known phosphorus ylide, trimethylmethylenephosphorane, has been studied in the mass range m/e=2 - 186 and the pressure range 10-7-10-4 Torr. The most abundant product ion, m/e = 104, (CH3)2C2H5PCH2'+ is formed by a methylene group transfer reaction of the molecular ion. Almost all of the other product ions formed from the molecular ion can be subsumed under the general formula (CH3)3PCHPRn+ (R = H, CH3; n=1,2,3). The reactions indicate that the molecular ion has lost its ylide character almost completely. The protonated molecule is formed almost exclusively by a reaction of the fragment ion m/e = 75. This reaction and the CH3PH group transfer reaction indicate a cyclic structure (CH3) HP(CH2)2+ for this ion. A cyclic structure is also assumed for the ion m/e = 73, PC3H6+, which undergoes P and PH transfer reactions. The reactions of the ion m/e = 47 are consistent with the structure CH3PH+. The ICR and mass spectra are given, some metastable decompositions are discussed.
The analysis of biomolecular macrocomplexes requires certain preconditions to be fulfilled. The preparation of biomolecular samples usually results in low yields. Due to this constraint of low availability any method should provide a sufficient sensitivity to cope with typical sample amounts. Biomolecules also often show a reduced stability, i.e. a propensity for fragmentation upon ionisation, which requires reasonable soft methods for the investigation. Furthermore macromolecular complexes usually are composed by means of non-covalent interactions presenting additional demands on the softness. This holds true for specific complexes like protein-ligand or DNA double strand binding. For the formation of non-covalent, specific complexes the biomolecules’ native structure and environment are a basic prerequisite and hence crucial. Therefore it is desirable during analysis to keep the biomolecules in a native environment to preserve their structure and weak interactions. One suitable method for analysing biomolecules is mass spectrometry. Mass spectrometry is capable of high throughput screening as well as determining masses with high accuracy and high sensitivity. Especially since the availability of MALDI-MS and ESI-MS mass spectrometry evolved to a versatile tool to investigate biomolecular complexes. Both, MALDI- and ESI-MS are sufficiently soft methods to observe fragile biomolecules. Yet both methods have their advantages and disadvantages. During the recent years an alternative mass spectrometric approach has been developed in our group, termed LILBID-MS (Laser Induced Liquid Bead Ionisation/Desorption). In LILBID microdroplets of aqueous solution containing buffer, salt and further additives among the analyte molecules are injected into vacuum and irradiated one-by-one by mid-IR laser pulses. The absorption of the energy by the water leads to a rapid ablation of the preformed analyte ions. LILBID is highly tolerant for the addition of salts and detergents allowing to study biomolecular complexes in a native environment. As LILBID-MS is soft enough to avoid fragmentation, specific non-covalent complexes can be analysed directly from their native environment by this method. In addition dissociation can be induced on demand by increasing the laser intensity which allows for the study of subunit compositions. A further prominent property of LILBID is the possibility to study hydrophobic membrane proteins due to the tolerated use of detergents. During the course of this work, several instrumental improvements mostly concerning ion focussing and beam steering were introduced. Together with refinements of different modes of measurement the result is a significantly improved signal-to-noise ratio as well as a further improvement in sensitivity. In addition the accessible m/z range for a given flight time has been vastly increased. The new possibilities that LILBID now offers for the study of biomolecular complexes were investigated. The ability to detect specific binding in LILBID-MS was investigated by means of nucleic acids and their interaction with proteins. It could be shown that the stability of a 16bp dsDNA corresponds to that in solution phase regarding the dependency on concentration and type of the salts used. In addition a competitive experiment with the well-known transcription factor p50 was used to demonstrate the detection of sequence-specific binding with LILBID. The improved sensitivity allowed to detect single stranded DNA at nanomolar concentrations and even the 2686bp plasmid pUC19 could be easily detected without fragmentation using a concentration of only 80nM. In case of the transcription factor p63 the mass spectrometric analysis could help to identify a new model of activation and inhibition. For the first time known quarternary structures of membrane proteins like the light-driven proton pump bacteriorhodopsin and the potassium channel KcsA could be detected with mass spectrometry. For the light-driven proton pump proteorhodopsin the type and the concentration of the used detergents significantly influenced the stability of this protein as well as the preferred quarternary structure.
The mass spectra and the ion molecule reactions of methylphosphine, dimethylphosphine and dimethyldeuterophosphine have been studied by ion cyclotron resonance spectrometry. About 50 ion molecule reaction are observed for each compound. The product ions can be classified as ions with two phosphorus atoms: P2R5+, P2R3+, P2R2+ and P2R+ (R = CH3 or H), as phosphonium and phosphinium ions and ions resulting from collision dissociations and charge exchange reactions. Tertiary ions with three phosphorus atoms like CH3P3H2+ (from CH3PH2) and (CH3)4P3H2 (from (CH3)2PH) have also been detected. The mechanisms of the ion molecule reactions, rearrangements, P -H- and C-H-reactivities and product ion structures are discussed, using in the case of dimethylphosphine the results obtained with the deuterated compound. Rate constants of formation of the more abundant product ions from the molecular ion and the CH3P+ ion, both odd electron particles, have been determined. The reactions with dimethylphosphine have much smaller rate constants than the reactions with methylphosphine.
Pulsed electron-electron double resonance (PELDOR) is a pulsed EPR method that can reliably and precisely provide structural information regarding duplex RNAs and DNAs by measuring long-range distances (1.5-7 nm) utilizing distance-dependent magnetic dipole-dipole interaction between two nitroxide spin labels. In this thesis the application field of PELDOR spectroscopy has been expanded. For the first time the global architecture of tertiary folded RNA has been mapped in vitro. Moreover, the first application of PELDOR for determining structural aspects of RNA and DNA molecules inside cells has been presented. RNA has the central role in cellular processes and gene regulation. It can adopt complex three dimensional structures, which in combination with its conformational dynamics is essential for its function as biological catalyst, structural scaffold and regulator of gene expression. Riboswitches are cis-acting RNA segments that modulate gene expression by direct binding of small molecules with high affinity and specificity. Neomycin-responsive riboswitch is an engineered riboswitch developed by combination of in vitro selection and in vivo screening. Upon insertion into the 5‟ untranslated region of mRNA and binding the cognate ligand it is able to inhibit translational initiation in yeast. Using enzymatic probing the secondary structure had been postulated comprising global stem-loop architecture with a terminal and an internal loop. In the first part of this thesis, the global conformational arrangement of this 27 nucleotides long RNA element has been studied by means of site-directed spin labeling and PELDOR spectroscopy. Spin-labeled neomycin-responsive riboswitch mutants were synthesized via a Sonogashira cross-coupling reaction between 5-membered pyrroline ring based nitroxide radical (TPA) and 5-iodo-uridine. The labeling positions were chosen outside of the binding pocket and UV melting curves revealed that spin-labeling neither disturbs the secondary structure nor interferes with ligand binding. Efficient ligand binding was proven by thermal stabilization of 20.3±3.3 oC upon addition of neomycin, as well as by cw EPR spectra. PELDOR time traces with long observation time windows and with good signal to noise ratio and modulation depth were recorded for all double-labeled samples allowing a reliable data analysis. The fact that there were no shifts in the measured distances upon addition of neomycin implied the existence of a prearranged tertiary structure of the neomycin-sensing riboswitch without a significant global conformational change induced by ligand binding. Measured distances were in very good agreement with the NMR structure of the ligand-bound state of the riboswitch indicating the intrinsic propensity of the global RNA architecture toward its energetically favored ligand-bound form at low temperature. The results harvested in this work represent the first application of PELDOR for mapping the global structure of a tertiary folded RNA. In the second part of this thesis the possibility of applying PELDOR on nucleic acids (NAs) in cellular environment has been investigated. It was shown before that global NA structure depends on matrix conditions, such as concentration of ions and small molecules, molecular crowding, viscosity and interactions with proteins. Therefore, PELDOR spectroscopy on a double-labeled 12-base pair DNA duplex, the 14-mer cUUCGg tetraloop hairpin RNA and the 27-mer neomycin-sensing riboswitch has been used to obtain long-range distance constraints on such systems in Xenopus laevis oocytes and to compare them with in vitro measurements. The reduced lifetime of nitroxide spin labels under cellular conditions has been a major challenge in these measurements. Investigation of nitroxide reduction kinetics in-cell has revealed that the 5-membered pyrrolidine and pyrroline rings are significantly slower reduced compared to 6-membered piperidine ring based nitroxides. Due to prolonged lifetime of the TPA nitroxides covalently attached to NA molecules PELDOR signals could be measured with good signal-to-noise ratios up to 70 minutes of incubation time. The partial loss of coupled spin labels due to nitroxide reduction only led to a decrease in the modulation depth upon increasing the incubation time. No alterations in the measured distances between in vitro and in-cell experiments implies the existence of stable overall conformations of the 14-mer cUUCGg tetraloop hairpin RNA and the 27-mer neomycin-sensing riboswitch, whereas the 12-bp duplex DNA experiences stacking in-cell but retaining the secondary structure. Thus, for the first time nanometer distance measurements were performed inside cells, clearly laying a foundation for the application of PELDOR spectroscopy to study biological processes in cells, such as diffusion, interaction with proteins and other factors or chemical reactions.
5-lipoxygenase (5-LO) is an enzyme with a substantial role in inflammatory processes. In vitro kinase assays using [32P]-ATP in combination with mutagenesis have revealed that serine residues 271, 523 and 663 can be phosphorylated by MK2, PKA and ERK2 kinases, respectively. A few available reports regarding 5-LO protein sequence have covered up to 30% of the sequence after amino acid sequencing including Ser663. In LCMS/MS analyses of 5-LO tryptic digests from different cellular sources different peptides have been detected; however, none of the three phosphorylations has been detected and only Ser663 was included in the covered sequence.
As there was no comprehensive mass spectrometric analysis of 5-LO, the purpose of this study was to optimize the experimental conditions under which detection of the aforementioned phosphorylation events, as well as other possible post-translational modifications (PTMs), would be feasible. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) was used for peptide analysis of 5-LO cleaved either by chemical reagents or by proteases. Sequence coverage of 5-LO could be enhanced to be close to completion by combination of results from digestions by trypsin, AspN and chymotrypsin. In-gel trypsin digestion followed by in-solution AspN digestion proved to be a useful sample treatment for reproducible detection of the Ser271-containing peptide.
Nevertheless, in none of the examined cleavage protocols the sequence around Ser523 was detected reproducibly or with acceptable signal intensity for subsequent peptide fragmentation. Propionic anhydride and sulfo-NHS-SS-biotin cross-linker (EZ-linkTM), were used for derivatization of lysine side chains and hindrance of lysine residue recognition by trypsin. Phosphopeptide enrichment became possible after tryptic digestion of these samples, not only due to formation of an individual Ser523-containing peptide, but also because TiO2-mediated enrichment, which is performed in acidic pH, was not impaired by positively charged free lysine side chains. Additionally, biotinylation of lysine residues was exploited for an intermediate enrichment step of the lysine containing peptides, prior to TiO2 phosphopeptide enrichment.
MALDI-MS analysis after in-vitro phosphorylation of 5-LO by the three kinases showed that Ser271 was phosphorylated in the MK2 and PKA kinase assays, while Ser523 was phosphorylated only in the PKA kinase assay. Surpisingly, no phosphopeptides were detected in the in-vitro kinase assays with ERK2, even though the unmodified counterpart of the Ser663-containing peptide was easily detected. The detection limit for each of the three phosphorylation sites was determined by the use of custom made phosphopeptides and an amount of 0.06 pmol of phosphopeptide in 1 μg 5-LO (representing 0.5% phosphorylation rate) was sufficient in all cases for successful enrichment and detection by MS.
In-vitro kinase assays with [32P]-ATP were performed for some kinases that were expected to phosphorylate 5-LO according to in-silico data. Three members of the Src tyrosine kinase family (Fgr, Hck and Yes) and the Ser/Thr specific kinase DNA-PK used 5-LO as their substrate and mainly residues at the N-terminal part of 5-LO were detected phosphorylated by MS (e.g. Y42, Y53). Additional in-vitro assays for recombinant 5-LO modification included incubation with glutathione or compound U73122, previously described as inhibitor of 5-LO.
Since in-vitro assays might have generated artifacts, a method for 5-LO purification from human cells was sought, in order to examine the modification state of the protein in the cellular context. ATP-agarose affinity purification and anti-5-LO immunoprecipitation proved inappropriate for sample purification for MALDI-MS analysis. Consequently, two human cell lines that are able to express 5-LO (Rec-1 Blymphocytes and MM6 monocytes) were transduced with a DNA cassette that contained recombinant human 5-LO sequence with an attached N-terminal FLAG-tag. Anti-FLAG immunoprecipitation was then performed effectively in cell lysates and the precipitated FLAG-5-LO was separated by SDS-PAGE before MALDI-MS analysis.
The examined cell stimuli were expected to result to phosphorylation of 5-LO at Ser523 by PKA in Rec-1 cells and to phosphorylation of Ser271 and/or Ser663 in MM6 cells by activated MK2 and ERK2, respectively. Additionally, under the conditions of MM6 cell stimulation, Fgr, Hck and Yes kinases, which phosphorylated 5-LO in vitro, were expected to be activated and the possibility of 5-LO phosphorylation on tyrosine was investigated. Although immunoblotting results indicated that all the aforementioned phosphorylation events existed in the examined samples, MALDI-MS analysis verified only phosphorylation on Ser271 in differentiated MM6 cells, interestingly regardless of cell stimulation.
Finally, the primary amine derivatization procedure by EZ-linkTM was utilized for MS analysis of lysine rich proteins. In the past, chemical propionylation of histones had been employed prior to trypsin digestion; however it was easily confused in MS with combinations of other PTMs (e.g. acetylation, methylation). Moreover, propionylation is a PTM for histone H3 and this information was lost. Consequently, the EZ-link reagent was more useful for analysis of histones, as unambiguous assignment of PTMs and detection of native propionylation on bovine H3 became possible.
The transcription factor ∆Np63 is a master regulator of epithelial cell identity and essential for the survival of squamous cell carcinoma (SCC) of lung, head and neck, oesophagus, cervix and skin. Here, we report that the deubiquitylase USP28 stabilizes ∆Np63 and maintains elevated ∆NP63 levels in SCC by counteracting its proteasome‐mediated degradation. Impaired USP28 activity, either genetically or pharmacologically, abrogates the transcriptional identity and suppresses growth and survival of human SCC cells. CRISPR/Cas9‐engineered in vivo mouse models establish that endogenous USP28 is strictly required for both induction and maintenance of lung SCC. Our data strongly suggest that targeting ∆Np63 abundance via inhibition of USP28 is a promising strategy for the treatment of SCC tumours.
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.
The low temperature IR stretching vibrations of difluorodisulfane (FSSF) and thiothionylfluoride (SSF2), in the solid phase and in a cyclohexane matrix, of the mixtures FSSF -SSF2, FSSF-OSF2 and SSF, -OSF2, and of solid difluorotrisulfane (FS3F) have been investigated. While SSF, forms no distinct oligomers, a dimer with absorption bands at 635 and 682 cm-1 has been detected in the case of FSSF. These differences between FSSF and SSF, are rationalized by the different S-F bond lengths. A structure of the FSSF dimer similar to that of the sulfur tetrafluoride dimer is proposed. The low temperature spectrum of FS3F shows 3 bands in the frequency range between 460 and 1000 cm-1: 590, 605 and 680 cm-1, due to associated molecules. FS3F decomposes on warming. The main decomposition products containing fluorine are FSSF and SSF2. Mechanisms for the rearrangement and decomposition of the three compounds studied are discussed.
Hydride transfers play a crucial role in a multitude of biological redox reactions and are mediated by flavin, deazaflavin or nicotinamide adenine dinucleotide cofactors at standard redox potentials ranging from 0 to –340 mV. 2-Naphthoyl-CoA reductase, a key enzyme of oxygen-independent bacterial naphthalene degradation, uses a low-potential one-electron donor for the two-electron dearomatization of its substrate below the redox limit of known biological hydride transfer processes at E°’ = −493 mV. Here we demonstrate by X-ray structural analyses, QM/MM computational studies, and multiple spectroscopy/activity based titrations that highly cooperative electron transfer (n = 3) from a low-potential one-electron (FAD) to a two-electron (FMN) transferring flavin cofactor is the key to overcome the resonance stabilized aromatic system by hydride transfer in a highly hydrophobic pocket. The results evidence how the protein environment inversely functionalizes two flavins to switch from low-potential one-electron to hydride transfer at the thermodynamic limit of flavin redox chemistry.
Complex I (proton-pumping NADH:ubiquinone oxidoreductase) is the largest enzyme of the mitochondrial respiratory chain and a significant source of reactive oxygen species (ROS). We hypothesized that during energy conversion by complex I, electron transfer onto ubiquinone triggers the concerted rearrangement of three protein loops of subunits ND1, ND3, and 49-kDa thereby generating the power-stoke driving proton pumping. Here we show that fixing loop TMH1-2ND3 to the nearby subunit PSST via a disulfide bridge introduced by site-directed mutagenesis reversibly disengages proton pumping without impairing ubiquinone reduction, inhibitor binding or the Active/Deactive transition. The X-ray structure of mutant complex I indicates that the disulfide bridge immobilizes but does not displace the tip of loop TMH1-2ND3. We conclude that movement of loop TMH1-2ND3 located at the ubiquinone-binding pocket is required to drive proton pumping corroborating one of the central predictions of our model for the mechanism of energy conversion by complex I proposed earlier.
Location and orientation of serotonin receptor 1a agonists in model and complex lipid membranes
(2008)
Magic angle spinning (MAS) NMR has been used to investigate the location and orientation of five serotonin receptor 1a agonists (serotonin, buspirone, quipazine, 8-OH-DPAT, and LY-163,165) in single component model lipid and brain lipid membranes. The agonist locations are probed by monitoring changes in the lipid proton chemical shifts and by MAS-assisted nuclear Overhauser enhancement spectroscopy, which indicates the orientation of the agonists with respect to the 1,2-dioleoyl-sn-glycero-3-phosphocholine lipids. In the single component bilayer, the membrane agonists are found predominantly in the top of the hydrophobic chain or in the glycerol region of the membrane. Most of the agonists orient approximately parallel to the membrane plane, with the exception of quipazine, whose piperazine ring is found in the glycerol region, whereas its benzene ring is located within the lipid hydrophobic chain. The location of the agonist in brain lipid membranes is similar to the 1,2-dioleoyl-sn-glycero-3-phosphocholine lipid bilayers; however, many of the agonists appear to locate close to the cholesterol in the membrane in preference to the phospholipids.
Live-cell labelling techniques to visualize proteins with minimal disturbance are important; however, the currently available methods are limited in their labelling efficiency, specificity and cell permeability. We describe high-throughput protein labelling facilitated by minimalistic probes delivered to mammalian cells by microfluidic cell squeezing. High-affinity and target-specific tracing of proteins in various subcellular compartments is demonstrated, culminating in photoinduced labelling within live cells. Both the fine-tuned delivery of subnanomolar concentrations and the minimal size of the probe allow for live-cell super-resolution imaging with very low background and nanometre precision. This method is fast in probe delivery (∼1,000,000 cells per second), versatile across cell types and can be readily transferred to a multitude of proteins. Moreover, the technique succeeds in combination with well-established methods to gain multiplexed labelling and has demonstrated potential to precisely trace target proteins, in live mammalian cells, by super-resolution microscopy.
Accurate labeling of endogenous proteins for advanced light microscopy in living cells remains challenging. Nanobodies have been widely used for antigen labeling, visualization of subcellular protein localization and interactions. To facilitate an expanded application, we present a scalable and high-throughput strategy to simultaneously target multiple endogenous proteins in living cells with micro- to nanometer resolution. For intracellular protein labeling, we advanced nanobodies by site-specific and stoichiometric attachment of bright organic fluorophores. Their fast and fine-tuned intracellular transfer by microfluidic cell squeezing enabled high-throughput delivery with less than 10% dead cells. This strategy allowed for the dual-color imaging of distinct endogenous cellular structures, and culminated in super-resolution imaging of native protein networks in genetically non-modified living cells. The simultaneous delivery of multiple engineered nanobodies does not only offer exciting prospects for multiplexed imaging of endogenous protein, but also holds potential for visualizing native cellular structures with unprecedented accuracy.
A simple and fast method of lipid analysis of isolated intact mitochondria by means of MALDI-TOF mass spectrometry is described. Mitochondria isolated from bovine heart and yeast have been employed to set up and validate the new method of lipid analysis. The mitochondrial suspension is directly applied over the target and, after drying, covered by a thin layer of the 9-aminoacridine matrix solution. The lipid profiles acquired with this procedure contain all peaks previously obtained by analyzing the lipid extracts of isolated mitochondria by TLC and/or mass spectrometry. The novel procedure allows the quick, simple, precise, and accurate analysis of membrane lipids, utilizing only a tiny amount of isolated organelle; it has also been tested with intact membranes of the bacterium Paracoccus denitrificans for its evolutionary link to present-day mitochondria. The method is of general validity for the lipid analysis of other cell fractions and isolated organelles.
LILBID and nESI : different native mass spectrometry techniques as tools in structural biology
(2018)
Native mass spectrometry is applied for the investigation of proteins and protein complexes worldwide. The challenge in native mass spectrometry is maintaining the features of the proteins of interest, such as oligomeric state, bound ligands, or the conformation of the protein complex, during transfer from solution to gas phase. This is an essential prerequisite to allow conclusions about the solution state protein complex, based on the gas phase measurements. Therefore, soft ionization techniques are required. Widely used for the analysis of protein complexes are nanoelectro spray ionization (nESI) mass spectrometers. A newer ionization method is laser induced liquid bead ion desorption (LILBID), which is based on the release of protein complexes from solution phase via infrared (IR) laser desorption. We use both methods in our lab, depending on the requirements of the biological system we are interested in. Here we benchmark the performance of our LILBID mass spectrometer in comparison to a nESI instrument, regarding sample conditions, buffer and additive tolerances, dissociation mechanism and applicability towards soluble and membrane protein complexes.
Light‐induced activation of biomolecules by uncaging of photolabile protection groups has found many applications for triggering biochemical reactions with minimal perturbations directly within cells. Such an approach might also offer unique advantages for solid‐state NMR experiments on membrane proteins for initiating reactions within or at the membrane directly within the closed MAS rotor. Herein, we demonstrate that the integral membrane protein E. coli diacylglycerol kinase (DgkA), which catalyzes the phosphorylation of diacylglycerol, can be controlled by light under MAS‐NMR conditions. Uncaging of NPE‐ATP or of lipid substrate NPE‐DOG by in situ illumination triggers its enzymatic activity, which can be monitored by real‐time 31P‐MAS NMR. This proof‐of‐concept illustrates that combining MAS‐NMR with uncaging strategies and illumination methods offers new possibilities for controlling biochemical reactions at or within lipid bilayers.
Channelrhodopsin-2 (ChR2) is a cation-selective light-gated channel from Chlamydomonas reinhardtii (Nagel G, Szellas T, Huhn W, Kateriya S, Adeishvili N, Berthold P, et al. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc Natl Acad Sci USA 2003;100:13940-5), which has become a powerful tool in optogenetics. Two-dimensional crystals of the slow photocycling C128T ChR2 mutant were exposed to 473 nm light and rapidly frozen to trap the open state. Projection difference maps at 6Å resolution show the location, extent and direction of light-induced conformational changes in ChR2 during the transition from the closed state to the ion-conducting open state. Difference peaks indicate that transmembrane helices (TMHs) TMH2, TMH6 and TMH7 reorient or rearrange during the photocycle. No major differences were found near TMH3 and TMH4 at the dimer interface. While conformational changes in TMH6 and TMH7 are known from other microbial-type rhodopsins, our results indicate that TMH2 has a key role in light-induced channel opening and closing in ChR2.
The full-length translation-regulating add adenine riboswitch (Asw) from Vibrio vulnificus has a more complex conformational space than its isolated aptamer domain. In addition to the predicted apo (apoA) and holo conformation that feature the conserved three-way junctional purine riboswitch aptamer, it adopts a second apo (apoB) conformation with a fundamentally different secondary structure. Here, we characterized the ligand-dependent conformational dynamics of the full-length add Asw by NMR and by single-molecule FRET (smFRET) spectroscopy. Both methods revealed an adenine-induced secondary structure switch from the apoB-form to the apoA-form that involves no tertiary structural interactions between aptamer and expression platform. This strongly suggests that the add Asw triggers translation by capturing the apoA-form secondary structure in the holo state. Intriguingly, NMR indicated a homogenous, docked aptamer kissing loop fold for apoA and holo, while smFRET showed persistent aptamer kissing loop docking dynamics between comparably stable, undocked and docked substates of the apoA and the holo conformation. Unraveling the folding of large junctional riboswitches thus requires the integration of complementary solution structural techniques such as NMR and smFRET.
The mfl-riboswitch is a transcriptional off-switch, which down-regulates expression of subunit ß of ribonucleotide reductase in Mesoplasma florum upon 2´-deoxyguanosine binding. We characterized binding of 2´-deoxyguanosine to the mfl-aptamer domain (WT aptamer) and a sequence-stabilized aptamer (MT aptamer) under in vitro and ‘in-cell-like’ conditions by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy. ‘In-celllike’ environment was simulated by Bacillus subtilis cell extract, in which both aptamers remained sufficiently stable to detect the resonances of structural elements and ligand binding in 2D NMR experiments. Under ‘in-cell-like’-environment, (i) the WT aptamer bound the endogenous metabolite guanosine and (ii) 2´-deoxyguanosine efficiently displaced guanosine from the WT aptamer. In contrast, MT aptamer exhibited moderate binding to 2´-deoxyguanosine and weak binding to guanosine. NMR experiments indicated that binding of guanosine was not limited to the aptamer domain of the riboswitch but also the full-length mfl-riboswitch bound guanosine, impacting on the regulation efficiency of the riboswitch and hinting that, in addition to 2´-deoxyguanosine, guanosine plays a role in riboswitch function in vivo. Reporter gene assays in B. subtilis demonstrated the regulation capacity of the WT aptamer, whereas the MT aptamer with lower affinity to 2´ -deoxyguanosine was not able to regulate gene expression.
Type 1 diabetes (T1D) is a chronic T cell-mediated autoimmune disorder that results in the destruction of insulin-producing pancreatic ß cells leading to life-long dependence on exogenous insulin. Attraction, activation and transmigration of inflammatory cells to the site of ß-cell injury depend on two major molecular interactions. First, interactions between chemokines and their receptors expressed on leukocytes result in the recruitment of circulating inflammatory cells to the site of injury. In this context, it has been demonstrated in various studies that the interaction of the chemokine CXCL10 with its receptor CXCR3 expressed on circulating cells plays a key role in the development of T1D. Second, once arrived at the site of inflammation adhesion molecules promote the extravasation of arrested cells through the endothelial cell layer to penetrate the site of injury. Here, the junctional adhesion molecule (JAM) JAM-C expressed on endothelial cells is involved in the process of leukocyte diabedesis. It was recently demonstrated that blocking of JAM-C efficiently attenuated cerulein-induced pancreatitis in mice. In my thesis I studied the influence of the CXCL10/CXCR3 interaction on the one hand, and of the adhesion molecule JAM-C on the other hand, on trafficking and transmigration of antigen-specific, autoaggressive T cells in the RIP-LCMV mouse model. RIP-LCMV mice express the glycoprotein (GP) or the nucleoprotein (NP) of the lymphocytic choriomeningitis virus (LCMV) as a target autoantigen specifically in the ß cells of the islets of Langerhans and turn diabetic after LCMV-infection. In my first project I found that pharmacologic blockade of CXCR3 during development of virus-induced T1D results in a significant delay but not in an abrogation of overt disease. However, neither the frequency nor the migratory properties of islet-specific T cells was significantly changed during CXCR3 blockade. In the second project I was able to demonstrate that JAM-C was upregulated around the islets in RIP-LCMV mice after LCMV infection and its expression correlated with islet infiltration and functional ß-cell impairment. Blockade with a neutralizing anti-JAM-C antibody slightly reduced T1D incidence, whereas overexpression of JAM-C on endothelial cells did not accelerate virus-induced diabetes. In summary, our data suggest that both CXCR3 as well as JAM-C are involved in trafficking and transmigration of antigen-specific autoaggressive T cells to the islets of Langerhans. However, the detection of only a moderate influence on the onset of clinical disease during CXCR3 or JAM-C blockade reflects the complex pathogenesis of T1D and indicates that several different inflammatory factors need to be neutralized in order to achieve a stable and persistent protection from disease.
It is shown that the kinetics of the back reaction of photosystem II in the seconds time range as derived from the luminescence decay curve in the presence of DCMU is controlled by the internal pH of the thylakoids. Modifications of the conformational state of the photosynthetic membrane while leaving the internal pH unchanged, however, reaction.
The catalytic mechanism, electron transfer coupled to proton pumping, of heme-copper oxidases is not yet fully understood. Microsecond freeze-hyperquenching single turnover experiments were carried out with fully reduced cytochrome aa(3) reacting with O(2) between 83 micros and 6 ms. Trapped intermediates were analyzed by low temperature UV-visible, X-band, and Q-band EPR spectroscopy, enabling determination of the oxidation-reduction kinetics of Cu(A), heme a, heme a(3), and of a recently detected tryptophan radical (Wiertz, F. G. M., Richter, O. M. H., Cherepanov, A. V., MacMillan, F., Ludwig, B., and de Vries, S. (2004) FEBS Lett. 575, 127-130). Cu(B) and heme a(3) were EPR silent during all stages of the reaction. Cu(A) and heme a are in electronic equilibrium acting as a redox pair. The reduction potential of Cu(A) is 4.5 mV lower than that of heme a. Both redox groups are oxidized in two phases with apparent half-lives of 57 micros and 1.2 ms together donating a single electron to the binuclear center in each phase. The formation of the heme a(3) oxoferryl species P(R) (maxima at 430 nm and 606 nm) was completed in approximately 130 micros, similar to the first oxidation phase of Cu(A) and heme a. The intermediate F (absorbance maximum at 571 nm) is formed from P(R) and decays to a hitherto undetected intermediate named F(W)(*). F(W)(*) harbors a tryptophan radical, identified by Q-band EPR spectroscopy as the tryptophan neutral radical of the strictly conserved Trp-272 (Trp-272(*)). The Trp-272(*) populates to 4-5% due to its relatively low rate of formation (t((1/2)) = 1.2 ms) and rapid rate of breakdown (t((1/2)) = 60 micros), which represents electron transfer from Cu(A)/heme a to Trp-272(*). The formation of the Trp-272(*) constitutes the major rate-determining step of the catalytic cycle. Our findings show that Trp-272 is a redox-active residue and is in this respect on an equal par to the metallocenters of the cytochrome c oxidase. Trp-272 is the direct reductant either to the heme a(3) oxoferryl species or to Cu (2+)(B). The potential role of Trp-272 in proton pumping is discussed.
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.
Expectation values of kinetic and potential energy are calculated for some lower antibonding orbital states of simple diatomic molecules using H2+ and HeH2+ as test cases. Common LCAO-MO theory and a scaling procedure are applied which allow an analysis of atomic orbital interactions in terms of RUEDENBERG'S1 promotion and interference effect at various internuclear distances. Contributions to the total energy at different regions of interatomic separations are discussed in detail. A characteristic increase of the kinetic energy is observed for antibonding linear combinations at distances where chemical bonding occurs.
C. elegans is used extensively as a model system in the neurosciences due to its well defined nervous system. However, the seeming simplicity of this nervous system in anatomical structure and neuronal connectivity, at least compared to higher animals, underlies a rich diversity of behaviors. The usefulness of the worm in genome-wide mutagenesis or RNAi screens, where thousands of strains are assessed for phenotype, emphasizes the need for computational methods for automated parameterization of generated behaviors. In addition, behaviors can be modulated upon external cues like temperature, O2 and CO2 concentrations, mechanosensory and chemosensory inputs. Different machine vision tools have been developed to aid researchers in their efforts to inventory and characterize defined behavioral “outputs”. Here we aim at providing an overview of different worm-tracking packages or video analysis tools designed to quantify different aspects of locomotion such as the occurrence of directional changes (turns, omega bends), curvature of the sinusoidal shape (amplitude, body bend angles) and velocity (speed, backward or forward movement).
Na(+)/H(+) exchangers are essential for regulation of intracellular proton and sodium concentrations in all living organisms. We examined and experimentally verified a kinetic model for Na(+)/H(+) exchangers, where a single binding site is alternatively occupied by Na(+) or one or two H(+) ions. The proposed transport mechanism inherently down-regulates Na(+)/H(+) exchangers at extreme pH, preventing excessive cytoplasmic acidification or alkalinization. As an experimental test system we present the first electrophysiological investigation of an electroneutral Na(+)/H(+) exchanger, NhaP1 from Methanocaldococcus jannaschii (MjNhaP1), a close homologue of the medically important eukaryotic NHE Na(+)/H(+) exchangers. The kinetic model describes the experimentally observed substrate dependences of MjNhaP1, and the transport mechanism explains alkaline down-regulation of MjNhaP1. Because this model also accounts for acidic down-regulation of the electrogenic NhaA Na(+)/H(+) exchanger from Escherichia coli (EcNhaA, shown in a previous publication) we conclude that it applies generally to all Na(+)/H(+) exchangers, electrogenic as well as electroneutral, and elegantly explains their pH regulation. Furthermore, the electrophysiological analysis allows insight into the electrostatic structure of the translocation complex in electroneutral and electrogenic Na(+)/H(+) exchangers.