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Biallelic mutations in TMEM126B cause severe complex i deficiency with a variable clinical phenotype
(2016)
Complex I deficiency is the most common biochemical phenotype observed in individuals with mitochondrial disease. With 44 structural subunits and over 10 assembly factors, it is unsurprising that complex I deficiency is associated with clinical and genetic heterogeneity. Massively parallel sequencing (MPS) technologies including custom, targeted gene panels or unbiased whole-exome sequencing (WES) are hugely powerful in identifying the underlying genetic defect in a clinical diagnostic setting, yet many individuals remain without a genetic diagnosis. These individuals might harbor mutations in poorly understood or uncharacterized genes, and their diagnosis relies upon characterization of these orphan genes. Complexome profiling recently identified TMEM126B as a component of the mitochondrial complex I assembly complex alongside proteins ACAD9, ECSIT, NDUFAF1, and TIMMDC1. Here, we describe the clinical, biochemical, and molecular findings in six cases of mitochondrial disease from four unrelated families affected by biallelic (c.635G>T [p.Gly212Val] and/or c.401delA [p.Asn134Ilefs∗2]) TMEM126B variants. We provide functional evidence to support the pathogenicity of these TMEM126B variants, including evidence of founder effects for both variants, and establish defects within this gene as a cause of complex I deficiency in association with either pure myopathy in adulthood or, in one individual, a severe multisystem presentation (chronic renal failure and cardiomyopathy) in infancy. Functional experimentation including viral rescue and complexome profiling of subject cell lines has confirmed TMEM126B as the tenth complex I assembly factor associated with human disease and validates the importance of both genome-wide sequencing and proteomic approaches in characterizing disease-associated genes whose physiological roles have been previously undetermined.
Hypoxia triggers several mechanisms to adapt cells to a low oxygen environment. Mitochondria are major consumers of oxygen and a potential source of reactive oxygen species (ROS). In response to hypoxia they exchange or modify distinct subunits of the respiratory chain and adjust their metabolism, especially lowering the citric acid cycle. Intermediates of the citric acid cycle participate in regulating hypoxia inducible factors (HIF), the key mediators of adaptation to hypoxia. Here we summarize how hypoxia conditions mitochondria with consequences for ROS-production and the HIF-pathway.
The development of single-photon-counting detectors, such as the PILATUS, has been a major recent breakthrough in macromolecular crystallography, enabling noise-free detection and novel data-acquisition modes. The new EIGER detector features a pixel size of 75 × 75 µm, frame rates of up to 3000 Hz and a dead time as low as 3.8 µs. An EIGER 1M and EIGER 16M were tested on Swiss Light Source beamlines X10SA and X06SA for their application in macromolecular crystallography. The combination of fast frame rates and a very short dead time allows high-quality data acquisition in a shorter time. The ultrafine φ-slicing data-collection method is introduced and validated and its application in finding the optimal rotation angle, a suitable rotation speed and a sufficient X-ray dose are presented. An improvement of the data quality up to slicing at one tenth of the mosaicity has been observed, which is much finer than expected based on previous findings. The influence of key data-collection parameters on data quality is discussed.
Biogenesis of mitochondrial cytochrome c oxidase (COX) is a complex process involving the coordinate expression and assembly of numerous subunits (SU) of dual genetic origin. Moreover, several auxiliary factors are required to recruit and insert the redox-active metal compounds, which in most cases are buried in their protein scaffold deep inside the membrane. Here we used a combination of gel electrophoresis and pull-down assay techniques in conjunction with immunostaining as well as complexome profiling to identify and analyze the composition of assembly intermediates in solubilized membranes of the bacterium Paracoccus denitrificans. Our results show that the central SUI passes through at least three intermediate complexes with distinct subunit and cofactor composition before formation of the holoenzyme and its subsequent integration into supercomplexes. We propose a model for COX biogenesis in which maturation of newly translated COX SUI is initially assisted by CtaG, a chaperone implicated in CuB site metallation, followed by the interaction with the heme chaperone Surf1c to populate the redox-active metal-heme centers in SUI. Only then the remaining smaller subunits are recruited to form the mature enzyme which ultimately associates with respiratory complexes I and III into supercomplexes.
We investigate complexes of two paramagnetic metal ions Gd3+ and Mn2+ to serve as polarizing agents for solid-state dynamic nuclear polarization (DNP) of 1H, 13C, and 15N at magnetic fields of 5, 9.4, and 14.1 T. Both ions are half-integer high-spin systems with a zero-field splitting and therefore exhibit a broadening of the mS = −1/2 ↔ +1/2 central transition which scales inversely with the external field strength. We investigate experimentally the influence of the chelator molecule, strong hyperfine coupling to the metal nucleus, and deuteration of the bulk matrix on DNP properties. At small Gd-DOTA concentrations the narrow central transition allows us to polarize nuclei with small gyromagnetic ratio such as 13C and even 15N via the solid effect. We demonstrate that enhancements observed are limited by the available microwave power and that large enhancement factors of >100 (for 1H) and on the order of 1000 (for 13C) can be achieved in the saturation limit even at 80 K. At larger Gd(III) concentrations (≥10 mM) where dipolar couplings between two neighboring Gd3+ complexes become substantial a transition towards cross effect as dominating DNP mechanism is observed. Furthermore, the slow spin-diffusion between 13C and 15N, respectively, allows for temporally resolved observation of enhanced polarization spreading from nuclei close to the paramagnetic ion towards nuclei further removed. Subsequently, we present preliminary DNP experiments on ubiquitin by site-directed spin-labeling with Gd3+ chelator tags. The results hold promise towards applications of such paramagnetically labeled proteins for DNP applications in biophysical chemistry and/or structural biology.
Die Geschichte der frühen Christenverfolgung wird in Hollywoodstreifen wesentlich dramatischer dargestellt, als sie in der jüngeren Forschung beurteilt wird. Auch andere Kulte wurden unterdrückt, denn es bestand kein Recht auf Religionsfreiheit. Die Gründe für solche Maßnahmen lagen nicht in Glaubensvorstellungen, sondern in Handlungen, die als gefährlich für das Gemeinwesen galten. So fürchtete man, dass die Weigerung der Christen, Opfer darzubringen, den Zorn der Götter heraufbeschwören könne oder ein Zeichen mangelnder Loyalität sei. Vor diesem Hintergrund forderten Christen Religionsfreiheit von den Heiden, doch wurde diese auch im später christlichen spätantiken Reich nicht praktiziert.
Antigenic and 3D structural characterization of soluble X4 and hybrid X4-R5 HIV-1 Env trimers
(2014)
Background: HIV-1 is decorated with trimeric glycoprotein spikes that enable infection by engaging CD4 and a chemokine coreceptor, either CCR5 or CXCR4. The variable loop 3 (V3) of the HIV-1 envelope protein (Env) is the main determinant for coreceptor usage. The predominant CCR5 using (R5) HIV-1 Env has been intensively studied in function and structure, whereas the trimeric architecture of the less frequent, but more cytopathic CXCR4 using (X4) HIV-1 Env is largely unknown, as are the consequences of sequence changes in and near V3 on antigenicity and trimeric Env structure.
Results: Soluble trimeric gp140 Env constructs were used as immunogenic mimics of the native spikes to analyze their antigenic properties in the context of their overall 3D structure. We generated soluble, uncleaved, gp140 trimers from a prototypic T-cell line-adapted (TCLA) X4 HIV-1 strain (NL4-3) and a hybrid (NL4-3/ADA), in which the V3 spanning region was substituted with that from the primary R5 isolate ADA. Compared to an ADA (R5) gp140, the NL4-3 (X4) construct revealed an overall higher antibody accessibility, which was most pronounced for the CD4 binding site (CD4bs), but also observed for mAbs against CD4 induced (CD4i) epitopes and gp41 mAbs. V3 mAbs showed significant binding differences to the three constructs, which were refined by SPR analysis. Of interest, the NL4-3/ADA construct with the hybrid NL4-3/ADA CD4bs showed impaired CD4 and CD4bs mAb reactivity despite the presence of the essential elements of the CD4bs epitope. We obtained 3D reconstructions of the NL4-3 and the NL4-3/ADA gp140 trimers via electron microscopy and single particle analysis, which indicates that both constructs inherit a propeller-like architecture. The first 3D reconstruction of an Env construct from an X4 TCLA HIV-1 strain reveals an open conformation, in contrast to recently published more closed structures from R5 Env. Exchanging the X4 V3 spanning region for that of R5 ADA did not alter the open Env architecture as deduced from its very similar 3D reconstruction.
Conclusions: 3D EM analysis showed an apparent open trimer configuration of X4 NL4-3 gp140 that is not modified by exchanging the V3 spanning region for R5 ADA.
Structural biology and life sciences in general, and NMR in particular, have always been associated with advanced computing. The current challenges in the post-genomic era call for virtual research platforms that provide the worldwide research community with both user-friendly tools, platforms for data analysis and exchange, and an underlying e-Infrastructure. WeNMR, a three-year European Commission co-funded project started in November 2010, groups different research teams into a worldwide virtual research community. It builds on the established eNMR e-Infrastructure and its steadily growing virtual organisation, which is currently the second largest VO in the area of life sciences. WeNMR provides an e-Infrastructure platform and Science Gateway for structural biology. It involves researchers from around the world and will build bridges to other areas of structural biology.
Background: In macrophages Toll-like receptor 4 (TLR4) is activated in response to lipopolysaccharide (LPS) and induces proinflammatory cytokine expression. Therefore, mechanisms terminating proinflammatory gene expression are important. Autophagy plays a central role in controlling innate immune responses by lysosomal degradation of signaling proteins, thus contributing to the resolution of inflammation. Autophagic proteins like p62 directly interact with molecules involved in the TLR4-signaling pathway, but a correlation with the IRAK E3 ligase and scaffold protein Pellino3 remains obscure. Hence, we are interested in elucidating the function of Pellino3 to prove our hypothesis that it is a key regulator in the TLR4-signaling cascade.
Methods: We used the cecal ligation and puncture (CLP) mouse model causing polymicrobial sepsis to analyze Pellino3 protein and mRNA expression. Furthermore, we induced endotoxemia in RAW264.7 mouse macrophages by LPS treatment to verify in vivo experiments. Lentiviral Pellino3 knockdown in RAW264.7 macrophages was used for cytokine measurements at mRNA level. To analyze potential Pellino3 binding partners in TLR4-signaling by mass spectrometry (MS), we overexpressed FLAG-tagged Pellino3 in RAW264.7 macrophages, treated cells for 3, 6 and 24 hours with LPS and immunoprecipitated Pellino3 via its FLAG-tag. To consider Pellino3 degradation as a result of p62-mediated autophagy, we transiently knocked down p62 by siRNA in RAW264.7 macrophages and also pharmacologically blocked LPS-induced autophagy by Bafilomycin A1.
Results: We demonstrated Pellino3 protein degradation in primary CD11b+ splenocytes after 24 hours following CLP operation and confirmed this in RAW264.7 macrophages after 24-hour LPS stimulation. Knockdown of Pellino3 attenuates proinflammatory cytokines, for example IL-6 mRNA, after 6 hours of LPS. Furthermore, we found by MS and verifying immunoprecipitation experiments that p62 is a Pellino3 binding partner, thus targeting Pellino3 for degradation. In line, both p62 knockdown and Bafilomycin A1 treatment prevent Pellino3 degradation, supporting an autophagic mechanism.
Conclusion: Our observations highlight a regulatory role of Pellino3 on TLR4 signaling. Thus, antagonism of Pellino3 in the hyperinflammatory phase of sepsis may counteract the cytokine storm. Furthermore, stabilization of Pellino3 by inhibition of autophagy in the hypoinflammatory phase of sepsis may improve immunity. In consideration of these two conflictive sepsis phases, modulation of Pellino3 may provide a new strategy for the development of a therapy approach in sepsis.
Attenuated NOX2 expression impairs ROS production during the hypoinflammatory phase of sepsis
(2012)
Background: The multicomponent phagocytic NADPH oxidase produces reactive oxygen species (ROS) after activation by microorganisms or inflammatory mediators. In the hypoinflammatory phase of sepsis, macrophages are alternatively activated by contact with apoptotic cells or their secretion products. This inhibits NADPH oxidase and leads to attenuated ROS production and furthermore contributes among others to a hyporeactive host defense. Due to this immune paralysis, sepsis patients suffer from recurrent and secondary infections. We focused on the catalytic subunit of NADPH oxidase, the transmembrane protein NOX2. We assume that after induction of sepsis the expression of NOX2 is reduced and hence ROS production is decreased.
Methods: We induced polymicrobial sepsis in mice by cecal ligation and puncture. The ability of peritoneal macrophages (PMs) to produce ROS was determined by FACS via hydroethidine assay. NOX2 expression of PMs was determined by western blot and qPCR. To elucidate the mechanism causing mRNA destabilization, we performed in vitro experiments using J774 macrophages. To obtain an alternatively activated phenotype, macrophages were stimulated with conditioned medium from apoptotic T cells (CM). By luciferase assays we figured out a 3'UTR-dependent regulation of NOX2 mRNA stability. Assuming that a protein is involved in the mRNA degradation, we performed a RNA pulldown with biotinylated NOX2-3'UTR constructs followed by mass spectrometry. We verified the role of SYNCRIP by siRNA approach. Additionally, we overexpressed NOX2 in J774 cells and analyzed the ROS production (w/wo CM treatment) by FACS.
Results: We found an impaired expression of NOX2 at RNA and protein level along with decreased ROS production after induction of sepsis in mice as well as stimulating J774 macrophages with CM of apoptotic T cells. This is due to a time-dependent NOX2 mRNA degradation depending on SYNCRIP, a RNA-binding protein, which stabilizes NOX2 mRNA through binding to its 3'UTR under normal conditions. In line, knockdown of SYNCRIP also decreases NOX2 mRNA expression. We assume that a CM-dependent modification or degradation of SYNCRIP prevents its stabilizing function. As the overexpression of NOX2 restores ROS production of CM-treated J774 cells, we assume that NOX2 expression is crucial for maintaining NADPH activity during the hypoinflammatory phase of sepsis.
Conclusion: Our data imply a regulatory impact of SYNCRIP on NOX2 stability during the late phase of sepsis. Therefore, further understanding of the regulation of NADPH oxidase could lead to the design of a therapy to reconstitute NADPH oxidase function, finally improving immune function in sepsis patients.
Background: The ligand-activated transcription factor, peroxisome-proliferator-activated receptor gamma (PPARγ), has been shown to play an essential role in immunosuppression during sepsis. PPARγ is upregulated in T cells of septic patients, sensitizing these cells to PPARγ-dependent apoptosis and thus contributing to T-cell depletion. In the polymicrobial cecum ligation and puncture (CLP) sepsis model in mice, both T-cell-specific gene knockout (Lck-Cre PPARγfl/fl) and systemic pharmacological PPARγ antagonism by GW9662 improved survival. Because GW9662 was only effective when applied 3 hours after CLP, we were interested to extend this time frame. For this reason we characterized the kinetics of SPPARγMs when administered before or in combination with the agonist thiazolidinedione, rosiglitazone.
Methods: A PPARγ-dependent transactivation assay was used in HEK293T cells. It is based on the vector pFA-PPARγ-LBD-GAL4-DBD encoding the hybrid protein PPARγ-LBD-GAL4-DBD and the reporter vector pFR-Luc, carrying a GAL4-responsive element in front of the Firefly luciferase gene. These two vectors were co-transfected, in combination with a control vector encoding Renilla luciferase (pRL-CMV) to normalize Firefly luciferase activity for transfection efficiency. Following transfection, cells were incubated with the SPPARγMs F-MOC and MCC-555 and the PPARγ antagonist GW9662 for different times (2 to 48 hours) and at increasing doses (0.01 to 10 μM), with or without rosiglitazone (0.01 to 10 μM). Transactivation was analyzed using a 96-well plate format.
Results: Rosiglitazone transactivated PPARγ in a time-dependent and dose-dependent manner, the response gradually increasing to a maximum at 48 hours with 10 μM. Low concentrations (0.01 to 0.1 μM) of SPPARγMs F-MOC and MCC-555 and the PPARγ antagonist GW9662 all exerted dose-independent antagonistic effects at an early incubation time point (2 hours). From 10 hours onwards, MCC-555 and GW9662, given alone, both exerted PPARγ agonistic effects, MCC-555 in parallel to responses to rosiglitazone, but GW9662 with characteristics of partial antagonism. F-MOC showed no dose-dependent effect at any concentration at later time points. Only GW9662 (1 to 10 μM) was able to inhibit rosiglitazone (0.1 to 1 μM)-induced PPARγ transactivation after 10 hours.
Conclusion: Our kinetic analysis reveals clear differences in the modulatory characteristics of PPARγ inhibitors, with previously unreported early inhibitory effects and late agonistic or partial agonistic activity. New SPPARγMs with extended inhibitory activity may prove useful in the therapy of sepsis.
5-Lipoxygenase contributes to PPAR [gamma] activation in macrophages in response to apoptotic cells
(2012)
Background: One hallmark contributing to immune suppression during the late phase of sepsis is macrophage polarization to an anti-inflammatory phenotype upon contact with apoptotic cells (AC). Taking the important role of the nuclear receptor PPARγ for this phenotype switch into consideration, it remains elusive how AC activate PPARγ in macrophages. Therefore, we were interested to characterize the underlying principle.
Methods: Apoptosis was induced by treatment of Jurkat T cells for 3 hours with 0.5 μg/ml staurosporine. Necrotic cells (NC) were prepared by heating cells for 20 minutes to 65°C. PPARγ activation was followed by stably transducing RAW264.7 macrophages with a vector encoding the red fluorescent protein mRuby after PPARγ binding to 4 × PPRE sites downstream of the reporter gene sequence. This readout was established by treatment with the PPARγ agonist rosiglitazone (1 μM) and AC (5:1). Twenty-four hours after stimulation, mRuby expression was analysed by fluorescence microscopy. Lipid rafts of AC, NC, as well as living cells (LC) were enriched by sucrose gradient centrifugation. Fractions were analysed for lipid raft-associated marker proteins. Lipid rafts were incubated with transduced RAW264.7 macrophages as described above. 5-Lipoxygenase (5-LO) involvement was verified by pharmacological inhibition (MK-866, 1 μM) and overexpression.
Results: Assuming that the molecule responsible for PPARγ activation in macrophages is localized in the cell membrane of AC, most probably associated to lipid rafts, we isolated lipid rafts from AC, NC and LC. Mass spectrometric analysis of lipid rafts of AC showed the expression of 5-LO, whereas lipid rafts of LC did not. Moreover, incubating macrophages with lipid rafts of AC induced mRuby expression. In contrast, lipid rafts of NC and LC did not. To verify the involvement of 5-LO in activating PPARγ in macrophages, Jurkat T cells were incubated for 30 minutes with the 5-LO inhibitor MK-866 (1 μM) before apoptosis induction. In line with our hypothesis, these AC did not induce mRuby expression. Finally, although living Jurkat T cells overexpressing 5-LO did not activate PPARγ in macrophages, mRuby expression was significantly increased when AC were generated from 5-LO overexpressing compared with wild-type Jurkat cells.
Conclusion: Our results suggest that induction of apoptosis activates 5-LO, localizing to lipid rafts, necessary for PPARγ activation in macrophages. Therefore, it will be challenging to determine whether 5-LO activity in AC, generated from other cell types, correlates with PPARγ activation, contributing to an immune-suppressed phenotype in macrophages.
Structured RNA regions are important gene control elements in prokaryotes and eukaryotes. Here, we show that the mRNA of a cyanobacterial heat shock gene contains a built-in thermosensor critical for photosynthetic activity under stress conditions. The exceptionally short 5´-untranslated region is comprised of a single hairpin with an internal asymmetric loop. It inhibits translation of the Synechocystis hsp17 transcript at normal growth conditions, permits translation initiation under stress conditions and shuts down Hsp17 production in the recovery phase. Point mutations that stabilized or destabilized the RNA structure deregulated reporter gene expression in vivo and ribosome binding in vitro. Introduction of such point mutations into the Synechocystis genome produced severe phenotypic defects. Reversible formation of the open and closed structure was beneficial for viability, integrity of the photosystem and oxygen evolution. Continuous production of Hsp17 was detrimental when the stress declined indicating that shutting-off heat shock protein production is an important, previously unrecognized function of RNA thermometers. We discovered a simple biosensor that strictly adjusts the cellular level of a molecular chaperone to the physiological need.
The mfl-riboswitch regulates expression of ribonucleotide reductase subunit in Mesoplasma florum by binding to 2´-deoxyguanosine and thereby promoting transcription termination. We characterized the structure of the ligand-bound aptamer domain by NMR spectroscopy and compared the mfl-aptamer to the aptamer domain of the closely related purine-sensing riboswitches. We show that the mfl-aptamer accommodates the extra 2´-deoxyribose unit of the ligand by forming a more relaxed binding pocket than these found in the purine-sensing riboswitches. Tertiary structures of the xpt-aptamer bound to guanine and of the mfl-aptamer bound to 2´-deoxyguanosine exhibit very similar features, although the sequence of the mfl-aptamer contains several alterations compared to the purine-aptamer consensus sequence. These alterations include the truncation of a hairpin loop which is crucial for complex formation in all purine-sensing riboswitches characterized to date. We further defined structural features and ligand binding requirements of the free mfl-aptamer and found that the presence of Mg2+ is not essential for complex formation, but facilitates ligand binding by promoting pre-organization of key structural motifs in the free aptamer.
We present here a set of 13C-direct detected NMR experiments to facilitate the resonance assignment of RNA oligonucleotides. Three experiments have been developed: (1) the (H)CC-TOCSY-experiment utilizing a virtual decoupling scheme to assign the intraresidual ribose 13C-spins, (2) the (H)CPC-experiment that correlates each phosphorus with the C40 nuclei of adjacent nucleotides via J(C,P) couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C10,H10 ribose signals. The experiments were applied to two RNA hairpin structures. The current set of 13C-direct detected experiments allows direct and unambiguous assignment of the majority of the hetero nuclei and the identification of the individual ribose moieties following their sequential assignment. Thus, 13C-direct detected NMR methods constitute useful complements to the conventional 1H-detected approach for the resonance assignment of oligonucleotides that is often hindered by the limited chemical shift dispersion. The developed methods can also be applied to large deuterated RNAs. Keywords: NMR spectroscopy , Direct carbon , detection , RNA
Metal-ion binding and metal-ion induced folding of the adenine-sensing riboswitch aptamer domain
(2007)
Divalent cations are important in the folding and stabilization of complex RNA structures. The adenine-sensing riboswitch controls the expression of mRNAs for proteins involved in purine metabolism by directly sensing intracellular adenine levels. Adenine binds with high affinity and specificity to the ligand binding or aptamer domain of the adenine-sensing riboswitch. The X-ray structure of this domain in complex with adenine revealed an intricate RNA-fold consisting of a three-helix junction stabilized by long-range base-pairing interactions and identified five binding sites for hexahydrated Mg2+-ions. Furthermore, a role for Mg2+-ions in the ligand-induced folding of this RNA was suggested. Here, we describe the interaction of divalent cations with the RNA–adenine complex in solution as studied by high-resolution NMR spectroscopy. Paramagnetic line broadening, chemical shift mapping and intermolecular nuclear Overhauser effects (NOEs) indicate the presence of at least three binding sites for divalent cations. Two of them are similar to those in the X-ray structure. The third site, which is important for the folding of this RNA, has not been observed previously. The ligand-free state of the RNA is conformationally heterogeneous and contains base-pairing patterns detrimental to ligand binding in the absence of Mg2+, but becomes partially pre-organized for ligand binding in the presence of Mg2+. Compared to the highly similar guanine-sensing riboswitch, the folding pathway for the adenine-sensing riboswitch aptamer domain is more complex and the influence of Mg2+ is more pronounced.
High-resolution NMR structure of an RNA model system : the 14-mer cUUCGg tetraloop hairpin RNA
(2009)
We present a high-resolution nuclear magnetic resonance (NMR) solution structure of a 14-mer RNA hairpin capped by cUUCGg tetraloop. This short and very stable RNA presents an important model system for the study of RNA structure and dynamics using NMR spectroscopy, molecular dynamics (MD) simulations and RNA force-field development. The extraordinary high precision of the structure (root mean square deviation of 0.3 Å) could be achieved by measuring and incorporating all currently accessible NMR parameters, including distances derived from nuclear Overhauser effect (NOE) intensities, torsion-angle dependent homonuclear and heteronuclear scalar coupling constants, projection-angle-dependent cross-correlated relaxation rates and residual dipolar couplings. The structure calculations were performed with the program CNS using the ARIA setup and protocols. The structure quality was further improved by a final refinement in explicit water using OPLS force field parameters for non-bonded interactions and charges. In addition, the 2'-hydroxyl groups have been assigned and their conformation has been analyzed based on NOE contacts. The structure currently defines a benchmark for the precision and accuracy amenable to RNA structure determination by NMR spectroscopy. Here, we discuss the impact of various NMR restraints on structure quality and discuss in detail the dynamics of this system as previously determined.
In prokaryotes, RNA thermometers regulate a number of heat shock and virulence genes. These temperature sensitive RNA elements are usually located in the 5'-untranslated regions of the regulated genes. They repress translation initiation by base pairing to the Shine–Dalgarno sequence at low temperatures. We investigated the thermodynamic stability of the temperature labile hairpin 2 of the Salmonella fourU RNA thermometer over a broad temperature range and determined free energy, enthalpy and entropy values for the base-pair opening of individual nucleobases by measuring the temperature dependence of the imino proton exchange rates via NMR spectroscopy. Exchange rates were analyzed for the wild-type (wt) RNA and the A8C mutant. The wt RNA was found to be stabilized by the extraordinarily stable G14–C25 base pair. The mismatch base pair in the wt RNA thermometer (A8–G31) is responsible for the smaller cooperativity of the unfolding transition in the wt RNA. Enthalpy and entropy values for the base-pair opening events exhibit linear correlation for both RNAs. The slopes of these correlations coincide with the melting points of the RNAs determined by CD spectroscopy. RNA unfolding occurs at a temperature where all nucleobases have equal thermodynamic stabilities. Our results are in agreement with a consecutive zipper-type unfolding mechanism in which the stacking interaction is responsible for the observed cooperativity. Furthermore, remote effects of the A8C mutation affecting the stability of nucleobase G14 could be identified. According to our analysis we deduce that this effect is most probably transduced via the hydration shell of the RNA.
Introduction: Immune paralysis with massive T-cell apoptosis is a central pathogenic event during sepsis and correlates with septic patient mortality. Previous observations implied a crucial role of peroxisome proliferator-activated receptor gamma (PPARγ) during T-cell apoptosis.
Methods: To elucidate mechanisms of PPARγ-induced T-cell depletion, we used an endotoxin model as well as the caecal ligation and puncture sepsis model to imitate septic conditions in wild-type versus conditional PPARγ knockout (KO) mice.
Results: PPARγ KO mice showed a marked survival advantage compared with control mice. Their T cells were substantially protected against sepsis-induced death and showed a significantly higher expression of the pro-survival factor IL-2. Since PPARγ is described to repress nuclear factor of activated T cells (NFAT) transactivation and concomitant IL-2 expression, we propose inhibition of NFAT as the underlying mechanism allowing T-cell apoptosis. Corroborating our hypothesis, we observed up-regulation of the pro-apoptotic protein BIM and downregulation of the anti-apoptotic protein Bcl-2 in control mice, which are downstream effector proteins of IL-2 receptor signaling. Application of a neutralizing anti-IL-2 antibody reversed the pro-survival effect of PPARγ-deficient T cells and confirmed IL-2-dependent apoptosis during sepsis.
Conclusion: Apparently antagonizing PPARγ in T cells might improve their survival during sepsis, which concomitantly enhances defence mechanisms and possibly provokes an increased survival of septic patients.
Die Podiumsdiskussion fand als öffentliche Abendveranstaltung im Rahmen des sozialwissenschaftlichen Workshops "Krise der 'Arbeitsgesellschaft' - Transformation zur 'Grundeinkommensgesellschaft'? Diskurse, Deutungsmuster und Habitusformen im Wandel" statt, der vom 14. bis 15. Juli 2006 an der Goethe-Universität in Frankfurt am Main abgehalten wurde. Veranstalter war das soziologische Forschungsprojekt "Praxis als Erzeugungsquelle von Wissen" (Leiter: Prof. Dr. Ulrich Oevermann) im Forschungskolleg und Sonderforschungsbereich 435 "Wissenskultur und gesellschaftlicher Wandel" der Deutschen Forschungsgemeinschaft an der Goethe-Universität Frankfurt am Main. Initiiert und organisiert wurde der Workshop von Manuel Franzmann mit Unterstützung zahlreicher Kollegen und Förderer.