570 Biowissenschaften; Biologie
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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.
Mountains, with their isolated position and altitudinal belts, are hotspots of biodiversity. Their flora and fauna have been observed worldwide since the days of Alexander von Humboldt, which has led to basic knowledge and understanding of species composition and the most important driving forces of ecosystem differentiation in such altitudinal gradients. Systematically designed analyses of changes in species composition with increasing elevation have been increasingly implemented since the 1990s. Since global climate change is one of the most important problems facing the world this century, a focus on such ecosystem studies is urgently needed. To identify the main future needs of such research we analyze the studies dealing with species changes of diverse taxonomical groups along altitudinal gradients (0 to 6,400 m a.s. l.) on all continents, published during the past one to two decades. From our study we can conclude that although mountains are powerful for climate change research most studies have to face the challenge of separating confounding effects driving species assemblages along altitudinal gradients. Our study therefore supports the view of the need of a global altitudinal concept including that (1) not only one or a few taxonomical groups should be analyzed, but rather different taxonomical groups covering all ecosystem functions simultaneously; (2) relevant site conditions should be registered to reveal direct environmental variables responsible for species distribution patterns and to resolve inconsistent effects along the altitudinal gradients; (3) transect design is appropriate for analyzing ecosystem changes in site gradients and over time; (4) both the study design and the individual methods should be standardized to compare the data collected worldwide; and (5) a long-term perspective is important to quantify the degree and direction of species changes and to validate species distribution models. (6) Finally we suggest to develop experimental altitudinal approaches to overcome the addressed problems of biodiversity surveys.
There have been many studies involving the use of hemispherical photographs to indirectly estimate canopy structures and forest light environments. A variety of commercial and free software packages are available for the analysis of hemispherical photographs. The costs of investment might represent an advantage of the free programmes over the commercial, but as yet little has been documented about the differences in their outputs and in the technical applications from a user (ecologist and forester) perspective. The objective of the study was to compare the canopy structure variables (canopy openness and effective plant area index) and solar radiation transmission estimates (direct, diffuse and global solar radiation transmittances) from digital hemispherical photographs taken under two forest canopy conditions (gap and closed canopy) in three different broadleaf forest regions (Chile, Germany, Venezuela) and calculated using four different programmes. The hemispherical photographs were analysed using one commercial (HemiView) and three free programmes (Gap Light Analyzer, hemIMAGE and Winphot). The results obtained revealed that all of the programmes computed similar estimates of both canopy structures and below-canopy solar radiation. Only the results relating to the effective plant area index with an ellipsoidal leaf angle distribution made with HemiView and Winphot deviated significantly. Other user aspects are also discussed, such as costs, image formats, computer system requirements, etc.
We found that the HMTase G9a, that catalyzes H3K9me2 in euchromatin, plays a key modulatory role in type I IFN expression. This finding raises the possibility of targeted intervention with type I IFN expression by using small synthetic inhibitors of G9a. Given the overall minimal negative effect of G9a-deficiency on differentiated cells, the short-term suppression of G9a could be used to potentiate type I IFN expression during chronic viral diseases such as hepatitis C. Accordingly, pharmacological enhancement of methylation, for example by inhibition of the H3K9me2 specific demethylases, could be potentially used to attenuate type I IFN expression and help to control chronic inflammatory and autoimmune conditions. The mechanism responsible for canvassing the epigenetic profile of type I IFN expressing cells are not known. It is plausible, that similar to neurons, where G9a is targeted to specific loci with the help of noncoding RNAs, IFN expressing cells possess similar mechanisms to target H3K9me2 demethylating enzymes to type I IFN loci, thus keeping these loci accessible for IFN-inducing transcription factors. Identification of non-coding RNAs that may contribute to the establishment of the epigenetic state of IFN producing cells will provide a further opportunity for targeted manipulation of IFN expression.
In my thesis, I describe the collaborative experiments that show the ability of synthetic compounds that interfere with the histone readers to suppress inflammation. Our results present a novel concept for the regulation of inflammatory gene expression. The diversity of histone readers and the combinatorial nature of regulation of gene transcription may provide an opportunity for highly selective interference with disease associated transcriptional programs by interfering with specific readers. In the future we plan to address the therapeutic potential of BET antagonists in autoimmune and chronic inflammatory conditions.In summary, the experiments described in my thesis provide an example of how the understanding of the basic mechanisms of chromatin control of gene expression can facilitate novel therapeutic approaches that target chromatin.
FIAS Scientific Report
(2011)
FIAS Scientific Report 2010
(2011)
In the year 2010 the Frankfurt Institute for Advanced Studies has successfully continued to follow its agenda to pursue theoretical research in the natural sciences. As stipulated in its charter, FIAS closely collaborates with extramural research institutions, like the Max Planck Institute for Brain Research in Frankfurt and the GSI Helmholtz Center for Heavy Ion Research, Darmstadt and with research groups at the science departments of Goethe University. The institute also engages in the training of young researchers and the education of doctoral students. This Annual Report documents how these goals have been pursued in the year 2010. Notable events in the scientific life of the Institute will be presented, e.g., teaching activities in the framework of the Frankfurt International Graduate School for Science (FIGSS), colloquium schedules, conferences organized by FIAS, and a full bibliography of publications by authors affiliated with FIAS. The main part of the Report consists of short one-page summaries describing the scientific progress reached in individual research projects in the year 2010...
Although it has been suggested that temperature increase may alter the toxic potential of environmental pollutants, few studies have investigated the potential risk of chemical stressors for wildlife under Global Climate Change (GCC) impact. We applied a bifactorial multigeneration study in order to test if GCC conditions alter the effects of low pesticide concentrations on life history and genetic diversity of the aquatic model organism Chironomus riparius. Experimental populations of the species were chronically exposed to a low concentration of the fungicide pyrimethanil (half of the no-observed-adverse-effect concentration: NOAEC/2) under two dynamic present-day temperature simulations (11.0–22.7°C; 14.0–25.2°C) and one future scenario (16.5–28.1°C). During the 140-day multigeneration study, survival, emergence, reproduction, population growth, and genetic diversity of C. riparius were analyzed. Our results reveal that high temperature and pyrimethanil act synergistically on the midge C. riparius. In simulated present-day scenarios, a NOAEC/2 of pyrimethanil as derived from a life-cycle toxicity test provoked only slight-to-moderate beneficial or adverse effects on C. riparius. In contrast, exposure to a NOAEC/2 concentration of pyrimethanil at a thermal situation likely for a summer under GCC conditions uncovered adverse effects on mortality and population growth rate. In addition, genetic diversity was considerably reduced by pyrimethanil in the future scenario, but only slightly under current climatic conditions. Our multigeneration study under near-natural (climatic) conditions indicates that not only the impact of climate change, but also low concentrations of pesticides may pose a reasonable risk for aquatic insects in future.
Nephronectin regulates atrioventricular canal differentiation via Bmp4-Has2 signaling in zebrafish
(2011)
The extracellular matrix is crucial for organogenesis. It is a complex and dynamic component that regulates cell behavior by modulating the activity, bioavailability and presentation of growth factors to cell surface receptors. Here, we determined the role of the extracellular matrix protein Nephronectin (Npnt) in heart development using the zebrafish model system. The vertebrate heart is formed as a linear tube in which myocardium and endocardium are separated by a layer of extracellular matrix termed the cardiac jelly. During heart development, the cardiac jelly swells at the atrioventricular (AV) canal, which precedes valve formation. Here, we show that Npnt expression correlates with this process. Morpholino-mediated knockdown of Npnt prevents proper valve leaflet formation and trabeculation and results in greater than 85% lethality at 7 days post-fertilization. The earliest observed phenotype is an extended tube-like structure at the AV boundary. In addition, the expression of myocardial genes involved in cardiac valve formation (cspg2, fibulin 1, tbx2b, bmp4) is expanded and endocardial cells along the extended tube-like structure exhibit characteristics of AV cells (has2, notch1b and Alcam expression, cuboidal cell shape). Inhibition of has2 in npnt morphants rescues the endocardial, but not the myocardial, expansion. By contrast, reduction of BMP signaling in npnt morphants reduces the ectopic expression of myocardial and endocardial AV markers. Taken together, our results identify Npnt as a novel upstream regulator of Bmp4-Has2 signaling that plays a crucial role in AV canal differentiation.
The genome sequence of Haloferax volcanii is available and several comparative genomic in silico studies were performed that yielded novel insight for example into protein export, RNA modifications, small non-coding RNAs, and ubiquitin-like Small Archaeal Modifier Proteins. The full range of functional genomic methods has been established and results from transcriptomic, proteomic and metabolomic studies are discussed. Notably, Hfx. volcanii is together with Halobacterium salinarum the only prokaryotic species for which a translatome analysis has been performed. The results revealed that the fraction of translationally-regulated genes in haloarchaea is as high as in eukaryotes. A highly efficient genetic system has been established that enables the application of libraries as well as the parallel generation of genomic deletion mutants. Facile mutant generation is complemented by the possibility to culture Hfx. volcanii in microtiter plates, allowing the phenotyping of mutant collections. Genetic approaches are currently used to study diverse biological questions–from replication to posttranslational modification—and selected results are discussed. Taken together, the wealth of functional genomic and genetic tools make Hfx. volcanii a bona fide archaeal model species, which has enabled the generation of important results in recent years and will most likely generate further breakthroughs in the future.
Riboswitch RNAs fold into complex tertiary structures upon binding to their cognate ligand. Ligand recognition is accomplished by key residues in the binding pocket. In addition, it often crucially depends on the stability of peripheral structural elements. The ligand-bound complex of the guanine-sensing riboswitch from Bacillus subtilis, for example, is stabilized by extensive interactions between apical loop regions of the aptamer domain. Previously, we have shown that destabilization of this tertiary loop–loop interaction abrogates ligand binding of the G37A/C61U-mutant aptamer domain (Gswloop) in the absence of Mg2+. However, if Mg2+ is available, ligand-binding capability is restored by a population shift of the ground-state RNA ensemble toward RNA conformations with pre-formed loop–loop interactions. Here, we characterize the striking influence of long-range tertiary structure on RNA folding kinetics and on ligand-bound complex structure, both by X-ray crystallography and time-resolved NMR. The X-ray structure of the ligand-bound complex reveals that the global architecture is almost identical to the wild-type aptamer domain. The population of ligand-binding competent conformations in the ground-state ensemble of Gswloop is tunable through variation of the Mg2+ concentration. We quantitatively describe the influence of distinct Mg2+ concentrations on ligand-induced folding trajectories both by equilibrium and time-resolved NMR spectroscopy at single-residue resolution.
Protein translocation across the chloroplast membrane is mediated by molecular machinery composed of protein complexes termed the TOC/TIC (the outer/inner envelope chloroplasts translocases). This translocation process is regulated by metabolic energy in form of GTP and ATP and is influenced by the lipid composition of the membrane. The ability to study the function of a single complex “TOC” in vitro using purified protein or purified chloroplast outer envelope vesicles has been instrumental for our understanding of the mechanism underlying this process.
Indeed, the TOC complex has been purified by previously established procedures. However its functional and structural analyses are impaired by the limited yield of purified protein. Therefore, protocols for native TOC complex purification are described here. The complex isolation is achieved by direct biochemical treatment of biological membrane hosting this complex or by tandem affinity purification of modified protein complex components from generated transgenic plants.
Furthermore, in this thesis, radioactive based in vitro import assays are described, namely those that allow monitoring translocation activity across the outer envelope of chloroplast. Based on the analysis of knock-out plants and isolated complexes it was previously suggested that lipid dependence of protein translocation might exist. Thus, the question was raised whether the lipid composition of the membrane has a direct influence on the behavior and functionality of the TOC translocon, or whether additional components of the chloroplast membrane account for the observed effect in vivo. To answer this question, a technique for vesicle fusion was developed. The principal aim was to explore the effect of an exchange of the lipid environment surrounding the complex translocon. This method helped to demonstrate that the SQDG and PI act stimulatory on the translocation across the outer envelope of chloroplast, whereas DGDG exhibits an inhibitory effect on TOC complex functionality.
Sponges are one of the major components of benthic communities and are considered to be a
key role organism in marine ecosystems. In addition to their importance in terms of
biodiversity, sponges are becoming increasingly attractive to the industry, as they themselves
or associated symbionts, produce various kinds of secondary metabolites of pharmaceutical
properties. Some of them have already been clinically applied.
The taxonomic characters of Porifera are limited to only a few morphological and
histological characters. In addition, sponges of the same species often show a wide
morphological variability, whereas the latter depends on different ecological parameters such
as water depth and current conditions. Thus, the taxonomic classification of sponges often
becomes a scientific challenge.
The fauna of the Yellow Sea rates among the least studied worldwide. At the same time,
according to the UN Atlas of the Ocean, the Yellow Sea is one of the most intensively
exploited marine areas in the world. This is not least due to the dense human population living
in the entire catchment area of the Yellow Sea region. In order to compile medium- and longterm
conclusions about the anthropogenic impact on biota of the Yellow Sea, the knowledge
of species and their distribution is of crucial importance, as these data form the baseline for all
future conservation efforts.
Until now the sponge fauna of the Chinese Yellow Sea is insufficiently investigated.
Thus, there is only one publication on sponges from this region that has been released
hitherto. This paper is dealing with only a view species. However, there is no reference
concerning the present location of the voucher material, on which this publication is based on.
Consequently, no scientific collection on Porifera from the Chinese part of the Yellow Sea
exists to date.
In order to compile a documentation of the recent sponge community of the Chinese
Yellow Sea, 12 study sites along the coast of the Liaoning Peninsula, China, Northeast
Yellow Sea, were investigated with focus on sponge distribution. The corresponding habitats
were characterized in regard to their topographical features, abiotic parameters, and common
composition of benthic megafaunal and macroalgal assemblages.
Due to the lack of comparable studies, a comprehensive literature research on sponges of the
shallow Northwest Pacific Ocean was required. As a result the first compilation of
publications is presented, dealing with sponges from shallow depths of the northwestern
Pacific Ocean.
Abstract
2
In the course of this study, 31 sponge species in total were recorded, which are scientifically
processed. With the exception of four all specimens were determined to species- level.
Twelve out of the total number of species are new to science and are described and classified
according to the recent taxonomic system of the phylum Porifera.
The results of this study indicate considerable differences in species composition between
investigated sites. It is shown that physical factors (particularly current regime, sedimentation,
seasonally related variations in temperatures), as well the availability of suitable substrates are
directly related to the diversity and abundance of investigated sponge communities. In this
context possible adaptation strategies of the corresponding sponges were discussed in detail.
Two sponge species, Clathria (Clathria) asodes and Antho (Acarnia) lithophoenix, formerly
known exclusively from the northeastern Pacific Ocean, are now recorded from the Northwest
Pacific Ocean for the first time. Furthermore, Penares hongdoensis, Clathria (Clathria)
hongdoensis and Celtodoryx girardae were synonymized with Penares cortius, Clathria
(Clathria) acanthostyli, and Celtodoryx ciocalyptoides respectively. Moreover, the occurrence
of eight sponge species, which were known from previous records from the Yellow Sea, could
be confirmed.
As a result of this study the Asian origin of a sponge species that is invasive to the French and
Dutch coasts of the Northeast Atlantic Ocean since the 1990s could be established. Moreover,
it is demonstrated that Celtodoryx girardae from the northeastern Atlantic is in fact
conspecific with Cornulum ciocalyptoides described by Burton (1935) from the Posiet Bay,
Sea of Japan. Apart from taxonomic remarks, variations between populations from both
oceans are examined and discussed thoroughly in regard to possible ecological implications.
The community of documented sponges shows overlapping with the one from the Sea of
Japan. According to the results it is assumed that the endemic degree of the sponges from the
Chinese Yellow Sea is rather low to moderate.
The material obtained in the course of this study was integrated in the collection of the
Senckenbergischen Naturforschenden Sammlungen. Therefore, it is the first scientific
collection of sponges from the Chinese Yellow Sea that can be consulted as a basis for all
further studies on sponges of this region.
The present study is the only investigation of sponges from Dalian and adjacent waters before
the spill occurred in the Dalian harbour in July 2010. Therefore, it provides an essential
baseline needed to assess the impact of the oil spill on benthic communities.
PaCATB : a secreted catalase protecting Podospora anserina against exogenous oxidative stress
(2011)
A differential mass spectrometry analysis of secreted proteins from juvenile and senescentPodospora anserina cultures revealed age-related differences in protein profiles. Among other proteins with decreased abundance in the secretome of senescent cultures a catalase, termed PaCATB, was identified. Genetic modulation of the abundance of PaCATB identified differential effects on the phenotype of the corresponding strains. Deletion of PaCatB resulted in decreased resistance, over-expression in increased resistance against hydrogen peroxide. While the lifespan of the genetically modified strains was found to be unaffected under standard growth conditions, increased exogenous hydrogen peroxide stress in the growth medium markedly reduced the lifespan of the PaCatB deletion strain but extended the lifespan of PaCatB over-expressors. Overall our data identify a component of the secretome of P. anserina as a new effective factor to cope with environmental stress, stress that under natural conditions is constantly applied on organisms and influences aging processes.
Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1
(2011)
The respiratory chain in the inner mitochondrial membrane contains three large multi-enzyme complexes that together establish the proton gradient for ATP synthesis, and assemble into a supercomplex. A 19-Å 3D map of the 1.7-MDa amphipol-solubilized supercomplex I1III2IV1 from bovine heart obtained by single-particle electron cryo-microscopy reveals an amphipol belt replacing the membrane lipid bilayer. A precise fit of the X-ray structures of complex I, the complex III dimer, and monomeric complex IV indicates distances of 13 nm between the ubiquinol-binding sites of complexes I and III, and of 10–11 nm between the cytochrome c binding sites of complexes III and IV. The arrangement of respiratory chain complexes suggests two possible pathways for efficient electron transfer through the supercomplex, of which the shorter branch through the complex III monomer proximal to complex I may be preferred.
Background: Hydrolysates of plant biomass used for the production of lignocellulosic biofuels typically contain sugar mixtures consisting mainly of D-glucose and D-xylose, and minor amounts of L-arabinose. The yeast Saccharomyces cerevisiae is the preferred microorganism for the fermentative production of ethanol but is not able to ferment pentose sugars. Although D-xylose and L-arabinose fermenting S. cerevisiae strains have been constructed recently, pentose uptake is still a limiting step in mixed sugar fermentations.
Results: Here we described the cloning and characterization of two sugar transporters, AraT from the yeast Scheffersomyces stipitis and Stp2 from the plant Arabidopsis thaliana, which mediate the uptake of L-arabinose but not of D-glucose into S. cerevisiae cells. A yeast strain lacking all of its endogenous hexose transporter genes and expressing a bacterial L-arabinose utilization pathway could no longer take up and grow with L-arabinose as the only carbon source. Expression of the heterologous transporters supported uptake and utilization of L-arabinose especially at low L-arabinose concentrations but did not, or only very weakly, support D-glucose uptake and utilization. In contrast, the S. cerevisiae D-galactose transporter, Gal2, mediated uptake of both L-arabinose and D-glucose, especially at high concentrations.
Conclusions: Using a newly developed screening system we have identified two heterologous sugar transporters from a yeast and a plant which can support uptake and utilization of L-arabinose in L-arabinose fermenting S. cerevisiae cells, especially at low L-arabinose concentrations.
NOSTRIN belongs to the recently defined F-BAR protein family. F-BAR proteins are
multi-domain proteins, which serve as adaptors between plasma membrane and
cytoskeleton components in processes such as membrane protrusion formation,
endocytosis and migration. NOSTRIN encompasses a F-BAR domain at the N-terminus,
which mediates membrane association, followed by a HR1 motif and an intermediate
domain (ID) domain in the middle, and a SH3 domain at the C-terminus. The domain
architecture and ability to form oligomers enable NOSTRIN to coordinate several
interaction partners namely dynamin, caveolin, N-WASP and endothelial nitric oxide
synthase (eNOS) in the process of eNOS trafficking. In this context NOSTRIN was
originally identified and hence termed eNOS traffick inducer. NOSTRIN is expressed in
vascularized tissues (e.g. liver and lung) and in primary endothelial cells.
Aims of the present work were (1) to investigate if NOSTRIN is involved in other
processes besides eNOS trafficking, (2) to analyse the function of NOSTRIN in vivo
through knockdown of NOSTRIN in developing zebrafish and (3) to study the
consequences of the loss of NOSTRIN on signal transduction in a primary cell culture
model derived from NOSTRIN knockout mice.
To study the possible involvement of NOSTRIN in other processes besides eNOS
trafficking a yeast two-hybrid screen was performed in which fibroblast growth factor
receptor 1 (FGFR1) was identified as a putative novel interaction partner of NOSTRIN. In
a series of yeast two-hybrid, pulldown and co-immunoprecipitation experiments the
interaction between NOSTRIN and FGFR1 was confirmed to occur between
endogenously expressed proteins and determined to be direct and to depend on the ID
domain of NOSTRIN and the 130 C-terminal amino acid residues of FGFR1. FGFR1 is
activated by binding of fibroblast growth factors (FGFs) and induces several different
signal transduction pathways (e.g. MAPK and Akt pathway). Overexpression of
NOSTRIN in HeLa cells specifically enhanced FGF2-dependent MAPK activation.
Accordingly, depletion of NOSTRIN attenuated FGF2-dependent MAPK activation and
did not affect FGF2-induced Akt activation.
In summary, NOSTRIN has been identified as a novel interaction partner of FGFR1
involved in FGF2-dependent signal transduction.
The morpholino oligonucleotide-mediated knockdown of NOSTRIN in developing
zebrafish caused vascular leakage and irregular vascular patterning e.g. a loss of the
proper trajectory of intersegmental vessel and interruptions of the dorsal longitudinal
anastomotic vessel. The vascular phenotype was consistent upon use of two different
morpholinos and could be rescued in a dose dependent manner by the injection of
zebrafish NOSTRIN mRNA. Detailed analysis involving confocal and time lapse
microscopy in zebrafish with endothelial specific expression of EGFP revealed that the
knockdown of NOSTRIN impacts in vivo on the migration and morphology of endothelial
tip cells and leads to a reduction of filopodia number and length.
Additionally a NOSTRIN knockout mouse was generated. The analysis of FGFR1 signal
transduction in primary mouse lung endothelial cells (MLECs) from NOSTRIN knockout
and wild type mice revealed that FGF2-dependent MAPK activation was attenuated in
MLECs isolated from NOSTRIN knockout mice when compared to MLECs isolated from
wild type mice. The effect of NOSTRIN on FGF2-dependent signal transduction seems to
be specific, since VEGF-induced MAPK activation was not affected in NOSTRIN
knockout MLECs. The importance of NOSTRIN for FGF2 signal transduction in vivo is
demonstrated by the greatly impaired angiogenic response to FGF2 in NOSTRIN
knockout mice in matrigel plug assay. In a detailed biochemical analysis it was
discovered that NOSTRIN interacts with the activated small GTPase Rac1 and that
overexpression of NOSTRIN enhances Rac1 activation. Furthermore, the interactions of
NOSTRIN with both Rac1 and its GEF Sos1 are required for NOSTRIN-mediated
activation of Rac1. In accordance, activation of Rac1 was not detected upon FGF2
stimulation in NOSTRIN knockout MLECs.
In conclusion, the present work describes a novel function of the F-BAR protein
NOSTRIN in FGFR1 signal transduction. Data presented in this work demonstrate that
NOSTRIN is required for the assembly of a complex consisting of FGFR1, Sos1 and
Rac1 and subsequently for the FGF2-dependent activation of Rac1 in endothelial cells.
Using an electrophysiological assay the activity of NhaA was tested in a wide pH range from pH 5.0 to 9.5. Forward and reverse transport directions were investigated at zero membrane potential using preparations with inside-out and right side-out-oriented transporters with Na+ or H+ gradients as the driving force. Under symmetrical pH conditions with a Na+ gradient for activation, both the wt and the pH-shifted G338S variant exhibit highly symmetrical transport activity with bell-shaped pH dependences, but the optimal pH was shifted 1.8 pH units to the acidic range in the variant. In both strains the pH dependence was associated with a systematic increase of the Km for Na+ at acidic pH. Under symmetrical Na+ concentration with a pH gradient for NhaA activation, an unexpected novel characteristic of the antiporter was revealed; rather than being down-regulated, it remained active even at pH as low as 5. These data allowed a transport mechanism to advance based on competing Na+ and H+ binding to a common transport site and a kinetic model to develop quantitatively explaining the experimental results. In support of these results, both alkaline pH and Na+ induced the conformational change of NhaA associated with NhaA cation translocation as demonstrated here by trypsin digestion. Furthermore, Na+ translocation was found to be associated with the displacement of a negative charge. In conclusion, the electrophysiological assay allows the revelation of the mechanism of NhaA antiport and sheds new light on the concept of NhaA pH regulation.
Although essential for T cell function, the identity of the T cell receptor (TCR) “inside-out” pathway for the activation of lymphocyte function-associated antigen 1 (LFA-1) is unclear. SKAP1 (SKAP-55) is the upstream regulator needed for TCR-induced RapL-Rap1 complex formation and LFA-1 activation. In this paper, we show that SKAP1 is needed for RapL binding to membranes in a manner dependent on the PH domain of SKAP1 and the PI3K pathway. A SKAP1 PH domain-inactivating mutation (i.e. R131M) markedly impaired RapL translocation to membranes for Rap1 and LFA-1 binding and the up-regulation of LFA-1-intercellular adhesion molecule 1 (ICAM-1) binding. Further, N-terminal myr-tagged SKAP1 for membrane binding facilitated constitutive RapL membrane and Rap1 binding and effectively substituted for PI3K and TCR ligation in the activation of LFA-1 in T cells.
The NS5B protein of the hepatitis C virus (HCV) is a RNA-dependent RNA polymerase, which is the key enzyme for viral replication. It is recognized as one of the promising targets for antiviral intervention within the new HCV treatment approach of direct-acting antivirals (DAA). However, several of the known non-nucleoside HCV polymerase inhibitors (NNIs) identified by screening approaches show limitations in the coverage of all six major HCV genotypes (GT). Genotypic profiling therefore has to be implemented early in the screening cascade to discover new broadly active NNIs. This implies knowledge of the specific individual biochemical properties of polymerases from all GTs which is to date limited to GT 1 only. The work submitted here gives a comprehensive overview of the biochemical properties of HCV polymerases derived from all major GTs 1 - 6. Biochemical analysis of polymerases from 38 individual sequences revealed that the optima for monovalent cations, pH and temperature were similar between the GTs, whereas significant differences concerning concentration of the preferred cofactor Mg2+ were identified. Implementing the optimal requirements for the polymerases from each individual GT led to significant improvements in their enzymatic activities. However, the specific activity was distributed unequally across the GTs and could be ranked in the following descending order: 1b, 6a > 2a, 3a, 4a, 5a > 1a. Furthermore, the optimized assay conditions for GT profiling were confirmed by testing the inhibitory activity of four known prototype NNIs, each addressing one of the four NNI binding sites. Additionally, a novel NNI chemotype - identified by screening - is described, the substituted N-phenyl-benzenesulphonamides (SPBS). This inhibitor class showed reversible inhibition of NS5B from HCV 1b Con1 with IC50 values up to 39 nM. Based on the decreased inhibitory activity against a recombinant NS5B protein carrying the mutation L419M, it was assumed that the SPBS inhibitors bound to the thumb site II as it has been described for the carboxy thiophene inhibitors. The postulated binding site was consequently confirmed by analysing a provided co-crystal structure of NS5B in complex with a SPBS analogue. Notably, the two SPBS analogues SPBS-1 and SPBS-2 reported here revealed significant differences in addressing the NH-group of the main chain Y477 by hydrogen-bonds, watermediated or directly, which provoked a shift of the carboxyphenyl group of the inhibitors towards the H475 position for the water-mediated binding mode. Interestingly, the differences observed in the binding mode led to a different cross resistance profile at positions M423 and I482. Using the previously optimized biochemical primer-dependent transcription assay, inhibitory activity of the SPBS could be demonstrated against polymerases from HCV GTs 1a and 1b whereas the inhibitor class failed to inhibit any of the non-GT 1 polymerases. Furthermore, initial antiviral activity for SPBS was demonstrated against the subgenomic replicons of HCV GTs 1a and 1b, respectively, and no considerable cytotoxic potential against a panel of ten different cell types. Finally, concerning a possible future treatment without PEG-IFN α or ribavirin, the SPBS analogues were found to display additive to synergistic effects in combination with the benzothiadiazine, the benzofuran and the indole - representative inhibitors for the binding sites palm I, palm II and thumb I, repectively - in the biochemical assay. Within the same binding site as the SPBS, the reference compound hydroxydihydropyranone displayed additive interactions only with the benzothiadiazine (palm I) in the biochemical assay as well as in cell culture. Hence it could be concluded that, having characterized one individual NNI, no universal predication is possible concerning the combinatory behaviour of NNIs binding to the same binding site. As synergistic, antagonistic or additive interactions are inhibitor-dependent (not binding sitedependent) each novel NNI has to be characterized individually in one-to-one combinations.