Biologische Hochschulschriften (Goethe-Universität; nur lokal zugänglich)
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In this thesis I have investigated the regulation of eicosanoid synthesizing-enzymes by cannabinoid receptor agonists. Rat renal mesangial cells were used as a model system. I could show that all three (CB1, CB2, and GPR55) cannabinoid receptors are expressed on the mRNA level in rat renal mesangial cells – but with differing expression profiles. The CB1 and GPR55 receptors are expressed in comparable amounts, whereas the CB2 receptor is considerably less expressed than the CB1 and the GPR55 receptors. Furthermore I could show that stimulation of renal mesangial cells with CB1 receptor agonists, such as R(+)MA or ACEA, increased IL-1β-induced cPLA2, sPLA2-IIa, and COX2 protein and mRNA expression which subsequently led to an enhanced IL-1β-induced PGE2 formation. Additionally, the IL-1β- induced sPLA2-IIa promoter activity was also increased by CB1 receptor stimulation. Besides the modulated expression of the eicosanoid synthesizing enzymes, I could show that CB1 agonists also led to an increase of IL-1β-induced iNOS expression and subsequent NO formation. In contrast, stimulation with CB2 selective agonists led to a decrease in IL-1β- induced sPLA2-IIa protein expression and PGE2 formation. Accordingly, the IL-1β-induced sPLA2-IIa promoter activity was also reduced by CB2 receptor agonists. IL-1β-induced iNOS expression and subsequent NO formation were not influenced by CB2 recptor activation. Matching the results I obtained with CB1 receptor agonists on IL-1β-induced PGE2 formation, I could observe an increased cPLA2 protein and mRNA expression with a subsequent increase in IL-1β-induced PGE2 formation by GPR55 stimulation. Stimulation with THC, an unselective CB agonist, increased the IL-1β-induced sPLA2-IIa protein expression and subsequently led to an enhanced IL-1β-induced PGE2 formation. Subjecting the cells to higher THC concentrations surprisingly led to a reduction of the IL-1b-induced sPLA2-IIa protein expression and PGE2 formation. A possible explanation may be the differential expression of the three CB receptors. At low concentrations THC may predominantly activate CB1 and GPR55 and with increasing concentration CB2 receptors may also be activated, slightly reversing the enhancing effect. Moreover, I could show that the CB1 receptor stimulation mediated phosphorylation and hence the activation of ERK1/2 MAPK. Additionally to ERK1/2, there was also a phosphorylation and activation of NFkB observed by CB1 receptor stimulation. In my thesis I could show for the first time that PPARα was activated by IL-1β in rMC. The IL-1β-induced PPARα promoter activity was completely inhibited by addition of the CB2 receptor agonist, JWH015. These findings were confirmed by inhibition of the IL-1β-induced PGE2 formation by a PPARα antagonist (MK-886). In summary, I could show that activation of CB1 receptors in our system led to a worsening of an inflammatory condition, whereas activation of the CB2 receptors led to the complete opposite; namely a reduction of the inflammatory response by reducing the sPLA2-IIa expression and PGE2 formation. GPR55 activation did not display any alteration of inflammatory conditions, since the classical inflammatory pathway was not influenced.
The research presented in this thesis characterizes U2AF homology motifs (UHM) and their interactions with UHM ligand motifs (ULM) in the context of splicing regulation. UHM domains are a subgroup of RNA recognition motifs (RRM) originally discovered in the proteins U2AF65 and U2AF35. Whereas canonical RRMs are usually involved in binding of RNA, UHM domains bind tryptophan containing linear protein motifs (ULM) instead. In the first article, we analyze the complex network of interactions between splicing factors and RNA that initiate the assembly of the spliceosome at the 3´ splice site of an intron. The protein U2AF65 binds a pyrimidine-rich element in introns and recruits U2snRNP by binding its protein component SF3b155. My contribution was to define the binding site of the protein U2AF65 to the intrinsically unstructured N-terminus of the scaffolding protein SF3b155. I could show that the UHM domain of U2AF65 recognizes a ULM in SF3b155, and that this binding site is not overlapping with the binding sites of other splicing factors, like p14, to SF3b155. As the U2AF65-UHM:SF3b155-ULM interaction is mutually exclusive with an interaction between U2AF65-UHM and a ULM in the splicing factor SF1, which was reported to initially recognize the branch point sequence, my results provide the molecular details on how SF3b155 replaces SF1 during spliceosomal reorganizations. In the second article, we show that overexpression of the UHM domain of the splicing factor SPF45 induces exon 6 skipping in the pre-mRNA of Fas (CD95/APO-1). I provide evidence for in vitro binding of SPF45-UHM to ULM sequences in the splicing factors U2AF65, SF1, and SF3b155. I crystallized free and SF3b155-bound SPF45 UHM and solved both structures by X-ray crystallography. The analysis of the complex interface and sequence differences in the ULMs allowed me to design mutations of SPF45-UHM, which selectively inhibit binding to distinct ULMs. After assessing the ULM binding properties in vitro, we could show that the activity of SPF45-UHM in influencing the splicing pattern of Fas relies on interactions with SF3b155 and/or SF1, but that an interaction with U2AF65 is dispensable. A mechanism for the activity of SPF45-UHM could thus be engaging in ULM interactions and thus interfering with the network of interactions that initiate the assembly of the spliceosome at the 3´splice site, as described above. In the third article, we describe an unusual flexible homodimerization mode of the UHM in the splicing factor Puf60, which enables simultaneous interactions with ULM sequences on other splicing factors. I could show that the NMR relaxation properties of Puf60-UHM are inconsistent with a model of a rigid dimer, but rather indicate a dimerization via a flexible linker. I identified a flexible loop in the peptide backbone of Puf60-UHM, and showed that mutiation of acidic residues in this loop impairs the dimerization. To analyze the dimerization interface in further detail, I solved the structure of Puf60-UHM by X-ray crystallography. The acidic residues in the flexible loop of one UHM dimer subunit mediate the dimerization by contacting basic residues on the β-sheet surface of the other dimer subunit. Differences in the four dimer interfaces observed for the eight molecules in the asymmetric unit of the crystal support the model of an undescribed, flexible mode of dimerization, and thus complement the NMR relaxation data. Furthermore, I could show that the Puf60-UHM dimer and U2AF65-UHM contact different ULM sequences on the SF3b155 N-terminus in vitro, thus providing a possible explanation for the mutual cooperative activation of Puf60 and U2AF65 in splicing assays described in the literature. The fourth article is a review about recent research on the recognition of DNA double strand breaks (DSB) by covalent histone modifications. The p53 binding protein 1 (53BP1) is a DSB sensor and a checkpoint protein for mitosis. Recent crystallographic evidence indicates that 53BP1 recognizes DSB sites by binding histone H4 dimetylated at lysine 20 (H4-K20). We provide a comprehensive overview of the atomic resolution structures that revealed how proteins can specifically recognize histone tail modifications, especially methylated lysines, to read the information stored in what is called the histone code.
Reggie-1 (flotillin-2) and reggie-2 (flotillin-1) are membrane microdomain proteins which are associated with the membrane by means of acylation. They influence different cellular signaling processes, such as neuronal, T-cell and insulin signaling. Upon stimulation of the EGF receptor, reggie-1 becomes phosphorylated and undergoes tyrosine 163 dependent translocation from the plasma membrane to endosomal compartments. In addition, reggie-1 was shown to influence actindependent processes. Reggie-2 has been demonstrated to affect caveolin- and clathrin-independent endocytosis. Both proteins form homo- and hetero-oligomers, but the function of these oligomers has remained elusive. Moreover, it has not been clarified if functions of reggie-1 are also influenced by reggie-2 and vice versa. The first aim of the study was to further investigate the interplay and the heterooligomerization of reggie proteins and their functional effects. Both reggie proteins were individually depleted by means of siRNA. In different siRNA systems and various cell lines, reggie-1 depleted cells showed reduced protein amounts of reggie-1 and reggie-2, but reggie-2 knock down cells still expressed reggie-1 protein. The decrease of reggie-2 in reggie-1 depleted cells was only detected at protein but not at mRNA level. Furthermore, reggie-2 expression could be rescued by expression of siRNA resistant wild type reggie-1-EGFP constructs, but not by the soluble myristoylation mutant G2A. This mutant was also not able to associate with endogenous reggie-1 or reggie-2, which demonstrates that membrane association of reggie-1 is necessary for hetero-oligomerization. In addition, fluorescence microscopy studies and membrane fractionations showed that correct localization of overexpressed reggie-2 was dependent on co-overexpressed reggie-1. Thus, hetero-oligomerization is crucial for membrane association of reggie-2 and for its protein stability or protein expression. Moreover, the binding of reggie-2 to reggie-1 required tyrosine 163 of reggie-1 which was previously shown to be important for endosomal translocation of reggie-1. Since reggie-2 was implicated to function in clathrin- and caveolin-independent endocytosis pathways, the effect of reggie-2 depletion on reggie-1 endocytosis was investigated. Indeed, reggie-1 was dependent on reggie-2 for endosomal localization and EGF-induced endocytosis. By FRET-FLIM analysis it could be shown that reggie heterooligomers are dynamic in size or conformation upon EGF stimulation. Thus, it can be concluded that reggie proteins are interdependent in different aspects, such as protein stability or expression, membrane association and subcellular localization. In addition, these results demonstrate that the hetero-oligomers are dynamic and reggie proteins influence each other in terms of function. A further aim was the characterization of reggie-1 and reggie-2 function in actindependent processes, where so far only reggie-1 was known to play a role. Depletion of either of the proteins reduced cell migration, cell spreading and the number of focal adhesions in steady state cells. Thus, also reggie-2 affects actin-dependent processes. Further investigation of the focal adhesions during cell spreading revealed that depletion of reggie-1 displayed different effects as compared to reggie-2 knock down. Reggie-1 depleted cells had elongated cell-matrix-adhesions and showed reduced activation of FAK and ERK2. On the other hand, depletion of reggie-2 resulted in a restricted localization of focal adhesion at the periphery of the cell and decreased ERK2 phosphorylation, but it did not affect FAK autophosphorylation. Hence, reggie proteins influence the regulation of cell-matrix-adhesions differently. A link between reggie proteins and focal adhesions is the actin cross-linking protein -actinin. The interaction of -actinin with reggie-1 could be verified by means of co-immunoprecipitations and FRET-FLIM analysis. Reggie-1 binds -actinin especially in membrane ruffles and in other locations where actin remodeling takes place. Moreover, -actinin showed a different localization pattern during cell spreading in reggie-1 depleted cells, as compared to the control cells. These results provide further insights into the function of both reggie proteins. Their interplay and hetero-oligomerization was shown to be crucial for their role in endocytosis. In addition, both reggie proteins influence actin-dependent processes and differentially affect focal adhesion regulation.
G-protein coupled receptors (GPCRs) comprise the largest superfamily of cell surface receptors and possess a signature motif of seven transmembrane helices. The endothelin B (ETB) receptor is a member of rhodopsin like GPCR family. It plays an important role in vasodilation and is found in the membranes of the endothelial cells enveloping blood vessels. Knowledge of the three-dimensional structure of G-protein coupled receptors in general would significantly add to our understanding of their molecular mechanisms and would be useful in the search for new specific drugs. However, three-dimensional structural analysis will require milligram quantities of pure and homogeneous protein. This dissertation is a study of the production, biochemical characterization and preliminary structural studies of the human ETB G-protein coupled receptor. The present work aimed at elucidating the structure and mechanistic details of function of the receptor by using a combination of X-ray crystallographic and NMR methods for collecting structural data. To obtain homogenous and monodisperse receptor protein preparation for structural and functional studies, we implemented the baculovirus expression system for the production of ETB receptor for the present work. The two step affinity purification ensured capture of full-length receptor. Silver stained SDS-PAGE of the purified receptor-ligand complex indicated greater than 90% protein purity. Based on previous reports, we used the high affinity ligand (endothelin -1) binding to the receptor for co-crystallization of receptor-ligand complex by locking the receptor in the activated conformation. As a prerequisite for 3D crystallization trials, the stability of the detergent solubilized receptor-ligand complex was assessed with respect to pH, temperature and time. Receptor-ligand complex did not show any degradation and aggregation over 6 days at 4°C and 18°C. Interestingly, change of pH suggested that receptor-ligand complex is unstable at lower pH due to possible charge induced conformational changes. In our work, we introduced the idea of using fluorophore labeled ligand for simple visual recognition of the receptor-ligand complex during purification and crystallization. On the other hand, we alternatively used biotinylated endothelin-1 to produce an adequate amount of ligand bound receptor complex, thus ensuring homogeneity of the purified complex for use in structural studies. Thus far, preliminary crystals have been obtained for both the unlabelled ET-1 and fluorophore labeled ET-1 complexed with ETB receptor. Moreover, we performed the systematic investigation of the protein/peptide binding partner for the receptor-ligand complex with the chief aims of stabilizing structure and increasing the possibilities of 3D-crystal contacts. Thus subsequent to formation of receptor-ligand complex, the additional in vitro formation of a ternary arrestin-receptor-ligand complex was also attempted for use in structural studies. We successfully demonstrated that arrestin mutant (R169E) forms a tight complex with ETB receptor regardless of its phosphorylation state. A second approach to get insight into the ETB receptor ligand binding site relied on the use of spin isotope labeled ET-1 ligand peptide by employing solid state MAS NMR method. Preliminary data provided compelling evidence that the C-terminal region of the peptide is immobilized in an ordered environment and presumably bound to the receptor. This indicates that the approach is feasible, although there are difficulties in sample preparation for further spectral measurements and data collection which are currently being discussed in ongoing investigations. At this point of our research work, we initiated a collaborative effort to obtain high yields of pure, active receptor without post translational modifications, from an E. coli cell lysate based in vitro expression system. We successfully optimized the production of homogenous and monodisperse endothelin B receptor in mg amounts. Thus this could potentially provide an alternative source of high quality receptor production in large quantities for immediate crystallization trials. Thus we hope that the results from these investigations can be applied in a more general sense to the production and crystallization of other G protein-coupled receptors.
The objective of this study is the avifauna of the North American Green River Formation. Five new Green River bird species as well as several new specimens of already known species are described. * Galliformes: Gallinuloides wyomingensis EASTMAN 1900 A second specimen of the galliform Gallinuloides wyomingensis could be identified. Gallinuloides wyomingensis resembles closely Paraortygoides MAYR 1999, which is known from Messel and the London Clay. The new specimen exhibits characters such as a cup-like cotyla scapularis of the coracoid that clearly indicate that Gallinuloides is a stem-group representative of galliforms. * Eurypygidae: Eoeurypyga olsoni gen. et sp. nov. Eoeurypyga is the only fossil representative of the Eurypygidae. Eoeurypyga and the modern sunbittern Eurypyga helias share the typical long bill, the caudally situated neck and the elongated vertebrae cervicales. Additional synapomorph characters were found. The new species indicates a North American origin for the Eurypygidae. * Messelornithidae: Messelornis nearctica HESSE 1992 The original description of Messelornis nearctica was based on a single specimen. Ten new specimens, described in this study, reveal additional information. Messelornis nearctica shows the same large intraspecific size range as Messelornis cristata HESSE 1988 from Messel, the type species of the genus. * Apodidae: Wyomingcypselus pohli gen. nov. sp. nov. Wyomingcypselus pohli is the first described fossil apodiform bird for North American. Due to characters of the wing, especially the position of the processus musculi extensor metacarpi radialis, Wyomingcypselus is referrred to the Apodidae. * Trogoniformes: unnamed species The Green River birds include a poorly preserved, but apparently heterodactyl specimen, which also resembles trogons in overall appearance. * Primobucconidae: Primobucco mcgrewi BRODKORB 1970 Originally, Primobucco mcgrewi was only known from a partial skeleton consisting of the right wing. Three new specimens could be referred to the species. Primobucco mcgrewi clearly exhibits an anisodactyl foot, which makes the assignment to the zygodactyl Bucconidae highly doubtful. Instead, Primobucco mcgrewi is referrred to the Coraciiformes s.s. Thus, Primobucconidae are the first New World representatives of stem-group Coraciiformes. * ?Leptosomidae: Plesiocathartes wyomingensis sp. nov. and Plesiocathartes major sp. nov. Plesiocathartes wyomingensis and Plesiocathartes major represent the first North American record for the genus. Both species exhibit the diagnostic characters for the Leptosomidae as listed by MAYR (2002a, b). * Primoscenidae: Eozygodactylus americanus gen. et sp. nov. and unnamed species Eozygodactylus americanus is the first North American member of this taxon. Both Eozygodactylus americanus and the unnamed species show the zygodactyl foot and the large processus intermetacarpalis of the carpometacarpus, which are typical for Primoscendiae. Due to differences mainly of the humerus, it was placed in a new genus. Besides the descriptionof new species, the avifauna of the Green River Formatin was studied and compared with the avifauna of Messel. The formations show a high concordance, more than 60 % of the Green River taxa also occur in Messel. Such a high concordance is also found for mammals. This is due to the existence of two landbridges, the Thule landbridge and the de Geer landbridge, between Europe and North America during the early Eocene.
Shrew-1 wurde bei der Suche invasivitätsassoziierter Gene mittels eines DDRT-PCR-Ansatzes aus invasiven Zellen isoliert. Wie computergestützte Analysen der Sequenz ergaben, wies das bis dahin unbekannte Protein keinerlei Ähnlichkeiten mit bereits bekannten Proteinen auf und homologe Proteine wurden bisher nur in Vertebraten gefunden. Expressionsanalysen mit einem GFP-markierten shrew-1 zeigten, dass es an der basolateralen Plasmamembran lokalisiert, wo es mit dem E-Cadherin vermittelten Adhäsions-Komplex kolokalisiert. Eine Integration in diesen Komplex geschieht höchstwahrscheinlich durch direkte Interaktion mit β-Catenin. Ein weiteres Molekül das als potenzieller Interaktionspartner von shrew-1 identifiziert wurde und das in der Literatur oft als Tumorsuppressor diskutiert wird, ist Caveolin-1. Ferner konnten Überexpressionexperimente bereits zeigen, dass shrew-1 die Invasivität von HT1080-Zellen erhöhen kann. Das Ziel dieser Arbeit war es, zum einen mit Hilfe des Hefe-Split-Ubiquitin-Systems eine Interaktion von shrew-1 und Caveolin-1 zu bestätigen und zum anderen neue Interaktionspartner zu identifizieren, die helfen könnten, die Rolle von shrew-1 in invasiven Vorgängen zu erklären. Um eine mögliche Verbindung von shrew-1 und einem neuen Interaktionspartner in Bezug auf die Zellinvasivität zu untersuchen, sollten sowohl shrew-1 als auch der potenzielle Interaktionspartner mittels RNAi ausgeschaltet werden. Mit Hilfe des Split-Ubiquitin-Systems war es möglich, die Interaktion zwischen shrew-1 und caveolin-1 zu bestätigen und zu zeigen, dass diese durch die zytoplasmatische Domäne von shrew-1 vermittelt wird. Weiterhin konnte CD147 als neuer Interaktionpartner identifiziert werden. Eine Interaktion beider Proteine konnte ferner mit Hilfe des Bimolekularen-Fluoreszens-Komplementations-Systems (BIFC), des Fluoreszens-Resonanz-Energie-Transfers (FRET) und Coimmunoprezipitationen bestätigt werden. Die Interaktion von shrew-1 und CD147 scheint allerdings abhängig vom zellulären Kontext zu sein, wie die FRET-Analysen vermuten lassen. So konnte nämlich mit diesen Analysen eine starke Interaktion in MCF7-Zellen gezeigt werden, wohingegen die Interaktion in MDCK-Zellen schwächer war. Einer der auffälligsten Unterschiede dieser beiden Zelllinien im Bezug auf diese Interaktion könnte sein, dass MCF7-Zellen im Gegensatz zu MDCK-Zellen kein Caveolin-1 exprimieren. Caveolin-1 konnte seinerseits als Interaktionspartner von shrew-1 mit Hilfe des Hefe-Split-Ubiquitin-Systems bestätigt werden und andererseits wurde von einer anderen Arbeitsgruppe eine Interaktion von CD147 mit Caveolin-1 publiziert. Um dies näher zu untersuchen, wurde Caveolin-1 in MCF7-Zellen exprimiert und die FRET-Analysen in diesen wiederholt. Wie vermutet kam es zu einer Reduktion der Interaktion in Caveolin-1 exprimierenden MCF7-Zellen. CD147 ist neben vielen anderen Funktionen auch maßgeblich an der Regulation von Matrix-Metalloproteinasen beteiligt und kann somit die Invasivität von Zellen beeinflussen. Um einen Einfluß von shrew-1 und CD147 auf die Invasivität zu untersuchen, wurden beide Proteine mittels RNAi in HeLa-Zellen ausgeschaltet. Nachdem ein negativer Einfluss dieses Ansatzes auf das Proliferationsverhalten der Zellen ausgeschlossen werden konnte, wurde ein möglicher Effekt auf die Invasivität der Zellen untersucht. Durch die Analyse in Matrigel-Invasionsassays konnte gezeigt werden, dass das unabhängige Ausschalten beider Proteine die Invasivität der Zellen auf 35-55% im Vergleich zu Kontrollzellen reduziert. Die Ergebnisse dieser Arbeit untermauern die Annahme, dass shrew-1 eine Rolle bei invasiven Vorgängen spielt und weisen darauf hin, dass dies möglicherweise durch eine Interaktion mit CD147 geschieht. Die Interaktion mit CD147 und damit eine mögliche Funktion von shrew-1 bei invasiven Vorgängen scheinen dabei abhängig vom zellulären Kontext zu sein.
The generation of O2- by NADPH oxidaes was mainly attributed to immune cells that kill invading bacteria or cancer cells. But importantly, in the past several years, several homologs of the catalytic subunit gp91phox (Nox2) of the phagocytic NADPH oxidase have been identified in non-immune cells and tissues. Superoxide production derived from NADPH oxidaes has been shown to play a role not only in host defense but also in defined signaling cascades mediating growth and apoptosis. The aim of this work was to study the expression and the regulation of the”new” Nox isoforms in rat renal mesangial cells (MC). In particular the following results were achieved. 1) mRNA’s for both Nox1 and Nox4 were detected by RT-PCR. 2) Nox1 mRNA levels were increased upon exposure to basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and fetal calf serum (FCS) in a time- and dose-dependent manner. Exposure of MC to bFGF and FCS increased also basal production of reactive oxygen species (ROS) by MC. By contrast, Nox4 mRNA levels were not significantly affected by bFGF treatment, but were markedly down-regulated by PDGF and FCS. 3) To study the regulation of Nox1 on the protein level, an anti-Nox1 antibody was generated and characterized using affinity chromatography. Up-regulation of Nox1 expression by growth factors was confirmed also on the protein level. 4) Based on the already known cDNA sequence for Nox1, the transcriptional start site was determined by the “gene RACE” technique. 2547 bp of the genomic sequence of the 5´-flanking region of the Nox1 gene were cloned and sequenced using the „Genome-Walking“ method. To study the regulation of Nox1 transcription functional Nox1 promoter/luciferase fusions were be established. MC were transiently transfected with different promoter/luciferase constructs and stimulated with growth factors. By measuring luciferase activity it was determined that growth factors induced the Nox1 transcription and that the Nox1 core promoter is sufficient for the activation. 5) By measurement of superoxide radicals and analysis of Nox1 mRNA expression by quantitative RT-PCR (TaqMan) as well as protein level by Western blotting it could be shown that treatment of MC with NO donors inhibited the expression of Nox1 in a time- and dose-dependent manner. Moreover, using activators and inhibitors of the soluble guanylyl cyclase (sGC) it could be shown, that the activation of sGC mediates the effect of NO on Nox1 expression. However, NO had no inhibitory effect on Nox1 promoter activity. Experiments with the inhibitor of transcription, actinomycin D, suggest that NO-mediated regulation of Nox1 is triggered probably via post-transcriptional mechanisms. Nox4 is regulated on the mRNA levels in a similar manner as Nox1. 6) To analyze the sub-cellular localization of the Nox isoforms, coding sequences for Nox1 and Nox4 were fused together with green fluorescent protein into the pEGFP-N1 demonstrated that both isoforms are localized predominantly in the plasma membrane, but also in the perinuclear region and cytoplasm. However, the localization of Nox1 in the plasma membrane was more pronounced. 7) In addition to Nox1 and Nox4, mRNA of the newly identified NOXA1 that is a homolog of the p67phox subunit of NADPH oxidase was detected in MC by RT-PCR.
The heat stress response is characterized by the presence of heat stress transcription factors (Hsfs) which mediate transcription of heat stress genes. In tomato (Lycopersicon peruvianum) cell cultures the simultaneous expression of four Hsfs, which are either constitutively (HsfA1 and HsfA3) or heat-stress inducible (HsfA2 and HsfB1) expressed, results in a complex network with dynamically changing cellular levels, intracellular localization and functional interactions. In order to examine the relevance of their multiplicity as well as to get more insights into the complexity of the plant heat stress response, the individual tomato Hsfs were investigated with respect to their protein interactions in vitro and in vivo. To this aim, I used pull-down assays as well as yeast assays to study the following aspects: 1. Oligomeric state of Hsfs: the results show that all class A Hsfs (HsfA1, HsfA2 and HsfA3) are trimeric proteins and interact with each other via the oligomerization (HR-A/B) domain. The similarity of their HRA/B regions allows formation of homo- and heterooligomeric complexes between all class A Hsfs. This special property was investigated by mutational studies with HsfA2 indicating that the linker and the HR-B regions are the minimal part required for Hsf/Hsf interactions. The conserved hydrophobic amino acid residues of the HR-B region are most important whereas the amino acid residues of the linker may provide higher flexibility to the HR-B region. Another investigated factor was HsfB1. HsfB1 is a member of class B Hsfs, which are characterized by an oligomerization domain without the 21 amino acid residues linker inserted between the HR-A and HR-B regions. It has a low activator potential and exists exclusively as dimer. HsfB1 can not physically interact with class A Hsfs. However, HsfB1 and HsfA1, binding to adjacent HSE sites, are assumed to cause strong synergistic effects in gene activation. 2. Potential HsfB1 interacting proteins: we searched for HsfB1 interacting proteins by using recombinant His-tagged proteins with HsfB1 as baits in pull-down assays. Histones H2A, H2B and H4 were identified by means of Peptide Mass Finger Printing and N-terminal sequencing analyses. The three histones represent the major proteins in tomato whole cell extracts retrieved by HsfB1. 3. HsfA2/small heat stress proteins (sHsps) interaction: pull-down and yeast two-hybrid assays were used to study the specific interaction of HsfA2 with tomato class II sHsp. This interaction occurs via the oligomerization domain of HsfA2. Other members of the plant Hsp20 family, including class I sHsp, do not interact with HsfA2. Heterooligomers of HsfA2 with class II sHsp may represent precursor forms of the plant higher molecular weight cytoplasmic complexes of heat stress granules, which form during heat stress. The findings presented in this thesis are a contribution to support the concept of a Hsfs network via protein-protein interactions. These data, together with information obtained from other studies, are used to propose a tentative model of the complex Hsfs network controlling the plant heat stress response.
Studies in particular of the last decade showed that active neurogenesis continuously takes place in the subventricular zone (SVZ) of the lateral ventricles of the adult rodent brain. Neurogenesis in the SVZ leads to migration of neuroblasts within the rostral migratory stream (RMS) and mature neuron formation mainly in the olfactory bulb (OB). According to present understanding, glial cells with astrocytic properties represent the actual adult neural stem cells. The cell types representing the various cellular transition states leading to the formation of mature neurons as well as the mechanisms controlling adult neurogenesis and neuroblast migration are poorly understood. A previous study from this laboratory demonstrated that the ATP-hydrolyzing enzyme nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) is associated with type B cells, the presumptive neural stem cells. NTPDase2 is a protein of the plasma membrane with its catalytic site facing the extracellular space. It hydrolyzes extracellular nucleoside triphosphates to their respective nucleoside diphosphates. This raises the possibility that the signaling pathway via extracellular nucleotides is involved in the control of adult neurogenesis. Neurons as well as glial cells express several subtypes of receptors (P2 receptors) that are responsive to the nucleotides ATP, ADP, UTP, or UDP. P2X receptors are ATP-gated Na+, K+ and Ca2+ permeable ion channels, P2Y receptors are coupled to trimeric G-proteins. In order to probe for a functional role of nucleotides in adult neurogenesis, the present study referred to an in vitro system (neurospheres). Neurospheres produced from isolates of the mouse SVZ and cultured in the presence of EGF and bFGF expressed the neural stem cell marker nestin and also GFAP, S100β, NTPDase2 and tissue non-specific alkaline phosphatase. Neurospheres generated from the cells of the subventricular zone were multipotenital. This was revealed by immunostaining of differentiated cells with markers for astrocytes, neurons and oligodendrocytes. The presence of ecto-nucleotidase was verified by analyzing the free phosphate released from nucleotides. The tissue non-specific form of alkaline phosphatase was the predominant enzyme. Both NTPDase2 and TNAP could be identified by immunocytochemistry and Western blotting. Hydrolysis was not observed for p-nitrophenyl thymidine monophosphate, a substrate of members of the ectonucleotide pyrophosphatase/phosphodiesterase family (NPP1 to NPP3). Since ecto-nucleotidases control the availability of extracellular nucleotide agonists, neurospheres were studied for the potential expression and functional role of nucleotide receptors. Neurospheres responded to extracellular nucleotides with a transient rise in Ca2+ (ATP = ADP > UTP). The rise in Ca2+ was due to P2Y receptors. The Ca2+ response was unaltered in the absence of extracellular Ca2+ and strongly reduced by thapsigargin, a blocker of internal Ca2+ stores. The P2Y1 antagonist MRS2179 strongly reduced the ATP- or ADP-induced increase in Ca2+, suggesting the involvement of a P2Y1 receptor. In addition, suramin and PPADS, non-selective antagonists for P2 receptors, inhibited most of the Ca2+ response. The agonistic activity of UTP and the lack of response to UDP implied the additional presence of a P2Y2 and/or a P2Y4 receptors and the absence of a functional P2Y6 receptor. RT-PCR experiments demonstrated that neurospheres expressed P2Y1 and P2Y2 receptors but not P2Y4 receptor. That the majority of the Ca2+ response to ATP was mediated via P2Y1 receptors was also confirmed by analysis of P2Y1 knockout mice and by application of the P2Y1 receptor-specific antagonist MRS2179. In addition, agonists of P2Y1 and P2Y2 receptors and low concentrations of adenosine augmented cell proliferation inspite of the presence of mitogenic growth factors. Neurosphere cell proliferation was attenuated after application of MRS2179 and in neurospheres from P2Y1 receptor knockout mice. These results infer a nucleotide receptor-mediated synergism that augments growth factor-mediated cell proliferation. Taken together these results suggest that P2Y-mediated nucleotidergic signalling is involved in neurosphere function and possibly also in adult neurogenesis in situ.
Active neurogenesis continuously takes place in the dentate gyrus of the adult mammalian brain. The dentate gyrus of the adult rodent hippocampus contains an astrocytelike cell population that is regarded as residual radial glia. These cells reside with their cell bodies in the subgranular layer (SGL). Radial processes traverse the granule cell layer (GCL) and form bushy ramifications in the inner molecular layer (IML). The residual radial glial cells apparently represent neuronal progenitor cells that can give rise to functionally integrated granule cells. To date the cellular and molecular events driving a subpopulation of these cells into neurogenesis as well as the cellular transition states are poorly understood. The present study shows, that in the mouse dentate gyrus, this cell type selectively expresses surfacelocated ATPhydrolyzing activity and is immunopositive for nucleoside triphosphate diphosphohydrolase 2 (NTPDase2). NTPDase2 is an ectoenzyme and hydrolyzes extracellular nucleoside triphosphates such as ATP or UTP to their respective nucleoside diphosphates. The enzyme becomes expressed in the hippocampus during late embryogenesis from E17 onwards, and is thus not involved in early brain development. Its embryonicpattern of expression mirrors dentate migration of neuroblasts and the formation of the primary and finally the tertiary dentate matrix. NTPDase2 is also expressed by a transient population of cortical radial glia from late embryonic development until postnatal day 5. NTPDase2 can be employed as a novel markerfor defining cellular transition states along the neurogenic pathway. It is associated with subpopulations of GFAP and nestinpositive cells. These intermediate filaments are typically expressed by the progenitor cells of the dentate gyrus. In addition there is a considerable overlap with doublecortinand PSANCAM positive cells. The expression of the microtubuleassociated protein doublecortin and of PSANCAM which are expressed by migrating neuroblasts is indicative of a transition of progenitors to a neural phenotype or an immature form of granule cell. NTPDase2 is no longer associated with young neurons and with maturegranule cells, as indicated by the lack of doubleimmunostaining for III tubulin and NeuN, respectively. Furthermore, β S100positive astrocytes do not express NTPDase2 validating that NTPDase2 is also not associated with later stages of gliogenesis. Experiments with the Sphase marker bromodeoxyuridine (BrdU) demonstrate that NTPDase2positive cell proliferate. Postmitotic BrdU-labeled cells preferentially acquire an NTPDase2positive phenotype. Many of these cells were also positive for GFAP. The contribution of BrdUlabeled cells positive for NTPDase2 increased with time from 2 h to 72 h, validating a strong association of NTPDase2 with proliferating cells of the dentate gyrus. The colocalization studies with various markers and the results of the experiments suggestthat NTPDase2 is associated with cell types of varying maturation states but not with mature neurons or astrocytes. Studies on the formation of neurospheres from the dentate gyrus validate previous data suggesting that the hippocampal progenitors have little capacity for self renewal in vitro. In situ hybridization results indicate the presence of one of the metabotropic purinergic receptor subtypes (the P2Y1 receptor) within the adult neurogenic regions, the dentate gyrus and the lateral walls of the lateral ventricles. A patchclamp analysis demonstrates the presence of functional ionotropic nucleotide receptor (P2X receptors) in progenitor cells expressing nestin promotordriven GFP. They suggest that the signaling pathway via extracellular nucleotides and nucleotide receptors may play a role in the control of adult hippocampal neurogenesis.
Zwei der wichtigsten Leistungen eines sich entwickelnden Embryos sind der Aufbau des Blutkreislauf- und des Nervensystems. Beide Systeme sind hierarchisch organisierte Strukturen, deren Verzweigungen nahezu alle Teile des Körpers erreichen. Es gibt eine zunehmende Zahl von Hinweisen darauf, dass ihre Entwicklung eng miteinander verknüpft ist, nach ähnlichen Prinzipien verläuft und verwandte molekulare Mechanismen verwendet. Die Entstehung eines funktionellen vaskulären Netzwerks erfordert Signale, die Prozesse wie die Lenkung und die Verzweigung von Gefäßen in den Zielgeweben kontrollieren. Ähnliche Anforderungen werden an wachsende Axone bei der Knüpfung der Verbindungen des Nervensystems während der Embryonalentwicklung gestellt. Einige der Faktoren, die die Lenkung der Axone kontrollieren, spielen auch eine ähnliche Rolle in der vaskulären Entwicklung. Lenkungsmoleküle, die eine Richtungsinformation vermitteln, sind für die Wegfindung der Axone besonders wichtig. Die größte Familie solcher Lenkungsmoleküle wird durch die Semaphorine gebildet. Semaphorine können in acht Klassen unterteilt werden, deren gemeinsames Merkmal eine konservierte Semaphorin-Domäne ist und die unterschieden werden anhand ihrer Klassen-spezifischen carboxyterminalen Domänen. Die Semaphorin-Familie umfasst sowohl sekretierte als auch membrangebundene Proteine. Die am besten charakterisierten hiervon sind die sekretierten Klasse 3 Semaphorine. Eine Kombination von in vitro und in vivo Ansätzen zeigte, dass die Klasse 3 Semaphorine an der Steuerung der Axon- und Dendritenlenkung, der Bildung von Axonbündeln und der neuronalen Migration während der Entwicklung des Nervensystems beteiligt sind. Sie agieren hauptsächlich als repulsiv wirkende Signale, die Axone aus Regionen ausschließen, von den Geweben weg, in denen sie exprimiert sind. Diese Wirkung wird über die Semaphorin-Domäne vermittelt. Verschiedene Hinweise deuten auf eine Beteiligung von Semaphorinen an der Entwicklung des vaskulären Systems. Sowohl homozygote Sema3a- als auch Sema3c-Mausnullmutanten sterben nach der Geburt aufgrund kardiovaskulärer Defekte. Darüber hinaus binden die Rezeptoren für die Klasse 3 Semaphorine, Neuropilin-1 (Nrp-1) und –2 (Nrp-2), einige Isoformen des vaskulären endothelialen Wachstumsfaktors (Vascular Endothelial Growth Factor, VEGF). Neuropilin-1 und Neuropilin-2-defiziente Mäuse und Neuropilin-1/-2-Doppelmutanten weisen Defekte des Gefäßsystems auf, wie z.B. eine Rückbildung der neuralen Vaskularisierung und Abweichungen in der Entwicklung des Herzens und der großen Gefäße. Die membrangebundenen Semaphorine sind bisher nur wenig untersucht, da zuverlässige in vitro Assays fehlen. Somit ist ein genetischer Ansatz der beste Weg, die physiologische Funktion dieser Proteine zu untersuchen. Aus diesen Gründen war die Zielsetzung dieser Arbeit, durch homologe Rekombination in embryonalen Stammzellen eine Mauslinie herzustellen, die ein Nullallel des membrangebundenen Sema5a-Gens trägt. Für diesen Ansatz wurde ein Mitglied der Klasse 5 Semaphorine gewählt, da es nur zwei Mitglieder dieser Klasse im Mausgenom gibt, die weitgehend komplementäre Expressionsmuster aufweisen. Damit unterscheiden sie sich von den anderen Klassen der Semaphorine, deren Mitglieder stark überlappende Expressionsmuster zeigen. Dies verringert die Wahrscheinlichkeit einer gegenseitigen funktionellen Kompensation nach Mutation eines Gens. Die Klasse 5 Semaphorine sind auch deshalb besonders interessant, da sie die einzigen sind, die sowohl in Vertebraten als auch in Invertebraten vertreten sind. Sie sind gekennzeichnet durch sieben carboxyterminale Typ 1-Thrombospondinmodule (TSP) in ihrer extrazellulären Domäne. TSPs wurden ursprünglich in den Proteinen Thrombospondin 1 und 2 gefunden, in denen sie das Auswachsen von Neuriten verschiedener Nervenzelltypen fördern. Dies lässt vermuten, dass Klasse 5 Semaphorine sowohl inhibierende als auch stimulierende Effekte haben könnten, in dem sie unterschiedliche Rezeptoren mit der Semaphorin-Domäne oder der TSPs aktivieren. Das Expressionsmuster von Sema5A und die bekannte Funktion von Semaphorinen in der Ausbildung neuronaler Verbindungen lassen es sinnvoll erscheinen, bei der Untersuchung der mutanten Tiere den Schwerpunkt auf die Entwicklung des Nerven- und des Gefäßsystems zu legen. Aufgrund technischer Schwierigkeiten konnte innerhalb der Bearbeitungszeit dieser Doktorarbeit nur der Phänotyp des vaskulären Systems untersucht werden. Die Inaktivierung des Sema5a-Gens wurde durch die Verwendung eines ‚Targeting’-Vektors erreicht, welcher die Exone 4 und 5 des Sema5a-Gens durch eine Neomycin-Selektionskassette ersetzte. Aus 144 untersuchten ES-Zellklonen wurden drei ES-Zellinien mit einem rekombinierten Sema5a-Locus identifiziert. Zwei der positiven Klone wurden zur Herstellung einer chimären Maus durch die Morula-Aggregationsmethode verwendet. Mit einem der Klone konnte eine männliche Chimäre erzeugt werden, die nach Kreuzung mit NMRI-Wildtyptieren die Mutation an die Nachkommen weitergab. Der Verlust der Proteinexpression in homozygoten Sema5a-Mutanten wurde durch Westernblot-Analyse von Zellmembranpräparationen homozygoter Embryonen unter Verwendung eines Antikörpers gegen das zytoplasmatische Ende von Sema5A bestätigt. Dieses Ergebnis bestätigte, dass die Deletion des vierten und fünften Exons des Sema5a-Gens ein Nullallel hervorbringt. Nach Verpaarungen heterozygoter Mutanten konnten keine Neugeborenen identifiziert werden, die homozygot für das mutierte Allel waren. Homozygte Mutanten starben zwischen E11,5 und E12,5 der Embryonalentwicklung, der Verlust von Sema5A ist also embryonal letal. Die Morphologie der homozygoten Tiere zeigte keinen offensichtlichen Unterschied zu den heterozygoten Embryonen oder zu Wildtyp-Geschwistern auf. Frühe embryonale Musterbildungsprozesse in Sema5a-Nullmutanten sind also nicht gestört. Ein Tod bei dieser Entwicklungsstufe deutet auf einen Defekt in der Entwicklung des Blutgefäßsystems hin, da die Embryonalstadien zwischen E9 und E13 besonders wichtig für die Ausbildung dieser Gefäße sind und viele Mutationen, die Herz und Blutgefäßen beeinträchtigen, den Tod der Embryonen in diesem Stadium bewirken. Das embryonale Blutgefäßsystem in E10,5 und E11,5 Embryonen wurde durch immunhistochemische Färbungen ganzer Embryonen unter Verwendung eines spezifischen gegen das Platelet Endothelial Cell Adhesion Molecule (PECAM) gerichteten Antikörpers dargestellt, welches in vaskulären Endothelzellen exprimiert ist. Die allgemeine Architektur des Gefäßsystems war in homo- und heterozygoten Mutanten ähnlich und wies weder an E10,5 noch an E11,5 besondere Abweichungen auf. Es wurden bei der Lage und der Anzahl intersomitischer Gefäße, der Entwicklung der dorsalen Aorta oder der Vaskularisierung der Extremitätenanlagen keine Abweichungen festgestellt. Morphologische Defekte konnten jedoch bei E10,5 in den Verästelungen der Blutgefäße detektiert werden, die von den Hauptvenen der Cranialregion abzweigen. Die Verzweigungen waren geringer ausgeprägt als in heterozygoten oder Wildtyp-Vergleichstieren. Insbesondere zeigte sich eine Verringerung der Anzahl sekundärer und tertiärer Verzweigungen. In dem sich entwickelnden Embryo führt die wiederholte Verzweigung von Ästen der Hauptvenen zu einem hierarchisch gegliederten Netzwerk großer Gefäße in der Region des medialen Kopfes. Während die Ausbildung dieses Netzwerkes in den Sema5a-/--Tieren beeinträchtigt ist, erscheint die Organisation der kleinen Gefäße in den mehr dorsal und peripher gelegenen Regionen des Kopfes normal. In heterozygoten und homozygoten Mutanten bilden die kleineren Gefäße ein dicht verzweigtes Netzwerk. Die Verminderung der Komplexität der größeren Gefäße konnte in allen untersuchten Nullmutanten beobachtet werden. Es variierte jedoch die Penetranz des Phänotyps. In allen Fällen war die Anzahl primärer Verzweigungen unverändert, während die Anzahl der sekundären und der tertiären Verzweigungen zu unterschiedlichen Graden reduziert war. Im Gegensatz dazu zeigte sich im Verzweigungsmuster von heterozygoten Mutanten und beim Wildtyp nur eine geringe Variabilität zwischen individuellen Embryonen. Dies belegt, dass die Verminderung des Verzweigungsgrades größerer Gefäße nicht innerhalb der normalen Variabilität liegt, sondern durch die Inaktivierung des Sema5a-Gens verursacht wird. Dieser Phänotyp ist in späteren Stadien sogar deutlicher ausgeprägt. In E11,5 Embryonen waren die Stämme der großen Blutgefäße in den Nullmutanten weniger komplex und in einigen Fällen trat sogar eine Reduzierung der Anzahl primärer Verzweigungen auf. Diese spätere Verminderung der Anzahl bereits ausgebildeter primärer Verzweigungen legt nahe, dass der Phänotyp durch eine Rückbildung von Verzweigungen aufgrund möglicher Defizite in deren Reifung und/oder Stabilisierung erfolgt. Die interessanteste Besonderheit der vaskulären Defekte in den Nullmutanten liegt in ihrer regionalen Spezifität. Bis hier ist das Netzwerk großer Gefäße, welches der anterioren Hauptvene entspringt, das einzige Gefäßsystem, in dem Abweichungen entdeckt wurden. Dieses Netzwerk wird durch die strukturelle Umbildung des primären kapillaren Plexuses gebildet. Zwischen E9,5 und E12 sprießen Zweige rostral aus der Hauptvene, um ein hierarchisch organisiertes Netzwerk von Gefäßen zu bilden. Die Umbildung des primären kapillaren Plexus in den mehr rostral und ventral gelegenen Kopfregionen führt zu der Bildung eines hochverzweigten vaskulären Netzwerkes, welches jedoch bei E10,5 noch nicht hierarchisch organisiert erscheint. Die Signale, die für diesen unterschiedlichen Ablauf der Musterbildung während der Entwicklung des Gefäßsystems des Kopfes verantwortlich sind, sind noch unbekannt. Die besonderen Defekte in der stereotypischen Organisation der cranialen Gefäße in Sema5a-Mutanten legt nahe, dass Sema5A eines dieser Signale sein könnte. Es könnte Teil eines Rezeptor/Ligandenkomplexes sein, welcher positionelle Signale für das Verzweigen und das Wachstum großer Gefäße in rostraler Richtung liefert. Sema5A könnte die Bildung von Verzweigungen durch die Regulierung der Wanderung endothelialer Zellen, ihrer Proliferation oder ihrer Interaktion mit unterstützenden Zellen oder der extrazellulären Matrix kontrollieren. Sema5A könnte Teil eines neuen Signalweges sein oder als Teil eines der bekannten Signalwegs wirken, welcher die Entwicklung des Gefäßsystems reguliert. Einer der Signalwege, die essentiell für die Gefäßbildung sind, wird durch VEGF und Angiopoietin (Ang-1) reguliert. Sowohl in VEGF-, als auch in Ang-1-Mutanten ist die Gefäßumbildung im Kopf beeinträchtigt. Insbesondere erscheint das Netzwerk kleiner Gefäße in den Ang-1 Nullmutanten als nur nur teilweise restrukturiert und die großen Gefäße als weniger komplex. Das Verzweigungsmuster der großen Gefäße in den Ang-1- Nullmutanten ähnelt auffallend dem der Sema5a-Nullmutanten. Eine zweite Ähnlichkeit in den Phänotypen von Ang-1- und Sema5a-Mutanten zeigt sich in der Reduzierung der primären Verzweigungen, welche in den Sema5a-Nullmutanten bei E11,5 beobachtet wird. Hier könnte die Verminderung aus einer Rückbildung von Gefäßen resultieren, wie sie auch typischerweise in Mutanten für Ang-1 oder dessen Rezeptor auftritt. Diese Beobachtung legt nahe, dass Sema5A ein neuer Teilnehmer innerhalb des Ang-1-Signalweges ist, welcher die Auswirkung von Ang-1 auf die endothelialen Zellen der großen Gefäße entweder vermittelt oder moduliert und dadurch das spezifische Muster der Blutgefäße des Kopfes beeinflußt. Mit dieser Doktorarbeit wird zum ersten Mal eine funktionelle Untersuchung des Klasse 5 Semaphorins Sema5A vorgestellt. Die phänotypische Untersuchung von Mäusen, die Nullallele für Sema5a-Gens tragen ergab, dass dieses membrangebundene Protein essentiell für die embryonale Entwicklung ist. Es ist an der Musterbildung des Gefäßsystems beteiligt. Seine Aufgabe besteht möglicherweise darin, die Bereitstellung positioneller Signale für die Ausbildung von Gefäßverzweigungen zu gewährleisten. Einige grundlegende Fragen werden durch diesen Phänotyp aufgeworfen. Sowohl die Ursache für die embryonale Sterblichkeit als auch die zellulären Prozesse, welche in den Sema5a-Nullmutanten beeinträchtigt sind, müssen noch beschrieben werden. Unbekannt ist ebenfalls, ob zusätzlich zu der hier beschriebenen Rolle von Sema5A in der Gefäßbildung dieses an der Entwicklung des Nervensystems beteiligt ist. Die ersten Daten über die physiologische Rolle von Sema5A, welche mit dieser Arbeit vorgelegt werden, öffnen den Weg für weitergehende Untersuchungen über die Funktion des Proteins während der Embrionalentwicklung. Das hier erstmals vorgestellte Modellsystem ermöglicht es, Sema5A regulierte zelluläre Mechanismen zu untersuchen. Zusätzlich stellt es ein Werkzeug zur Verfügung, um die funktionelle Beziehung zwischen der Entwicklung des kardiovaskulären Systems und des Nervensystems zu untersuchen. Damit können die Aufgaben der Semaphorin-Proteinfamilie, die an diesen beiden wichtigen Prozessen beteiligt sind, näher charakterisiert werden.
The technique of site-specific fluorescence labelling with Tetramethylrhodaminemaleimide (TMRM) in combination with two electrode voltage-clamp technique (TEVC), an approach that has been named voltage clamp fluorometry (VCF), has been used in this work to study the Na,K-ATPase. The TMRM dye has the ability to attach covalently to cysteine residues and it responds to changes in the hydrophobicity of its local environment. We exploited this property using a construct of the Na-pump in which the native, extracellularly accessible cysteines were removed and cysteine residues were introduced by site-directed mutagenesis in specific positions of the Na-pump. In this way it was possible to detect site-specific conformational rearrangements of the Na-pump in a time-resolved fashion within a native membrane environment. In particular this technique allows to resolve reactions with low electrogenicity that cannot be satisfactorily analyzed with purely electrophysiological techniques and to identify the conformations of the enzyme under specific ionic composition of the measuring buffers. We used VCF to study the influence that several cations like Na+, K+, NMG+, TEA+ and BTEA+ exert on the distribution of the Na,K-ATPase between several enzymatic intermediates and on some of the reactions related to cation transport. To this end we utilized the mutants N790C in the loop M5-M6 and the mutant E307C, T309C, L311C and E312C in the loop M3-M4. From the correspondence of the fluorescence changes with the activation and inhibition of pumping current, by K+ and ouabain respectively, and from the fact that in Na+/Na+ exchange conditions the voltage distribution of charge movement and fluorescence changes evoked by voltage jumps are in reasonable agreement we conclude that through the fluorescence signals measured from these mutants, we can indeed monitor conformational changes linked to transport activity of the enzyme. For the mutants N790 and L311, it was found that the Na+ dependence of the amplitude and kinetics of the fluorescence signal associated with the E1P-E2P transition is in agreement with the prediction of an access channel model describing the regulation of the access of extracellular Na+ to its binding site. In particular for the mutants E307 and T309 it was found that in Na+/Na+ exchange conditions, the conformational change tracked by the fluorescence was much slower than the charge relaxation at hyperpolarized potentials while the kinetics was very similar at depolarized potentials. This implies that at hyperpolarized potentials the conformational change connected to the E1P-E2P transition does not give a large contribution to the electrogenicity of the process which is also consistent with the access channel model. On the mutant N790C it was found that the external pH does not seem to have any effect on the E1P-E2P equilibrium even if it seems to modulate the fluorescence quantum yield of the dye. Fluorescence quenching experiments with iodide and D2O indicate that at hyperpolarized potentials the local environment of the mutant N790C, experiences a small change in the accessibility to water without major changes in the local electrostatic field ...
Sodium proton antiporters are ubiquitous membrane proteins found in the cytoplasmic and organelle membranes of cells of many different origins, including plants, animals and microorganisms. They are involved in cell energetics, and play primary roles in the homeostasis of intracellular pH, cellular Na+ content and cell volume. Adaptation to high salinity and/or extreme pH in plants and bacteria or in human heart muscles requires the action of such Na+/H+ antiporters. NhaA is the essential Na+/H+ antiporter for pH and Na+ homeostasis (at alkaline pH) in Escherichia coli and many other enterobacteria. NhaA is an electrogenic Na+/H+ antiporter that exchanges 2H+ for 1Na+ (or Li+). NhaA shares with many other prokaryotic and eukaryotic antiporters a very strong dependence on pH. In order to achieve three-dimensional structure of NhaA, the previously described NhaA protein preparation was modified: (i) the wild type bacterial strain (TA16) used for homologous over-expression of NhaA was replaced with a delta nhaA strain (RK20). As a result, the purity and homogeneity of the sample was significantly improved; (ii) the previously two-step purification procedure was shortened to a single step affinity chromatography purification; (iii) a wide-range screening of crystallisation conditions, more than 20,000, was performed; (iv) a Seleno-L-methionine (SeMet) NhaA derivative was produced in order to solve the phases during structure determination. In parallel, attempts of production and crystallisation of co-complexes composed of NhaA and antibody fragments have been made. Four different monoclonal antibodies were available against NhaA. Selected antibody fragments were produced and the stability of the complex analysed. Here, the crystal structure of the pH down-regulated secondary transporter NhaA of Escherichia coli is presented at 3.45 Å resolution. A negatively charged ion funnel opens to the cytoplasm and ends in the middle of the membrane at the putative ion-binding site. There, a unique assembly of two pairs of short helices connected by crossed, extended chains creates a balanced electrostatic environment. A possible mechanism is proposed: the binding of charged substrates causes electric imbalance inducing movements, which allow for a rapid alternating access mechanism. This ion exchange machinery is regulated by a conformational change elicited by a pH signal perceived at the cytoplasmic funnel entry. The structure represents a novel fold that provides two major insights: it reveals the structural basis for the mechanism of Na+/H+ exchange and its unique regulation by pH in NhaA and in many other similar antiporters. Furthermore, it is also important for the understanding of the architecture of membrane proteins in general. However, although many aspects of the ion-translocation mechanism and pH regulation are clarified by the NhaA structure, higher resolution structures with Li+ or Na+ bound are required for understanding the ligand binding and the translocation mechanism at the atomic level. The alkaline pH-induced conformation is essential to further understand the pH-control and proton access to the binding site.