Nitric oxide sensing in plants is mediated by proteolytic control of group VII ERF transcription factors

Nitric oxide (NO) is an important signaling compound in prokaryotes and eukaryotes. In plants, NO regulates critical developmental transitions and stress responses. Here, we identify a mechanism for NO sensing that coordinates responses throughout development based on targeted degradation of plant-specific transcriptional regulators, the group VII ethylene response factors (ERFs). We show that the N-end rule pathway of targeted proteolysis targets these proteins for destruction in the presence of NO, and we establish them as critical regulators of diverse NO-regulated processes, including seed germination, stomatal closure, and hypocotyl elongation. Furthermore, we define the molecular mechanism for NO control of germination and crosstalk with abscisic acid (ABA) signaling through ERF-regulated expression of ABSCISIC ACID INSENSITIVE5 (ABI5). Our work demonstrates how NO sensing is integrated across multiple physiological processes by direct modulation of transcription factor stability and identifies group VII ERFs as central hubs for the perception of gaseous signals in plants

Ubiquitin- and Proteasome-dependent protein turnover in Arabidopsis thaliana

Stickstoffmonoxid (NO) ist ein kleines Molekül, welches in allen höheren Organismen die eine Rolle als Signalmolekül spielt. Es ist bereits bekannt, dass NO in Pflanzen nicht nur die Antwort auf Umweltstress, sondern auch Entwicklungsprozesse reguliert. Einige Mechanismen, wie die S-Nitrosylierung von regulatorischen Proteinen in Pflanzen, wurden bereits beschrieben, viele molekulare Mechanismen sind aber immer noch rätselhaft. Hier wurde gezeigt, dass NO Proteine mit N-terminalem Cystein in den Ubiquitin-abhängigen Proteinabbaubbau einschleust und dass dieser Prozess O2 erfordert. Diese Experimente deuten darauf hin, dass der durch NO eingeleitete Abbau von Proteinen eine Rolle bei der NO-vermittelten Signaltransduktion spielt. Die Expression einer Ubiquitin-Variante mit Arg anstelle von Lys in Position 48 (ubK48R) führt in der Arabidopsis Linie RV86-5 zum Zelltod. Um diesen Zelltodprozess zu verstehen, muss ubK48R Expression in RV86-5 frei von Effekten sein, die auf das Expressionssystem zurückzuführen sind. Da vom verwendeten Aktivierungssystem unerwünschte Effekte berichtet worden waren, wurde versucht, herauszufinden, ob solche Effekte die Zelltodprozesse in RV96-5 Pflanzen beeinflussen. Die Ergebnisse lieferten Informationen über die funktionelle Relevanz des Systems. Ein weiterer Teil der Diplomarbeit knüpfte an das Thema Zelltod an mit der Untersuchung von Suppressormutanten, welche die Expression von ubK48R überleben. 5 Mutantenlinien waren zuvor durch EMS-Mutagenese erzeugt worden, sud2 (SUPPRESSOR OF UBIQUITIN UBK48R-INDUCED CELL DEATH 2) war die interessanteste dieser Mutanten. Die Mutation war auf Chromosom 3 kartiert worden, aber weitere Experimente waren notwendig, um nach der partiellen Genomsequenzierung Mutationen in der relevanten Region des Genoms zu bestätigen. Basierend auf einer neuen Validierungsmethode (Sedlazeck et al. 2013) wurden Kandidatenmutationen mit der Sanger-Sequenzierungsmethode bestätigt. Diese Bemühungen führten zu potentiellen Kandidaten für das SUD2 Gen.Nitric Oxide (NO) is a small molecule present in all higher organisms that plays a role as a significant signaling compound. It is already known that plants use NO as a regulatory compound not only in response to the stress coming from the environment, but also in developmental processes. Although some mechanisms in plants have been reported, such as S-nitrosylation of regulatory proteins, many molecular mechanisms are still an enigma. Here we show that NO targets protein substrates with N-terminal Cysteine for ubiquitin-dependent degradation and that this process requires O2. These experiments suggest that NO-mediated protein turnover plays a role in plant NO sensing and signaling. Expression of a ubiquitin variant with Arg instead of Lys at position 48 (ubK48R) in the Arabidopsis plant line RV86-5 leads to cell death. In order to understand the downstream effects of this process in a precise manner, ubK48R expression in line RV86-5 has to be devoid of any side effects due to the expression system. Since the GVG activation system, which is present in line RV86-5, has already been reported as having side effects, we tried to find out whether it has any effect on cell death in RV86-5. These results provided reliable information about the functional relevance of the system. Another part of the Thesis is related to the previous topic; analyzing a suppressor mutant that survives the lethal effects of ubK48R. 5 mutant lines had previously been generated by EMS mutagenesis and sud2 (SUPPRESSOR OF UBIQUITIN UBK48R-INDUCED CELL DEATH 2) was the most promising among them. The position of SUD2 had been mapped to chromosome 3, but there were still experiments necessary for validation after next generation sequencing of the relevant region. Based on scoring developed by Sedlazeck et al. (2013), we tried to validate candidate mutations using Sanger sequencing methods for confirmation. These efforts provided us with potential candidate(s) for the SUD2 gene

Identification and characterization of novel Fasciola Hepatica Sodium Chloride dependent Taurine transporter

Die wirtschaftliche Belastung durch den Leberegel Fasciola hepatica resultiert aus dem Befall von Wiederkäuern, die als Nutztiere gehalten werden. Die Tatsache, dass mehrere Millionen Menschen weltweit von diesem Trematoden befallen sind, hat die Weltgesundheitsorganisation (WHO) dazu veranlasst, die menschliche Fascioliasis als eine Krankheit von globaler Bedeutung für die öffentliche Gesundheit zu klassifizieren. Da keine effektive Impfung gegen diesen Parasiten existiert, beruht die Behandlung von Tieren und Menschen nur auf Anthelminthika. Von diesen ist Triclabendazol seit mehr als 25 Jahren das Mittel der Wahl zur Bekämpfung dieses Parasiten. Triclabendazol wirkt sowohl gegen adulte als auch juvenile Egel und ist gut verträglich. Das zunehmende Auftreten von Triclabendazol- resistenten Egeln macht die Suche nach alternativen Ansatzpunkten erforderlich. Adulte Egel persistieren in den Gallengängen des Wirtsorganismus, wo sie dem aggressiven Milieu der Galle ausgesetzt sind. Gallensäuren werden im Normalfall mit Taurin (2- Aminoethansulfonsäure) konjugiert, wodurch sie weniger toxisch werden. In der vorliegenden Arbeit wurde Transporter aus der solute carrier-6 (SLC6) -Familie kloniert, der homolog zum menschlichen Turintransporter ist. Die phlygenetische Analyse zeigte, dass dieser Transporter am nächsten mit dem GABA-Transporter-2 von Invertebraten und unklassfizierten Transportern anderer Nematoden verwandt ist. Nach heterologer Expression in HEK 293 Zellen, vermittelte dieser F. hepatica-Transporter eine hochaffine zelluläre Aufnahme von Taurin (KM = 12,0 0,5 M), nicht jedoch von GABA. Die Substrataufnahme war abhängig von Na+ - und Cl- (berechnete Stöchiometrie 2: 1). Übereinstimmend mit einer relativ geringen Chloridkonzentration in der Galle von Säugertieren zeigte der F. hepatica Transporter eine offensichtlich höhere Affinität für Cl- (EC50 = 14 3 mM) als der menschliche Taurintransporter (EC50 = 55 7 mM). Experimentell konnte gezeigt werden, dass unkonjugierte Gallensäuren rasch zum Tod von Fasciola hepatica führen. Die Zugabe von Taurin ermöglichte hingegen das Überleben der Egel in Gegenwart von Gallensäuren. Guanidinoethansulfonsäure, ein Inhibitor des Taurintransporters, hob diese schützende Wirkung von Taurin wieder auf. Diese Beobachtungen Untersuchungen legen den Schluss nahe, dass der Taurintransporter für das Überleben von Leberegeln in der Galle essentiell ist. Somit stellt der Taurintransporter einen möglichen Angriffstpunkt für neue anthelminthische Wirkstoffe dar.The economic burden imposed by the liver fluke Fasciola hepatica is the result of its infestation of domestic ruminants. The fact that several million people worldwide are infested by this trematode has led the World Health Organization (WHO) to classify human fascioliasis as a disease of vital global public health importance. Because a vaccine against this parasite is not available anthelmintic drugs are the only option, for both animals and people. As an anthelminthic chemotherapeutic, triclabendazole has been the drug of choice in fighting this parasite for more than 25 years. Triclabendazole is active against both adult and juvenile flukes and is well tolerated by the host organisms. However, the increasing prevalence of flukes resistant to triclabendazole has made the identification of alternative drug targets a priority. In this study, I focused on the fact that adult F. hepatica persists in the hostile environment of the bile ducts of infected organisms. In mammals, bile acids are rendered less toxic by conjugation to taurine (2-aminoethanesulfonic acid). This reaction is thus also contingent on cellular uptake of taurine. In my thesis, I cloned the Fasciola hepatica taurine transporter, a member of the solute carrier-6 (SLC6) family. A comparative analysis of the sequence indicated its close relation to unknown SLC6 transporters from related nematodes (i.e., Clonorchis sinensis and Schistosoma spp.) analysis When heterologously expressed in HEK293 cells, this F. hepatica transporter supported the high-affinity cellular uptake of taurine (KM=12.0 0.5 M) but not of GABA. Substrate uptake was dependent on Na+ and Cl- (calculated stoichiometry 2:1). Consistent with the low chloride concentration in mammalian bile, the F. hepatica transporter had a higher apparent affinity for Cl- (EC50 =143 mM) than the human taurine transporter (EC50=557 mM). I incubated flukes with unconjugated bile acids in the presence and absence of taurine and found that taurine promoted the survival of flukes, while the taurine transporter inhibitor guanidinoethansulfonic acid abolished this protective effect of taurine. These observations support the conclusion that the taurine transporter is critical for the survival of liver flukes in the bile. Thus, the taurine transporter represents a viable drug target candidate.submitted by Bulut HamaliAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Diss., 2018OeBB(VLID)251646

Regulation of the heterochromatin spreading reaction by trans-acting factors.

Heterochromatin is a gene-repressive protein-nucleic acid ultrastructure that is initially nucleated by DNA sequences. However, following nucleation, heterochromatin can then propagate along the chromatin template in a sequence-independent manner in a reaction termed spreading. At the heart of this process are enzymes that deposit chemical information on chromatin, which attracts the factors that execute chromatin compaction and transcriptional or co/post-transcriptional gene silencing. Given that these enzymes deposit guiding chemical information on chromatin they are commonly termed writers. While the processes of nucleation and central actions of writers have been extensively studied and reviewed, less is understood about how the spreading process is regulated. We discuss how the chromatin substrate is prepared for heterochromatic spreading, and how trans-acting factors beyond writer enzymes regulate it. We examine mechanisms by which trans-acting factors in Suv39, PRC2, SETDB1 and SIR writer systems regulate spreading of the respective heterochromatic marks across chromatin. While these systems are in some cases evolutionarily and mechanistically quite distant, common mechanisms emerge which these trans-acting factors exploit to tune the spreading reaction

PLOS Neglected Tropical Diseases / Identification and characterization of the Fasciola hepatica sodium- and chloride-dependent taurine transporter

The parasitic liver fluke Fasciola hepatica infests mainly ruminants, but it can also cause fasciolosis in people, who ingest the metacercariae encysted on plants. The drug of choice to treat fasciolosis is triclabendazole (TBZ), which has been on the market for several decades. This is also true for the other available drugs. Accordingly, drug-resistant flukes have been emerging at an increasing rate making it desirable to identify alternative drug targets. Here, we focused on the fact that adult F. hepatica persists in the hostile environment of the bile ducts of infected organisms. A common way to render bile acids less toxic is to conjugate them to taurine (2-aminoethanesulfonic acid). We cloned a transporter from the solute carrier-6 (SLC6) family, which was most closely related to the GABA-transporter-2 of other organisms. When heterologously expressed, this F. hepatica transporter supported the high-affinity cellular uptake of taurine (KM = 12.0 0.5 M) but not of GABA. Substrate uptake was dependent on Na+- and Cl- (calculated stoichiometry 2:1). Consistent with the low chloride concentration in mammalian bile, the F. hepatica transporter had a higher apparent affinity for Cl- (EC50 = 143 mM) than the human taurine transporter (EC50 = 557 mM). We incubated flukes with unconjugated bile acids in the presence and absence of taurine: taurine promoted survival of flukes; the taurine transporter inhibitor guanidinoethansulfonic acid abolished this protective effect of taurine. Based on these observations, we conclude that the taurine transporter is critical for the survival of liver flukes in the bile. Thus, the taurine transporter represents a candidate drug target.(VLID)479007

Identification and characterization of the <i>Fasciola hepatica</i> sodium- and chloride-dependent taurine transporter - Fig 3

<p><b>Cellular localization of (A, B) and substrate uptake (C, D) by the <i>F</i>. <i>hepatica</i> (A, C) and the human taurine transporter (B, D) after heterologous expression in HEK293 cells.</b> HEK293 cells were stably transfected with plasmids encoding fluorescently tagged versions of the <i>F</i>. <i>hepatica</i> (YFP-FhTauT, panel A) and the human (CFP-HsTauT, panel B) taurine transporter. The cellular distribution of the tagged transporters was visualized by confocal microscopy (left-hand images). The cell surface was delineated by staining with trypan blue (images in the middle). The captured confocal images were overlaid (right-hand images). <i>C & D</i>: HEK 293 cells (1*10⁵ cells/well) expressing the YFP-tagged <i>F</i>. <i>hepatica</i> taurine transporter (circles in C) and the YFP-tagged human taurine transporter (circles in D) or untransfected HEK 293 cells (open triangles in C) were incubated for 10 min in the presence of the indicated concentration of [³H]taurine. The accumulated radioactivity was determined as described under <i>Materials and Methods</i>. Data are means ± S.D. of at least three independent experiments, which were carried out in duplicate. The solid lines were drawn by fitting the data to the equation of a rectangular hyperbola.</p

Identification and characterization of the <i>Fasciola hepatica</i> sodium- and chloride-dependent taurine transporter - Fig 4

<p><b>GABA- (A) and β-alanine-induced (B) inhibition of [</b>^<b>3</b><b>H]taurine uptake by the <i>F</i>. <i>hepatica</i> and the human taurine transporter after heterologous expression in HEK293 cells.</b> Stably transfected HEK 293 cells (1*10⁵ cells/well) expressing the YFP-tagged F. hepatica taurine transporter (circles, YFP-FhTauT) or the YFP-tagged human taurine transporter (triangles, CFP-HsTauT) were incubated for 10 min in the presence of 0.1 μM [3H]taurine and the indicated concentration of GABA (A) and β-alanine (B). The accumulated radioactivity was determined as described under Materials and Methods. Uptake observed in the absence of any inhibitor was set at 100% to normalize for interassay variability. These 100% control values were 0.89 ± 0.05 and 1.102 ± 0.142 pmol.min^-1.10⁻⁶ cells for the <i>F</i>. <i>hepatica</i> and the human transporter, respectively. Data are means ± S.D. from three independent experiments. The solid lines were drawn by fitting the data to the equation for a monophasic inhibition curve.</p

Inhibition of [³H]taurine uptake by unlabeled taurine in the absence and presence of GES.

<p>HEK293 cells (1*10⁵ cells/well) stably expressing the <i>F</i>. <i>hepatica</i> (A, B) or the human taurine transporter (C, D) were incubated for 10 min in the presence of 0.1 μM [³H]taurine and of the indicated concentrations of unlabeled taurine and of GES. Data represent means ± S.D. from three independent experiments carried out in duplicate. The solid lines in panels A and C were drawn by fitting the data to a monophasic inhibition curve. In panels, B and D, the data shown in panels A and C, respectively, were replotted in Dixon plots to visualize the non-competitive and the competitive inhibitory action of GES by the intersecting lines (B) and the parallel lines (D), respectively.</p

Amino acid alignment of the <i>F</i>. <i>hepatica</i> taurine transporter (Fh TauT) with the human serotonin transporter (Hs SERT), the GABA-transporter-2 of <i>C</i>. <i>sinensis</i> (Cs GAT2), the human GABA-transporter-2 (Hs GAT2) and the human taurine transporter (Hs TauT).

<p>Amino acids are represented in their single-letter code. Invariant sequences are denoted by white letters boxed in red, sequences with conservative substitutions are denoted by red letters. The red and blue arrow heads mark amino acids contributing to the first and second Na⁺ binding sites, respectively, in the crystal structure of Hs SERT. The green arrow heads highlight residues involved in the coordination of Cl^- in SERT. The cysteine residues in the second extracellular loop, which form a putative disulfide bridge, are indicated by magenta arrow heads. The brown arrow heads indicate candidate sites for N-linked glycosylation predicted by NetNGlyc 1.0 (<a href="http://www.cbs.dtu.dk/services/NetNGlyc/" target="_blank">http://www.cbs.dtu.dk/services/NetNGlyc/</a>).</p

Survival of adult <i>F</i>. <i>hepatica</i> in bile acid-containing medium in the presence and absence of taurine.

<p><b>A:</b> Adult flukes (12 flukes/condition in individual wells) were incubated at 37°C and 5% CO₂ in a humidified atmosphere in Hédon Feig medium in the absence (control, closed circles) or presence of 50 μM taurine (closed circle), of 2 mM GES (GES, closed triangle) or of bile acids (closed triangle, 5 mM cholic acid, 1.5 mM deoxycholic acid and 0.5 mM chenodeoxycholic acid). The number of dead flukes was recorded at the indicated time points. <b>B:</b> Adult flukes were incubated at 37°C and 5% CO₂ in a humidified atmosphere in Hédon Feig medium in the absence (control) or presence of bile acids (5 mM cholic acid, 1.5 mM deoxycholic acid and 0.5 mM chenodeoxycholic acid). Where indicated, the medium contained, in addition, 50 μM taurine or the combination of 50 μM taurine and 2 mM GES (GES) for 4h. The number of dead flukes was recorded at the end of the incubation period. The data are from three independent experiments with 11 to 12 flukes/condition. Survival in the presence of bile acids and taurine differed significantly from that observed in the sole presence of bile acids (p = 0.0006, Fisher's exact test) and in the presence of the combination of bile acids, taurine and GES (p = 0.0068, Fisher's exact test), but there was no significant difference between survival in the presence of bile acids and of the combination of bile acids, taurine and GES (p = 0.464). Thus, after Bonferroni correction for multiple testing (with α≤0.016), taurine afforded a statistically significant protection against bile acids.</p