Lipoic acid protein ligases in Plasmodium spp.

Protozoan parasites of the genus Plasmodium are the causative agent of malaria. The four human pathogenic species infect more than 500 million people each year, causing the death of at least 1 million people. The most severe form of human malaria is caused by P. falciparum, which is responsible for 90% of the malaria deaths. A major problem in the treatment of this disease is resistance of the parasites against most of the existing chemotherapies. Therefore, there is an urgent need to identify, validate and assess potential new drug targets. The prerequisite of a potential drug target is that it should not be of significance for the human host or it should be sufficiently different from the human counterpart, so that parasite-specific inhibition is feasible. Lipoic acid metabolism in Plasmodium differs from that of mammals in some ways and therefore it might be a promising target for the development of new antimalarials. This study investigated the importance of lipoic acid ligation in P. falciparum using reverse genetic approaches, to assess whether this pathway has potential for drug design. In addition, a spectrophotometric assay system was developed that allowed the biochemical characterisation of lipoic acid ligases and can be adapted to high-throughput screening approaches of inhibitors for these enzymes. Lipoic acid, also known as 6,8-thioctic acid, is an essential cofactor of alpha-keto acid dehydrogenase complexes (KADH) and the glycine cleavage system (GCV). The KADH include the pyruvate dehydrogenase (PDH), branched chain alpha-keto acid dehydrogenase (BCDH) and alpha-ketoglutarate dehydrogenase (KGDH), which are an integral part for any cell's metabolism. In Plasmodium spp. the lipoic acid dependent enzyme complexes are found in the apicoplast, a plastid related organelle, and in the mitochondrion and thus two organelle specific lipoylation pathways are present in these parasites. Biosynthesis of the cofactor occurs in the apicoplast. Octanoyl-[acyl carrier protein]: protein N-octanoyltransferase (LipB) catalyses the attachment of octanoyl-acyl carrier protein (octanoyl-ACP) to the PDH and lipoic acid synthase (LipA) then catalyses the insertion of two sulfurs into the octanoyl-chain to form lipoamide. In the mitochondrion, scavenged lipoic acid is ligated to the enzyme complexes by the action of lipoic acid protein ligase A (LplA1), in an ATP-dependent reaction. However, a second lipoate protein ligase A (LplA2) was identified in the genome of P. falciparum, but its subcellular localisation could not be predicted using the available prediction programs. To further analyse its localisation, parasites were generated expressing full length LplA2 in frame with green fluorescent protein (GFP). In addition, immunofluorescence analyses on wild-type parasites using LplA2 specific antibodies were performed. These studies showed that LplA2 is dually targeted to the apicoplast as well as to the mitochondrion, raising the question about potential redundancy between the ligases present in the parasites. To further analyse this possibility, knock-out studies of lplA1 and lplA2 were performed in the human and rodent malaria parasites P. falciparum and P. berghei, respectively. Knock-out studies showed that LplA1 and LplA2 are non-redundant and strongly suggested that LplA1 is crucial for intraerythrocytic development, whereas LplA2 is essential for sexual development in the mosquito. According to these results it appears that (1) a key regulator of lipoic acid metabolism in Plasmodium spp. is stage specific expression of the relevant proteins and (2) both ligases are potential drug targets as knock-out of lplA1 appeared impossible in the blood stages and knock-out of lplA2 resulted in the interruption of parasite sexual development in the mosquito, and thus transmission of the parasites would be blocked if LplA2 was inhibited. To further analyse the biochemical properties of P. falciparum LplA1 and LplA2, a spectrophotometric assay system was developed, which is also suitable for the development of a high-throughput assay system. The spectrophotometric assay monitors the first part of the LplA reaction - the activation of lipoic acid by ATP. The released pyrophosphate is converted to phosphate which is detected by acidic ammonium molybdate. Using the Escherichia coli LplA protein as a positive control, kinetic parameters for the bacterial protein were determined that are in reasonable agreement with the published data. The results validate the assay and suggest that it might be suitable for inhibitor screening in the future

Abschätzung der Meereisproduktion in der Laptev-See mit dem Ozean-Meereismodell NAOSIM

Fernerkundungsdaten haben eine kontinuierliche Abnahme des Meereises in den vergangenen 30 Jahren gezeigt, Klimamodelle prognostizieren eine anhaltende Abnahme für die Zukunft. Dies erfordert eine genauere Analyse der verursachenden Prozesse, der Trendentwicklung und der regionalen Variabilität. Dabei spielt die Laptev-See in der sibirischen Arktis eine bedeutende Rolle, da es hier, bedingt durch eine große Polynja-Aktivität, zur vermehrten Eisproduktion kommt. Zur näheren Untersuchung der verursachenden thermodynamischen und dynamischen Prozesse nutzen wir eine mit täglichen NCEP/NCAR-Daten angetriebene Simulation mit dem gekoppelten Ozean-Meereismodell NAOSIM (North Atlantic/Arctic Ocean-Sea Ice Model) von 1990-2008 mit 0.08° Auflösung. Aufgrund seiner realitätsnahen Wiedergabe des mittleren Jahresgangs und des negativen Trends der Eisbedeckung ist dieses Modell für die Auswertung gut geeignet. Die getrennte Analyse der thermodynamischen Eisproduktion bzw. Eisschmelze und der dynamischen Umverteilung für die gesamte Arktis bestätigt, dass im Bereich der Laptev-See die Eisproduktion im Mittel 850km3/a größer ist als die Eisschmelze. Dieses Eis wird von der Laptev-See in die zentrale Arktis exportiert. In der gesamten Arktis nimmt das Eisvolumen im Mittel um -450km3/a von 1990-2008 ab. usammenhänge zwischen der Eisproduktion der Laptev-See und des Eisvolumens der Arktis werden mittels einer Zeitreihenananlyse untersucht. Die Entstehungsgründe für Extremjahre (Bsp.: Minimum 2007, Maximum 1996) werden aufgezeigt und ihre regionalen Folgen in der Arktis diskutiert

Responsibility in supply chains: Germany's due diligence act is a good start

On 3 March, the Federal Cabinet adopted an act on corporate due diligence in supply chains. This represents an important step towards German businesses assuming full and proper responsibility for the supply chains associated with their goods and ser­vices. The move puts Germany in a group of European countries like France and the Netherlands that have already instituted legal frameworks of their own. However, by choosing to exclude civil liability the German government has left aside a powerful tool for applying targeted pressure to companies that fail to fulfil their obligations. In order to maximise the law’s impact, the German Bundestag and government should therefore adopt additional flanking measures. At the European and international levels, Germany can also contribute to making companies assume greater respon­si­bility for their own supply chains. (author's abstract

Extreme Warming in the Kara Sea and Barents Sea during the Winter Period 2000–16

The regional climate model COSMOin Climate Limited-AreaMode (COSMO-CLM or CCLM) is used with a high resolution of 15km for the entire Arctic for all winters 2002/03–2014/15. The simulations show a high spatial and temporal variability of the recent 2-m air temperature increase in the Arctic. The maximum warming occurs north of Novaya Zemlya in the Kara Sea and Barents Sea between March 2003 and 2012 and is responsible for up to a 208C increase. Land-based observations confirm the increase but do not cover the maximum regions that are located over the ocean and sea ice.Also, the 30-km version of theArctic SystemReanalysis (ASR) is used to verify the CCLM for the overlapping time period 2002/03–2011/12. The differences between CCLM and ASR 2-m air temperatures vary slightly within 18C for the ocean and sea ice area. Thus,ASR captures the extreme warming as well. The monthly 2-m air temperatures of observations and ERA-Interim data show a large variability for the winters 1979–2016. Nevertheless, the air temperature rise since the beginning of the twenty-first century is up to 8 times higher than in the decades before. The sea ice decrease is identified as the likely reason for the warming. The vertical temperature profiles show that the warming has a maximum near the surface, but a 0.58Cyr21 increase is found up to 2 km. CCLM, ASR, and also the coarser resolved ERA-Interim data show that February and March are the months with the highest 2-m air temperature increases, averaged over the ocean and sea ice area north of 708N; for CCLM the warming amounts to an average of almost 58C for 2002/03–2011/12

Verantwortung in Lieferketten: das Sorgfaltspflichtgesetz ist ein erster Schritt

Mitte Februar haben sich die beteiligten Bundesministerien auf einen Entwurf für ein Gesetz über die unternehmerischen Sorgfaltspflichten in Lieferketten geeinigt. Dies ist ein wichtiger Schritt, damit deutsche Unternehmen umfassende Verantwortung für die Lieferketten ihrer Waren und Dienstleistungen übernehmen. Deutschland hat sich damit in die Riege europäischer Länder wie Frankreich und die Niederlande ein­gereiht, die verbindliche Regulierungsrahmen schon gesetzt haben. Gleich­wohl hat die Bundesregierung mit der Absage an eine zivilrechtliche Haftung auf einen ent­scheidenden Hebel verzichtet, um Unternehmen, die ihrer Sorgfaltspflicht nicht nachkommen, gezielt zu sanktionieren. Um dem Gesetz die größtmögliche Wirkung zu verleihen, sollten Bundestag und Bundesregierung da­her weitere flankierende Maßnahmen beschließen, die über die rechtlichen Regelungen im Gesetzentwurf hinausgehen. Deutschland kann zudem auf europäischer und internationaler Ebene dazu beitragen, dass Unternehmen in der EU und im globalen Maßstab mehr Ver­antwortung in Lieferketten übernehmen. (Autorenreferat

Роль метода электрофоретического осаждения в создании биокомпозита на основе слоев гидроксиапатити и наночастиц серебра

Работа посвящена созданию многофункционального биокомпозита, состоящего из покрытия на основе гидроксиапатита (ГА) и наночастиц серебра с использованием высокотехнологичных методов обработки поверхности. Высокочастотное магнетронное распыление использовалось для получения слоев ГА покрытия с различной толщиной и структурой на титане и наночастицах серебра. Для получения антибактериального слоя наночастиц серебра использовался метод электрофоретического осаждения. Наночастицы серебра имели сферическую форму с диаметром 70±20 нм и[zeta] -потенциалом -20 мВ. Дифракционные картины биокомпозитов выявили пики кристаллического ГА и серебра (Ag). Так же установлено, что наночастицы серебра являются кристаллическими с размером кристаллитов 14 нм

Knockout studies reveal an important role of <i>plasmodium</i> lipoic acid protein ligase a1 for asexual blood stage parasite survival

Lipoic acid (LA) is a dithiol-containing cofactor that is essential for the function of a-keto acid dehydrogenase complexes. LA acts as a reversible acyl group acceptor and 'swinging arm' during acyl-coenzyme A formation. The cofactor is post-translationally attached to the acyl-transferase subunits of the multienzyme complexes through the action of octanoyl (lipoyl): &lt;i&gt;N&lt;/i&gt;-octanoyl (lipoyl) transferase (LipB) or lipoic acid protein ligases (LplA). Remarkably, apicomplexan parasites possess LA biosynthesis as well as scavenging pathways and the two pathways are distributed between mitochondrion and a vestigial organelle, the apicoplast. The apicoplast-specific LipB is dispensable for parasite growth due to functional redundancy of the parasite's lipoic acid/octanoic acid ligases/transferases. In this study, we show that &lt;i&gt;LplA1&lt;/i&gt; plays a pivotal role during the development of the erythrocytic stages of the malaria parasite. Gene disruptions in the human malaria parasite &lt;i&gt;P.falciparum&lt;/i&gt; consistently were unsuccessful while in the rodent malaria model parasite &lt;i&gt;P. berghei&lt;/i&gt; the &lt;i&gt;LplA1&lt;/i&gt; gene locus was targeted by knock-in and knockout constructs. However, the &lt;i&gt;LplA1&lt;/i&gt; &lt;sup&gt;(-)&lt;/sup&gt; mutant could not be cloned suggesting a critical role of LplA1 for asexual parasite growth &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt;. These experimental genetics data suggest that lipoylation during expansion in red blood cells largely occurs through salvage from the host erythrocytes and subsequent ligation of LA to the target proteins of the malaria parasite

Repression of human papillomavirus oncogene expression under hypoxia is mediated by PI3K/mTORC2/AKT signaling

Oncogenic HPV types are major human carcinogens. Under hypoxia, HPV-positive cancer cells can repress the viral E6/E7 oncogenes and induce a reversible growth arrest. This response could contribute to therapy resistance, immune evasion, and tumor recurrence upon reoxygenation. Here, we uncover evidence that HPV oncogene repression is mediated by hypoxia-induced activation of canonical PI3K/mTORC2/AKT signaling. AKT-dependent downregulation of E6/E7 is only observed under hypoxia and occurs, at least in part, at the transcriptional level. Quantitative proteome analyses identify additional factors as candidates to be involved in AKT-dependent E6/E7 repression and/or hypoxic PI3K/mTORC2/AKT activation. These results connect PI3K/mTORC2/AKT signaling with HPV oncogene regulation, providing new mechanistic insights into the cross talk between oncogenic HPVs and their host cells.Hypoxia is linked to therapeutic resistance and poor clinical prognosis for many tumor entities, including human papillomavirus (HPV)-positive cancers. Notably, HPV-positive cancer cells can induce a dormant state under hypoxia, characterized by a reversible growth arrest and strong repression of viral E6/E7 oncogene expression, which could contribute to therapy resistance, immune evasion and tumor recurrence. The present work aimed to gain mechanistic insights into the pathway(s) underlying HPV oncogene repression under hypoxia. We show that E6/E7 downregulation is mediated by hypoxia-induced stimulation of AKT signaling. Ablating AKT function in hypoxic HPV-positive cancer cells by using chemical inhibitors efficiently counteracts E6/E7 repression. Isoform-specific activation or downregulation of AKT1 and AKT2 reveals that both AKT isoforms contribute to hypoxic E6/E7 repression and act in a functionally redundant manner. Hypoxic AKT activation and consecutive E6/E7 repression is dependent on the activities of the canonical upstream AKT regulators phosphoinositide 3-kinase (PI3K) and mechanistic target of rapamycin (mTOR) complex 2 (mTORC2). Hypoxic downregulation of E6/E7 occurs, at least in part, at the transcriptional level. Modulation of E6/E7 expression by the PI3K/mTORC2/AKT cascade is hypoxia specific and not observed in normoxic HPV-positive cancer cells. Quantitative proteome analyses identify additional factors as candidates to be involved in hypoxia-induced activation of the PI3K/mTORC2/AKT signaling cascade and in the AKT-dependent repression of the E6/E7 oncogenes under hypoxia. Collectively, these data uncover a functional key role of the PI3K/mTORC2/AKT signaling cascade for viral oncogene repression in hypoxic HPV-positive cancer cells and provide new insights into the poorly understood cross talk between oncogenic HPVs and their host cells under hypoxia

Inhibition of Plasmepsin V activity demonstrates its essential role in protein export, PfEMP1 display, and survival of malaria parasites

The malaria parasite Plasmodium falciparum exports several hundred proteins into the infected erythrocyte that are involved in cellular remodeling and severe virulence. The export mechanism involves the Plasmodium export element (PEXEL), which is a cleavage site for the parasite protease, Plasmepsin V (PMV). The PMV gene is refractory to deletion, suggesting it is essential, but definitive proof is lacking. Here, we generated a PEXEL-mimetic inhibitor that potently blocks the activity of PMV isolated from P. falciparum and Plasmodium vivax. Assessment of PMV activity in P. falciparum revealed PEXEL cleavage occurs cotranslationaly, similar to signal peptidase. Treatment of P. falciparum-infected erythrocytes with the inhibitor caused dose-dependent inhibition of PEXEL processing as well as protein export, including impaired display of the major virulence adhesin, PfEMP1, on the erythrocyte surface, and cytoadherence. The inhibitor killed parasites at the trophozoite stage and knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of PMV increased resistance. This provides the first direct evidence that PMV activity is essential for protein export in Plasmodium spp. and for parasite survival in human erythrocytes and validates PMV as an antimalarial drug target