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Parasitology Section Nematode-specific regulation of S-adenosylmethionine decarboxylase offers a novel strategy for enzyme inhibition Zusammenfassung Onchocercose (Flussblindheit) stellt weiterhin ein bedeutendes Gesundheitsproblem in zahlreichen afrikanischen Staaten dar. Da mit Ivermectin zur Zeit nur ein Medikament für die Massentherapie zur Verfügung steht, ist die Identifizierung neuer Ziele für die Chemotherapie im Stoffwechsel des Erregers Onchocerca volvulus dringend erforderlich. Polyamine sind für Wachstums- und Differenzierungsprozesse essenziell, weshalb ihr Stoffwechsel als ein vielversprechender Angriffspunkt für die Chemotherapie gilt. Im Polyamin-Syntheseweg ist die S-Adenosylmethionin-Decarboxylase (AdoMetDC) ein Schlüsselenzym, das decarboxyliertes S-Adenosylmethionin für die Synthese von Spermidin und Spermin bereitstellt. Für das humane Enzym ist bekannt, dass seine enzymatische Aktivität durch das Vorläufermolekül Putrescin stimuliert wird. Vergleichende Studien zeigen nun, dass sich die AdoMetDC der parasitischen Filarie O. volvulus und des freilebenden Modellnematoden Caenorhabditis elegans hinsichtlich dieses Regulationsmechanismus vom humanen Enzym unterscheiden. Die ermittelten spezifischen Eigenschaften der Nematoden-AdoMetDC sind eine geringe Aktivität des nichtstimulierten Enzyms und eine geringere Spezifität gegenüber dem Stimulatormolekül. Diese Besonderheiten sollen ausgenutzt werden, um mit einer neuen Strategie die Nematodenenzyme spezifisch über die Stimulator-Bindungstasche zu inhibieren. Dazu wurden erste Inhibitionstests mit Polyaminanaloga durchgeführt. Introduction The human filarial parasite Onchocerca volvulus causes onchocerciasis. The disease is endemic in 37 African countries affecting about 15 million people. To date the only existing drug against this parasitic nematode is ivermectin. However, ivermectin kills only the microfilariae of O. volvulus and has hardly any efficacy on adult worms that survive in their human host for up to 14 years, with females producing constantly microfilariae. Due to the lack of a laboratory host for O. volvulus, the worm is inaccessible for physiological studies. However, in recent years the model nematode Caenorhabditis elegans has become a useful system to study certain aspects of the metabolism of parasitic nematodes. The naturally occuring polyamines putrescine, spermidine and spermine are aliphatic polycations found in all organisms. Since polyamines are essential for growth and developmental processes, targeting their metabolism is a promising approach in anti-parasite research. The polyamine synthetic pathway contains two regulatory steps catalysed by ornithine decarboxylase and 36 S-adenosylmethionine decarboxylase (AdoMetDC). The latter provides decarboxylated S-adenosylmethionine that is used to form the polyamines spermidine and spermine, respectively. The AdoMetDC from the nematodes O. volvulus and C. elegans have been previously cloned and characterised in our department (Da’dara et al. 1996, Biochem J 320: 519-30; Da’dara and Walter, 1998, Biochem J 336: 545-50). In the present study we have detected a nematode-specific regulation of AdoMetDC. Based on these findings a novel strategy to inhibit nematode AdoMetDC is proposed. Figure 1: Different effects of natural polyamines on nematode and human AdoMetDC activity Dose-dependent stimulation of A) O. volvulus, B) C. elegans and C) human AdoMetDC by putrescine (�), spermidine (�) and spermine (�). The basis activity of the human enzyme is indicated by the dotted blue line. Nematode AdoMetDCs highly depend on an activator and their enzyme activity is stimulated not only by putrescine but also by spermidine and spermine.
Project Description and Results AdoMetDCs are highly regulated at the transcriptional, at the translational as well as at the posttranslational level. The catalytic activity of mammalian AdoMetDC, e.g., is stimulated by putrescine which is thought to be a regulatory mechanism to relate putrescine abundance with the synthesis of higher polyamines in order to avoid unbalanced consumption of S-adenosylmethionine. AdoMetDCs from the nematodes O. volvulus and C. elegans resemble to a large extent the biochemical and biophysical properties of their mammalian counterparts. However, while the human AdoMetDC exhibits a high basic activity even in the absence of the activator putrescine, both nematode enzymes are nearly inactive under these conditions (Figure 1). We conclude that they highly depend on an activator to achieve significant catalytic activity. Accordingly, activation of the C. elegans and O. volvulus AdoMetDC by putrescine is much more pronounced (maximal 350- and 63-fold, respectively) than of the human enzyme (maximal 3-fold). Moreover, the human AdoMetDC responses exclusively to putrescine. In contrast to that, the enzymatic activity of the nematode AdoMetDC increases also in the presence of a number of other polyamines including the naturally occuring spermidine and spermine (Figure 1). The recently resolved crystal structure of the human AdoMetDC revealed the topology of the putrescinebinding site responsible for the putrescine stimulation. Interestingly, the amino acid residues that were found to be involved in putrescine binding are well conserved in the nematode enzymes despite their lower specificity for stimulator molecules. Mutagenic studies indicate that at least three of these residues most likely also contribute to activator binding of C. elegans AdoMetDC. Furthermore, the C. elegans AdoMetDC mutant Glu 194Gln has a 100-fold enhanced basic activity in the absence of any stimulator, suggesting that this mutant protein mimics the conformational change naturally induced by activator molecules. Figure 2: Synthetic polyamine analogue BW-1 evaluated for inhibitory activity against O. volvulus, C.elegans and human AdoMetDC in vitro. Usually, AdoMetDCs are translated as pro-proteins, that are subsequently cleaved by an autocatalytic process resulting in a heteroterameric enzyme complex. Both recombinantly expressed nematode AdoMetDC are correctly posttranslationally processed in Escherichia coli resulting in the formation of a small β- and a large α-subunit. Mutations of amino acid residues that are part of the putative activator binding site affect this cleavage step, indicative for a putrescine responsiveness of this process like it has been reported also for mammalian AdoMetDC. 37 Parasitology Section As shown above, at least two features distinguish nematode AdoMetDCs from their mammalian counterparts. First, the unstimulated enzyme exhibits a very low specific activity. Second, its polyamine binding pocket has a lower specificity for activator binding. To examine if these differences might be exploitable to specifically inhibit nematode AdoMetDC activity, we looked for polyamine analogues that bind to the pocket but do not stimulate the enzyme. The effects of several new polyamine analogues were tested on the O. volvulus, C. elegans and human AdoMetDC activity. Tetramines with a 3-7-3 backbone and terminal bulky ring systems like BW-1 (Figure 2) do not stimulate nematode AdoMetDC. On the contrary, they reduce the basic activity of the unstimulated AdoMetDCs and, in addition, have an inhibitory effect on the maximal stimulated enzymes (Table 1). At this, both nematode enzymes are generally more sensitive than the human AdoMetDC. Future studies will elucidate whether the found unspecificity of nematode AdoMetDCs towards stimulator molecules may be exploitable for the development of novel chemotherapeutic agents (polyamine analogues) against nematodes. O. volvulus C. elegans H. sapiens Addition Specific activity 2 mM [nmol min-1 mg-1] Putrescine 79 ± 3 240 ± 24 410 ± 34 Putrescine + BW-1 0.3 ± 0.1 0.5 ± 0.4 63 ± 6 Table 1: Inhibition of putrescine-stimulated O. volvulus, C. elegans and human AdoMetDC by the polyamine analogue BW-1. Specific activities were determined under standard assay conditions in the presence of 2 mM putrescine with or without the addition of 2 mM of the polyamine analogue. Results are given as the mean (± SD) of at least three independent duplicate determinations. Selected Publications • Ndjonka D et al. (2003) Biol Chem 384: 1195-201 • Ndjonka D et al. (2003) Biol Chem 384: 83-91 Funding • Deutsche Forschungsgemeinschaft • Deutscher Akademischer Austauschdienst Cooperating Partners • P. Woster, Wayne State University, Detroit, USA • A. Da’dara, Harvard School of Public Health, Boston, USA Investigators • Kai Lüersen • Dieudonné Ndjonka • Rolf D. Walter
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