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The spermidine synthase of the malaria parasite Plasmodium falciparum: Molecular and biochemical characterisation of the polyamine synthesis enzyme
Abstract
The gene encoding spermidine synthase was cloned from the human malaria parasite Plasmodium falciparum. Northern and Western blot analyses revealed a stage specific expression during the erythrocytic schizogony with the maximal amount of transcript and protein in mature trophozoites. Immunofluorescence assays (IFAs) suggest a cytoplasmatic localisation of the spermidine synthase in P. falciparum. The spermidine synthase polypeptide of 321 amino acids has a molecular mass of 36.6kDa and contains an N-terminal extension of unknown function that, similarly, is also found in certain plants but not in animal or bacterial orthologues. Omitting the first 29 amino acids, a truncated form of P. falciparum spermidine synthase has been recombinantly expressed in Escherichia coli. The enzyme catalyses the transfer of an aminopropyl group from decarboxylated S-adenosylmethionine (dcAdoMet) onto putrescine with Km values of 35 and 52microM, respectively. In contrast to mammalian spermidine synthases, spermidine can replace to some extent putrescine as the aminopropyl acceptor. Hence, P. falciparum spermidine synthase has the capacity to catalyse the formation of spermine that is found in small amounts in the erythrocytic stages of the parasite. Among the spermidine synthase inhibitors tested against P. falciparum spermidine synthase, trans-4-methylcyclohexylamine (4MCHA) was found to be most potent with a Ki value of 0.18microM. In contrast to the situation in mammals, where inhibition of spermidine synthase has no or only little effect on cell proliferation, 4MCHA was an efficient inhibitor of P. falciparum cell growth in vitro with an IC50 of 35microM, indicating that P. falciparum spermidine synthase represents a putative drug target.
Supplementary resource (1)
Nucleotide Sequence
January 2000
... In P. falciparum the polyamine pathway is less complex than in higher eukaryotes, with several features that might be exploited for drug development [9,10]). Relevant to the present work is that P. falciparum lacks a specific SpmS and that its spermidine synthase (PfSpdS) can also catalyze the formation of spermine from spermidine to a small extent [11]. Several inhibitors of mammalian SpdS that also inhibit PfSpdS have been identified during the last decades. ...
... additional aminopropyl cavity extending beyond the distal nitrogen of putrescine, presumably accounting for the reported ability of PfSpdS to convert spermidine to spermine [11]. The dcA-doMet site is subdivided into a small central cavity accommodating the donor aminopropyl group, and a larger cavity accommodating the MTA portion of the dcAdoMet substrate. ...
... The structures of compounds discussed in this work are shown in Fig 2, and their inhibitory properties are given in Table 1. AdoDATO (S-adenosyl-3-thio-1,8-diaminooctane, [21]) and 4MCHA (trans-4-methylcyclohexylamine) are among the most potent PfSpdS inhibitors discovered yet [11,18,20]. AdoDATO is a multi-substrate analog that occupies both the dcAdoMet and putrescine substrate binding sites of PfSpdS, whereas 4MCHA occupies only the putrescine site, and only in the presence of dcAdoMet [18). ...
The aminopropyltransferase spermidine synthase (SpdS) is a promising drug target in cancer and in protozoan diseases including malaria. Plasmodium falciparum SpdS (PfSpdS) transfers the aminopropyl group of decarboxylated S-adenosylmethionine (dcAdoMet) to putrescine or to spermidine to form spermidine or spermine, respectively. In an effort to understand why efficient inhibitors of PfSpdS have been elusive, the present study uses enzyme activity assays and isothermal titration calorimetry with verified or predicted inhibitors of PfSpdS to analyze the relationship between binding affinity as assessed by KD and inhibitory activity as assessed by IC50. The results show that some predicted inhibitors bind to the enzyme with high affinity but are poor inhibitors. Binding studies with PfSpdS substrates and products strongly support an ordered sequential mechanism in which the aminopropyl donor (dcAdoMet) site must be occupied before the aminopropyl acceptor (putrescine) site can be occupied. Analysis of the results also shows that the ordered sequential mechanism adequately accounts for the complex relationship between IC50 and KD and may explain the limited success of previous efforts at structure-based inhibitor design for PfSpdS. Based on PfSpdS active-site occupancy, we suggest a classification of ligands that can help to predict the KD?IC50 relations in future design of new inhibitors. The present findings may be relevant for other drug targets that follow an ordered sequential mechanism.
... CQ resistance mechanisms were identified as pfmdr1 gene overexpression [54,56]. Moreover, mutation of an D 86 residue to Y 86 in Pgh1 has been reported to confer both CQ and mefloquine resistance [44,56]. The P. falciparum multidrug resistant protein (PfMDR1) is yet another transport protein predicted to occur on the parasite food vacuole membrane and is associated with CQ and quinine resistance [54]. ...
... catalyzes this reaction (Fig 1.7). A enzyme of this nature has not been identified in P. falciparum, however, it was found that the PfSpdSyn has extended substrate accommodative properties, allowing additional formation of low levels of spermine from spermidine [86]. ...
... DFMO treated P. falciparum have marginal increased spermine levels, and this might be a compensatory mechanism due to the rapid decline in available putrescine, resulting in dcSAM accumulation [80,82]. A spermine aminopropyltansferase (SAPT, SpmSyn) has not yet been identified in P. falciparum, and the formation of the spermine metabolite was therefore attributed to PfSpdSyn, which has been shown to have extended substrate binding capability [86]. A substantial increase in dcSAM, predicted in DFMO-treated cultures, is thought to drive this reaction and lead to accumulation of spermine. ...
... SpdSyn has also been shown to be able to catalyse the addition of an extra propylamine to spermidine, resulting in spermine. 166 The polyamines are essential for eukaryotic cell growth and differentiation. [167][168][169][170][171][172] In this, their binding to and stabilisation of the negatively charged DNA is believed to be important. ...
... In 2005, Haider et al. published a study in which some simple amines were tested with regard to their in vitro inhibition of SpdSyn as well as Pf growth in cell cultures. 166 The most potent SpdSyn inhibitor was 4-methyl-cyclohexylamine (4MCHA), with an enzyme inhibition IC 50 of 1.4 μM and a growth inhibition EC 50 of 34 μM. ...
... NMR binding experiments were used to confirm active-site binding as we did not have access to an enzymatic assay to measure SpdSyn inhibition. The known binder MTA (IC 50 = 159 ± 27 μM) 166 was used as both positive control and competitor. For this purpose, Pf SpdSyn was expressed in E. coli and purified according to existing protocols. ...
... Moreover, a decrease was observed in spermidine and spermine concentrations. P. falciparum lacks a spermine synthase activity but PfSpdSyn has been shown to uniquely metabolize its product, spermidine, to additionally provide spermine (10% of its activity, [12]). The observation of a decrease in spermine levels after PfSpdSyn inhibition with cyclohexylamine therefore confirms this secondary activity of the protein. ...
... A number of compounds have been shown to inhibit spermidine synthase activity in P. falciparum [12,16]. Some of these drugs include dicyclohexylamine, agmatine, 1,7-diaminoheptane, 4 MCHA, APE, (S, R)-dcAdoMet, 1-aminoxy-3-aminopropane (APA) and cyclohexylamine. ...
... Spermidine is synthesized from putrescine and decarboxylated S-adenosylmethionine (dcAdoMet) through the aminopropyltransferase action of spermidine synthase (SpdSyn) [11]. In P. falciparum, this protein has the additional and unique function of being responsible for the low level production of spermine [12,13]. The relative paucity of polyamine studies focused on PfSpdSyn may belie the importance and essential nature of this enzyme, reflected by the need for spermidine in the synthesis of hypusine, eukaryotic initiation factor 5A and its involvement in P. falciparum DNA polymerase and topoisomerase I and II [14]. ...
Plasmodium falciparum, the causative agent of severe human malaria, has evolved to become resistant to previously successful antimalarial chemotherapies, most notably chloroquine and the antifolates. The prevalence of resistant strains has necessitated the discovery and development of new chemical entities with novel modes-of-action. Although much effort has been invested in the creation of analogues based on existing drugs and the screening of chemical and natural compound libraries, a crucial shortcoming in current Plasmodial drug discovery efforts remains the lack of an extensive set of novel, validated drug targets. A requirement of these targets (or the pathways in which they function) is that they prove essential for parasite survival. The polyamine biosynthetic pathway, responsible for the metabolism of highly abundant amines crucial for parasite growth, proliferation and differentiation, is currently under investigation as an antimalarial target. Chemotherapeutic strategies targeting this pathway have been successfully utilized for the treatment of Trypanosomes causing West African sleeping sickness. In order to further evaluate polyamine depletion as possible antimalarial intervention, the consequences of inhibiting P. falciparum spermidine synthase (PfSpdSyn) were examined on a morphological, transcriptomic, proteomic and metabolic level.
Morphological analysis of P. falciparum 3D7 following application of the PfSpdSyn inhibitor cyclohexylamine confirmed that parasite development was completely arrested at the early trophozoite stage. This is in contrast to untreated parasites which progressed to late trophozoites at comparable time points. Global gene expression analyses confirmed a transcriptional arrest in the parasite. Several of the differentially expressed genes mapped to the polyamine biosynthetic and associated metabolic pathways. Differential expression of corresponding parasite proteins involved in polyamine biosynthesis was also observed. Most notably, uridine phosphorylase, adenosine deaminase, lysine decarboxylase (LDC) and S-adenosylmethionine synthetase were differentially expressed at the transcript and/or protein level. Several genes in associated metabolic pathways (purine metabolism and various methyltransferases) were also affected. The specific nature of the perturbation was additionally reflected by changes in polyamine metabolite levels.
This study details the malaria parasite's response to PfSpdSyn inhibition on the transcriptomic, proteomic and metabolic levels. The results corroborate and significantly expand previous functional genomics studies relating to polyamine depletion in this parasite. Moreover, they confirm the role of transcriptional regulation in P. falciparum, particularly in this pathway. The findings promote this essential pathway as a target for antimalarial chemotherapeutic intervention strategies.
... We further used the 1,2-DAB/ -ME-APT assay, to determine the APT activity and kinetic properties the P. falciparum spermidine synthase, PfSPDS, using a recombination MBP-PfSPDS enzyme (Fig. 5). The enzyme was previously examined for its catalytic activity using 14 Cputrescine 24 , and shares high sequence similarity with APT enzymes from E. coli, S. cerevisiae, and H. sapiens, especially in the catalytic site with the key residues aspartate 127, glutamate 147 and aspartate 196 in PfSPDS all found to be conserved among other well-known APT enzymes ( Fig. 5A and S4). Using the DAB-APT assay, PfSPDS was found to catalyze the conversion of putrescine to spermidine as determined by the time-dependent increase in fluorescence following incubation with DAB (Fig. 5B). ...
... Two clones from each were selected and tested for expression of MBP-tagged Spe3, Spe4, and PfSPDS. Briefly, each clone was inoculated into 5 mL of Luria broth 24 containing ampicillin (50 g/ml) and allowed to grow overnight at 37°C incubator shaker (200 rpm). The following day, secondary cultures were initiated using the primary cultures and grown to 0.6 OD600. ...
Polyamines are polycationic molecules that are crucial in a wide array of cellular functions. Their biosynthesis is mediated by aminopropyl transferases (APTs), promising targets in antimicrobial, antineoplastic and antineurodegenerative therapies. A major limitation, however, is the lack of high-throughput assays to measure their activity. We developed the first fluorescence-based assay, DAB-APT, for measurement of APT activity using 1,2-diacetyl benzene, which forms fluorescent conjugates with putrescine, spermidine and spermine with fluorescence intensity increasing with increasing carbon chain length. The assay has been validated using APT enzymes from S. cerevisiae and P. falciparum and is suitable for high-throughput screening of large chemical libraries. Given the importance of APTs in infectious diseases, cancer and neurobiology, our DAB-APT assay has broad applications, holding promise for advancing research and drug discovery efforts.
... The proteins were expressed and purified as described previously with slight modifications [18]. The protein sequences of PfSpdsyn with a 29-residue N-terminal deletion [19] and PfEGF1-α were obtained from Plas-moDB database. The genes were cloned into the pET28a vector, transfected into Escherichia coli BL21, and cultured in LB medium. ...
... As shown above, PfSpdsyn was identified as a potential antimalarial target of Cel. We expressed and purified the recombinant PfSpdsyn protein in vitro and performed a series of experiments to validate its interaction with Cel [19]. The fluorescence labeling experiments showed that Cel-P binds the P. falciparum protein in a dose-dependent manner and can be specifically competed away by excess Cel (Fig. 4A-B). ...
Background
Malaria remains a global health burden, and the emergence and increasing spread of drug resistance to current antimalarials poses a major challenge to malaria control. There is an urgent need to find new drugs or strategies to alleviate this predicament. Celastrol (Cel) is an extensively studied natural bioactive compound that has shown potentially promising antimalarial activity, but its antimalarial mechanism remains largely elusive.
Methods
We first established the Plasmodium berghei ANKA-infected C57BL/6 mouse model and systematically evaluated the antimalarial effects of Cel in conjunction with in vitro culture of Plasmodium falciparum . The potential antimalarial targets of Cel were then identified using a Cel activity probe based on the activity-based protein profiling (ABPP) technology. Subsequently, the antimalarial mechanism was analyzed by integrating with proteomics and transcriptomics. The binding of Cel to the identified key target proteins was verified by a series of biochemical experiments and functional assays.
Results
The results of the pharmacodynamic assay showed that Cel has favorable antimalarial activity both in vivo and in vitro. The ABPP-based target profiling showed that Cel can bind to a number of proteins in the parasite. Among the 31 identified potential target proteins of Cel, Pf Spdsyn and Pf EGF1-α were verified to be two critical target proteins, suggesting the role of Cel in interfering with the de novo synthesis of spermidine and proteins of the parasite, thus exerting its antimalarial effects.
Conclusions
In conclusion, this study reports for the first time the potential antimalarial targets and mechanism of action of Cel using the ABPP strategy. Our work not only support the expansion of Cel as a potential antimalarial agent or adjuvant, but also establishes the necessary theoretical basis for the development of potential antimalarial drugs with pentacyclic triterpenoid structures, as represented by Cel.
... There are two types of enzymatic mechanisms for SpdS: ping-pong and sequential. SpdS in Glycine max (soybean) and E. coli follow a ping-pong mechanism [19,20], whereas SpdS in Thermotoga maritima, Plasmodium falciparum, Rattus rattus (rat), and Homo sapiens utilize a sequential mechanism [1,[21][22][23]. ...
... Although the molecular mechanism underlying SpdS function is well-established, its structure-based evolutionary relationships remain to be fully understood; moreover, very few structural studies have been conducted on SpdS from fungal species. Here, we found that the first structure of fungal KlSpdS, which was determined at 1.9 Å resolution, exhibited highly similar to that of HsSpdS, suggesting that KlSpdS is phylogenetically closer to HsSpdS than SpdS from E. coli and could therefore utilize a sequential mechanism rather than a ping-pong mechanism [19][20][21][22][23]. In KlSpdS, the carboxylate group of Asp167 plays a major role in substrate deprotonation along with the aid of the backbone carbonyl of Ser168 and hydroxyl groups of conserved residues Tyr73 and Tyr235 [1,29]. ...
Spermidine is a polyamine molecule that performs various cellular functions, such as DNA and RNA stabilization, autophagy modulation, and eIF5A formation, and is generated from putrescine by aminopropyltransferase spermidine synthase (SpdS). During synthesis, the aminopropyl moiety is donated from decarboxylated S-adenosylmethionine to form putrescine, with 5′-deoxy-5′-methylthioadenosine being produced as a byproduct. Although the molecular mechanism of SpdS function has been well-established, its structure-based evolutionary relationships remain to be fully understood. Moreover, only a few structural studies have been conducted on SpdS from fungal species. Here, we determined the crystal structure of an apo-form of SpdS from Kluyveromyces lactis (KlSpdS) at 1.9 Å resolution. Structural comparison with its homologs revealed a conformational change in the α6 helix linked to the gate-keeping loop, with approximately 40° outward rotation. This change caused the catalytic residue Asp170 to move outward, possibly due to the absence of a ligand in the active site. These findings improve our understanding of the structural diversity of SpdS and provide a missing link that expands our knowledge of the structural features of SpdS in fungal species.
... This again confirms the importance of the de novo biosythesis of Spermidine and possibly also Spermine by SpdS in the progression of the malaria parasite life cycle. Earlier studies showed a contradictory result on PfSpdS inhibition rescue experiment by exogenous supply of polyamines where rescue was observed against di-cyclohexylamine which is a Putrescine analog, however no rescue was found against 5-Amino-1-Pentene (APE) or Trans-4-Methylcyclohexylamine (4 MCHA) inhibitors (Kaiser et al., 2001;Haider et al., 2005). Cellular localization of proteins is an important characteristic which is useful in determining the functionality of the protein, help in the identification of molecular interaction and play significant role in defining the cellular physiological processes (Huh et al., 2003). ...
... A study on cancer cells reported cytoplasmic localization of Spermine synthase in breast cancer and neuroblastoma cell lines (Dolce et al., 2019). In Plasmodium falciparum, a study suggested a cytoplasmic localization of SpdS based on Immunofluorescence Assay (IFA) with anti-SpdS rabbit serum and nuclear staining, without any co-localization analysis to confirm the cytoplasmic localization (Haider et al., 2005). Herein, the C-terminal tagging of PySpdS with eGFP enabled analysing its precise temporal and spatial expression profile in blood and mosquito stages. ...
Positively-charged polyamines are essential molecules for the replication of eukaryotic cells and are particularly important for the rapid proliferation of parasitic protozoa and cancer cells. Unlike in Trypanosoma brucei, the inhibition of the synthesis of intermediate polyamine putrescine caused only partial defect in malaria parasite blood-stage growth. In contrast, reducing the intracellular concentrations of Spermidine and Spermine by polyamine analogs caused significant defects in blood-stage growth in Plasmodium yoelii and P. falciparum. However, little is known about the synthesizing enzyme of Spermidine and Spermine in the malaria parasite. Herein, malaria parasite conserved Spermidine Synthase (SpdS) gene was targeted for deletion/complementation analyses by knockout/knock-in constructs in P. yoelii. SpdS was found to be essential for blood-stage growth. Live fluorescent imaging in blood-stages and sporozoites confirmed a specific mitochondrial localization, which is not known for any polyamine-synthesizing enzyme so far. This study identifies SpdS as an excellent drug targeting candidate against the malaria parasite, which is localized to the parasite mitochondrion.
... . ........ 20 (Casero and Woster, 2001;Haider et al., 2005;Molitor et al., 2004;Müller et al., 2001;Schramm et al., 1996) (Birkholtz et al., 2003;Wells et al., 2006) (Rosenberg et al., 1996) (Birkholtz, 2002;Roux, 2006 1967. Unfortunately, this statement has proven to be overly optimistic, especially in view of the failure of the 1950s mosquito eradication campaign in third world countries by the World Health Organization (WHO). ...
... This bifunctional protein decarboxylates both ornithine and S-adenosylmethionine to form putrescine and dcAdoMet, from which spermidine is formed by spermidine synthase (SpdSyn). It has been suggested that the low levels of spermine present in the parasite is due to the fact that this enzyme can also transfer an aminopropyl moiety to spermidine to form spermine (Burger et al., 2007;Haider et al., 2005). The 5'-methylthioadenosine (MTA) that is also formed by this reaction enters the methionine recycling pathway (MR) . ...
... Die beiden für sich funktionellen Domänen sind über eine "Scharnierregion" (hinge) miteinander verbunden (Müller et al., 2000). Weiterhin besitzt P. falciparum keine SPMS (Haider et al., 2005) (Takusagawa et al., 1996;Berg et al., 2003). ...
... Gyrationsradius Rg, mit dessen Hilfe sich Aussagen über Form und Größe des Partikels treffen lassen. A2 ist der zweite Virialkoeffizient der Allgemeinen Gasgleichung für reale Gase und beschreibt die zwischen realen Teilchen stattfindende Interaktion (Zimm, 1948a;Zimm, 1948b;Santos und Castanho, 1996 (Haider et al., 2005). Dieses bestand aus dem um die ersten 87 bp verkürzten ORF der plasmodialen Spermidin-Synthase ...
Malaria ist mit bis zu zwei Millionen Todesfällen jährlich und 300 Millionen erkrankten Menschen die bedeutendste Tropenkrankheit. Ungefähr zwei Drittel aller Erkrankten leben in Afrika. Dabei werden über die Hälfte aller Infektionen durch den Erreger Plasmodium falciparum verursacht, der zunehmend Resistenzen gegen etablierte Chemotherapeutika entwickelt. Dies macht es dringend erforderlich, neue Angriffspunkte für die Entwicklung wirksamer Chemotherapeutika zu identifizieren. Wie sich auch bei Erregern anderer parasitärer Erkrankungen zeigt, stellt der Polyamin-Stoffwechsel solch einen Angriffspunkt dar. Polyamine spielen eine essentielle Rolle bei Proliferation und Differenzierung und sind ubiquitär verbreitet. Da sich der Biosyntheseweg der Polyamine bei P. falciparum in wichtigen Punkten vom menschlichen Syntheseweg unterscheidet, lassen sich Substanzen entwickeln, die ohne toxische Wirkung für den Menschen mit hoher Spezifität plasmodiale Enzyme inhibieren. Die Biosynthese des S-Adenosylmethionins wird durch die S-Adenosylmethionin-Synthetase katalysiert. Sie setzt Methionin und ATP zu S-Adenosylmethionin, Pyrophosphat und Phosphat um. S-Adenosylmethionin fließt in seiner decarboxylierten Form in den Polyamin-Stoffwechsel und dient dort als Aminopropylgruppendonor. Von großer Bedeutung ist die Rolle des S-Adenosylmethionins als wichtigster Methylgruppendonor vieler Methylierungsreaktionen und als Zwischenstufe im Transsulfurierungsweg zu Cystein. Diese zentrale Position der S-Adenosylmethionin-Synthetase macht sie als mögliches Ziel für die Entwicklung wirksamer Chemotherapeutika interessant. Im Rahmen dieser Diplomarbeit wurde die plasmodiale S-Adenosylmethionin-Synthetase molekularbiologisch und biochemisch charakterisiert. Die kinetischen Daten des Enzyms liegen im Rahmen der Werte, die für S-Adenosylmethionin-Synthetasen anderer Organismen beschrieben wurden. Das plasmodiale Enzym ist im Vergleich zu anderen S-Adenosylmethionin-Synthetasen jedoch ein Monomer. Durch Mutationsanalyse konnte ein für das plasmodiale Enzym einzigartiges Cystein bestimmt werden, das für die Aktivität der PfSAMS von großer Bedeutung ist. Die Lokalisierung der plasmodialen S-Adenosylmethionin-Synthetase in Blutstadien von P. falciparum ergab eine cytosolische Verteilung des Enzyms innerhalb des Parasiten. Um die Bedeutung der PfSAMS für P. falciparum genauer zu untersuchen, wurden Transfektionen mit einem reprimierbaren Überexpressionskonstrukt des PfSAMS-Gens (pT-Pfsams) durchgeführt. Es konnte gezeigt werden, dass das verwendete Überexpressionssystem im Gegensatz zu den Angaben des publizierten Systems durch AHT nicht reprimiert, sondern induziert wird. Zudem wurden mehrere transgene Zelllinien von P. falciparum erzielt und die episomale Etablierung des Konstrukts pT-Pfsams nachgewiesen. Durch Inhibierung des Wachstums von P. falciparum mit Seleno-L-Methionin, einem klassischen SAMS-spezifischen Inhibitor, konnte gezeigt werden, dass dieses Enzym für den Parasiten essentiell ist und die Hemmung der plasmodialen S-Adenosylmethionin-Synthetase einen antiproliferativen Effekt hat. Die bedeutend bessere Inhibierung der Proliferation durch CGP 40215A beruht jedoch weniger auf der spezifischen Hemmung der PfSAMS. Hier muss davon ausgegangen werden, dass CGP 40215A durch Bindung an AT-reiche DNA-Sequenzen das Wachstum der Parasiten inhibiert.
... provides activated aminopropyl donor, decarboxylated Sadenosylmethionine (dcAdoMet), for the synthesis of spermidine. No spermine synthase activity has been described in plasmodia, but uniquely, SpdSyn is able to produce low levels of spermine (Haider et al., 2005). Most importantly and interestingly, the two rate-limiting decarboxylase activities are found in a bifunctional arrangement in a single protein complex (AdoMetDC/ODC), a property unique to plasmodia (Müller et al., 2000). ...
... Investigations of CHA derivatives 4-MCHA (trans-1,4-methylcyclohexylamine), dicyclohexylamine as well as the ODC inhibitor APA and its derivative, 5-amino-1-pentene (APE), revealed 4- MCHA to exhibit the best in vitro growth IC 50 at 34 µM (Table 1 ). Moreover, 4- MCHA was also the most potent direct inhibitor of SpdSyn activity with a Ki of 0.18 µM (Haider et al., 2005). APA seems to be a more potent inhibitor of ODC activity and only inhibits SpdSyn at an IC 50 of 84 µM. ...
Inhibition of polyamine biosynthesis and/or the perturbation of polyamine functionality have been exploited with success against parasitic diseases such as Trypanosoma infections. However, when the classical polyamine biosynthesis inhibitor, alpha-difluoromethylornithine, is used against the human malaria parasite, Plasmodium falciparum, it results in only a cytostatic growth arrest. Polyamine metabolism in this parasite has unique properties not shared by any other organism. These include the bifunctional arrangement of the catalytic decarboxylases and an apparent absence of the typical polyamine interconversion pathways implying different mechanisms for the regulation of polyamine homeostasis that includes the uptake of exogenous polyamines at least in vitro. These properties make polyamine metabolism an enticing drug target in P. falciparum provided that the physiological and functional consequences of polyamine metabolism perturbation are understood. This review highlights our current understanding of the biological consequences of inhibition of the biosynthetic enzymes in the polyamine pathway in P. falciparum as revealed by several global analytical approaches. Ultimately, the evidence suggests that polyamine metabolism in P. falciparum is a validated drug target worth exploiting.
... The polyamine biosynthetic pathway in P. falciparum consists of two regulatory key enzymes, i.e. ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC). While ODC catalyzes the decarboxylation of ornithine to putrescine, AdoMetDC regenerates decarboxylated S-adenosylmethionine which is the aminopropyl moiety donor for the biosynthesis of spermidine (Haider et al. 2005). In P. falciparum, spermidine synthase can also catalyze the reaction of spermidine to spermine (Haider et al. 2005). ...
... While ODC catalyzes the decarboxylation of ornithine to putrescine, AdoMetDC regenerates decarboxylated S-adenosylmethionine which is the aminopropyl moiety donor for the biosynthesis of spermidine (Haider et al. 2005). In P. falciparum, spermidine synthase can also catalyze the reaction of spermidine to spermine (Haider et al. 2005). This result strongly supported previous findings that no separate spermine synthase locus has been isolated from the parasite. ...
An important issue facing global health today is the need for new, effective and affordable drugs against malaria, particularly in resource-poor countries. Moreover, the currently available antimalarials are limited by factors ranging from parasite resistance to safety, compliance, cost and the current lack of innovations in medicinal chemistry. Depletion of polyamines in the intraerythrocytic phase of P. falciparum is a promising strategy for the development of new antimalarials since intracellular levels of putrescine, spermidine and spermine are increased during cell proliferation. S-adenosyl-methionine-decarboxylase (AdoMETDC) is a key enzyme in the biosynthesis of spermidine. The AdoMETDC inhibitor CGP 48664A, known as SAM486A, inhibited the separately expressed plasmodial AdoMETDC domain with a Kmi
of 3 μM resulting in depletion of spermidine. Spermidine is an important precursor in the biosynthesis of hypusine. This prompted us to investigate a downstream effect on hypusine biosynthesis after inhibition of AdoMETDC. Extracts from P. falciparum in vitro cultures that were treated with 10 μM SAM 486A showed suppression of eukaryotic initiation factor 5A (eIF-5A) in comparison to the untreated control in two-dimensional gel electrophoresis. Depletion of eIF-5A was also observed in Western blot analysis with crude protein extracts from the parasite after treatment with 10 μM SAM486A. A determination of the intracellular polyamine levels revealed an approximately 27% reduction of spemidine and a 75% decrease of spermine while putrescine levels increased to 36%. These data suggest that inhibition of AdoMetDc provides a novel strategy for eIF-5A suppression and the design of new antimalarials.
... Plasmodium has a specialized bifunctional enzyme to produce dcAdoMet and putrescine, ODC-AdoMetDC (Fig. 6). Yet, presence of an Arginine decarboxylase and Agmatinase could not be shown (8,(22)(23)(24). ...
A frequent side effect of chemotherapy against malaria parasite blood infections is a dramatic induction of the sexual blood stages, thereby enhancing the risk of future malaria transmissions. The polyamine biosynthesis pathway has been suggested as a candidate target for transmission-blocking anti-malarial drug development. Herein, we describe the role of a bacterial-type amino acid decarboxylase (AAD) in the life cycle of the malaria model parasite Plasmodium yoelii. Hallmarks of AAD include a conserved catalytic lysine residue and high-level homology to arginine/lysine/ornithine decarboxylases of pathogenic bacteria. By targeted gene deletion, we show that AAD plays an essential role in the exflagellation of microgametes, resulting in complete absence of sporozoites in the mosquito vector. These data highlight the central role of the biosysthesis of polyamines in the final steps of male gamete sexual development of the malaria parasite and, hence, onward transmission to mosquitoes.
... Catabolic enzymes such as SAT1, APAO, and SMOX are absent in the parasite. Peculiarities of the core biosynthetic route leading to the triamine Spd are a bifunctional ODC/AdoMetDC [45] and the occurrence of a SpdS which can catalyze the formation of Spm [46]. Spd is an essential substrate for hypusine formation in Plasmodium [47]. ...
The triamine spermidine is a key metabolite of the polyamine pathway. It plays a crucial role in many infectious diseases caused by viral or parasitic infections. Spermidine and its metabolizing enzymes, i.e., spermidine/spermine-N1-acetyltransferase, spermine oxidase, acetyl polyamine oxidase, and deoxyhypusine synthase, fulfill common functions during infection in parasitic protozoa and viruses which are obligate, intracellular parasites. The competition for this important polyamine between the infected host cell and the pathogen determines the severity of infection in disabling human parasites and pathogenic viruses. Here, we review the impact of spermidine and its metabolites in disease development of the most important, pathogenic human viruses such as SARS-CoV-2, HIV, Ebola, and in the human parasites Plasmodium and Trypanosomes. Moreover, state-of-the-art translational approaches to manipulate spermidine metabolism in the host and the pathogen are discussed to accelerate drug development against these threatful, infectious human diseases.
... kDa). 63 Unlikely common targets are the plasmodial pyruvate kinases (MW = 55.6 and 86.6 kDa) 64 and lactate deshydrogenase (MW = 33 kDa). 65 In comparison to probe 2 labeling, probe 10, used as a negative control due to its lack of endoperoxide, showed no protein alkylation in the same conditions and was indistinguishable from the DMSO control ( Figure S2 in the SI). ...
... Few among the first potent and selective inhibitors of SPDS was sadenosyl-1,8-diamino-3-thio-octane (AdoDATO). Another compound, trans-4-methylcyclohexylamine (4MCHA), was also demonstrated to stop the development of P. falciparum (IC 50 of 35 µM) and exhibit plasmodicidal activity [128]. To get an insight into the structure-activity properties of P. falciparum SPDS (pfSPDS), recently, Dufe et al. have determined the structure of the apo-pfSPDS as well as the structures of the complexes with the substrate dcAdoMet and the inhibitors AdoDATO and 4MCHA [129]. ...
The malaria parasite resistance to the existing drugs is a serious problem to currently used anti-malarials and, thus, highlights the urgent need to develop new and effective anti-malarial molecules. This could be achieved either by the identification of the new drugs for the validated targets or by further refining/improving the existing antimalarials; or by combining previously effective agents with new/existing drugs to have synergistic effect that counter parasite resistance; or by identifying novel targets for the malarial chemotherapy. In this review article, a comprehensive collection of some of the novel molecular targets has been enlisted for the antimalarial drugs. The targets which could be deliberated for developing new anti-malarial drugs could be: membrane biosynthesis, mitochondrial system, apicoplasts, parasite transporters, shikimate pathway, hematin crystals, parasite proteases, glycolysis, isoprenoid synthesis, cell cycle control/cycline dependent kinase, redox system, nucleic acid metabolism, methionine cycle and the polyamines, folate metabolism, the helicases, erythrocyte G-protein, and farnesyl transferases. Modern genomic tools approaches such as structural biology and combinatorial chemistry, novel targets could be identified followed by drug development for drug resistant strains provide wide ranges of novel targets in the development of new therapy. The new approaches and targets mentioned in the manuscript provide a basis for the development of new unique strategies for antimalarial therapy with limited off- target effects in the near future.
... Intracellular polyamine levels in P. falciparum are nearly 2-orders of magnitude higher than levels in uninfected erythrocytes suggesting that the parasite requires the biosynthetic enzymes to provide sufficient polyamines for growth (54). Plasmodium species synthesize both putrescine and spermidine de novo, and while spermine synthase is not encoded in the genome 3 , characterization of recombinant P. falciparum SpdSyn showed it also converted spermidine to spermine, though at a ~10fold lower rate, explaining the presence of spermine in the organism (55). While individual knockouts have not been generated for pathway enzymes in Plasmodium, two genome-wide genetic screens have demonstrated that ODC/AdoMetDC, SpdSyn and DHS are all essential enzymes for survival of P. falciparum based on saturation transposon mutagenesis (56), and for P. berghei infection in mice based on analysis of the ability to recover barcoded knockout mutants (57). ...
Polyamines are polycationic organic amines that are required for all eukaryotic life, exemplified by the polyamine spermidine, which plays an essential role in translation. They also play more specialized roles that differ across species, and their chemical versatility has been fully exploited during the evolution of protozoan pathogens. These eukaryotic pathogens, which cause some of the most globally widespread infectious diseases, have acquired species-specific polyamine-derived metabolites with essential cellular functions and have evolved unique mechanisms that regulate their core polyamine biosynthetic pathways. Many of these parasitic species have lost enzymes and or transporters from the polyamine metabolic pathway that are found in the human host. These pathway differences have prompted drug discovery efforts to target the parasite polyamine pathways, and indeed the only clinically approved drug targeting the polyamine biosynthetic pathway is used to manage human African trypanosomiasis. This review will primarily focus on polyamine metabolism and function in Trypanosoma, Leishmania,and Plasmodium species, which are the causative agents of human African trypanosomiasis (HAT) and Chagas disease, Leishmaniasis, and malaria, respectively. Aspects of polyamine metabolism across a diverse group of protozoan pathogens will also be explored.
... The pH optimum of Synechococcus Spds measured in this study was similar to that of Spds from E. coli (Lee et al. 2013), P. falciparum , rat (Samejima and Yamanoha 1982) and human (Wu et al. 2007) but different from Spds of soybean (Yoon et al. 2000) and Chinese cabbage (Sindhu and Cohen 1984). The Synechococcus Spds K m values for putrescine (111 µM) and dcSAM (20 µM) were in similar range with those from other organisms such as animals, parasite and bacteria (52-158 µM for putrescine, and 10-35 µM for dcSAM) (Dufe et al. 2005;Haider et al. 2005;Lee et al. 2013;Samejima and Yamanoha 1982). Similar to Synechococcus enzyme, the Spds proteins in many organisms, including human Spds are highly specific for putrescine (Tabor and Tabor 1984;Sindhu and Cohen 1984), whereas the promiscuous T. maritima Spds allows the accommodation of longer chain length diamine substrates (Korolev et al. 2002). ...
Spermidine synthase (Spds) catalyzes the formation of spermidine by transferring the aminopropyl group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine. The Synechococcus spds gene encoding Spds was expressed in Escherichia coli. The purified recombinant enzyme had a molecular mass of 33 kDa and showed optimal activity at pH 7.5, 37 °C. The enzyme had higher affinity for dcSAM (Km, 20 µM) than for putrescine (Km, 111 µM) and was highly specific towards the diamine putrescine with no activity observed towards longer chain diamines. The three-dimensional structural model for Synechococcus Spds revealed that most of the ligand binding residues in Spds from Synechococcus sp. PCC 7942 are identical to those of human and parasite Spds. Based on the model, the highly conserved acidic residues, Asp89, Asp159 and Asp162, are involved in the binding of substrates putrescine and dcSAM and Pro166 seems to confer substrate specificity towards putrescine.
... S-adenosylmethioninamine is then converted to spermidine by spermidine synthase (EC 2.5.1.16). No spermine synthase activity has been found, but low levels of spermine were produced by spermidine synthase (Haider 2005). ...
The last overview of the biochemistry of the malaria parasite was provided by Irwin Sherman's monograph, which, adhering to his good tradition, summarized this important field for the community. This chapter deals with metabolism as a set of chemical reactions organized in cellular metabolic pathways. The description of the various pathways relies heavily on the schemes of the website on the functional genomics of Plasmodium falciparum, Malaria Parasite Metabolic Pathways (MPMP). The MPMP project organizes data from the genome project of the parasite into assemblies with shared physiological and biological functions. The chapter deals with the metabolic pathways of carbohydrates, lipids, nucleotides, amino acids, redox metabolism, pathways in the mitochondrion, apicoplast, and hemoglobin (Hb) digestion. Phosphoenolpyruvate and glycerone-phosphate produced by glycolysis enter the apicoplast and serve as substrates for the biosynthesis of isoprenoids. A heme detoxification protein has been identified that is shown to enhance heme-to-hemozoin transformation and to be essential for parasite life.
... Two inhibitors, Sadenosyl-1,8-diamino-3-thiooctane (AdoDATO) and trans-4-methylcyclohexylamine (4MCHA) have been shown to bind to the active pocket of P. falciparum SPDS in crystallization studies ( Fig. 9) (Dufe et al., 2007). 4MCHA inhibitory capacity was confirmed in in vitro P. falciparum cultures, although the effect of AdoDATO is unexplored (Haider et al., 2005). Still, the therapeutic value of these polyamine synthesis inhibitors has not been validated in vivo. ...
... The polyamine biosynthetic pathway in P. falciparum depends on PfADA and PfPNP to recycle 59-methylthioadenosine (MTA), a product of polyamine synthesis (Figure 1). Its removal is necessary for the cell to perform polyamine metabolism, since MTA is a strong inhibitor of spermine synthase, spermidine synthase and of ornithine decarboxylase [5,6,7]. Primates encode a specific MTA phosphorylase for this purpose, but this activity is not found in P. falciparum. ...
Plasmodium falciparum causes most of the one million annual deaths from malaria. Drug resistance is widespread and novel agents against new targets are needed to support combination-therapy approaches promoted by the World Health Organization. Plasmodium species are purine auxotrophs. Blocking purine nucleoside phosphorylase (PNP) kills cultured parasites by purine starvation. DADMe-Immucillin-G (BCX4945) is a transition state analogue of human and Plasmodium PNPs, binding with picomolar affinity. Here, we test BCX4945 in Aotus primates, an animal model for Plasmodium falciparum infections. Oral administration of BCX4945 for seven days results in parasite clearance and recrudescence in otherwise lethal infections of P. falciparum in Aotus monkeys. The molecular action of BCX4945 is demonstrated in crystal structures of human and P. falciparum PNPs. Metabolite analysis demonstrates that PNP blockade inhibits purine salvage and polyamine synthesis in the parasites. The efficacy, oral availability, chemical stability, unique mechanism of action and low toxicity of BCX4945 demonstrate potential for combination therapies with this novel antimalarial agent
... In a previous study, we showed that decarboxylated AdoMet levels were inversely proportional to AdoMetDC prozyme levels, suggesting that elevated decarboxylated AdoMet levels lead to reduced prozyme expression (30). SpdSyn from human and Plasmodium has been shown to be feedback inhibited by methylthioadenosine, a reaction product of SpdSyn chemistry (51,52), and both spermidine and methylthioadenosine have been demonstrated by crystallographic studies to bind SpdSyn (53). Thus, a working model that explains the available data suggests that, when T. brucei cells no longer require spermidine for trypanothione biosynthesis, spermidine or methylthioadenosine might inhibit SpdSyn by feedback, leading to buildup of dcAdoMet and to downregulation of prozyme expression. ...
Human African trypanosomiasis (HAT) is a debilitating and fatal vector-borne disease. Polyamine biosynthesis is the target
of one of the key drugs (eflornithine) used for the treatment of late-stage disease, suggesting that the pathway might be
exploited for the identification of additional drug targets. The polyamine spermidine is required in trypanosomatid parasites
for formation of a unique redox cofactor termed trypanothione, which is formed from the conjugation of glutathione to spermidine.
Here we characterize recombinant Trypanosoma brucei glutathione synthetase (TbGS) and show that depletion of TbGS in blood-form parasites using a regulated knockout strategy leads to loss of trypanothione and to cell death as quantified
by fluorescence-activated cell sorter (FACS) analysis. These data suggest that >97% depletion of TbGS is required before trypanothione is depleted and cell growth arrest is observed. Exogenous glutathione was able to partially
compensate for the loss of TbGS, suggesting that parasites are able to transport intact glutathione. Finally, reduced expression of TbGS leads to increased levels of upstream glutathione biosynthetic enzymes and decreased expression of polyamine biosynthetic
enzymes, providing evidence that the cells cross regulate the two branches of the trypanothione biosynthetic pathway to maintain
spermidine and trypanothione homeostasis.
... The parasites could not be significantly rescued by the addition of exogenous spermidine, which was not the case with 3-aminooxy-1- aminopropane inhibition of ODC that could be reversed by addition of putrescine. [17,262728 ...
... Since the terminal amino group of Put must be deprotonated in SpdSy active center before reacting with dcAdoMet, APA may be considered as an analogue of the intermediate (transition) monocationic form of Put. Respectively, APA is a potent competitive inhibitor (K i 2.3 M) of homogeneous bovine brain SpdSy [171] and in the case of SpdSy from P.falciparum APA has IC 50 of 84 M [192]. ...
Mitochondrial dysfunction, either inherited or acquired, is associated with several diseases in humans. Depending on the cell type and location, cells are prone to multiple types of insults that may compromise their proper function. Generally, these insults are overcome by defensive mechanisms but sometimes they lead to sustained damage, requiring the action of scavenging and repair machineries to retain the viability of the cells. As a final measure, severely damaged cells are targeted to a controlled cell death pathway in order not to compromise the well-being of the whole tissue. The polyamines spermine and spermidine are essential cellular constituents, participating in many vital functions such as proliferation and differentiation, immune response and scavenging of reactive oxygen species. Therefore, dysregulation of polyamine metabolism is often associated with different pathological states. Polyamine acetylating enzyme spermidine/spermine-N1-acetyltransferase is induced by inflammation, drugs and by several other environmental insults. Resulting accelerated polyamine acetylation with accompanying polyamine biosynthesis induction i.e. activation of polyamine futile cycle generates excessive amount of hydrogen peroxide, hampers cell energy metabolism and induces mitochondrial dysfunction and biogenesis. Therefore, the drugs inhibiting polyamine metabolism are valuable in protecting mitochondria and cell energy metabolism. Here we review the current literature focusing on the applicability of charge-deficient polyamine analogues as drugs to modulate polyamine metabolism. Alteration of pKa of amino group(s) in a respective analogue is achieved by fluorine substitution of hydrogen atom, hydroxylamine substitution of methylamine or by reducing the numbers of carbon atoms between amine groups to two instead of three or four.
... This bifunctional arrangement of AdoMetDC and ODC is unique to Plasmodium and is postulated to be necessary for the regulation of polyamine production (Williams et al., 2011). Furthermore , in contrast to other species, both spermidine and spermine are synthesized by spermidine synthase (Haider et al., 2005) with no spermine synthase encoded in the P. falciparum genome. Under in vitro conditions, inhibition of this enzyme by compounds such as DFMO results in growth arrest of the intraerythrocytic P. falciparum parasite. ...
Polyamines and the enzymes involved in their biosynthesis are present at high levels in rapidly prolifer-ating cells, including cancer cells and protozoan parasites. Inhibition of polyamine biosynthesis in asexual blood-stage malaria parasites causes cytostatic arrest of parasite development under in vitro conditions, but does not cure infections in vivo. This may be due to replenishment of the parasite's intracellular poly-amine pool via salvage of exogenous polyamines from the host. However, the mechanism(s) of polyamine uptake by the intraerythrocytic parasite are not well understood. In this study, the uptake of the poly-amines, putrescine and spermidine, into Plasmodium falciparum parasites functionally isolated from their host erythrocyte was investigated using radioisotope flux techniques. Both putrescine and spermidine were taken up into isolated parasites via a temperature-dependent process that showed cross-competi-tion between different polyamines. There was also some inhibition of polyamine uptake by basic amino acids. Inhibition of polyamine biosynthesis led to an increase in the total amount of putrescine and sper-midine taken up from the extracellular medium. The uptake of putrescine and spermidine by isolated parasites was independent of extracellular Na + but increased with increasing external pH. Uptake also showed a marked dependence on the parasite's membrane potential, decreasing with membrane depo-larization and increasing with membrane hyperpolarization. The data are consistent with polyamines being taken up into the parasite via an electrogenic uptake process, energised by the parasite's inwardly negative membrane potential.
... There is no evidence of trypanothione synthesis in these apicomplexan parasites. Possibly the most distinctive feature of polyamine biosynthesis in plasmodia is that a single open reading frame encodes both ODC and AdoMetDC in a unique bifunctional protein of ∼330 kDa molecular mass (AdoMetDC/ODC) (Figure 5) [110]. The advantage of this bifunctional AdoMetDC/ODC protein probably is not a metabolic channelling of substrates to optimize downstream synthesis, because AdoMetDC and ODC do not catalyse consecutive reactions in the polyamine biosynthetic pathway. ...
New drugs are urgently needed for the treatment of tropical and subtropical parasitic diseases, such as African sleeping sickness, Chagas' disease, leishmaniasis and malaria. Enzymes in polyamine biosynthesis and thiol metabolism, as well as polyamine transporters, are potential drug targets within these organisms. In the present review, the current knowledge of unique properties of polyamine metabolism in these parasites is outlined. These properties include prozyme regulation of AdoMetDC (S-adenosylmethionine decarboxylase) activity in trypanosomatids, co-expression of ODC (ornithine decarboxylase) and AdoMetDC activities in a single protein in plasmodia, and formation of trypanothione, a unique compound linking polyamine and thiol metabolism in trypanosomatids. Particularly interesting features within polyamine metabolism in these parasites are highlighted for their potential in selective therapeutic strategies.
... The SPDS gene is widely conserved in a variety of organisms and encodes a compact globular protein (Dufe et al., 2007). A number of diverse plants possess unique motifs that are attached to the 59 end of the open reading frame that encodes the conserved enzymatic portion of the protein (Haider et al., 2005; Dufe et al., 2007). The 59 motifs exhibit no conserved structure but are possible regulatory domains for the enzyme. ...
Here, we show that the polyamine spermidine plays a key role as a morphogenetic determinant during spermatid development in the water fern Marsilea vestita. Spermidine levels rise first in sterile jacket cells and then increase dramatically in spermatogenous cells as the spermatids mature. RNA interference and drug treatments were employed to deplete spermidine in the gametophyte at different stages of gametogenesis. Development in spermidine-depleted gametophytes was arrested before the completion of the last round of cell divisions. In spermidine-depleted spermatogenous cells, chromatin failed to condense properly, basal body positioning was altered, and the microtubule ribbon was in disarray. When cyclohexylamine, a spermidine synthase (SPDS) inhibitor, was added at the start of spermatid differentiation, the spermatid nuclei remained round, centrin failed to localize into basal bodies, thus blocking basal body formation, and the microtubule ribbon was completely abolished. In untreated gametophytes, spermidine made in the jacket cells moves into the spermatids, where it is involved in the unmasking of stored SPDS mRNAs, leading to substantial spermidine synthesis in the spermatids. We found that treating spores directly with spermidine or other polyamines was sufficient to unmask a variety of stored mRNAs in gametophytes and arrest development. Differences in patterns of transcript distribution after these treatments suggest that specific transcripts reside in different locations in the dry spore; these differences may be linked to the timing of unmasking and translation for that mRNA during development.
... In the first case, the model was not able to synthesize spermidine. The synthesis of spermidine from putrescine by spermidine synthase was accompanied by the production of 5methylthioadenosine (MTA) (Haider et al, 2005), which was a metabolic dead end in the initial model. Consequently, the spermidine synthesis caused MTA to accumulate, violating the steady-state assumption of the constraint-based approach. ...
Genome-scale metabolic reconstructions can serve as important tools for hypothesis generation and high-throughput data integration. Here, we present a metabolic network reconstruction and flux-balance analysis (FBA) of Plasmodium falciparum, the primary agent of malaria. The compartmentalized metabolic network accounts for 1001 reactions and 616 metabolites. Enzyme-gene associations were established for 366 genes and 75% of all enzymatic reactions. Compared with other microbes, the P. falciparum metabolic network contains a relatively high number of essential genes, suggesting little redundancy of the parasite metabolism. The model was able to reproduce phenotypes of experimental gene knockout and drug inhibition assays with up to 90% accuracy. Moreover, using constraints based on gene-expression data, the model was able to predict the direction of concentration changes for external metabolites with 70% accuracy. Using FBA of the reconstructed network, we identified 40 enzymatic drug targets (i.e. in silico essential genes), with no or very low sequence identity to human proteins. To demonstrate that the model can be used to make clinically relevant predictions, we experimentally tested one of the identified drug targets, nicotinate mononucleotide adenylyltransferase, using a recently discovered small-molecule inhibitor. © 2010 EMBO and Macmillan Publishers Limited All rights reserved.
... Arginase is thus part of one of two alternative biosynthetic routes to putrescine. Polyamine biosynthesis enzymes characterized in the malaria parasite, Plasmodium falciparum, include the bifunctional S-adenosylmethionine decarboxylase ⁄ ornithine decarboxylase [18][19][20][21][22], spermidine synthase [23] and arginase [24]. In P. falciparum, the agmatinase route to putrescine has not been identified, thus making arginase the sole biosynthetic route to putrescine in the malaria parasite [24]. ...
A recent study implicated a role for Plasmodium falciparum arginase in the systemic depletion of arginine levels, which in turn has been associated with human cerebral malaria pathogenesis. Arginase (EC 3.5.3.1) is a multimeric metallo-protein that catalyses the hydrolysis of arginine to ornithine and urea by means of a binuclear spin-coupled Mn(2+) cluster in the active site. A previous report indicated that P. falciparum arginase has a strong dependency between trimer formation, enzyme activity and metal co-ordination. Mutations that abolished Mn(2+) binding also caused dissociation of the trimer; conversely, mutations that abolished trimer formation resulted in inactive monomers. By contrast, the monomers of mammalian (and therefore host) arginase are also active. P. falciparum arginase thus appears to be an obligate trimer and interfering with trimer formation may therefore serve as an alternative route to enzyme inhibition. In the present study, the mechanism of the metal dependency was explored by means of homology modelling and molecular dynamics. When the active site metals are removed, loss of structural integrity is observed. This is reflected by a larger equilibration rmsd for the protein when the active site metal is removed and some loss of secondary structure. Furthermore, modelling revealed the existence of a novel inter-monomer salt-bridge between Glu295 and Arg404, which was shown to be associated with the metal dependency. Mutational studies not only confirmed the importance of this salt-bridge in trimer formation, but also provided evidence for the independence of P. falciparum arginase activity on trimer formation.
Apicomplexan parasite diseases, including malaria (Plasmodium) and theileriosis (Theileria), pose a significant threat to global health and the socioeconomic well-being of low-income countries. Despite recent advances, the common host metabolic proteins essential for these highly auxotrophic pathogens remain elusive. Here, we present a comprehensive investigation integrating a metabolic model of P. falciparum parasites in hepatocytes and a genome-wide CRISPR screen targeting Theileria schizont-infected macrophages. We reveal unifying host metabolic enzymes critical for the intracellular survival of these related hematozoa. We show that pathways such as host purine and heme biosynthesis are essential for both Theileria survival and Plasmodium liver development, while genes involved in glutathione and polyamine biosynthesis are predicted to be essential for Plasmodium only under certain metabolic conditions. Our work highlights the importance of host porphyrins for the viability of liver-stage Plasmodium. Shared parasite vulnerabilities provide a resource for exploring alternative therapeutic approaches to combat these crippling diseases.
Malaria, an infectious disease caused by protozoan parasites from the genus Plasmodium, represents a serious global health threat. The continued emergence of drug resistant strains has severely decreased current antimalarial drug efficacy and led to a perpetual race for drug discovery. Most protozoan parasites, including Plasmodium spp., are unable to synthesize purines de novo and instead rely on an essential purine salvage pathway for acquisition of purines from the infected host. Because purines are essential for Plasmodium growth and survival, the enzymes of the purine salvage pathway represent promising targets for drug discovery. Target-based high-throughput screening (HTS) assays traditionally focus on a single target, which severely limits the screening power of this type of approach. To circumvent this limitation, we have reconstituted the purine salvage pathway from Plasmodium falciparum in an assay combining four drug targets. This assay was developed for HTS and optimized to detect partial inhibition of any of the four enzymes in the pathway. Inhibitors of several enzymes in the pathway were identified in a pilot screen, with several compounds exhibiting effective inhibition when provided in micromolar amounts.
Polyamines are ubiquitous molecules that are involved in a number of important cellular processes. Aberrations in their function or metabolism play a role in diseases such as cancer and parasitic infection. A number of validated drug targets have been identified, including enzymes in the polyamine biosynthetic and catabolic pathways and the S-adenosylmethionine synthetic and salvage pathways. Polyamine Drug Discovery is the first comprehensive volume to cover all aspects of the design and development of potential therapeutics targeting polyamine metabolism. The book details research progress from 1975 to the present date and discusses the design and use of polyamine metabolism inhibitors as therapeutic agents. Various polyamine-containing drugs are described that can be used in chemotherapy, and as treatments for infections including trypanosomiasis, leishmaniasis and malaria. Finally, the roles of polyamine analogues in chemoprevention, polyamine-containing vectors for gene delivery, and the design of polyamine-based epigenetic modulators are detailed. Each chapter addresses a different aspect of polyamine drug discovery and all are written by medicinal and biological chemists with particular expertise in developing agents that modulate polyamine metabolism or function. The book will increase the visibility of polyamine drug discovery among pharmaceutical researchers and provide a valuable reference for everyone working in the field.
Twenty-two compounds belonging to several classes of polyamine analogs have been examined for their ability to inhibit the growth of the human malaria parasite Plasmodium falciparum in vitro and in vivo. Four lead compounds from the thiourea sub-series and one compound from the urea-based analogs were found to be potent inhibitors of both chloroquine-resistant (Dd2) and chloroquine-sensitive (3D7) strains of Plasmodium with IC50 values ranging from 150 to 460 nM. In addition, the compound RHW, N1,N7-bis (3-(cyclohexylmethylamino) propyl) heptane-1,7-diamine tetrabromide was found to inhibit Dd2 with an IC50 of 200 nM. When RHW was administered to P. yoelii-infected mice at 35 mg/kg for 4 days, it significantly reduced parasitemia. RHW was also assayed in combination with the ornithine decarboxylase inhibitor difluoromethylornithine, and the two drugs were found not to have synergistic antimalarial activity. Furthermore, these inhibitors led to decreased cellular spermidine and spermine levels in P. falciparum, suggesting that they exert their antimalarial activities by inhibition of spermidine synthase.
Appendix A. Table S1: the gold standard. This consists of reactions which are biologically proven to be targets of already accepted/tested drugs. Table S2: knocked-out reactions for reactions a–g. Appendix B. Table S3: all predicted essential reactions in the network used in this study. The predicted essential reactions based on our analysis in the network are presented in this table, most of which were identified to be found in the gold standards or have been predicted to be essential to the network in previous study. Figure S1: graph to show number of reactions considered and essentiality of reactions. Figure S2: graph to represent essentiality of reaction.
Visceral leishmaniasis caused by the protozoan Leishmania donovani is the most severe form of leishmaniasis and it is potentially lethal if untreated. Despite the availability of drugs for treating the disease, the current drug regime suffers from drawbacks like antibiotic resistance and toxicity. New drugs have to be discovered in order to overcome these limitations. Our aim is to identify natural compounds from plant sources as putative inhibitors considering the occurrence of structural diversity in plant sources. Spermidine Synthase (SpdS) was chosen as the target enzyme as it plays a vital role in growth, survival and due to its contribution in virulence. Our initial investigation started with a literature survey in identifying natural compounds that showed antileishmanial activity. Subsequently, we identified two monoterpenoid compounds, namely Geraniol and Linalool that was structurally analogous to one of the substrates (putrescine) of SpdS. In the present study homology model of L. donovani SpdS was generated and the binding affinity of the identified compounds was analyzed and also compared with the putrescine through molecular docking and dynamic studies. The pharmacokinetic properties of the identified compounds were validated and the binding efficiency of these ligands over the original substrate has been demonstrated. Based on these studies, Geraniol and Linalool can be considered as lead molecules for future investigations targeting SpdS. This study further emphasizes the choice of natural compounds as a good source of therapeutic agents.
Trotz weltweiter Bemühungen, die Tropenkrankheit Malaria zurückzudrängen, erkranken jährlich bis zu einer halben Milliarde Menschen an Malaria mit der Folge von über einer Million Todesopfern. Da zur Zeit eine wirksame Impfung nicht in Sicht ist und sich Resistenzen gegen gängige Medikamente ausbreiten, werden dringend neue Antimalariamittel benötigt. Um die Suche nach neuen Angriffsorten für Medikamente zu unterstützen, untersucht die vorliegende Arbeit mit einem rechnergestützten Ansatz den Stoffwechsel von Plasmodium falciparum, dem tödlichsten Malaria-Erreger. Basierend auf einem aus dem aktuellen Forschungsstand rekonstruierten metabolischen Netzwerk des Parasiten werden metabolische Flüsse für die einzelnen Stadien des Lebenszyklus von P. falciparum berechnet. Dabei wird ein im Rahmen dieser Arbeit entwickelter Fluss-Bilanz-Analyse-Ansatz verwendet, der ausgehend von in den jeweiligen Entwicklungsstadien gemessenen Genexpressionsprofilen entsprechende Flussverteilungen ableitet. Für das so ermittelte stadienspezifische Flussgeschehen ergibt sich eine gute Übereinstimmung mit bekannten Austauschprozessen von Stoffen zwischen Parasit und infiziertem Erythrozyt. Knockout Simulationen, die mit Hilfe einer ähnlichen Vorhersagemethode durchgeführte werden, decken essentielle metabolische Reaktionen im Netzwerk auf. Fast 90% eines Sets von experimentell bestimmten essentiellen Enzymen wird wiedergefunden, wenn die Annahme getroffen wird, dass Nährstoffe nur begrenzt aus der Wirtszelle aufgenommen werden können. Die als essentiell vorhergesagten Enzyme stellen mögliche Angriffsorte für Medikamente dar. Anhand der Flussverteilungen, die für die einzelnen Entwicklungsstadien berechnet wurden, können diese potenziellen Targets nach Relevanz für Malaria Prophylaxe und Therapie sortiert werden, je nachdem, in welchem Stadium die Enzyme als aktiv vorhergesagt wurden. Dies bietet einen vielversprechenden Startpunkt für die Entwicklung von neuen Antimalariamitteln.
The enzymes of the polyamine-biosynthesis pathway have been proposed to be
promising drug targets in the treatment of malaria. Spermidine synthase (SpdS;
putrescine aminopropyltransferase) catalyzes the transfer of the aminopropyl
moiety from decarboxylated S-adenosylmethionine to putrescine, leading to
the formation of spermidine and 50-methylthioadenosine (MTA). In this work,
X-ray crystallography was used to examine ligand complexes of SpdS from the
malaria parasite Plasmodium falciparum (PfSpdS). Five crystal structures were
determined of PfSpdS in complex with MTA and the substrate putrescine, with
MTA and spermidine, which was obtained as a result of the enzymatic reaction
taking place within the crystals, with dcAdoMet and the inhibitor 4-methylaniline,
with MTA and 4-aminomethylaniline, and with a compound predicted in
earlier in silico screening to bind to the active site of the enzyme, benzimidazol-
(2-yl)pentan-1-amine (BIPA). In contrast to the other inhibitors tested, the
complex with BIPA was obtained without any ligand bound to the dcAdoMetbinding
site of the enzyme. The complexes with the aniline compounds and
BIPA revealed a new mode of ligand binding to PfSpdS. The observed binding
mode of the ligands, and the interplay between the two substrate-binding sites
and the flexible gatekeeper loop, can be used in the design of new approaches in
the search for new inhibitors of SpdS.
Background:
Plasmodium falciparum is the most pathogenic of the human malaria parasite species and a major cause of death in Africa. It's resistance to most of the current drugs accentuates the pressing need for new chemotherapies. Polyamine metabolism of the parasite is distinct from the human pathway making it an attractive target for chemotherapeutic development. Plasmodium falciparum spermidine synthase (PfSpdS) catalyzes the synthesis of spermidine and spermine. It is a major polyamine flux-determining enzyme and spermidine is a prerequisite for the post-translational activation of P. falciparum eukaryotic translation initiation factor 5A (elF5A). The most potent inhibitors of eukaryotic SpdS's are not specific for PfSpdS.
Methods:
'Dynamic' receptor-based pharmacophore models were generated from published crystal structures of SpdS with different ligands. This approach takes into account the inherent flexibility of the active site, which reduces the entropic penalties associated with ligand binding. Four dynamic pharmacophore models were developed and two inhibitors, (1R,4R)-(N1-(3-aminopropyl)-trans-cyclohexane-1,4-diamine (compound 8) and an analogue, N-(3-aminopropyl)-cyclohexylamine (compound 9), were identified.
Results:
A crystal structure containing compound 8 was solved and confirmed the in silico prediction that its aminopropyl chain traverses the catalytic centre in the presence of the byproduct of catalysis, 5'-methylthioadenosine. The IC50 value of compound 9 is in the same range as that of the most potent inhibitors of PfSpdS, S-adenosyl-1,8-diamino-3-thio-octane (AdoDATO) and 4MCHA and 100-fold lower than that of compound 8. Compound 9 was originally identified as a mammalian spermine synthase inhibitor and does not inhibit mammalian SpdS. This implied that these two compounds bind in an orientation where their aminopropyl chains face the putrescine binding site in the presence of the substrate, decarboxylated S-adenosylmethionine. The higher binding affinity and lower receptor strain energy of compound 9 compared to compound 8 in the reversed orientation explained their different IC50 values.
Conclusion:
The specific inhibition of PfSpdS by compound 9 is enabled by its binding in the additional cavity normally occupied by spermidine when spermine is synthesized. This is the first time that a spermine synthase inhibitor is shown to inhibit PfSpdS, which provides new avenues to explore for the development of novel inhibitors of PfSpdS.
The biogenic polyamines spermine, spermidine, and their precursor putrescine are present in micro-to-millimolar concentrations in all cell types and are vitally important for their normal growth. High intracellular content of spermine and spermidine determines the multiplicity of the cellular functions of the polyamines. Many of these functions are not well characterized at the molecular level, ensuring the ongoing development of this field of biochemistry. Tumor cells have elevated polyamine level if compared with normal cells, and this greatly stimulates the search for new opportunities to deplete the intracellular pool of spermine and spermidine resulting in decrease in cell growth and even cell death. O-Substituted hydroxylamines occupy their own place among chemical regulators of the activity of the enzymes of polyamine metabolism. Varying the structure of the alkyl substituent made it possible to obtain within one class of chemical compounds highly effective inhibitors and regulators of the activity of all the enzymes of putrescine, spermine and spermidine metabolism (with the exception of FAD-dependent spermine oxidase and acetylpolyamine oxidase), effectors of the polyamine transport system, and even actively transported in cells "proinhibitor" of ornithine decarboxylase. Some principles for the design of specific inhibitors of these enzymes as well as the peculiarities of cellular effects of corresponding O-substituted hydroxylamines are discussed.
Spermidine synthase catalyzes the production of spermidine from putrescine and decarboxylated S-adenosylmethionine (dcSAM), and plays a crucial role in cell proliferation and differentiation. The gatekeeping loop identified in the structure of spermidine synthase was predicted to contain residues important for substrate binding, but its correlation with enzyme catalysis has not been fully understood. In this study, recombinant Escherichia coli spermidine synthase (EcSPDS) was produced and its enzyme kinetics was characterized. Site-directed mutants of EcSPDS were obtained to demonstrate the importance of the amino acid residues in the gatekeeping loop. Substitution of Asp158 and Asp161 with alanine completely abolished EcSPDS activity, suggesting that these residues are absolutely required for substrate interaction. Reduction in enzyme activity was observed in the C159A, T160A, and P165Q variants, indicating that hydrophobic interactions contributed by Cys159, Thr160, and Pro165 are important for enzyme catalysis as well. On the other hand, replacement of Pro162 and Ile163 had no influence on EcSDPS activity. These results indicate that residues in the gatekeeping loop of spermidine synthase are indispensable for the catalytic reaction of EcSPDS. To the best of our knowledge, this is the first functional study on the gatekeeping loop of EcSPDS by site-directed mutagenesis.
Among various tropical diseases, malaria is a major life-threatening disease caused by Plasmodium parasite. Plasmodium falciparum is responsible for the deadliest form of malaria, so-called cerebral malaria. Purine nucleoside phosphorylase from P. falciparum is a homohexamer containing single tryptophan residue per subunit that accepts inosine and guanosine but not adenosine for its activity. This enzyme has been exploited as drug target against malaria disease. It is important to draw together significant knowledge about inherent properties of this enzyme which will be helpful in better understanding of this drug target. The enzyme shows disorder to order transition during catalysis. The single tryptophan residue residing in conserved region of transition loop is present in purine nucleoside phosphorylases throughout the Plasmodium genus. This active site loop motif is conserved among nucleoside phosphorylases from apicomplexan parasites. Modification of tryptophan residue by N-bromosuccinamide resulted in complete loss of activity showing its importance in catalysis. Inosine was not able to protect enzyme against N-bromosuccinamide modification. Extrinsic fluorescence studies revealed that tryptophan might not be involved in substrate binding. The tryptophan residue localised in electronegative environment showed collisional and static quenching in the presence of quenchers of different polarities.
The polyamines putrescine, spermidine, and spermine are simple-structured cationic molecules which fulfill essential functions in cell growth and differentiation. Consequently, the obstruction of synthesis, uptake, or interconversion of polyamines has been proposed as a potent drug target in cancer therapy, as well as against various infections caused by protozoan parasites. Although polyamines are found throughout the Apicomplexa, members of this group differ widely in their polyamine supply, which either derives via their own biosynthesis or from the host. In this chapter, attention is focused on changes in, and differences between, the parasite and host polyamine metabolism. The use of specific enzyme inhibitors may serve as an excellent tool for the validation of their potential in chemotherapeutic interventions.
The Plasmodium falciparum food vacuole (FV) is a lysosome-like organelle where erythrocyte hemoglobin digestion occurs. It is a favorite target in the development of antimalarials. We have used a tandem mass spectrometry approach to investigate the proteome of an FV-enriched fraction and identified 116 proteins. The electron microscopy analysis and the Western blot data showed that the major component of the fraction was the FV and, as expected, the majority of previously known FV markers were recovered. Of particular interest, several proteins involved in vesicle-mediated trafficking were identified, which are likely to play a key role in FV biogenesis and/or FV protein trafficking. Recovery of parasite surface proteins lends support to the cytostomal pathway of hemoglobin ingestion as a FV trafficking route. We have identified 32 proteins described as hypothetical in the databases. This insight into FV protein content provides new clues towards understanding the biological function of this organelle in P. falciparum.
Despite enormous efforts to combat malaria the disease still afflicts up to half a billion people each year of which more than one million die. Currently no approved vaccine is available and resistances to antimalarials are widely spread. Hence, new antimalarial drugs are urgently needed.
Here, we present a computational analysis of the metabolism of Plasmodium falciparum, the deadliest malaria pathogen. We assembled a compartmentalized metabolic model and predicted life cycle stage specific metabolism with the help of a flux balance approach that integrates gene expression data. Predicted metabolite exchanges between parasite and host were found to be in good accordance with experimental findings when the parasite's metabolic network was embedded into that of its host (erythrocyte). Knock-out simulations identified 307 indispensable metabolic reactions within the parasite. 35 out of 57 experimentally demonstrated essential enzymes were recovered and another 16 enzymes, if additionally the assumption was made that nutrient uptake from the host cell is limited and all reactions catalyzed by the inhibited enzyme are blocked. This predicted set of putative drug targets, shown to be enriched with true targets by a factor of at least 2.75, was further analyzed with respect to homology to human enzymes, functional similarity to therapeutic targets in other organisms and their predicted potency for prophylaxis and disease treatment.
The results suggest that the set of essential enzymes predicted by our flux balance approach represents a promising starting point for further drug development.
Enzymes have evolved to catalyse reactions, reducing the activation barrier by lowering transition state energy. Many reaction types are represented in enzymology; however, it is noticeable that SN2-type nucleophilic reactions at carbon are not common. The activation barrier of an SN2 reaction is generally high, as it progresses through a trigonal bipyramidal transition state, and this presents a challenge to efficient catalysis. This review summarises those enzyme reactions which almost certainly take place by a SN2 reaction mechanism, although it is recognised that the S N2 terminology, which derives from the bimolecular kinetics of a reaction in solution, is compromised to some extent in enzymes as they all display Michealis-Menten kinetics. Nonetheless, the SN2 terminology is used here to classify enzymes which catalyse nucleophilic reactions at sp3-hybridised carbon. There is a particular focus on highlighting the active-site residues involved in catalysis where known, information that comes primarily from a combination of structural and mutagenesis studies. Predictably, methyl transfer reactions are most widely represented; however, there are a number of enzymes that halogenate/dehalogenate, as well as epoxide hydrolases and inverting sulfatases. Although most of the enzymes have been known for some time, recent advances in structural biology are providing more details on how such enzymes function.
In order to find novel bioactive molecules regulating differentiation and hormone secretion of pancreatic endocrine cells, the effects of various substances including purinergic receptor agonists and inhibitors of polyamine biosynthesis were examined in pancreatic islets and several pancreatic cell lines. The nicotinic alpha3beta4 receptor was found to be present and capable of increasing cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) and insulin secretion in mouse pancreatic Beta-TC6 cells. Activation of both nicotinic and muscarinic M(3)/M(4) receptors resulted in reduction of insulin release when compared with stimulation of muscarinic receptor alone in Beta-TC6 cells. In mouse islets, purinergic P2Y(1) and P2Y(6) receptors, which are coupled to Gq proteins, were expressed and appeared to regulate insulin secretion through Ca(2+) mobilization from intracellular stores. Similar results were observed in Beta-TC6 cells. Spermidine, one of polyamines, was found to modulate insulin synthesis and [Ca(2+)](i) in Beta-TC6 cells by use of a specific spermidine synthesis inhibitor, trans-4-methylcyclohexylamine (MCHA). Antizyme, which binds to ornithine decarboxylase (ODC) and thereby reduces the cellular polyamine level, was found to be necessary for conversion of ASPC-1 cells, a pancreatic ductal tumor cell line, into alpha-cells forming the islet-like structure and expressing glucagon gene. These findings help advance our understanding of the complex mechanisms involved in the regulation of pancreatic endocrine cell function and develop new therapeutic agents in diabetes mellitus.
In order to assess the functional role of the polyamines spermidine and spermine in pancreatic beta-cells, we examined the effect of spermidine and spermine synthase inhibitors, trans-4-methylcyclohexylamine (MCHA) and N-(3-aminopropyl)cyclohexylamine (APCHA), on cellular polyamine and insulin contents, insulin secretion, and cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in mouse insulin-secreting Beta-TC6 cells. The cellular spermidine and spermine contents were reduced 90% and 64% by cultivation of cells in the presence of MCHA and APCHA for 3 days, respectively. Addition of spermidine or spermine reversed the polyamine level reduced by MCHA or APCHA, respectively. Insulin secretion was decreased 40~60% in the cells treated with MCHA or APCHA. The reduction by MCHA was reversed to the untreated level by adding spermidine exogenously, while the effect of APCHA was not reversed by treatment with spermine. The cellular insulin content was also reduced by treatment with MCHA but not the expression of insulin 1 and 2 genes, suggesting that spermidine was involved in the translation of insulin mRNAs. The elevation of [Ca(2+)](i), a key event triggering insulin secretion induced by glucose, was reduced in Beta-TC6 cells by MCHA treatment. The spermidine synthase inhibitor also augmented the sustained [Ca(2+)](i) rise induced by carbamylcholine but not by a high concentration of KCl or nicotine. These results suggested that spermidine rather than spermine plays an important role in the regulation of insulin synthesis and the glucose-induced [Ca(2+)](i) rise in Beta-TC6 cells.
Putrescine N-methyltransferase (PMT) catalyses S-adenosylmethionine (SAM) dependent methylation of the diamine putrescine. The product N-methylputrescine is the first specific metabolite on the route to nicotine, tropane, and nortropane alkaloids. PMT cDNA sequences were cloned from tobacco species and other Solanaceae, also from nortropane-forming Convolvulaceae and enzyme proteins were synthesised in Escherichia coli. PMT activity was measured by HPLC separation of polyamine derivatives and by an enzyme-coupled colorimetric assay using S-adenosylhomocysteine. PMT cDNA sequences resemble those of plant spermidine synthases (putrescine aminopropyltransferases) and display little similarity to other plant methyltransferases. PMT is likely to have evolved from the ubiquitous enzyme spermidine synthase. PMT and spermidine synthase proteins share the same overall protein structure; they bind the same substrate putrescine and similar co-substrates, SAM and decarboxylated S-adenosylmethionine. The active sites of both proteins, however, were shaped differentially in the course of evolution. Phylogenetic analysis of both enzyme groups from plants revealed a deep bifurcation and confirmed an early descent of PMT from spermidine synthase in the course of angiosperm development.
Polyamines are widespread distributed all over in living organisms. In Thalassiosira pseudonana 10 N-aminopropyl transferase like nucleotide sequences exists. It is assumed that these sequences are involved in the biomineralization of the diatom shell. The cDNA of the sequences were cloned, recombinant overexpressed and assayed with decarboxylated S-adenosylmethionine and several radioactive labelled polyamines. However, only a spermidine synthase and a thermospermine synthase were found to be enzymatically active in an in vitro assay. Both enzyme activities could be recognized in the crude extracts of Thalassiosira pseudonana and Cyclotella meneghiana. In further investigations the kinetics of the thermospermine synthase was determined and a site-specific mutagenesis of the bindig cavity of decarboxylated S-adenosylmethionine was carried out.
Polyamines are ubiquitous components of all living cells, and their depletion usually causes cytostasis, a strategy employed for treatment of West African trypanosomiasis. To evaluate polyamine depletion as an anti-malarial strategy, cytostasis caused by the co-inhibition of S-adenosylmethionine decarboxylase/ornithine decarboxylase in Plasmodium falciparum was studied with a comprehensive transcriptome, proteome, and metabolome investigation. Highly synchronized cultures were sampled just before and during cytostasis, and a novel zero time point definition was used to enable interpretation of results in lieu of the developmentally regulated control of gene expression in P. falciparum. Transcriptome analysis revealed the occurrence of a generalized transcriptional arrest just prior to the growth arrest due to polyamine depletion. However, the abundance of 538 transcripts was differentially affected and included three perturbation-specific compensatory transcriptional responses as follows: the increased abundance of the transcripts for lysine decarboxylase and ornithine aminotransferase and the decreased abundance of that for S-adenosylmethionine synthetase. Moreover, the latter two compensatory mechanisms were confirmed on both protein and metabolite levels confirming their biological relevance. In contrast with previous reports, the results provide evidence that P. falciparum responds to alleviate the detrimental effects of polyamine depletion via regulation of its transcriptome and subsequently the proteome and metabolome.
After invading human erythrocytes, the malarial parasite Plasmodium falciparum, initiates a remarkable process of secreting proteins into the surrounding erythrocyte cytoplasm and plasma membrane. One of these exported proteins, the knob-associated histidine-rich protein (KAHRP), is essential for microvascular sequestration, a strategy whereby infected red cells adhere via knob structures to capillary walls and thus avoid being eliminated by the spleen. This cytoadherence is an important factor in many of the deaths caused by malaria. Green fluorescent protein fusions and fluorescence recovery after photobleaching were used to follow the pathway of KAHRP deployment from the parasite endomembrane system into an intermediate depot between parasite and host, then onwards to the erythrocyte cytoplasm and eventually into knobs. Sequence elements essential to individual steps in the pathway are defined and we show that parasite-derived structures, known as Maurer's clefts, are an elaboration of the canonical secretory pathway that is transposed outside the parasite into the host cell, the first example of its kind in eukaryotic biology.
Species of malaria parasite that infect rodents have long been used as models for malaria disease research. Here we report the whole-genome shotgun sequence of one species, Plasmodium yoelii yoelii, and comparative studies with the genome of the human malaria parasite Plasmodium falciparum clone 3D7. A synteny map of 2,212 P. y. yoelii contiguous DNA sequences (contigs) aligned to 14 P. falciparum chromosomes reveals marked conservation of gene synteny within the body of each chromosome. Of about 5,300 P. falciparum genes, more than 3,300 P. y. yoelii orthologues of predominantly metabolic function were identified. Over 800 copies of a variant antigen gene located in subtelomeric regions were found. This is the first genome sequence of a model eukaryotic parasite, and it provides insight into the use of such systems in the modelling of Plasmodium biology and disease.
Metabolic studies on Phytomonas sp. isolated from the lactiferous tubes of the latex-bearing spurge Euphorbia characias indicate that glucose is the preferred energy and carbon substrate during logarithmic growth. In stationary phase cells glucose consumption was dramatically reduced. Glucose consumption and end-product formation were measured on logarithmically growing cells, both under aerobic (air and 95% O2/5% CO2) and anerobic (95% N2/5% CO2 and 100% N2) conditions. The rate of glucose consumption slightly increased anerobic conditions indicating that Phytomonas facks a ‘reverse Pasteur’ effect contrary to the situation encountered in Leishmania major. Major end-products of glucose catabolism under aerobic conditions, detected by enzymatic and NMR measurements, were acetate, ethanol and carbon dioxide and under anaerobic conditions ethanol, glycerol and carbon dioxide. Smaller amounts of pyruvate, succinate, L-malate, L-lactate, phosphenolpyruvate, alanine and aspartate were also detected.
A Primer of Population Genetics and Genomics , 4th edition, has been completely revised and updated to provide a concise but comprehensive introduction to the basic concepts of population genetics and genomics. Recent textbooks have tended to focus on such specialized topics as the coalescent, molecular evolution, human population genetics, or genomics. This primer bucks that trend by encouraging a broader familiarity with, and understanding of, population genetics and genomics as a whole. The overview ranges from mating systems through the causes of evolution, molecular population genetics, and the genomics of complex traits. Interwoven are discussions of ancient DNA, gene drive, landscape genetics, identifying risk factors for complex diseases, the genomics of adaptation and speciation, and other active areas of research. The principles are illuminated by numerous examples from a wide variety of animals, plants, microbes, and human populations. The approach also emphasizes learning by doing, which in this case means solving numerical or conceptual problems. The rationale behind this is that the use of concepts in problem-solving lead to deeper understanding and longer knowledge retention. This accessible, introductory textbook is aimed principally at students of various levels and abilities (from senior undergraduate to postgraduate) as well as practising scientists in the fields of population genetics, ecology, evolutionary biology, computational biology, bioinformatics, biostatistics, physics, and mathematics.
P‐glycoproteins can cause resistance of mammalian tumor cells to chemotherapeutic drugs. They belong to an evolutionarily well‐conserved family of ATP binding membrane transporters. Four P‐glycoprotein gene homologs have been found in the nematode Caenorhabditis elegans; this report describes the functional analysis of two. We found that PGP‐3 is expressed in both the apical membrane of the excretory cell and in the apical membrane of intestinal cells, whereas PGP‐1 is expressed only in the apical membrane of the intestinal cells and the intestinal valve. By transposon‐mediated deletion mutagenesis we generated nematode strains with deleted P‐glycoprotein genes and found that the pgp‐3 deletion mutant, but not the pgp‐1 mutant, is sensitive to both colchicine and chloroquine. Our results suggest that soil nematodes have P‐glycoproteins to protect themselves against toxic compounds made by plants and microbes in the rhizosphere.
Plasmodium falciparum can now be maintained in continuous culture in human erythrocytes incubated at 38°C in RPMI 1640 medium with human serum under an atmosphere with 7 percent carbon dioxide and low oxygen (1 or 5 percent). The original parasite material, derived from an infected Aotus trivirgatus monkey, was diluted more than 100 million times by the addition of human erythrocytes at 3- or 4-day intervals. The parasites continued to reproduce in their normal asexual cycle of approximately 48 hours but were no longer highly synchronous. They have remained infective to Aotus.
Gossypol has been shown to exhibit antifertility, spermicidal antiviral, antitrypanosomal and antimalarial activity. However, gossypol has toxic properties which appear to result from reactions involving its aldehyde functional groups. It was therefore of interest to examine a derivative of gossypol for biological activity. Gossylic nitrile diacetate is a derivative of gossypol in which the aldehyde groups have been converted into nitriles. Gossylic nitrile diacetate was tested for its antimalarial activity in vitro, against the human malarial parasite P. falciparum. Gossylic nitrile diacetate, 10-4 M, prevented growth of the parasites. Although this derivative is not as effective as gossypol itself, these results do suggest that biological activity can be separated from toxicity. Gossylic nitrile diacetate, like gossypol, was shown to inhibit the activity of parasite lactate dehydrogenase, the putative site of action.
Gossypol, a polyphenolic triterpene obtained from cottonseed oil, has been reported to exhibit spermicidal, antiviral and antitrypanosomal activity. It is known to inhibit sperm lactate dehydrogenase X. In the present study, we examined the antimalarial properties of gossypol on the in vitro growth of the human malarial parasite Plasmodium falciparum, and examined the effects of gossypol on parasite lactate dehydrogenase. The growth of the parasite was monitored both by 3H-hypoxanthine incorporation and by differential cell counts. At concentrations above 1 μM gossypol inhibited the growth of all stages of parasites; even at concentrations as low as 0.1 μM, it slowed the development of young parasites. 20 μM Gossypol irreversibly inactivated parasite lactate dehydrogenase within 20 min.
Trichomonads are deep-branching protists that are thought to be early-diverging eukaryotes (Sogin, 1991). These organisms belong to the phylum Parabasalia which encompasses both non-parasitic and parasitic trichomonads. The two best studied parasitic trichomonads are the cattle-infective parasite, Tritrichomonas foetus and the human-infective parasite, Trichomonas vaginalis. These parasites are flagellated, extracellular organisms that are sexually transmitted and reside in the urogenital tracts of their hosts. Over 150 million cases of human trichomoniasis are reported each year and significant financial losses are frequently suffered due to trichomoniasis in cattle, making these parasites important in both the medical and agricultural communities. Aside from their medical and agricultural importance, a number of unusual biochemical properties of Trichomonas have captured the attention of scientists. The appeal of trichomonads from a biological viewpoint stems, in large part, from properties that reflect both their primitive nature and parasitic lifestyle. For example, trichomonads lack two organelles typically found in eukaryotes, the mitochondrion and the peroxisome, but instead contain an organelle involved in carbohydrate metabolism called the hydrogenosome.
Resistance to ivermectin and related drugs is an increasing problem for parasite control. The mechanism of ivermectin resistance in nematode parasites is currently unknown. Some P-glycoproteins and multidrug resistance proteins have been found to act as membrane transporters which pump drugs from the cell. A disruption of the mdr1a gene, which encodes a P-glycoprotein in mice, results in hypersensitivity to ivermectin. Genes encoding members of the P-glycoprotein family are known to exist in nematodes but the involvement of P-glycoprotein in nematode ivermectin-resistance has not been described. Our data suggest that a P-glycoprotein may play a role in ivermectin resistance in the sheep nematode parasite Haemonchus contortus. A full length P-glycoprotein cDNA from H. contortus has been cloned and sequenced. Analysis of the sequence showed 61 – 65% homology to other P-glycoprotein/multidrug resistant protein sequences, such as mice, human and Caenorhabditis elegans. Expression of P-glycoprotein mRNA was higher in ivermectin-selected than unselected strains of H. contortus. An alteration in the restriction pattern was also found for the genomic locus of P-glycoprotein derived from ivermectin-selected strains of H. contortus compared with unselected strains. P-glycoprotein gene structure and/or its transcription are altered in ivermectin-selected H. contortus. The multidrug resistance reversing agent, verapamil, increased the efficacy of ivermectin and moxidectin against a moxidectin-selected strain of this nematode in jirds (Meriones unguiculatus). These data indicate that a P-glycoprotein may be involved in resistance to ivermectin and other macrocyclic lactones in H. contortus.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
The following five principles were deduced from the accumulated evidence
on molecular evolution and theoretical considerations of the population
dynamics of mutant substitutions: (i) for each protein, the rate of
evolution in terms of amino acid substitutions is approximately
constant/site per year for various lines, as long as the function and
tertiary structure of the molecule remain essentially unaltered. (ii)
Functionally less important molecules or parts of a molecule evolve (in
terms of mutant substitutions) faster than more important ones. (iii)
Those mutant substitutions that disrupt less the existing structure and
function of a molecule (conservative substitutions) occur more
frequently in evolution than more disruptive ones. (iv) Gene duplication
must always precede the emergence of a gene having a new function. (v)
Selective elimination of definitely deleterious mutants and random
fixation of selectively neutral or very slightly deleterious mutants
occur far more frequently in evolution than positive Darwinian selection
of definitely advantageous mutants.
The Symposium, `Mectizan™ and Onchocerciasis—A Decade of Accomplishment and Prospects for the Future: The Evolution of the Donation of a Drug into a Development Concept', was hosted at the Liverpool School of Tropical Medicine, UK, 8–9 December 1997, by David Molyneux, Director of the School, with assistance from the Mectizan™ Donation Program, Atlanta, USA, to mark the tenth anniversary of the 1987 decision of Merck & Co. to donate Mectizan™ (ivermectin, MSD), its effective microfilaricide, to treat all persons with onchocerciasis who could benefit from the drug for as long as needed. Merck & Co. provided financial support. The Proceedings have been published in Vol. 92 of the Annals of Tropical Medicine and Parasitology.
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Investigations were undertaken aimed at distinguishing and clarifying the reactivities of the Fe(SCys)4 and diironoxo sites of rubrerythrin (Rr) during its catalysis of substrate oxidations by either dioxygen or hydrogen peroxide. Three Rr-catalyzed reactions were investigated: (1) the ferroxidase reaction: Feaq2++O2→Feaq3++[O]red; (2) the NADH peroxidase reaction: NADH+H++H2O2→NAD++2H2O; and (3) the aromatic diamine peroxidase reaction exemplified with o-dianisidine as substrate: o-dianisidine+H2O2→o-dianisidine quinonediimine+2H2O. A non-native bacterial oxidoreductase was used as a co-catalyst for the NADH peroxidase reaction. Residues at or near both metal sites of Rr, including those furnishing iron ligands, were mutated to assist in clarifying the metal-site reactivities. In addition a Rr with Zn2+ substituted for iron in the Fe(SCys)4 site was examined. The results indicate that, in reactions 1 and 2, electrons from the reductant flow initially into the Fe(SCys)4 site of Rr, then out through the diferrous site into O2 or H2O2. In reaction 3 oxidized Rr appears to weakly activate H2O2 for oxidation of the aromatic diamine substrate. The highest turnover occurs for the NADH peroxidase reaction. It is proposed that an extra carboxylate ligand not present in other diironoxo enzymes shifts the reactivity of the diferrous site of Rr towards hydrogen peroxide and away from dioxygen.
The gene encoding spermidine synthase (spsA) was isolated from Dictyostelium discoideum using the technique of insertional mutagenesis. Northern blot analysis showed that the spsA mRNA is expressed maximally during the vegetative stage and decreases gradually during the 24 h of development. Sequencing of the genomic DNA and a full-length cDNA clone indicated the presence of one intron in a gene coding for a predicted protein (SpsA) with 284 amino acids. The sequence is highly conserved, with amino acid identities compared to spermidine synthases of humans, 59.5%, to mouse, 61.3%, and to yeast, 58.1%. A null mutant of the spsA gene is unable to grow in the absence of exogenous spermidine. Development of spsA null cells grown in the absence of spermidine produced fruiting bodies that have abnormally short stalks.
The amitochondriate sexually-transmitted human parasitic protozoanTrichomonas vaginalis (Bushby strain) grown anaerobically on complex medium containing cysteine and ascorbic acid consumed O2 avidly (6.9 μM min−1 per 106 organisms) with an apparentK
m
value of 5.1 μM O2 : O2 uptake was inhibited by O2 > 120 μM. Spectrophotometric assays in the presence of microperoxidase (419-407 nm) indicated that H2O2 was produced and that inhibition by high O2 concentrations was again evident. Hydrogenosomes oxidizing pyruvate in the presence of ADP and succinate showed similar patterns
of O2 consumption, H2O2 production (33.5 pmol min−1 per mg protein), and O2 inhibition. Cytosolic NADH oxidase gave no detectable H2O2, whereas the cytosolic NADPH oxidase produced H2O2 at a rate (43 pmol min−1 per mg protein) greater than that of hydrogenosomes. These results are discussed in relation to the oxidative stress experienced
by the pathogen in its natural habitat.
The addition of arginine to cultures of Escherichia coli K12 deficient in agmatine ureohydrolase (EC 3.5.3.7) results in polyamine depletion and a striking inhibition of nucleic
acid accumulation and growth. The omission of lysine from these cultures leads to a further decrease in growth rate and nucleic
acid synthesis. In arginine-inhibited cells the addition of putrescine or spermidine, in the presence or absence of lysine,
restores the control rate of growth and nucleic acid accumulation. Under the same conditions of arginine inhibition in the
absence of lysine, the addition of cadeverine alone stimulates growth rate and RNA synthesis. The addition of lysine to polyamine-depleted
cultures results in cadaverine production and in the appearance of a new spermidine analogue, containing lysine carbon. The
new compound has been identified as N-3-aminopropyl-1,5-diaminopentane.
Infection of this arginine-inhibited, polyamine-depleted mutant with T4D results in markedly decreased amounts of DNA accumulation, as compared to infected cells uninhibited by arginine. Supplementation
of arginine-inhibited infected cells by putrescine or spermidine restores DNA synthesis to the uninhibited level.
A critical step in infection by Plasmodium falciparum, the microorganism
that causes the most severe form of malaria, is the adhesion of parasitized
red blood cells to capillary endothelium. The human protein CD36 is a major
receptor for P. falciparum-infected red blood cells1, 2
and may contribute to the disease by sequestering infected red blood cells1, 2 and inhibiting the immune response to the parasite3.
We have found that African populations contain an exceptionally high frequency
of mutations in CD36. Unexpectedly, these mutations that cause CD36 deficiency
are associated with susceptibility to severe malaria, suggesting that the
presence of distinct CD36 mutations in Africans and Asians4, 5, 6
is due to some selection pressure other than malaria.
We probed DNA from all trypanosomatid genera by slot blot hybridization with an oligonucleotide (SL3′) complementary to a sequence of thePhytomonasspliced-leader or mini-exon RNA. The 19-nucleotide probe target site was previously shown to be highly conserved among a limited number ofPhytomonasisolates, but diverges in other kinetoplastid genera. Our examination of 84 isolates of various genera of trypanosomatids showed hybridization of this probe exclusively with isolates from plants or insects which could, by morphological, biochemical, and molecular criteria, be considered to belong to the genusPhytomonas. In contrast, no hybridization was observed with flagellates of the generaBlastocrithidia, Crithidia, Endotrypanum, Herpetomonas, Leptomonas, Leishmania,andTrypanosoma. The method detected DNA quantities as low as 50 ng using either radioactive or nonradioactive probes, and was effective with as few as 104intact flagellates. Together, these results suggest that this probe will serve as a convenient marker for taxonomic and epidemiological studies requiring reliable identification ofPhytomonasspp. in plants or in putative insect vectors.
In a previous publication, we described the purification of a membrane-bound acid phosphatase of Leishmania mexicana as a heterogeneously N-glycosylated protein of an apparent molecular mass of 70 000–72 000 expressed in both the promastigote and the amastigote stage of the parasite [19]. Screening of a genomic DNA library of L. mexicana with degenerate oligonucleotides designed according to the NH2-terminus of the protein led to the cloning of the lmmbap gene, which is present in one copy per haploid genome. The open reading frame predicts a protein of 516 amino acids composed of a signal sequence, a large hydrophilic region, a trans-membrane α-helix and a short cytoplasmic tail. The sequence of the hydrophilic region is homologous to acid phosphatases from other organisms. While in wild-type promastigotes, the acid phosphatase is located in the endosomal/lysosomal compartment between the flagellar pocket and the nucleus, overexpression leads to its abundant exposure on the cell surface. In cells transfected with a construct lacking the region corresponding to the trans-membrane and the cytoplasmic parts, the resulting altered acid phosphatase is efficiently secreted into the culture medium. The potential of this system for studies on membrane trafficking in kinetoplastid organisms is discussed.
Lactate dehydrogenase (LDH) interconverts pyruvate and lactate with concomitant interconversion of NADH and NAD+. Although crystal structures of a variety of LDH have previously been described, a notable absence has been any of the three known human forms of this glycolytic enzyme. We have now determined the crystal structures of two isoforms of human LDH—the M form, predominantly found in muscle; and the H form, found mainly in cardiac muscle. Both structures have been crystallized as ternary complexes in the presence of the NADH cofactor and oxamate, a substrate-like inhibitor. Although each of these isoforms has different kinetic properties, the domain structure, subunit association, and active-site regions are indistinguishable between the two structures. The pKa that governs the KM for pyruvate for the two isozymes is found to differ by about 0.94 pH units, consistent with variation in pKa of the active-site histidine. The close similarity of these crystal structures suggests the distinctive activity of these enzyme isoforms is likely to result directly from variation of charged surface residues peripheral to the active site, a hypothesis supported by electrostatic calculations based on each structure. Proteins 2001;43:175–185. © 2001 Wiley-Liss, Inc.
Studies were made on the binding mode of the nucleosome-assembly protein AP-I with histones H2A + H2B and/or H3 + H4. Histones H2A + H2B bound with AP-I to form a 7-S complex which contained twice as much AP-I (by weight) as histones. Histone H3 + H4 formed an 8-S complex with AP-I. The 7-S and 8-S complexes did not form a new complex when mixed, but significant amounts of two histone pairs were assembled into a 12-S complex on mixing the (H2A + H2B)–AP-I complex (7-S) with free H3 + H4. In contrast, when the (H3 + H4)–AP-I complex (8-S) was incubated with free H2A + H2B, almost no assembly occurred, but the 7-S complex of H2A + H2B was newly formed. Binding studies by enzyme-linked immunosorbent assay showed that AP-I bound with H2A + H2B faster than with H3 + H4.
From these results, it is suggested that AP-I has a higher binding affinity for histone H2A + H2B than for H3 + H4, and that the 7-S complex is an intermediate in formation of the 12-S octamer complex (H2A + H2B + H3 + H4)2
Four out of five Desulfovibrio strains tested were able to oxidize l-malate to acetate in the presence of sulfate. Fumarate and succinate were also oxidized to acetate by these strains, but growth with the latter substrate was marginal. During growth on malate high NADP-dependent malic enzyme and NADPH DH activities were found in all strains. These activities were lower in lactate-or pyruvate-grown cells. An NADPH DH from D. gigas was partially purified. It was oxygen-labile, very sensitive to heavy metal ions and highly specific for NADPH. Growth yield studies indicated that energy conservation occurred during the transport of reducing equivalents from NADPH to the sulfate reduction pathway.
The expression of the pfemp3 gene and the corresponding PfEMP3 knob-associated protein in the pre-erythrocytic stages of Plasmodium falciparum was demonstrated by RT-PCR, Western blots, IFAT and IEM. The antigen was found on the surface of the sporozoite and in the cytoplasm of mature hepatic stage parasites. Immunological cross-reactivity was observed with sporozoites from the rodent malaria parasites Plasmodium yoelii yoelii and Plasmodium berghei and was exploited to assess a potential role of this protein at the pre-erythrocytic stages. Specific antibodies from immune individuals were found to inhibit P. yoelii yoelii and P. berghei sporozoite invasion of primary hepatocyte cultures. PfEMP3 should now be added to the small list of proteins expressed at the pre-erythrocytic stages of P. falciparum, and its vaccine potential now deserves to be investigated.
Glucokinase (GK) and glucosephosphate isomerase (GPI), the first two enzymes of the glycolytic pathway of the diplomonads Giardia intestinalis and Spironucleus barkhanus, Type I amitochondriate eukaryotes, were sequenced. GPI of the parabasalid Trichomonas vaginalis was also sequenced. The diplomonad GKs belong to a family of specific GKs present in cyanobacteria, in some proteobacteria and also in T. vaginalis, a Type II amitochondriate protist. These enzymes are not part of the hexokinase family, which is broadly distributed among eukaryotes, including the Type I amitochondriate parasite Entamoeba histolytica. G. intestinalis GK expressed in Escherichia coli was specific for glucose and glucosamine, as are its eubacterial homologs. The sequence of diplomonad and trichomonad GPIs formed a monophyletic group more closely related to cyanobacterial and chloroplast sequences than to cytosolic GPIs of other eukaryotes and prokaryotes. The findings show that certain enzymes of the energy metabolism of these amitochondriate protists originated from sources different than those of other eukaryotes. The observation that the two diplomonads and T. vaginalis share the same unusual GK and GPI is consistent with gene trees that suggest a close relationship between diplomonads and parabasalids. The intriguing relationships of these enzymes to cyanobacterial (and chloroplast) enzymes might reflect horizontal gene transfer between the common ancestor of the diplomonad and parabasalid lineages and the ancestor of cyanobacteria.
Ever since the discovery that the human tryptophanyl-tRNA synthetase (TrpRS)-encoding gene is induced by interferon (IFN) [J. Fleckner et al., Proc. Natl. Acad. Sci. USA 88 (1991) 11520 11524] and contains IFN-response regulatory elements [Frolova et al., Gene 128 (1993) 237-245], the biological rationale for this induction has remained unresolved. A survey of immune system proteins in this study reveals that the human major histocompatibility complex (MHC) antigens, β-2-microglobulin (βMG) and complement factor B, which are known to be induced by IFN, together with immunoglobulins (Ig) are all exceptionally enriched in Trp residues, as compared to human proteins in general. It also reveals the conservation of a sequence motif, CX10-17WX26-62C, in Ig domains. The conservation of this sequence motif and the utility of Trp residues within antigen-binding sites clearly contribute to the Trp enrichment in Ig. These observations suggest a biological rationale for the induction of TrpRS by IFN in safeguarding Trp incorporation for the IFN-enhanced synthesis of immunological molecules.
Onchocerca volvulus, the filarial parasite that causes onchocerciasis or river blindness, contains three distinct genomes. These include the nuclear genome, the mitochondrial genome and the genome of an intracellular endosymbiont of the genus Wolbachia. The nuclear genome is roughly 1.5×108 bp in size, and is arranged on four chromosome pairs. Analysis of expressed sequence tags from different life-cycle stages has resulted in the identification of transcripts from roughly 4000 O. volvulus genes. Several of these transcripts are highly abundant, including those encoding collagen and cuticular proteins. Analysis of several gene sequences from O. volvulus suggests that the nuclear genes of O. volvulus are relatively compact and are interrupted relatively frequently by small introns. The intron–exon boundaries of these genes generally follow the GU–AG rule characteristic of the splice donor and acceptors of other vertebrate organisms. The nuclear genome also contains at least one repeated sequence family of a 150 bp repeat which is arranged in tandem arrays and appears subject to concerted evolution. The mitochondrial genome of O. volvulus is remarkably compact, only 13 747 bp in size. Consistent with the small size of the genome, four gene pairs overlap, eight contain no intergenic regions and the remaining gene pairs are separated by small intergenic domains ranging from 1 to 46 bp. The protein-coding genes of the O. volvulus mitochondrial genome exhibit a striking codon bias, with 15/20 amino acids having a single codon preference greater than 70%.Intraspecific variation in both the nuclear and mitochondrial genomes appears to be quite limited, consistent with the hypothesis that O. volvulus has suffered a genetic bottleneck in the recent past.
Human lactate dehydrogenases (LDH-A4, -B4, and -C4) are highly homologous with 84–89% sequence similarities and 69–75% amino acid identities. Active site residues are especially conserved. Gossypol, a natural product from cotton seed, is a non-selective competitive inhibitor of NADH binding to LDH, with Ki values of 1.9, 1.4, and 4.2 μM for LDH-A4, -B4, and -C4, respectively. However, derivatives of gossypol and structural analogs of gossypol in the substituted 2,3-dihydroxy-1-naphthoic acid family exhibited markedly greater selectivity and, in many cases, greater potency. For gossypol derivatives, greater than 35-fold selectivity was observed. For dihydroxynaphthoic acids with substituents at the 4- and 7-positions, greater than 200-fold selectivity was observed. Inhibition was consistently competitive with the binding of NADH, with dissociation constants as low as 30 nM. By comparison, a series of N-substituted oxamic acids, which are competitive inhibitors of the binding of pyruvate to LDH, exhibited very modest selectivity. These results suggest that substituted dihydroxynaphthoic acids are good lead compounds for the development of selective LDH inhibitors. Selective inhibitors of LDH-C4 targeted to the dinucleotide fold may hold promise as male antifertility drugs. Selective inhibitors of LDH-A4 and -B4 may be useful for studies of lactic acidemia associated with ischemic events. More broadly, the results raise the question of the general utility of drug design targeted at the dinucleotide binding sites of dehydrogenases/reductases.
Differential screening of cDNA libraries constructed from knobby and predominantly knobless Plasmodium falciparum isolates, identified the sequence SD17. Chromosome blotting experiments have shown that this sequence, which is located on chromosome 2 of most isolates, was deleted in the cloned parasite line E12 of the isolate. Here we show that erythrocytes infected with the SD17-containing cloned line D10 have typical knob structures on their surfaces, whereas those infected with the line E12 lack knobs. An expression clone was constructed from SD17 and used to affinity purify antibodies from the sera of individuals living in areas of Papua New Guinea where malaria is endemic. The antibodies reacted in immunoblotting experiments with a single polypeptide that varied in Mr from 85,000 to 105,000 among different isolates. The antigen was not expressed in the knobless clone E12. Postembedding immunoelectron microscopy showed localization of the antigen over the knobs of FC27 and two other isolates, largely on the cytoplasmic side. We conclude that the parasite antigen corresponding to clone SD17 is a knob protein.