Regulation and function of polyamines in African trypanosomes

The polyamine biosynthetic pathway is an important drug target for the treatment of human African trypanosomiasis (HAT), raising interest in understanding polyamine function and their mechanism of regulation. Polyamine levels are tightly controlled in mammalian cells, but similar regulatory mechanisms appear absent in trypanosomes. Instead trypanosomatid S-adenosylmethionine decarboxylase (AdoMetDC), which catalyzes a key step in the biosynthesis of the polyamine spermidine, is activated by dimerization with an inducible protein termed prozyme. Prozyme is an inactive paralog of the active AdoMetDC enzyme that evolved by gene duplication and is found only in the trypanosomatids. In Trypanosoma brucei, AdoMetDC activity appears to be controlled by regulation of prozyme protein levels, potentially at the translational level.     

Trypanosoma brucei S-Adenosylmethionine Decarboxylase N Terminus Is Essential for Allosteric Activation by the Regulatory Subunit Prozyme

Human African trypanosomiasis is caused by a single-celled protozoan parasite, Trypanosoma brucei. Polyamine biosynthesis is a clinically validated target for the treatment of human African trypanosomiasis. Metabolic differences between the parasite and the human polyamine pathway are thought to contribute to species selectivity of pathway inhibitors. S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes a key step in the production of the polyamine spermidine. We previously showed that trypanosomatid AdoMetDC differs from other eukaryotic enzymes in that it is regulated by heterodimer formation with a catalytically dead paralog, designated prozyme, which binds with high affinity to the enzyme and increases its activity by up to 10³-fold. Herein, we examine the role of specific residues involved in AdoMetDC activation by prozyme through deletion and site-directed mutagenesis. Results indicate that 12 key amino acids at the N terminus of AdoMetDC are essential for prozyme-mediated activation with Leu-8, Leu-10, Met-11, and Met-13 identified as the key residues. These N-terminal residues are fully conserved in the trypanosomatids but are absent from other eukaryotic homologs lacking the prozyme mechanism, suggesting co-evolution of these residues with the prozyme mechanism. Heterodimer formation between AdoMetDC and prozyme was not impaired by mutation of Leu-8 and Leu-10 to Ala, suggesting that these residues are involved in a conformational change that is essential for activation. Our findings provide the first insight into the mechanisms that influence catalytic regulation of AdoMetDC and may have potential implications for the development of new inhibitors against this enzyme.     

Polyamine metabolism in <Emphasis Type="Italic">Leishmania</Emphasis>: from arginine to trypanothione

Polyamines (PAs) are essential metabolites in eukaryotes, participating in a variety of proliferative processes, and in trypanosomatid protozoa play an additional role in the synthesis of the critical thiol trypanothione. The PAs are synthesized by a metabolic process which involves arginase (ARG), which catalyzes the enzymatic hydrolysis of l-arginine (l-Arg) to l-ornithine and urea, and ornithine decarboxylase (ODC), which catalyzes the enzymatic decarboxylation of l-ornithine in putrescine. The S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the irreversible decarboxylation of S-adenosylmethionine (AdoMet), generating the decarboxylated S-adenosylmethionine (dAdoMet), which is a substrate, together with putrescine, for spermidine synthase (SpdS). Leishmania parasites and all the other members of the trypanosomatid family depend on spermidine for growth and survival. They can synthesize PAs and polyamine precursors, and also scavenge them from the microenvironment, using specific transporters. In addition, Trypanosomatids have a unique thiol-based metabolism, in which trypanothione (N1-N8-bis(glutathionyl)spermidine, T(SH)₂) and trypanothione reductase (TR) replace many of the antioxidant and metabolic functions of the glutathione/glutathione reductase (GR) and thioredoxin/thioredoxin reductase (TrxR) systems present in the host. Trypanothione synthetase (TryS) and TR are necessary for the protozoa survival. Consequently, enzymes involved in spermidine synthesis and its utilization, i.e. ARG, ODC, AdoMetDC, SpdS and, in particular, TryS and TR, are promising targets for drug development.     

Polyamine depletion by ODC-AdoMetDC antisense adenovirus impairs human colorectal cancer growth and invasion in vitro and in vivo

BACKGROUND: Polyamine biosynthesis is controlled primarily by ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC). Polyamine concentrations are elevated in colorectal cancer. Depletion of polyamine content in colorectal cancer by chemotherapy is related to tumor regression and impaired tumorigenicity. The current study evaluates the therapeutic effects of antisense ODC and AdoMetDC sequences on colorectal cancer in vitro and in vivo. METHODS: Antisense ODC and AdoMetDC sequences were cloned into an adenoviral vector (Ad-ODC-AdoMetDCas). The human colon cancer cell lines, HT-29 and Caco-2, were infected with Ad-ODC-AdoMetDCas as well as with control vector. Viable cell counting, determination of polyamine concentrations, cell cycle analysis, and Matrigel invasion assays were performed in order to assess properties of tumor growth and invasiveness. Furthermore, the antitumor effects of Ad-ODC-AdoMetDCas were also evaluated in vivo in a nude mouse tumor model. RESULTS: Our study demonstrated that adenovirus-mediated ODC and AdoMetDC antisense expression inhibits tumor cell growth through a blockade of the polyamine synthesis pathway. This inhibitory effect cannot be reversed by the administration of putrescine. Tumor cells were arrested at the G1 phase of the cell cycle after gene transfer and had reduced invasiveness. The adenovirus also induced tumor regression in established tumors in nude mice. CONCLUSIONS: Our study suggests that Ad-ODC-AdoMetDCas has antitumor activity and therapeutic potential for the treatment of colorectal cancer.     

Changes in polyamine metabolism during glucocorticoid-induced programmed cell death in mouse thymus

When mice are injected with dexamethasone, cortical thymocytes are deleted through programmed cell death (PCD). We have used this in vivo model system to investigate the kinetics of PCD and cell proliferation in relation to polyamine metabolism for 16 h after injection of dexamethasone. As a marker for PCD, we used the appearance of a sub-G(1)peak in the DNA histogram. When a sub-G(1)peak appeared at 4 h after dexamethasone treatment, the activity of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) was significantly increased and the activity of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC) was significantly decreased compared to the activities found in the thymi of control mice. Despite the significant changes in the activities of SSAT and AdoMetDC, the only change in the polyamine pool during the experimental period was that of putrescine. Presumably the complexity of this in vivo system masks changes in the spermidine and spermine pools that were expected in relation to the increased SSAT activity and decreased AdoMetDC activity.     

Diethylglyoxal bis(guanylhydrazone), a potent inhibitor of mammalian S-adenosylmethionine decarboxylase. Effects on cell proliferation and polyamine metabolism in L1210 leukemia cells

The polyamines are cell constituents essential for growth and differentiation. S-Adenosylmethionine decarboxylase (AdoMetDC) catalyzes a key step in the polyamine biosynthetic pathway. Methylglyoxal bis(guanylhydrazone) (MGBG) is an anti-leukemic agent with a strong inhibitory effect against AdoMetDC. However, the lack of specificity limits the usefulness of MGBG. In the present report we have used an analog of MGBG, diethylglyoxal bis(guanylhydrazone) (DEGBG), with a much greater specificity and potency against AdoMetDC, to investigate the effects of AdoMetDC inhibition on cell proliferation and polyamine metabolism in mouse L1210 leukemia cells. DEGBG was shown to effectively inhibit AdoMetDC activity in exponentially growing L1210 cells. The inhibition of AdoMetDC was reflected in a marked decrease in the cellular concentrations of spermidine and spermine. The concentration of putrescine, on the other hand, was greatly increased. Treatment with DEGBG resulted in a compensatory increase in the synthesis of AdoMetDC demonstrating an efficient feedback control. Cells seeded in the presece of DEGBG ceased to grow after a lag period of 1–2 days, indicating that the cells contained an excess of polyamines which were sufficient for one or two cell cycles in the absence of polyamine synthesis. The present results indicate that analogs of MGBG, having a greater specificity against AdoMetDC, might be valuable for studies concerning polyamines and cell proliferation.     

ODC, AdoMetDC双反义腺病毒对肺癌增殖和侵袭抑制作用的体外和体内研究

鸟氨酸脱羧酶(ODC)和S-甲硫氨酸脱羧酶(AdoMetDC)是多胺体内合成的2个关键酶.研究腺病毒Ad-ODC-AdoMetDCas介导的ODC和AdoMetDC反义RNA对肺癌多胺合成,细胞增殖以及侵袭的抑制作用.用活细胞计数和流式细胞术分别检测Ad-ODCas和Ad-ODC-AdoMetDCas对肺癌A-549细胞增殖的影响,蛋白质印迹和HPLC方法分别检测腺病毒对肺癌A-549细胞中ODC和AdoMetDC蛋白表达以及胞内多胺含量的抑制作用,TUNEL标记检测法观察Ad-ODC-AdoMetDCas对肺癌细胞凋亡的影响,Matrigel侵袭实验分析腺病毒对肺癌A-549细胞侵袭活性的改变,裸鼠皮下移植瘤模型研究Ad-ODC-AdoMetDCas对体内肺癌生长的抑制作用.实验结果显示,Ad-ODC-AdoMetDCas明显抑制肺癌A-549细胞的增殖,导致细胞凋亡,显著降低肺癌A-549细胞的体外侵袭能力,肺癌A-549细胞感染Ad-ODC-AdoMetDCas后细胞内3种多胺含量都明显降低,Ad-ODC-AdoMetDCas对已形成的裸鼠皮下移植瘤具有明显的抑制作用.实验表明,ODC和AdoMetDC双反义腺病毒具有显著抑制肺癌增殖和侵袭的作用,对于肺癌的防治研究具有一定的前景.     

ODC,AdoMetDC双反义腺病毒对肺癌增殖和侵袭制作用的体外和体内研究

鸟氨酸脱羧酶(ODC)和S-甲硫氨酸脱羧酶(AdoMetDC)是多胺体内合成的2个关键酶.研究腺病毒Ad-ODC-AdoMetDCas介导的ODC和AdoMetDC反义RNA对肺癌多胺合成,细胞增殖以及侵袭的抑制作用.用活细胞计数和流式细胞术分别检测Ad-ODCas和Ad-ODC-AdoMetDCas对肺癌A-549细胞增殖的影响,蛋白质印迹和HPLC方法分别检测腺病毒对肺癌A-549细胞中ODC和AdoMetDC蛋白表达以及胞内多胺含量的抑制作用,TUNEL标记检测法观察Ad-ODC-AdoMetDCas对肺癌细胞凋亡的影响,Matrigel侵袭实验分析腺病毒对肺癌A-549细胞侵袭活性的改变,裸鼠皮下移植瘤模型研究Ad-ODC-AdoMetDCas对体内肺癌生长的抑制作用.实验结果显示,Ad-ODC-AdoMetDCas明显抑制肺癌A-549细胞的增殖,导致细胞凋亡,显著降低肺癌A-549细胞的体外侵袭能力,肺癌A-549细胞感染Ad-ODC-AdoMetDCas后细胞内3种多胺含量都明显降低,Ad-ODC-AdoMetDCas对已形成的裸鼠皮下移植瘤具有明显的抑制作用.实验表明,ODC和AdoMetDC双反义腺病毒具有显著抑制肺癌增殖和侵袭的作用,对于肺癌的防治研究具有一定的前景.     

Concomitant Changes in Polyamine Pools and DNA Methylation during Growth Inhibition of Human Colonic Cancer Cells

The effects of CGP 48664 and DFMO, selective inhibitors of the key enzymes of polyamine biosynthesis, namely, ofS-adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), were investigated on growth, polyamine metabolism, and DNA methylation in the Caco-2 cell line. Both inhibitors caused growth inhibition and affected similarly the initial expression of the differentiation marker sucrase. In the presence of the AdoMetDC inhibitor, ODC activity and the intracellular pool of putrescine were enhanced, whereas the spermidine and spermine pools were decreased. In the presence of the ODC inhibitor, the AdoMetDC activity was enhanced and the intracellular pools of putrescine and spermidine were decreased. With both compounds, the degree of global DNA methylation was increased. Spermine and spermidine (but not putrescine) selectively inhibited cytosine–DNA methyltransferase activity. Our observations suggest that spermidine (and to a lesser extent spermine) controls DNA methylation and may represent a crucial step in the regulation of Caco-2 cell growth and differentiation.     

Targeting the polyamine biosynthetic enzymes: a promising approach to therapy of African sleeping sickness, Chagas’ disease, and leishmaniasis

Summary. Trypanosomatids depend on spermidine for growth and survival. Consequently, enzymes involved in spermidine synthesis and utilization, i.e. arginase, ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetDC), spermidine synthase, trypanothione synthetase (TryS), and trypanothione reductase (TryR), are promising targets for drug development. The ODC inhibitor α-difluoromethylornithine (DFMO) is about to become a first-line drug against human late-stage gambiense sleeping sickness. Another ODC inhibitor, 3-aminooxy-1-aminopropane (APA), is considerably more effective than DFMO against Leishmania promastigotes and amastigotes multiplying in macrophages. AdoMetDC inhibitors can cure animals infected with isolates from patients with rhodesiense sleeping sickness and leishmaniasis, but have not been tested on humans. The antiparasitic effects of inhibitors of polyamine and trypanothione formation, reviewed here, emphasize the relevance of these enzymes as drug targets. By taking advantage of the differences in enzyme structure between parasite and host, it should be possible to design new drugs that can selectively kill the parasites.     

Selective regulation of S-adenosylmethionine decarboxylase activity by the spermine analogue 6-spermyne.

Polyamine-biosynthesis activity is known to be negatively regulated by intracellular polyamine pools. Accordingly, treatment of cultured L1210 cells with 10 microM-spermine rapidly and significantly lowered ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) activities in a sequential manner. By contrast, treatment for 48 h with 10 microM of the unsaturated spermine analogue 6-spermyne lowered AdoMetDC activity, but not ODC activity. An initial decrease in ODC activity at 2 h was attributed to a transient increase in free intracellular spermidine and spermine brought about through their displacement by the analogue. Thereafter, ODC activity recovered steadily to control values as 6-spermyne pools increased and spermidine and spermine pools decreased owing to analogue suppression of AdoMetDC activity. The apparent ability of 6-spermyne to regulate AdoMetDC, but not ODC, activity suggests an interesting structure-function correlation and demonstrates that the typical co-regulation of these enzyme activities can be dissociated. This, in turn, may reflect the existence of independent regulatory binding sites for the two enzymes.     

Translational regulation of ornithine decarboxylase and other enzymes of the polyamine pathway

It has long been known that polyamines play an essential role in the proliferation of mammalian cells, and the polyamine biosynthetic pathway may provide an important target for the development of agents that inhibit carcinogenesis and tumor growth. The rate-limiting enzymes of the polyamine pathway, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are highly regulated in the cell, and much of this regulation occurs at the level of translation. Although the 5' leader sequences of ODC and AdoMetDC are both highly structured and contain small internal open reading frames (ORFs), the regulation of their translation appears to be quite different. The translational regulation of ODC is more dependent on secondary structure, and therefore responds to the intracellular availability of active eIF-4E, the cap-binding subunit of the eIF-4F complex, which mediates translation initiations. Cell-specific translation of AdoMetDC appears to be regulated exclusively through the internal ORF, which causes ribosome stalling that is independent of eIF-4E levels and decreases the efficiency with which the downstream ORF encoding AdoMetDC protein is translated. The translation of both ODC and AdoMetDC is negatively regulated by intracellular changes in the polyamines spermidine and spermine. Thus, when polyamine levels are low, the synthesis of both ODC and AdoMetDC is increased, and an increase in polyamine content causes a corresponding decrease in protein synthesis. However, an increase in active eIF-4E may allow for the synthesis of ODC even in the presence of polyamine levels that repress ODC translation in cells with lower levels of the initiation factor. In contrast, the amino acid sequence that is encoded by the upstream ORF is critical for polyamine regulation of AdoMetDC synthesis and polyamines may affect synthesis by interaction with the putative peptide, MAGDIS.     

An endosymbiont positively modulates ornithine decarboxylase in host trypanosomatids

Some trypanosomatids, such as Crithidia deanei, are endosymbiont-containing species. Aposymbiotic strains are obtained after antibiotic treatment, revealing interesting aspects of this symbiotic association. Ornithine decarboxylase (ODC) promotes polyamine biosynthesis and contributes to cell proliferation. Here, we show that ODC activity is higher in endosymbiont-bearing trypanosomatids than in aposymbiotic cells, but isolated endosymbionts did not display this enzyme activity. Intriguingly, expressed levels of ODC were similar in both strains, suggesting that ODC is positively modulated in endosymbiont-bearing cells. When the aposymbiotic strain was grown in conditioned medium, obtained after cultivation of the endosymbiont-bearing strain, cellular proliferation as well as ODC activity and localization were similar to that observed in the endosymbiont-containing trypanosomatids. Furthermore, dialyzed-heated medium and trypsin treatment reduced ODC activity of the aposymbiont strain. Taken together, these data indicate that the endosymbiont can enhance the protozoan ODC activity by providing factors of protein nature, which increase the host polyamine metabolism.     

Trypanothione: A unique bis-glutathionyl derivative in trypanosomatids

Background

Trypanosomatids are early-diverging eukaryotes devoid of the major disulfide reductases – glutathione reductase and thioredoxin reductase – that control thiol-redox homeostasis in most organisms. These protozoans have evolved a unique thiol-redox system centered on trypanothione, a bis-glutathionyl conjugate of spermidine. Notably, the trypanothione system is capable to sustain several cellular functions mediated by thiol-dependent (redox) processes.

Scope of review

This review provides a summary of some historical and evolutionary aspects related to the discovery and appearance of trypanothione in trypanosomatids. It also addresses trypanothione's biosynthesis, physicochemical properties and reactivity towards biologically-relevant oxidants as well as its participation as a cofactor for metal binding. In addition, the role of the second most abundant thiol of trypanosomatids, glutathione, is revisited in light of the putative glutathione-dependent activities identified in these organisms.

Major conclusions

Based on biochemical and genome data, the occurrence of a thiol-redox system that is strictly dependent on trypanothione appears to be a feature unique to the order Kinetoplastida. The properties of trypanothione, a dithiol, are the basis for its unique reactivity towards a wide diversity of oxidized and/or electrophilic moieties in proteins and low molecular weight compounds from endogenous or exogenous sources. Novel functions have emerged for trypanothione as a potential cofactor in iron metabolism.

General significance

The minimalist thiol-redox system, developed by trypanosomatids, is an example of metabolic fitness driven by the remarkable physicochemical properties of a glutathione derivative. From a pharmacological point of view, such specialization is the Achilles' heel of these ancient and deadly parasites. This article is part of a Special Issue entitled Cellular functions of glutathione.     

Oxidative stress and antioxidant defenses: a target for the treatment of diseases caused by parasitic protozoa

Parasitic protozoa cause several diseases, affecting hundreds of millions, particularly in underdeveloped countries. Although these organisms are eukaryotic cells, some of them present major differences with their mammalian host in selected metabolic pathways. These differences may be exploited as targets for developing better pharmacological agents for the treatment of specific parasitic diseases. This review describes some of the differences in terms of antioxidant defenses between these organisms and their mammalian host, which may provide useful targets for the treatment of these diseases. Some of the potential targets are: (i). iron metabolism in Plasmodium, (ii). the presence of a Fe-containing form of superoxide dismutase in trypanosomatids and malaria-causing parasites, (iii). the unique trypanothione-dependent antioxidant metabolism in trypanosomatids, (iv). the ascorbate peroxidase found in Trypanosoma cruzi and perhaps present in other trypanosomatids.     

The activator-binding site of Onchocerca volvulus S-adenosylmethionine decarboxylase,a potential drug target

S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in polyamine biosynthesis. In many eukaryotes its activity is stimulated specifically by putrescine. The AdoMetDC of the filarial parasite Onchocerca volvulus, however, is not only stimulated by putrescine but also by the naturally occuring polyamines spermidine and spermine. Several diamines, acetylated polyamines and polyamine analogues were used to analyse what molecular prerequisites are needed to stimulate nematode AdoMetDC activity. In the absence of an activator, the O. volvulus enzyme exhibits an extremely low specific activity. This fact, together with the unspecificity of activator binding, was thought to be useful for a new strategy to inhibit nematode AdoMetDC activity. Therefore, different polyamine analogues were tested as competitive inhibitors towards the stimulatory effect putrescine has on the O. volvulus and, in comparison, on the Caenorhabditis elegans and human AdoMetDC. Bis(aralkyl)- and bis(alkyl)-substituted polyamine analogues with a 3-7-3 backbone were found to inhibit AdoMetDC activities, however, probably without interfering with the putrescine stimulation. The best inhibitor, BW-1, was about 10-fold more effective against O. volvulus AdoMetDC than against the human enzyme. Unexpectedly, BW-1 was determined to be a competitive inhibitor with respect to AdoMet, having a Ki value of 310 microM for the putrescine-stimulated human AdoMetDC. Furthermore, we show for the O. volvulus and the human enzyme that the degree of inhibition by BW-1 depends on the actual putrescine concentration.