RESUMO
Trypanosoma cruzi, the agent of the American Trypanosomiasis, Chagas disease, and Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, the agents of Sleeping sickness (Human African Trypanosomiasis, HAT), as well as Trypanosoma brucei brucei, the agent of the cattle disease nagana, contain cysteine, serine, threonine, aspartyl and metallo peptidases. The most abundant among these enzymes are the cysteine proteases from the Clan CA, the Cathepsin L-like cruzipain and rhodesain, and the Cathepsin B-like enzymes, which have essential roles in the parasites and thus are potential targets for chemotherapy. In addition, several other proteases, present in one or both parasites, have been characterized, and some of them are also promising candidates for the developing of new drugs. Recently, new inhibitors, with good selectivity for the parasite proteasomes, have been described and are very promising as lead compounds for the development of new therapies for these neglected diseases. This article is part of a Special Issue entitled: "Play and interplay of proteases in health and disease".
Assuntos
Peptídeo Hidrolases/genética , Trypanosoma brucei brucei/genética , Trypanosoma cruzi/genética , Tripanossomíase Africana/genética , Animais , Catepsina B/genética , Catepsina B/isolamento & purificação , Bovinos , Cisteína Endopeptidases/química , Cisteína Endopeptidases/uso terapêutico , Cisteína Proteases/genética , Inibidores de Cisteína Proteinase/uso terapêutico , Humanos , Proteínas de Protozoários/química , Proteínas de Protozoários/uso terapêutico , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/patogenicidade , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/patogenicidade , Tripanossomíase Africana/enzimologia , Tripanossomíase Africana/parasitologiaRESUMO
The essential SAS2-related acetyltransferase 1 (Esa1), as a acetyltransferase of MYST family, is indispensable for the cell cycle and transcriptional regulation. The Tudor domain consists of 60 amino acids and belongs to the Royal family, which serves as a module interacting with methylated histone and/or DNA. Although Tudor domain has been widely studied in higher eukaryotes, its structure and function remain unclear in Trypanosoma brucei (T. brucei), a protozoan unicellular parasite causing sleeping sickness in human and nagana in cattle in sub-Saharan Africa. Here, we determined a high-resolution structure of TbEsa1 presumed Tudor domain from T. brucei by X-ray crystallography. TbEsa1 Tudor domain adopts a conserved Tudor-like fold, which is comprised of a five-stranded ß-barrel surrounded by two short α-helices. Furthermore, we revealed a non-specific DNA binding pattern of TbEsa1 Tudor domain. However, TbEsa1 Tudor domain showed no methyl-histone binding ability, due to the absence of key aromatic residues forming a conserved aromatic cage.
Assuntos
Histona Acetiltransferases/ultraestrutura , Trypanosoma brucei brucei/ultraestrutura , Tripanossomíase Africana/microbiologia , Domínio Tudor/genética , Sequência de Aminoácidos/genética , Animais , Sítios de Ligação/genética , Bovinos , Cristalografia por Raios X , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Humanos , Modelos Moleculares , Ligação Proteica/genética , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética , Tripanossomíase Africana/enzimologia , Tripanossomíase Africana/genéticaRESUMO
To study the anatomo-biochemical substrates of brain inflammatory processes, Wistar male rats were infected with Trypanosoma brucei brucei. With this reproducible animal model of human African trypanosomiasis, brain cells (astrocytes, microglial cells, neurons) expressing the inducible nitric oxide synthase (iNOS) enzyme were revealed. Immunohistochemistry was achieved for each control and infected animal through eight coronal brain sections taken along the caudorostral axis of the brain (brainstem, cerebellum, diencephalon and telencephalon). Specific markers of astrocytes (anti-glial fibrillary acidic protein), microglial cells (anti-integrin alpha M) or neurons (anti-Neuronal Nuclei) were employed. The iNOS staining was present in neurons, astrocytes and microglial cells, but not in oligodendrocytes. Stained astrocytes and microglial cells resided mainly near the third cavity in the rostral part of brainstem (periaqueductal gray), diencephalon (thalamus and hypothalamus) and basal telencephalon. Stained neurons were scarce in basal telencephalon, contrasting with numerous iNOS-positive neuroglial cells. Contrarily, in dorsal telencephalon (neocortex and hippocampus), iNOS-positive neurons were plentiful, contrasting with the marked paucity of labelled neuroglial (astrocytes and microglial) cells. The dual distribution between iNOS-labelled neuroglial cells and iNOS-labelled neurons is a feature that has never been described before. Functionalities attached to such a divergent distribution are discussed.
Assuntos
Astrócitos/enzimologia , Cerebelo/enzimologia , Microglia/enzimologia , Neurônios/enzimologia , Óxido Nítrico Sintase Tipo II/metabolismo , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/enzimologia , Animais , Astrócitos/parasitologia , Células Cultivadas , Cerebelo/parasitologia , Masculino , Microglia/parasitologia , Neurônios/parasitologia , Ratos , Ratos Wistar , Tripanossomíase Africana/parasitologiaRESUMO
The human pathogenic parasite Trypanosoma brucei possess both de novo and salvage routes for the biosynthesis of pyrimidine nucleotides. Consequently, they do not require salvageable pyrimidines for growth. Thymidine kinase (TK) catalyzes the formation of dTMP and dUMP and is one of several salvage enzymes that appear redundant to the de novo pathway. Surprisingly, we show through analysis of TK conditional null and RNAi cells that TK is essential for growth and for infectivity in a mouse model, and that a catalytically active enzyme is required for its function. Unlike humans, T. brucei and all other kinetoplastids lack dCMP deaminase (DCTD), which provides an alternative route to dUMP formation. Ectopic expression of human DCTD resulted in full rescue of the RNAi growth phenotype and allowed for selection of viable TK null cells. Metabolite profiling by LC-MS/MS revealed a buildup of deoxypyrimidine nucleosides in TK depleted cells. Knockout of cytidine deaminase (CDA), which converts deoxycytidine to deoxyuridine led to thymidine/deoxyuridine auxotrophy. These unexpected results suggested that T. brucei encodes an unidentified 5'-nucleotidase that converts deoxypyrimidine nucleotides to their corresponding nucleosides, leading to their dead-end buildup in TK depleted cells at the expense of dTTP pools. Bioinformatics analysis identified several potential candidate genes that could encode 5'-nucleotidase activity including an HD-domain protein that we show catalyzes dephosphorylation of deoxyribonucleotide 5'-monophosphates. We conclude that TK is essential for synthesis of thymine nucleotides regardless of whether the nucleoside precursors originate from the de novo pathway or through salvage. Reliance on TK in the absence of DCTD may be a shared vulnerability among trypanosomatids and may provide a unique opportunity to selectively target a diverse group of pathogenic single-celled eukaryotes with a single drug.
Assuntos
Nucleotídeos/biossíntese , Timidina Quinase/metabolismo , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/enzimologia , Tripanossomíase Africana/parasitologia , Animais , Western Blotting , Cromatografia Líquida , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Pirimidinas/metabolismo , Espectrometria de Massas em Tandem , TransfecçãoRESUMO
Trypanosoma brucei is a protozoan that causes African sleeping sickness in humans. Many glycoconjugate compounds are present on the entire cell surface of Trypanosoma brucei to control the infectivity and survival of this pathogen. These gycoconjugates are anchored to the plasma membrane with the help of glycosyl phosphatidyl inositol (GPI) anchors. This type of anchor is much more common in protozoans than in other eukaryotes. The second step of glycosyl phosphatidyl inositol (GPI) anchor biosynthesis is catalyzed by an enzyme, which is GlcNAc-PI de-N-acetylase. GlcNAc-PI de-N-acetylase has a conserved GPI domain, which is responsible for the functionality of this enzyme. In this study, the three-dimensional structure of the target is modelled by I-TASSER and the ligand is modelled by PRODRG server. It is found that the predicted active site residues of the GPI domain are ultra-conserved for the Trypanosomatidae family. The predicted active site residues are His41, Pro42, Asp43, Asp44, Met47, Phe48, Ser74, Arg80, His103, Val144, Ser145, His147 and His150. Two hydrogen bond acceptors and four hydrogen bond donors are found in the modelled pharmacophore. All compounds of the Drugbank database and twenty three known inhibitors have been considered for structure based virtual screening. This work is focused on approved drugs because they are already tested for safety and effectiveness in humans. After the structure-based virtual screening, seventeen approved drugs and two inhibitors are found, which interact with the ligand on the basis of the designed pharmacophore. The docking has been performed for the resultant seventeen approved drugs and two known inhibitors. Two approved drugs have negative binding energy and their pKa values are similar to the selected known inhibitors. The result of this study suggests that the approved drugs Ethambutol (DB00330) and Metaraminol (DB00610) may prove useful in the treatment of African sleeping sickness.
Assuntos
Amidoidrolases/antagonistas & inibidores , Antimaláricos/farmacologia , Simulação por Computador , Reposicionamento de Medicamentos , Inibidores Enzimáticos/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico , Amidoidrolases/metabolismo , Antimaláricos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Etambutol/química , Etambutol/farmacologia , Humanos , Metaraminol/química , Metaraminol/farmacologia , Modelos Moleculares , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/enzimologia , Tripanossomíase Africana/metabolismoRESUMO
Human African trypanosomiasis (HAT) is a major health problem in sub-Saharan Africa caused by Trypanosoma brucei infection. Current HAT drugs are difficult to administer and not effective against all parasite species at different stages of the disease which indicates an unmet pharmaceutical need. TbRET2 is an indispensable enzyme for the parasite and is targeted here using a computational approach that combines molecular dynamics simulations and virtual screening. The compounds prioritized are then tested in T. brucei via Alamar blue cell viability assays. This work identified 20 drug-like compounds which are candidates for further testing in the drug discovery process.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , RNA Nucleotidiltransferases/antagonistas & inibidores , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/parasitologia , Desenho Assistido por Computador , Descoberta de Drogas , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , RNA Nucleotidiltransferases/metabolismo , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/enzimologiaRESUMO
African trypanosomes express three virtually identical non-selenium glutathione peroxidase (Px)-type enzymes which preferably detoxify lipid-derived hydroperoxides. As shown previously, bloodstream Trypanosoma brucei lacking the mitochondrial Px III display only a weak and transient proliferation defect whereas parasites that lack the cytosolic Px I and Px II undergo extremely fast lipid peroxidation and cell lysis. The phenotype can completely be rescued by supplementing the medium with the α-tocopherol derivative Trolox. The mechanism underlying the rapid cell death remained however elusive. Here we show that the lysosome is the origin of the cellular injury. Feeding the px I-II knockout parasites with Alexa Fluor-conjugated dextran or LysoTracker in the presence of Trolox yielded a discrete lysosomal staining. Yet upon withdrawal of the antioxidant, the signal became progressively spread over the whole cell body and was completely lost, respectively. T. brucei acquire iron by endocytosis of host transferrin. Supplementing the medium with iron or transferrin induced, whereas the iron chelator deferoxamine and apo-transferrin attenuated lysis of the px I-II knockout cells. Immunofluorescence microscopy with MitoTracker and antibodies against the lysosomal marker protein p67 revealed that disintegration of the lysosome precedes mitochondrial damage. In vivo experiments confirmed the negligible role of the mitochondrial peroxidase: Mice infected with px III knockout cells displayed only a slightly delayed disease development compared to wild-type parasites. Our data demonstrate that in bloodstream African trypanosomes, the lysosome, not the mitochondrion, is the primary site of oxidative damage and cytosolic trypanothione/tryparedoxin-dependent peroxidases protect the lysosome from iron-induced membrane peroxidation. This process appears to be closely linked to the high endocytic rate and distinct iron acquisition mechanisms of the infective stage of T. brucei. The respective knockout of the cytosolic px I-II in the procyclic insect form resulted in cells that were fully viable in Trolox-free medium.
Assuntos
Membrana Celular/metabolismo , Ferro/metabolismo , Peroxidases/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/enzimologia , Animais , Membrana Celular/genética , Feminino , Técnicas de Silenciamento de Genes , Lisossomos/enzimologia , Lisossomos/genética , Camundongos , Camundongos Endogâmicos BALB C , Peroxidases/genética , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética , Tripanossomíase Africana/genéticaRESUMO
The zinc-metalloenzyme GlcNAc-PI de-N-acetylase is essential for the biosynthesis of mature GPI anchors and has been genetically validated in the bloodstream form of Trypanosoma brucei, which causes African sleeping sickness. We screened a focused library of zinc-binding fragments and identified salicylic hydroxamic acid as a GlcNAc-PI de-N-acetylase inhibitor with high ligand efficiency. This is the first small molecule inhibitor reported for the trypanosome GPI pathway. Investigating the structure activity relationship revealed that hydroxamic acid and 2-OH are essential for potency, and that substitution is tolerated at the 4- and 5-positions.
Assuntos
Amidoidrolases/metabolismo , Ácidos Hidroxâmicos/química , Salicilatos/química , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/parasitologia , Amidoidrolases/antagonistas & inibidores , Humanos , Ligação Proteica , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Especificidade por Substrato , Tripanossomíase Africana/enzimologia , Zinco/química , Zinco/metabolismoRESUMO
A significant improvement in the treatment of trypanosomiases has been achieved with the recent development of nifurtimox-eflornithine combination therapy (NECT). As an alternative to drug combinations and as a means to overcome most of the antitrypanosomatid drug discovery challenges, a multitarget drug design strategy has been envisaged. To begin testing this hypothesis, we designed and developed a series of quinone-coumarin hybrids against glyceraldehyde-3-phosphate dehydrogenase/trypanothione reductase (GAPDH/TR). These enzymes belong to metabolic pathways that are vital to Trypanosoma brucei and Trypanosoma cruzi, and have thus been considered promising drug targets. The synthesized molecules were characterized for their dual-target antitrypanosomal profile, both in enzyme assays and in in vitro parasite cultures. The merged derivative 2-{[3-(3-dimethylaminopropoxy)-2-oxo-2H-chromen-7-yl]oxy}anthracene-1,4-dione (10) showed an IC50 value of 5.4 µM against TbGAPDH and a concomitant Ki value of 2.32 µM against TcTR. Notably, 2-{4-[6-(2-dimethylaminoethoxy)-2-oxo-2H-chromen-3-yl]phenoxy}anthracene-1,4-dione (compound 6) displayed a remarkable EC50 value for T.brucei parasites (0.026 µM) combined with a very low cytotoxicity toward mammalian L6 cells (7.95 µM). This promising low toxicity of compound 6 might be at least partially due to the fact that it does not interfere with human glutathione reductase.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , NADH NADPH Oxirredutases/antagonistas & inibidores , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/enzimologia , Trypanosoma cruzi/enzimologia , Animais , Antracenos/química , Antracenos/farmacologia , Linhagem Celular , Doença de Chagas/tratamento farmacológico , Doença de Chagas/enzimologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Modelos Moleculares , NADH NADPH Oxirredutases/metabolismo , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/enzimologiaRESUMO
Polyphosphate (polyP) is an anionic polymer of orthophosphate groups linked by high energy bonds that typically accumulates in acidic, calcium-rich organelles known as acidocalcisomes. PolyP synthesis in eukaryotes was unclear until it was demonstrated that the protein named Vtc4p (vacuolar transporter chaperone 4) is a long chain polyP kinase that localizes to the yeast vacuole. Here, we report that TbVtc4 (Vtc4 ortholog of Trypanosoma brucei) encodes, in contrast, a short chain polyP kinase that localizes to acidocalcisomes. The subcellular localization of TbVtc4 was demonstrated by fluorescence and electron microscopy of cell lines expressing TbVtc4 in its endogenous locus fused to an epitope tag and by purified polyclonal antibodies against TbVtc4. Recombinant TbVtc4 was expressed in bacteria, and polyP kinase activity was assayed in vitro. The in vitro growth of conditional knock-out bloodstream form trypanosomes (TbVtc4-KO) was significantly affected relative to the parental cell line. This mutant had reduced polyP kinase activity and short chain polyP content and was considerably less virulent in mice. The wild-type phenotype was recovered when an ectopic copy of the TbVtc4 gene was expressed in the presence of doxycycline. The mutant also exhibited a defect in volume recovery under osmotic stress conditions in vitro, underscoring the relevance of polyP in osmoregulation.
Assuntos
Chaperonas Moleculares/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/patogenicidade , Tripanossomíase Africana/enzimologia , Animais , Técnicas de Silenciamento de Genes , Camundongos , Chaperonas Moleculares/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética , Tripanossomíase Africana/genéticaRESUMO
Understanding African Trypanosomiasis (AT) host-pathogen interaction is the key to an "anti-disease vaccine", a novel strategy to control AT. Here we provide a better insight into this poorly described interaction by characterizing the activation of a panel of endothelial cells by bloodstream forms of four African trypanosome species, known to interact with host endothelium. T. congolense, T. vivax, and T. b. gambiense activated the endothelial NF-κB pathway, but interestingly, not T. b. brucei. The parasitic TS (trans-sialidases) mediated this NF-κB activation, remarkably via their lectin-like domain and induced production of pro-inflammatory molecules not only in vitro but also in vivo, suggesting a considerable impact on pathogenesis. For the first time, TS activity was identified in T. b. gambiense BSF which distinguishes it from the subspecies T. b. brucei. The corresponding TS were characterized and shown to activate endothelial cells, suggesting that TS represent a common mediator of endothelium activation among trypanosome species with divergent physiopathologies.
Assuntos
Células Endoteliais/metabolismo , Glicoproteínas/metabolismo , Mediadores da Inflamação/metabolismo , Neuraminidase/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma/enzimologia , Tripanossomíase Africana/enzimologia , Animais , Células Endoteliais/imunologia , Células Endoteliais/parasitologia , Feminino , Glicoproteínas/genética , Glicoproteínas/imunologia , Mediadores da Inflamação/imunologia , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/genética , NF-kappa B/imunologia , NF-kappa B/metabolismo , Neuraminidase/genética , Neuraminidase/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Trypanosoma/genética , Trypanosoma/imunologia , Tripanossomíase Africana/genética , Tripanossomíase Africana/imunologia , Tripanossomíase Africana/patologiaRESUMO
6-Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway (PPP), is essential for biosyntheses and oxidative stress defence. It also has the function of depleting 6PG, whose accumulation induces cell senescence. 6PGDH is a proposed drug target for African trypanosomiasis caused by Trypanosoma brucei and for other microbial infections and cancer. Gel filtration, density gradient sedimentation, cross-linking and dynamic light scattering were used to assay the oligomerization state of T. brucei 6PGDH in the absence and presence of several ligands. The enzyme displays a dimer-tetramer equilibrium and NADPH (but not NADP) reduces the rate of approach to equilibrium, while 6PG is able to antagonize the NADPH effect. The different behaviour of the two forms of coenzyme appears to be related to the differences in ΔCp, with NADP binding ΔCp closer to what is expected of crystallographic structures, while NADPH ΔCp is three times larger. The estimated dimer-tetramer association constant is 1.5·10(6)M(-1), and the specific activity of the tetramer is about 3 fold higher than the specific activity of the dimer. Thus, cellular conditions promoting tetramer formation could allow an efficient clearing of 6PG. Experiments carried out on sheep liver 6PGDH indicate that tetramerization is a specificity of the parasite enzyme.
Assuntos
NADP/química , Fosfogluconato Desidrogenase/química , Multimerização Proteica/fisiologia , Trypanosoma brucei brucei/enzimologia , Animais , NADP/metabolismo , Fosfogluconato Desidrogenase/metabolismo , Ovinos , Especificidade da Espécie , Tripanossomíase Africana/enzimologiaRESUMO
Trypanosoma brucei is the causing agent of African trypanosomiasis. These parasites possess a unique thiol redox system required for DNA synthesis and defense against oxidative stress. It includes trypanothione and trypanothione reductase (TryR) instead of the thioredoxin and glutaredoxin systems of mammalian hosts. Here, we show that the benzisothiazolone compound ebsulfur (EbS), a sulfur analogue of ebselen, is a potent inhibitor of T. brucei growth with a favorable selectivity index over mammalian cells. EbS inhibited the TryR activity and decreased non-protein thiol levels in cultured parasites. The inhibition of TryR by EbS was irreversible and NADPH-dependent. EbS formed a complex with TryR and caused oxidation and inactivation of the enzyme. EbS was more toxic for T. brucei than for Trypanosoma cruzi, probably due to lower levels of TryR and trypanothione in T. brucei. Furthermore, inhibition of TryR produced high intracellular reactive oxygen species. Hydrogen peroxide, known to be constitutively high in T. brucei, enhanced the EbS inhibition of TryR. The elevation of reactive oxygen species production in parasites caused by EbS induced a programmed cell death. Soluble EbS analogues were synthesized and cured T. brucei brucei infection in mice when used together with nifurtimox. Altogether, EbS and EbS analogues disrupt the trypanothione system, hampering the defense against oxidative stress. Thus, EbS is a promising lead for development of drugs against African trypanosomiasis.
Assuntos
NADH NADPH Oxirredutases/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Tiazóis/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/tratamento farmacológico , Animais , Masculino , Camundongos , NADH NADPH Oxirredutases/metabolismo , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/farmacologia , Proteínas de Protozoários/metabolismo , Tiazóis/química , Tripanossomicidas/química , Tripanossomíase Africana/enzimologiaRESUMO
In many bacteria, heat shock locus V (HslV) functions as a protease, which is activated by heat shock locus U (HslU). The primary sequence and structure of HslV are well conserved with those of the ß-subunit of the 20 S proteasome core particle in eukaryotes. To date, the HslVU complex has only been characterized in the prokaryotic system. Recently, however, the coexistence of a 20 S proteasome with HslV protease in the same living organism has been reported. In Trypanosoma brucei, a protozoan parasite that causes human sleeping sickness in Africa, HslV is localized in the mitochondria, where it has a novel function in regulating mitochondrial DNA replication. Although the prokaryotic HslVU system has been studied extensively, little is known regarding its eukaryotic counterpart. Here, we report the biochemical characteristics of an HslVU complex from T. brucei. In contrast to the prokaryotic system, T. brucei possesses two potential HslU molecules, and we found that only one of them activates HslV. A key activating residue, Tyr(494), was identified in HslU2 by biochemical and mutational studies. Furthermore, to our knowledge, this study is the first to report the crystal structure of a eukaryotic HslV, determined at 2.4 Å resolution. Drawing on our comparison of the biochemical and structural data, we discuss herein the differences and similarities between eukaryotic and prokaryotic HslVs.
Assuntos
Proteínas Mitocondriais/química , Peptídeo Hidrolases/química , Proteínas de Protozoários/química , Trypanosoma brucei brucei/enzimologia , Cristalografia por Raios X , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Estrutura Quaternária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/genética , Tripanossomíase Africana/enzimologia , Tripanossomíase Africana/genéticaRESUMO
Human African trypanosomes are blood parasites that cause sleeping sickness, a debilitating disease in sub-Saharan Africa. Glycerol kinase (GK) of these parasites additionally possesses a novel property of reverse catalysis. GK is essential to blood stream form trypanosome, and therefore a promising drug target. Here, utilizing recombinant DNA technology an optimized procedure for obtaining large amount of the purified protein was established. Furthermore, biochemical data on its enzymology are reported. The protein was maximally active at pH 6.8 over a temperature range of 25-70°C, with activation energy of 34.02 ± 0.31 kJ mol(-1). The enzyme catalyses a reversible bisubstrate [ADP and glycerol 3-phosphate (G3P)]-biproduct (ATP and glycerol) reaction. It has Km of 0.90 and 5.54 mM for ADP and G3P, respectively, and Vmax of 25.3 and 20.0 µmol min(-1) mg(-1), respectively. Unexpectedly, the enzyme lost more than 50% of its activity in 48 h at 4°C in 0.1 M sodium phosphate buffer pH 6.8 containing 10 mM MgSO4. However, perfect stabilization of the GK for more than 4 weeks was achieved in the presence of its natural ligands and cofactor. Using this stabilized protein, crystals of trypanosome GK with better resolution were obtained. This will accelerate the success of GK inhibitor development for drug design.
Assuntos
Glicerol Quinase/química , Proteínas de Protozoários/química , Trypanosoma brucei gambiense/enzimologia , Cristalização , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Genes de Protozoários , Glicerol Quinase/antagonistas & inibidores , Glicerol Quinase/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Trypanosoma brucei gambiense/genética , Trypanosoma brucei gambiense/patogenicidade , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/enzimologia , Tripanossomíase Africana/parasitologiaRESUMO
Nicotinamide, a soluble compound of the vitamin B3 group, has antimicrobial activity against several microorganisms ranging from viruses to parasite protozoans. However, the mode of action of this antimicrobial activity is unknown. Here, we investigate the trypanocidal activity of nicotinamide on Trypanosoma brucei, the causative agent of African trypanosomiasis. Incubation of trypanosomes with nicotinamide causes deleterious defects in endocytic traffic, disruption of the lysosome, failure of cytokinesis, and, ultimately, cell death. At the same concentrations there was no effect on a cultured mammalian cell line. The effects on endocytosis and vesicle traffic were visible within 3 h and can be attributed to inhibition of lysosomal cathepsin b-like protease activity. The inhibitory effect of nicotinamide was confirmed by a direct activity assay of recombinant cathepsin b-like protein. Taken together, these data demonstrate that inhibition of the lysosomal protease cathepsin b-like blocks endocytosis, causing cell death. In addition, these results demonstrate for the first time the inhibitory effect of nicotinamide on a protease.
Assuntos
Catepsina B/antagonistas & inibidores , Niacinamida/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Trypanosoma brucei brucei/enzimologia , Complexo Vitamínico B/farmacologia , Catepsina B/metabolismo , Endocitose/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/parasitologia , Humanos , Lisossomos/enzimologia , Transporte Proteico/efeitos dos fármacos , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/citologia , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/enzimologiaRESUMO
Polo-like kinases (Plks) play multiple roles in mitosis and cytokinesis in eukaryotes and are characterized by the C-terminal Polo-box domain (PBD), which is implicated in binding to Plk substrates, targeting Plk and regulating Plk activity. The Plk homolog in Trypanosoma brucei (TbPLK) possesses a similar architecture, but it lacks the crucial residues involved in substrate binding and regulates cytokinesis but not mitosis. Little is known about the regulation of TbPLK and the role of the PBD in TbPLK localization and function. Here, we addressed the requirement of the kinase activity and the PBD for TbPLK localization and function through coupling RNAi of endogenous TbPLK with ectopic expression of TbPLK mutants. We demonstrate that the kinase activity and phosphorylation of two threonine residues, Thr198 and Thr202, in the activation loop (T-loop) of the kinase domain are essential for TbPLK function but not for TbPLK localization. Deletion of the PBD abolishes TbPLK localization, but the PBD itself is not correctly targeted, indicating that TbPLK localization requires both the PBD and the kinase domain. Surprisingly, the kinase domain of TbPLK, but not the PBD, binds to its substrates TbCentrin2 and p110, suggesting that TbPLK might interact with its substrate through different mechanisms. Finally, the PBD interacts with the kinase domain of TbPLK and inhibits its activity, and this inhibition is relieved when Thr198 is phosphorylated. Together, these results suggest an essential role of T-loop phosphorylation in TbPLK activation and crucial roles of the PBD in regulating TbPLK activity and localization.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas de Protozoários/fisiologia , Trypanosoma brucei brucei/enzimologia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Células HeLa , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Relação Estrutura-Atividade , Trypanosoma brucei brucei/crescimento & desenvolvimento , Tripanossomíase Africana/enzimologia , Tripanossomíase Africana/parasitologia , Quinase 1 Polo-LikeRESUMO
Human African trypanosomiasis (HAT), caused by the protozoan parasite Trypanosoma brucei, is a neglected fatal disease. Leucyl-tRNA synthetase (LeuRS), which has been successfully applied in the development of antifungal agent, represents a potential antiprotozoal drug target. In this study, a 3D model of T. brucei LeuRS (TbLeuRS) synthetic active site was constructed and subjected to virtual screening using a combination of pharmacophore- and docking-based methods. A new 2-pyrrolinone scaffold was discovered and the structure-activity relationship (SAR) studies aided by the docking model and organic synthesis were carried out. Compounds with various substituents on R(1), R(2) and R(3) were synthesized and their SAR was discussed.
Assuntos
Antiprotozoários/química , Antiprotozoários/farmacologia , Desenho de Fármacos , Leucina-tRNA Ligase/antagonistas & inibidores , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/tratamento farmacológico , Sequência de Aminoácidos , Antiprotozoários/síntese química , Domínio Catalítico , Humanos , Leucina-tRNA Ligase/química , Leucina-tRNA Ligase/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Relação Estrutura-Atividade , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/enzimologiaRESUMO
BACKGROUND: Involvement of nitric oxide (NO) in the pathophysiology of human African trypanosomiasis (HAT) was analyzed in a HAT animal model (rat infected with Trypanosoma brucei brucei). With this model, it was previously reported that trypanosomes were capable of limiting trypanocidal properties carried by NO by decreasing its blood concentration. It was also observed that brain NO concentration, contrary to blood, increases throughout the infection process. The present approach analyses the brain impairments occurring in the regulations exerted by arginase and N(G), N(G)-dimethylarginine dimethylaminohydrolase (DDAH) on NO Synthases (NOS). In this respect: (i) cerebral enzymatic activities, mRNA and protein expression of arginase and DDAH were determined; (ii) immunohistochemical distribution and morphometric parameters of cells expressing DDAH-1 and DDAH-2 isoforms were examined within the diencephalon; (iii) amino acid profiles relating to NOS/arginase/DDAH pathways were established. METHODOLOGY/PRINCIPAL FINDINGS: Arginase and DDAH activities together with mRNA (RT-PCR) and protein (western-blot) expressions were determined in diencephalic brain structures of healthy or infected rats at various days post-infection (D5, D10, D16, D22). While arginase activity remained constant, that of DDAH increased at D10 (+65%) and D16 (+51%) in agreement with western-blot and amino acids data (liquid chromatography tandem-mass spectrometry). Only DDAH-2 isoform appeared to be up-regulated at the transcriptional level throughout the infection process. Immunohistochemical staining further revealed that DDAH-1 and DDAH-2 are contained within interneurons and neurons, respectively. CONCLUSION/SIGNIFICANCE: In the brain of infected animals, the lack of change observed in arginase activity indicates that polyamine production is not enhanced. Increases in DDAH-2 isoform may contribute to the overproduction of NO. These changes are at variance with those reported in the periphery. As a whole, the above processes may ensure additive protection against trypanosome entry into the brain, i.e., maintenance of NO trypanocidal pressure and limitation of polyamine production, necessary for trypanosome growth.
Assuntos
Amidoidrolases/metabolismo , Arginase/metabolismo , Encéfalo/enzimologia , Tripanossomíase Africana/enzimologia , Amidoidrolases/genética , Aminoácidos/sangue , Aminoácidos/química , Animais , Arginase/genética , Vias Biossintéticas , Encéfalo/parasitologia , Encéfalo/patologia , Modelos Animais de Doenças , Progressão da Doença , Regulação Enzimológica da Expressão Gênica , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Espectrometria de Massas , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Trypanosoma brucei brucei , Tripanossomíase Africana/sangue , Tripanossomíase Africana/parasitologia , Tripanossomíase Africana/patologiaRESUMO
The prodrug nifurtimox has been used for more than 40 years to treat Chagas disease and forms part of a recently approved combinational therapy that targets West African trypanosomiasis. Despite this, its mode of action is poorly understood. Detection of reactive oxygen and nitrogen intermediates in nifurtimox-treated extracts led to the proposal that this drug induces oxidative stress in the target cell. Here, we outline an alternative mechanism involving reductive activation by a eukaryotic type I nitroreductase. Several enzymes proposed to metabolize nifurtimox, including prostaglandin F2α synthase and cytochrome P450 reductase, were overexpressed in bloodstream-form Trypanosoma brucei. Only cells with elevated levels of the nitroreductase displayed altered susceptibility to this nitrofuran, implying a key role in drug action. Reduction of nifurtimox by this enzyme was shown to be insensitive to oxygen and yields a product characterized by LC/MS as an unsaturated open-chain nitrile. This metabolite was shown to inhibit both parasite and mammalian cell growth at equivalent concentrations, in marked contrast to the parental prodrug. These experiments indicate that the basis for the selectivity of nifurtimox against T. brucei lies in the expression of a parasite-encoded type I nitroreductase.