RESUMO
Trypanosoma cruzi, the etiological agent of Chagas disease, has a digenetic life cycle. In its passage from the insect vector to the mammalian host, and vice versa, it must be prepared to cope with abrupt changes in environmental conditions, such as carbon source, pH, temperature and osmolarity, in order to survive. Sensing and signaling pathways that allow the parasite to adapt, have unique characteristics with respect to their hosts and other free-living organisms. Many of the canonical proteins involved in these transduction pathways have not yet been found in the genomes of these parasites because they present divergences either at the functional, structural and/or protein sequence level. All of this makes these pathways promising targets for therapeutic drugs. The AMP-activated protein kinase (AMPK) is a serine/threonine kinase activated by environmental stresses such as osmotic stress, hypoxia, ischaemia and exercise that results in reduction of ATP and increase of AMP levels. Thus, AMPK is regarded as a fuel gauge, functioning both as a nutrient and an energy sensor, to maintain energy homeostasis and, eventually, to protect cells from death by nutrient starvation. In the present study we report the characterization of AMPK complexes for the first time in T. cruzi and propose the function of TcAMPK as a novel regulator of nutritional stress in epimastigote forms. We show that there is phosphotransferase activity specific for SAMS peptide in epimastigotes extracts, which is inhibited by Compound C and is modulated by carbon source availability. In addition, TcAMPKα2 subunit has an unprecedented functional substitution (Ser x Thr) at the activation loop and its overexpression in epimastigotes led to higher autophagic activity during prolonged nutritional stress. Moreover, the over-expression of the catalytic subunits resulted in antagonistic phenotypes associated with proliferation. Together, these results point to a role of TcAMPK in autophagy and nutrient sensing, key processes for the survival of trypanosomatids and for its life cycle progression.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/metabolismo , Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/genética , Autofagia , Metabolismo Energético , Proteínas Serina-Treonina Quinases/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Estresse Fisiológico , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Intracellular levels of cyclic nucleotide second messengers are regulated predominantly by a large superfamily of phosphodiesterases (PDEs). Most of the different PDE variants play specific physiological functions; in fact, PDEs can associate with other proteins allowing them to be strategically anchored throughout the cell. In this regard, precise cellular expression and compartmentalization of these enzymes produce the specific control of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) gradients in cells and enable their integration with other signaling pathways.In trypanosomatids, some PDEs are essential for their survival and play fundamental roles in the adaptation of these parasites to different environmental stresses, as well as in the differentiation between their different life cycle forms. Given that these enzymes not only are similar to human PDEs but also have differential biochemical properties, and due to the great knowledge of drugs that target human PDEs, trypanosomatid PDEs could be postulated as important therapeutic targets through the repositioning of drugs.In this chapter, we describe a simple and sensitive radioisotope-based method to measure cyclic 3',5'-nucleotide phosphodiesterase using [3H]cAMP.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/isolamento & purificação , Ensaios Enzimáticos/métodos , Marcação por Isótopo/métodos , Proteínas de Protozoários/isolamento & purificação , Trypanosoma cruzi/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/química , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , AMP Cíclico/química , AMP Cíclico/metabolismo , Estágios do Ciclo de Vida , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Trítio/químicaRESUMO
Among the many environmental challenges the parasite Trypanosoma cruzi has to overcome to complete its life cycle through different hosts, oxidative stress plays a central role. Different stages of this parasite encounter distinct sources of oxidative stress, such as the oxidative burst of the immune system, or the Heme released from hemoglobin degradation in the triatomine's midgut. Also, the redox status of the surroundings functions as a signal to the parasite, triggering processes coupled to differentiation or proliferation. Intracellular second messengers, like cAMP, are responsible for the transduction of environmental queues and initiating cellular processes accordingly. In trypanosomatids cAMP is involved in a variety of processes, including proliferation, differentiation, osmoregulation and quorum sensing. Trypanosomatid phosphodiesterases (PDE) show atypical pharmacological properties and some have been involved in key processes for the survival of the parasites, which validates them as attractive therapeutic targets. Our work here shows that cAMP modulates different processes according to parasite stage. Epimastigotes become more resistant to oxidative stress when pre-treated with cAMP analogs, while in trypomastigotes an increase in intracellular cAMP doesn't seem to aid in this response, although it does increase the number of amastigotes obtained 48 h after infection, compared to the control group. Also, we show that TcrPDEA1, a functionally enigmatic phosphodiesterase with very high Km, is involved in the epimastigotes response to oxidative stress.
Assuntos
AMP Cíclico/metabolismo , Citoplasma/metabolismo , Trypanosoma cruzi/fisiologia , Animais , Chlorocebus aethiops , Estágios do Ciclo de Vida , Oxirredução , Células VeroRESUMO
Trypanosomatids are a group of flagellated unicellular eukaryotes, causing serious human diseases including Chagas disease (Trypanosoma cruzi), sleeping sickness (Trypanosoma brucei spp.) and Leishmaniasis (Leishmania spp.). The second messenger cAMP is involved in numerous and fundamental processes in these parasites including differentiation between stages, proliferation, osmoregulation, oxidative stress and quorum sensing. Interestingly, its signaling pathway is quite different from that of mammals, including structurally different adenylyl cyclases, the shortage of orthologous effector proteins and the absence of G-protein-coupled-receptors, among others. These characteristics make the proteins involved in these transduction pathways good candidates for therapeutic targets. However, the identification of new unknown druggable targets involves extensive research time and is economically very expensive, making difficult the transition from basic research to the clinical phase. Trypanosomatid PDEs have characteristic binding pockets that allow for a differential inhibition from their human orthologs. Modification in the approved drugs for human to convert them into trypanocidal treatments could lead to more effective therapies, shorter lab time and lower costs. In view of the fact that kinetoplastid PDEs are highly conserved with their mammalian counterparts, and since there are already numerous drugs on the market against human PDEs, the drug repositioning approach is highly promising. The development of new technologies, higher government and industrial involvement and more scientists committed to basic investigation, are the key to ultimately find an effective treatment and cure for the neglected tropical diseases.
Assuntos
Inibidores de Fosfodiesterase/farmacologia , Transdução de Sinais/efeitos dos fármacos , Adenilil Ciclases/fisiologia , Cálcio/fisiologia , Doença de Chagas/tratamento farmacológico , Reposicionamento de Medicamentos , Humanos , Leishmania donovani/enzimologia , Leishmania donovani/fisiologia , Proteínas Quinases/fisiologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/fisiologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/fisiologiaRESUMO
The class III phosphatidylinositol 3-kinase (PI3K) Vps34 is an important regulator of key cellular functions, including cell growth, survival, intracellular trafficking, autophagy and nutrient sensing. In yeast, Vps34 is associated with the putative serine/threonine protein kinase Vps15, however, its role in signaling has not been deeply evaluated. Here, we have identified the Vps15 orthologue in Trypanosoma brucei, named TbVps15. Knockdown of TbVps15 expression by interference RNA resulted in inhibition of cell growth and blockage of cytokinesis. Scanning electron microcopy revealed a variety of morphological abnormalities, with enlarged parasites and dividing cells that often exhibited a detached flagellum. Transmission electron microscopy analysis of TbVps15 RNAi cells showed an increase in intracellular vacuoles of the endomembrane system and some cells displayed an enlargement of the flagellar pocket, a common feature of cells defective in endocytosis. Moreover, uptake of dextran, transferrin and Concanavalin A was impaired. Finally, TbVps15 downregulation affected the PI3K activity, supporting the hypothesis that TbVps15 and TbVps34 form a complex as occurs in other organisms. In summary, we propose that TbVps15 has a role in the maintenance of cytokinesis, endocytosis and intracellular trafficking in T. brucei.
Assuntos
Citocinese , Endocitose , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/fisiologia , Proteína VPS15 de Distribuição Vacuolar/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Transmissão de Doença Infecciosa , Técnicas de Silenciamento de Genes , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Fosfatidilinositol 3-Quinase/análise , Ligação Proteica , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/genética , Proteína VPS15 de Distribuição Vacuolar/genéticaRESUMO
Autophagy is a degradative process by which eukaryotic cells digest their own components to provide aminoacids that may function as energy source under nutritional stress conditions. There is experimental evidence for autophagy in parasitic protists belonging to the family Trypanosomatidae. However, few proteins implicated in this process have been characterized so far in these parasites. Moreover, it has been shown that autophagy is involved in Trypanosoma cruzi differentiation and thus might have a role in pathogenicity. Here, we report the cloning and biochemical characterization of TcVps15. In addition, we demonstrate that TcVps15 interact with the PI3K TcVps34 and that both proteins associate with cellular membranes. Under nutritional stress conditions, TcVps15 and TcVps34 modify their subcellular distribution showing a partial co-localization in autophagosomes with TcAtg8.1 and using an active site TcVps15-mutated version (TcVps15-K219D-HA) we demonstrated that this relocalization depends on the TcVps15 catalytic activity. Overexpression of TcVps15-HA and TcVps15-K219D-HA also leads to increased accumulation of monodansylcadaverine (MDC) in autophagic vacuoles under nutritional stress conditions compared to wild-type cells. In addition, the MDC-specific activity shows to be significantly higher in TcVps15-HA overexpressing cells when compared with TcVps15-K219D-HA. Our results reveal for the first time a role of TcVps15 as a key regulator of TcVps34 enzymatic activity and implicate the TcVps15-Vps34 complex in autophagy in T. cruzi, exposing a new key pathway to explore novel chemotherapeutic targets.