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1.
Biochim Biophys Acta Mol Cell Res ; 1867(7): 118694, 2020 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-32151656

RESUMO

Dot1 enzymes are histone methyltransferases that mono-, di- and trimethylate lysine 79 of histone H3 to affect several nuclear processes. The functions of these different methylation states are still largely unknown. Trypanosomes, which are flagellated protozoa that cause several parasitic diseases, have two Dot1 homologues. Dot1A catalyzes the mono- and dimethylation of lysine 76 during late G2 and mitosis, and Dot1B catalyzes trimethylation, which is a modification found in all stages of the cell cycle. Here, we generated Trypanosoma cruzi lines lacking Dot1B. Deletion of one allele resulted in parasites with increased levels of mono- and dimethylation and a reduction in H3K76me3. In the full knockout (DKO), no trimethylation was observed. Both the DKO and the single knockout (SKO) showed aberrant morphology and decreased growth due to cell cycle arrest after G2. This phenotype could be rescued by caffeine in the DKO, as caffeine is a checkpoint inhibitor of the cell cycle. The knockouts also phosphorylated γH2A without producing extensive DNA breaks, and Dot1B-depleted cells were more susceptible to general checkpoint kinase inhibitors, suggesting that a lack of H3K76 trimethylation prevents the initiation and/or completion of cytokinesis.

2.
Artigo | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: but-ib17532

RESUMO

Dot1 enzymes are histone methyltransferases that mono-, di- and trimethylate lysine 79 of histone H3 to affect several nuclear processes. The functions of these different methylation states are still largely unknown. Trypanosomes, which are flagellated protozoa that cause several parasitic diseases, have two Dot1 homologues. Dot1A catalyzes the mono- and dimethylation of lysine 76 during late G2 and mitosis, and Dot1B catalyzes trimethylation, which is a modification found in all stages of the cell cycle. Here, we generated Trypanosoma cruzi lines lacking Dot1B. Deletion of one allele resulted in parasites with increased levels of mono- and dimethylation and a reduction in H3K76me3. In the full knockout (DKO), no trimethylation was observed. Both the DKO and the single knockout (SKO) showed aberrant morphology and decreased growth due to cell cycle arrest after G2. This phenotype could be rescued by caffeine in the DKO, as caffeine is a checkpoint inhibitor of the cell cycle. The knockouts also phosphorylated ?H2A without producing extensive DNA breaks, and Dot1B-depleted cells were more susceptible to general checkpoint kinase inhibitors, suggesting that a lack of H3K76 trimethylation prevents the initiation and/or completion of cytokinesis.

3.
Trends Parasitol ; 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31708398
4.
J Leukoc Biol ; 106(3): 581-594, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31299112

RESUMO

Chronic lymphocytic leukemia (CLL) is a chronic form of leukemia that originates from an abnormal expansion of CD5+ B-1 cells. Deregulation in the BCR signaling is associated with B-cell transformation. Contrariwise to B-2 cells, BCR engagement in B-1 cells results in low proliferation rate and increased apoptosis population, whereas overactivation may be associated with lymphoproliferative disorders. It has been demonstrated that several transcription factors that are involved in the B cell development play a role in the regulation of BCR function. Among them, Ikaros is considered an essential regulator of lymphoid differentiation and activation. Several reports suggest that Ikaros expression is deregulated in different forms of leukemia. Herein, we demonstrated that CLL cells show decreased Ikaros expression and abnormal cytoplasmic cell localization. These alterations were also observed in radioresistant B-1 cells, which present high proliferative activity, suggesting that abnormal localization of Ikaros could determine its loss of function. Furthermore, Ikaros knockdown increased the expression of BCR pathway components in murine B-1 cells, such as Lyn, Blnk, and CD19. Additionally, in the absence of Ikaros, B-1 cells become responsive to BCR stimulus, increasing cell proliferation even in the absence of antigen stimulation. These results suggested that Ikaros is an important controller of B-1 cell proliferation by interfering with the BCR activity. Therefore, altered Ikaros expression in CLL or radioresistant B-1 cells could determine a responsive status of BCR to self-antigens, which would culminate in the clonal expansion of B-1 cells.

5.
Front Microbiol ; 9: 2286, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30333803

RESUMO

Sporotrichosis is a mycosis that affects the skin, lymphatic system and other organs in humans and animals. The disease has a worldwide distribution, with endemic areas in Brazil, and is caused by a complex of species, including Sporothrix brasiliensis. Some fungi release extracellular vesicles (EVs) that can interact with the host cell and modulate the host immune response. The aim of this study was to analyze the participation of S. brasiliensis EVs in the modulation of dendritic cells (DCs) and in the control of infection in vivo. Our results showed that in vitro, the EVs isolated from S. brasiliensis induced an increase in the phagocytic index and fungal burden in DCs. In addition, we observed a significant increase in IL-12p40 and TNF-α cytokine production. Then, the EVs were inoculated into BALB/c mice before subcutaneous infection with yeast, and the lesion was analyzed after 21, 35, and 42 days. An increase in fungal burden and lesion diameter were observed after 21 days in mice inoculated with a high concentration of EVs. However, after 35 days, we observed a regression of the lesion, which persisted until 42 days after infection. Interestingly, we observed an increase in fungal burden in these mice. In addition, we observed the presence of immunogenic components and proteins that could be related with virulence in EVs. These results suggest that EVs can play an important role in virulence and modulation of the host immune system during experimental S. brasiliensis infection.

6.
J Proteome Res ; 17(1): 374-385, 2018 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-29168382

RESUMO

Protein acetylation is a post-translational modification regulating diverse cellular processes. By using proteomic approaches, we identified N-terminal and ε-lysine acetylated proteins in Trypanosoma cruzi and Trypanosoma brucei, which are protozoan parasites that cause significant human and animal diseases. We detected 288 lysine acetylation sites in 210 proteins of procyclic form, an insect stage of T. brucei, and 380 acetylation sites in 285 proteins in the form of the parasite that replicates in mammalian bloodstream. In T. cruzi insect proliferative form we found 389 ε-lysine-acetylated sites in 235 proteins. Notably, we found distinct acetylation profiles according to the developmental stage and species, with only 44 common proteins between T. brucei stages and 18 in common between the two species. While K-ac proteins from T. cruzi are enriched in enzymes involved in oxidation/reduction balance, required for the parasite survival in the host, in T. brucei, most K-ac proteins are enriched in metabolic processes, essential for its adaptation in its hosts. We also identified in both parasites a quite variable N-terminal acetylation sites. Our results suggest that protein acetylation is involved in differential regulation of multiple cellular processes in Trypanosomes, contributing to our understanding of the essential mechanisms for parasite infection and survival.


Assuntos
Acetilação , Lisina/metabolismo , Proteômica/métodos , Proteínas de Protozoários/metabolismo , Trypanosoma/química , Proteínas de Protozoários/análise , Trypanosoma/enzimologia , Trypanosoma brucei brucei/metabolismo , Trypanosoma cruzi/metabolismo
7.
Immunobiology ; 223(2): 252-257, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29107383

RESUMO

Ikaros is a broad transcription factor pointed as a critical regulator of lymphocyte development. Recent reports have emphasized that distinct isoforms of Ikaros control the dichotomy of the hematopoietic system into lymphoid and myeloid lineages. In addition, expression of dominant-negative isoforms of Ikaros is linked to abnormal hematopoiesis, which could culminate in hematological disorders due to loss of function of the protein. B-1 cells are an intriguing subtype of B-lymphocytes that preserves some myeloid characteristics. These cells are able to differentiate into phagocytes (B-1CDP - B-1 cell derived phagocytes) in vitro and in vivo. During such process, reprogramming of gene expression occurs: lymphoid genes are turned off, while expression of myeloid genes is increased. This study aims to investigate whether Ikaros could be related to the control of B-1 cell plasticity. Interestingly, Ikaros expression by B-1CDP cells was found to be relatively low, and the protein is abnormally localized in the cytoplasm. Moreover, the isoforms expressed by B-1 cells are different from those expressed by other lymphocytes, with expression of active isoforms being almost absent in B-1CDP. Based on these findings, Ikaros could be an important factor driving the differentiation and proliferation of B-1 cells.


Assuntos
Linfócitos B/imunologia , Fator de Transcrição Ikaros/metabolismo , Fagócitos/imunologia , Isoformas de Proteínas/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Plasticidade Celular , Células Cultivadas , Regulação da Expressão Gênica , Hematopoese , Fator de Transcrição Ikaros/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Isoformas de Proteínas/genética
8.
Front. Microbiol. ; 9: 2286, 2018.
Artigo | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: but-ib15602

RESUMO

Sporotrichosis is a mycosis that affects the skin, lymphatic system and other organs in humans and animals. The disease has a worldwide distribution, with endemic areas in Brazil, and is caused by a complex of species, including Sporothrix brasiliensis. Some fungi release extracellular vesicles (EVs) that can interact with the host cell and modulate the host immune response. The aim of this study was to analyze the participation of S. brasiliensis EVs in the modulation of dendritic cells (DCs) and in the control of infection in vivo. Our results showed that in vitro, the EVs isolated from S. brasiliensis induced an increase in the phagocytic index and fungal burden in DCs. In addition, we observed a significant increase in IL-12p40 and TNF-alpha cytokine production. Then, the EVs were inoculated into BALB/c mice before subcutaneous infection with yeast, and the lesion was analyzed after 21, 35, and 42 days. An increase in fungal burden and lesion diameter were observed after 21 days in mice inoculated with a high concentration of EVs. However, after 35 days, we observed a regression of the lesion, which persisted until 42 days after infection. Interestingly, we observed an increase in fungal burden in these mice. In addition, we observed the presence of immunogenic components and proteins that could be related with virulence in EVs. These results suggest that EVs can play an important role in virulence and modulation of the host immune system during experimental S. brasiliensis infection.

9.
Parasitology ; 144(11): 1498-1510, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28653592

RESUMO

Trypanosoma cruzi is exposed to oxidative stresses during its life cycle, and amongst the strategies employed by this parasite to deal with these situations sits a peculiar trypanothione-dependent antioxidant system. Remarkably, T. cruzi's antioxidant repertoire does not include catalase. In an attempt to shed light on what are the reasons by which this parasite lacks this enzyme, a T. cruzi cell line stably expressing catalase showed an increased resistance to hydrogen peroxide (H2O2) when compared with wild-type cells. Interestingly, preconditioning carried out with low concentrations of H2O2 led untransfected parasites to be as much resistant to this oxidant as cells expressing catalase, but did not induce the same level of increased resistance in the latter ones. Also, presence of catalase decreased trypanothione reductase and increased superoxide dismutase levels in T. cruzi, resulting in higher levels of residual H2O2 after challenge with this oxidant. Although expression of catalase contributed to elevated proliferation rates of T. cruzi in Rhodnius prolixus, it failed to induce a significant increase of parasite virulence in mice. Altogether, these results indicate that the absence of a gene encoding catalase in T. cruzi has played an important role in allowing this parasite to develop a shrill capacity to sense and overcome oxidative stress.


Assuntos
Catalase/metabolismo , Estresse Oxidativo , Transdução de Sinais , Trypanosoma cruzi/metabolismo , Animais , Catalase/genética , Linhagem Celular , Doença de Chagas/parasitologia , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Camundongos , NADH NADPH Oxirredutases/metabolismo , Rhodnius/parasitologia , Superóxido Dismutase/metabolismo , Transfecção , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/patogenicidade
10.
Cell Biol Int ; 41(1): 2-7, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27862595

RESUMO

The advance in biochemical and microscopy techniques has revealed the complexity and intricate nucleoplasm structure. Several subcompartments were identified in nucleus and the importance of these subcompartments in processes crucial for normal nuclear activity has been demonstrated. In this mini-review, we will give an overview about the composition, function, and importance of the major nuclear subcompartments. Also, we will show the impact that perturbing these structures can cause in normal nuclear activity, and how these can contribute to the development of some human diseases.


Assuntos
Núcleo Celular/metabolismo , Animais , Ciclo Celular , Doença , Humanos , RNA/metabolismo , Ribonucleoproteínas/biossíntese , Frações Subcelulares/metabolismo
11.
Cell Chem Biol ; 23(5): 608-617, 2016 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-27133314

RESUMO

Kinetoplastids cause Chagas disease, human African trypanosomiasis, and leishmaniases. Current treatments for these diseases are toxic and inefficient, and our limited knowledge of drug targets and inhibitors has dramatically hindered the development of new drugs. Here we used a chemogenetic approach to identify new kinetoplastid drug targets and inhibitors. We conditionally knocked down Trypanosoma brucei inositol phosphate (IP) pathway genes and showed that almost every pathway step is essential for parasite growth and infection. Using a genetic and chemical screen, we identified inhibitors that target IP pathway enzymes and are selective against T. brucei. Two series of these inhibitors acted on T. brucei inositol polyphosphate multikinase (IPMK) preventing Ins(1,4,5)P3 and Ins(1,3,4,5)P4 phosphorylation. We show that IPMK is functionally conserved among kinetoplastids and that its inhibition is also lethal for Trypanosoma cruzi. Hence, IP enzymes are viable drug targets in kinetoplastids, and IPMK inhibitors may aid the development of new drugs.


Assuntos
Fosfatos de Inositol/metabolismo , Parasitos/efeitos dos fármacos , Parasitos/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Animais , Relação Dose-Resposta a Droga , Células HeLa , Células Hep G2 , Humanos , Fosfatos de Inositol/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Trypanosoma brucei brucei/crescimento & desenvolvimento , Células Tumorais Cultivadas
12.
J Proteome Res ; 15(6): 2039-51, 2016 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-27108550

RESUMO

Histones are well-conserved proteins that form the basic structure of chromatin in eukaryotes and undergo several post-translational modifications, which are important for the control of transcription, replication, DNA damage repair, and chromosome condensation. In early branched organisms, histones are less conserved and appear to contain alternative sites for modifications, which could reveal evolutionary unique functions of histone modifications in gene expression and other chromatin-based processes. Here, by using high-resolution mass spectrometry, we identified and quantified histone post-translational modifications in two life cycle stages of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. We detected 44 new modifications, namely: 18 acetylations, seven monomethylations, seven dimethylations, seven trimethylations, and four phosphorylations. We found that replicative (epimastigote stage) contains more histone modifications than nonreplicative and infective parasites (trypomastigote stage). Acetylations of lysines at the C-terminus of histone H2A and methylations of lysine 23 of histone H3 were found to be enriched in trypomastigotes. In contrast, phosphorylation in serine 23 of H2B and methylations of lysine 76 of histone H3 predominates in proliferative states. The presence of one or two methylations in the lysine 76 was found in cells undergoing mitosis and cytokinesis, typical of proliferating parasites. Our findings provide new insights into the role of histone modifications related to the control of gene expression and cell-cycle regulation in an early divergent organism.


Assuntos
Cromatina/química , Código das Histonas , Estágios do Ciclo de Vida , Proteômica/métodos , Acetilação , Ciclo Celular , Regulação da Expressão Gênica , Metilação , Fosforilação , Processamento de Proteína Pós-Traducional/fisiologia , Trypanosoma cruzi
13.
PLoS One ; 10(12): e0144507, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26659253

RESUMO

Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathogens rely on chitinases for successful parasitization. Here, we purified and characterized a chitinase from T. gondii. The enzyme, provisionally named Tg_chitinase, has a molecular mass of 13.7 kDa and exhibits a Km of 0.34 mM and a Vmax of 2.64. The optimal environmental conditions for enzymatic function were at pH 4.0 and 50 °C. Tg_chitinase was immunolocalized in the cytoplasm of highly virulent T. gondii RH strain tachyzoites, mainly at the apical extremity. Tg_chitinase induced macrophage activation as manifested by the production of high levels of pro-inflammatory cytokines, a pathogenic hallmark of T. gondii infection. In conclusion, to our knowledge, we describe for the first time a chitinase of T. gondii tachyzoites and provide evidence that this enzyme might influence the pathogenesis of T. gondii infection.


Assuntos
Quitinases/imunologia , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/imunologia , Proteínas de Protozoários/imunologia , Toxoplasma/imunologia , Sequência de Aminoácidos , Animais , Quitinases/genética , Quitinases/metabolismo , Cromatografia Líquida , Citocinas/imunologia , Citocinas/metabolismo , Citoplasma/enzimologia , Interações Hospedeiro-Parasita/imunologia , Concentração de Íons de Hidrogênio , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Cinética , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/parasitologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia Confocal , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Espectrometria de Massas em Tandem , Temperatura Ambiente , Toxoplasma/enzimologia , Toxoplasma/fisiologia
14.
Mol Biochem Parasitol ; 204(1): 1-10, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26602446

RESUMO

The histone H4 from Trypanosomatids diverged from other eukaryotes in the N-terminus, a region that undergoes post-translation modifications involved in the control of gene expression, DNA replication, and chromatin assembly. Nonetheless, the N-terminus of Trypanosoma cruzi histone H4 is mainly acetylated at lysine 4. The lysines 10 and 14 are also acetylated, although at less extent, increasing during the S-phase or after DNA damage, which suggests a regulatory function. Here, we investigated the roles of these acetylations by expressing non-acetylated forms of histone H4 in T. cruzi. We found that histone H4 containing arginines at positions 10 or 14, to prevent acetylation were transported to the nucleus and inserted into the chromatin. However, their presence, even at low levels, interfered with DNA replication and transcription, causing a significant growth arrest of the cells. The absence of acetylation also increased the amount of soluble endogenous histones H3 and H4 and affected the interaction with Asf1, a histone chaperone. Therefore, acetylation of lysines 10 and 14 of the histone H4 in trypanosomes could be required for chromatin assembly and/or remodeling required for transcription and replication.


Assuntos
Replicação do DNA , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Proteínas de Protozoários/metabolismo , Transcrição Genética , Trypanosoma cruzi/genética , Acetilação , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica , Histona Acetiltransferases/metabolismo , Lisina/química , Processamento de Proteína Pós-Traducional
15.
Antimicrob Agents Chemother ; 59(8): 4669-79, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26014945

RESUMO

Acetylation of lysine is a major posttranslational modification of proteins and is catalyzed by lysine acetyltransferases, while lysine deacetylases remove acetyl groups. Among the deacetylases, the sirtuins are NAD(+)-dependent enzymes, which modulate gene silencing, DNA damage repair, and several metabolic processes. As sirtuin-specific inhibitors have been proposed as drugs for inhibiting the proliferation of tumor cells, in this study, we investigated the role of these inhibitors in the growth and differentiation of Trypanosoma cruzi, the agent of Chagas disease. We found that the use of salermide during parasite infection prevented growth and initial multiplication after mammalian cell invasion by T. cruzi at concentrations that did not affect host cell viability. In addition, in vivo infection was partially controlled upon administration of salermide. There are two sirtuins in T. cruzi, TcSir2rp1 and TcSir2rp3. By using specific antibodies and cell lines overexpressing the tagged versions of these enzymes, we found that TcSir2rp1 is localized in the cytosol and TcSir2rp3 in the mitochondrion. TcSir2rp1 overexpression acts to impair parasite growth and differentiation, whereas the wild-type version of TcSir2rp3 and not an enzyme mutated in the active site improves both. The effects observed with TcSir2rp3 were fully reverted by adding salermide, which inhibited TcSir2rp3 expressed in Escherichia coli with a 50% inhibitory concentration (IC50) ± standard error of 1 ± 0.5 µM. We concluded that sirtuin inhibitors targeting TcSir2rp3 could be used in Chagas disease chemotherapy.


Assuntos
Doença de Chagas/tratamento farmacológico , Naftóis/farmacologia , Fenilpropionatos/farmacologia , Sirtuínas/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Macaca mulatta
16.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 5): 615-21, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25961325

RESUMO

Prior studies have highlighted the potential of superoxide dismutases as drug targets in eukaryotic pathogens. This report presents the structures of three iron-dependent superoxide dismutases (FeSODs) from Trypanosoma cruzi, Leishmania major and Babesia bovis. Comparison with existing structures from Plasmodium and other trypanosome isoforms shows a very conserved overall fold with subtle differences. In particular, structural data suggest that B. bovis FeSOD may display similar resistance to peroxynitrite-mediated inactivation via an intramolecular electron-transfer pathway as previously described in T. cruzi FeSOD isoform B, thus providing valuable information for structure-based drug design. Furthermore, lysine-acetylation results in T. cruzi indicate that acetylation occurs at a position close to that responsible for the regulation of acetylation-mediated activity in the human enzyme.


Assuntos
Babesia bovis/enzimologia , Eucariotos/enzimologia , Leishmania major/enzimologia , Superóxido Dismutase/química , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Apicomplexa/química , Apicomplexa/enzimologia , Apicomplexa/genética , Babesia bovis/química , Babesia bovis/genética , Cristalização , Cristalografia por Raios X , Eucariotos/química , Eucariotos/genética , Humanos , Leishmania major/química , Leishmania major/genética , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Superóxido Dismutase/genética , Trypanosoma cruzi/química , Trypanosoma cruzi/genética
17.
PLoS Pathog ; 11(2): e1004618, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25658109

RESUMO

Translation initiation has been described as a key step for the control of growth and differentiation of several protozoan parasites in response to environmental changes. This occurs by the activation of protein kinases that phosphorylate the alpha subunit of the translation initiation factor 2 (eIF2α), which decreases translation, and in higher eukaryotes favors the expression of stress remedial response genes. However, very little is known about the signals that activate eIF2α kinases in protozoan parasites. Here, we characterized an eIF2α kinase of Trypanosoma cruzi (TcK2), the agent of Chagas' disease, as a transmembrane protein located in organelles that accumulate nutrients in proliferating parasite forms. We found that heme binds specifically to the catalytic domain of the kinase, inhibiting its activity. In the absence of heme, TcK2 is activated, arresting cell growth and inducing differentiation of proliferative into infective and non-proliferative forms. Parasites lacking TcK2 lose this differentiation capacity and heme is not stored in reserve organelles, remaining in the cytosol. TcK2 null cells display growth deficiencies, accumulating hydrogen peroxide that drives the generation of reactive oxygen species. The augmented level of hydrogen peroxide occurs as a consequence of increased superoxide dismutase activity and decreased peroxide activity. These phenotypes could be reverted by the re-expression of the wild type but not of a TcK2 dead mutant. These findings indicate that heme is a key factor for the growth control and differentiation through regulation of an unusual type of eIF2α kinase in T. cruzi.


Assuntos
Endossomos/metabolismo , Heme/metabolismo , Trypanosoma cruzi/enzimologia , eIF-2 Quinase/metabolismo , Imunofluorescência , Immunoblotting , Imunoprecipitação , Dados de Sequência Molecular , Espécies Reativas de Oxigênio/metabolismo
18.
Eukaryot Cell ; 13(7): 855-65, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24813189

RESUMO

The phosphorylation of the carboxy-terminal heptapeptide repeats of the largest subunit of RNA polymerase II (Pol II) controls several transcription-related events in eukaryotes. Trypanosomatids lack these typical repeats and display an unusual transcription control. RNA Pol II associates with the transcription site of the spliced leader (SL) RNA, which is used in the trans-splicing of all mRNAs transcribed on long polycistronic units. We found that Trypanosoma cruzi RNA Pol II associated with chromatin is highly phosphorylated. When transcription is inhibited by actinomycin D, the enzyme runs off from SL genes, remaining hyperphosphorylated and associated with polycistronic transcription units. Upon heat shock, the enzyme is dephosphorylated and remains associated with the chromatin. Transcription is partially inhibited with the accumulation of housekeeping precursor mRNAs, except for heat shock genes. DNA damage caused dephosphorylation and transcription arrest, with RNA Pol II dissociating from chromatin although staying at the SL. In the presence of calyculin A, the hyperphosphorylated form detached from chromatin, including the SL loci. These results indicate that in trypanosomes, the unusual RNA Pol II is phosphorylated during the transcription of SL and polycistronic operons. Different types of stresses modify its phosphorylation state, affecting pre-RNA processing.


Assuntos
Cromatina/metabolismo , Resposta ao Choque Térmico , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/metabolismo , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , Trypanosoma cruzi/metabolismo , Fosforilação , Proteínas de Protozoários/genética , RNA Polimerase II/genética , Processamento de RNA , Transcrição Genética
19.
Cell Microbiol ; 15(5): 709-17, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23336291

RESUMO

Trypanosomatids are parasites of worldwide distribution with relevant importance in human and veterinary health, which inhabit invertebrate and vertebrate hosts, such that they are exposed to large environmental variations during their life cycle. The signalling mechanisms and molecular basis that lead these parasites to adjust to such distinct conditions are beginning to be understood, and are somehow related to modifications in gene expression. Although the control of gene expression in this group of organisms happens predominantly at the post-transcriptional level, they present modifications in chromatin that has been implicated in transcription initiation, replication and DNA repair. Here, we explore the current scenario of chromatin alterations in these protozoans and how these changes affect transcription, replication and DNA repair in response to environmental modifications.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Reparo do DNA/genética , Replicação do DNA/genética , Trypanosoma/genética , Cromatina/genética , Regulação da Expressão Gênica , Humanos , Transcrição Genética
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