Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 24
Filtrar
1.
J Immunol ; 207(10): 2551-2560, 2021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-34635586

RESUMO

The protozoan parasite Trypanosoma brucei is the causative agent of the neglected tropical disease human African trypanosomiasis, otherwise known as sleeping sickness. Trypanosomes have evolved many immune-evasion mechanisms to facilitate their own survival, as well as prolonging host survival to ensure completion of the parasitic life cycle. A key feature of the bloodstream form of T. brucei is the secretion of aromatic keto acids, which are metabolized from tryptophan. In this study, we describe an immunomodulatory role for one of these keto acids, indole-3-pyruvate (I3P). We demonstrate that I3P inhibits the production of PGs in activated macrophages. We also show that, despite the reduction in downstream PGs, I3P augments the expression of cyclooxygenase (COX2). This increase in COX2 expression is mediated in part via inhibition of PGs relieving a negative-feedback loop on COX2. Activation of the aryl hydrocarbon receptor also participates in this effect. However, the increase in COX2 expression is of little functionality, as we also provide evidence to suggest that I3P targets COX activity. This study therefore details an evasion strategy by which a trypanosome-secreted metabolite potently inhibits macrophage-derived PGs, which might promote host and trypanosome survival.


Assuntos
Ciclo-Oxigenase 2/metabolismo , Indóis/metabolismo , Macrófagos/imunologia , Prostaglandinas/metabolismo , Tripanossomíase Africana/imunologia , Animais , Humanos , Evasão da Resposta Imune/imunologia , Indóis/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Prostaglandinas/imunologia , Trypanosoma brucei brucei/imunologia , Trypanosoma brucei brucei/metabolismo , Tripanossomíase Africana/metabolismo
2.
Nature ; 501(7467): 430-4, 2013 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-23965626

RESUMO

The African parasite Trypanosoma brucei gambiense accounts for 97% of human sleeping sickness cases. T. b. gambiense resists the specific human innate immunity acting against several other tsetse-fly-transmitted trypanosome species such as T. b. brucei, the causative agent of nagana disease in cattle. Human immunity to some African trypanosomes is due to two serum complexes designated trypanolytic factors (TLF-1 and -2), which both contain haptoglobin-related protein (HPR) and apolipoprotein LI (APOL1). Whereas HPR association with haemoglobin (Hb) allows TLF-1 binding and uptake via the trypanosome receptor TbHpHbR (ref. 5), TLF-2 enters trypanosomes independently of TbHpHbR (refs 4, 5). APOL1 kills trypanosomes after insertion into endosomal/lysosomal membranes. Here we report that T. b. gambiense resists TLFs via a hydrophobic ß-sheet of the T. b. gambiense-specific glycoprotein (TgsGP), which prevents APOL1 toxicity and induces stiffening of membranes upon interaction with lipids. Two additional features contribute to resistance to TLFs: reduction of sensitivity to APOL1 requiring cysteine protease activity, and TbHpHbR inactivation due to a L210S substitution. According to such a multifactorial defence mechanism, transgenic expression of T. b. brucei TbHpHbR in T. b. gambiense did not cause parasite lysis in normal human serum. However, these transgenic parasites were killed in hypohaptoglobinaemic serum, after high TLF-1 uptake in the absence of haptoglobin (Hp) that competes for Hb and receptor binding. TbHpHbR inactivation preventing high APOL1 loading in hypohaptoglobinaemic serum may have evolved because of the overlapping endemic area of T. b. gambiense infection and malaria, the main cause of haemolysis-induced hypohaptoglobinaemia in western and central Africa.


Assuntos
Apolipoproteínas/sangue , Apolipoproteínas/metabolismo , Lipoproteínas HDL/sangue , Lipoproteínas HDL/metabolismo , Trypanosoma brucei gambiense/fisiologia , África , Animais , Animais Geneticamente Modificados , Apolipoproteína L1 , Apolipoproteínas/antagonistas & inibidores , Apolipoproteínas/toxicidade , Membrana Celular/química , Membrana Celular/metabolismo , Cisteína Proteases/metabolismo , Haptoglobinas/metabolismo , Hemoglobinas/metabolismo , Hemólise , Humanos , Interações Hidrofóbicas e Hidrofílicas , Metabolismo dos Lipídeos , Lipoproteínas HDL/antagonistas & inibidores , Lipoproteínas HDL/química , Lipoproteínas HDL/toxicidade , Parasitos/patogenicidade , Parasitos/fisiologia , Estrutura Secundária de Proteína , Soro/química , Soro/parasitologia , Trypanosoma brucei gambiense/efeitos dos fármacos , Trypanosoma brucei gambiense/patogenicidade , Tripanossomíase Africana/parasitologia , Glicoproteínas Variantes de Superfície de Trypanosoma/química , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo
3.
Proc Natl Acad Sci U S A ; 113(48): E7778-E7787, 2016 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-27856732

RESUMO

The parasite Trypanasoma brucei causes African trypanosomiasis, known as sleeping sickness in humans and nagana in domestic animals. These diseases are a major burden in the 36 sub-Saharan African countries where the tsetse fly vector is endemic. Untreated trypanosomiasis is fatal and the current treatments are stage-dependent and can be problematic during the meningoencephalitic stage, where no new therapies have been developed in recent years and the current drugs have a low therapeutic index. There is a need for more effective treatments and a better understanding of how these parasites evade the host immune response will help in this regard. The bloodstream form of T. brucei excretes significant amounts of aromatic ketoacids, including indolepyruvate, a transamination product of tryptophan. This study demonstrates that this process is essential in bloodstream forms, is mediated by a specialized isoform of cytoplasmic aminotransferase and, importantly, reveals an immunomodulatory role for indolepyruvate. Indolepyruvate prevents the LPS-induced glycolytic shift in macrophages. This effect is the result of an increase in the hydroxylation and degradation of the transcription factor hypoxia-inducible factor-1α (HIF-1α). The reduction in HIF-1α levels by indolepyruvate, following LPS or trypanosome activation, results in a decrease in production of the proinflammatory cytokine IL-1ß. These data demonstrate an important role for indolepyruvate in immune evasion by T. brucei.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Imunidade Inata , Macrófagos/metabolismo , Piruvatos/metabolismo , Trypanosoma brucei brucei/imunologia , Tripanossomíase Africana/imunologia , Animais , Linhagem Celular , Glicólise , Células HEK293 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Evasão da Resposta Imune , Indóis/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/parasitologia , Lipopolissacarídeos/farmacologia , Macrófagos/parasitologia , Camundongos Endogâmicos C57BL , Tripanossomíase Africana/parasitologia
4.
Nucleic Acids Res ; 43(Database issue): D637-44, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25300491

RESUMO

The metabolic network of a cell represents the catabolic and anabolic reactions that interconvert small molecules (metabolites) through the activity of enzymes, transporters and non-catalyzed chemical reactions. Our understanding of individual metabolic networks is increasing as we learn more about the enzymes that are active in particular cells under particular conditions and as technologies advance to allow detailed measurements of the cellular metabolome. Metabolic network databases are of increasing importance in allowing us to contextualise data sets emerging from transcriptomic, proteomic and metabolomic experiments. Here we present a dynamic database, TrypanoCyc (http://www.metexplore.fr/trypanocyc/), which describes the generic and condition-specific metabolic network of Trypanosoma brucei, a parasitic protozoan responsible for human and animal African trypanosomiasis. In addition to enabling navigation through the BioCyc-based TrypanoCyc interface, we have also implemented a network-based representation of the information through MetExplore, yielding a novel environment in which to visualise the metabolism of this important parasite.


Assuntos
Bases de Dados de Compostos Químicos , Trypanosoma brucei brucei/metabolismo , Mineração de Dados , Internet , Redes e Vias Metabólicas , Proteômica , Trypanosoma brucei brucei/genética
5.
Antioxidants (Basel) ; 11(1)2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-35052669

RESUMO

The extracellular parasite and causative agent of African sleeping sickness Trypanosoma brucei (T. brucei) has evolved a number of strategies to avoid immune detection in the host. One recently described mechanism involves the conversion of host-derived amino acids to aromatic ketoacids, which are detected at relatively high concentrations in the bloodstream of infected individuals. These ketoacids have been shown to directly suppress inflammatory responses in murine immune cells, as well as acting as potent inducers of the stress response enzyme, heme oxygenase 1 (HO-1), which has proven anti-inflammatory properties. The aim of this study was to investigate the immunomodulatory properties of the T. brucei-derived ketoacids in primary human immune cells and further examine their potential as a therapy for inflammatory diseases. We report that the T. brucei-derived ketoacids, indole pyruvate (IP) and hydroxyphenylpyruvate (HPP), induce HO-1 expression through Nrf2 activation in human dendritic cells (DC). They also limit DC maturation and suppress the production of pro-inflammatory cytokines, which, in turn, leads to a reduced capacity to differentiate adaptive CD4+ T cells. Furthermore, the ketoacids are capable of modulating DC cellular metabolism and suppressing the inflammatory profile of cells isolated from patients with inflammatory bowel disease. This study therefore not only provides further evidence of the immune-evasion mechanisms employed by T. brucei, but also supports further exploration of this new class of HO-1 inducers as potential therapeutics for the treatment of inflammatory conditions.

6.
Curr Opin Immunol ; 72: 13-20, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33721725

RESUMO

Human African trypanosomiasis, or sleeping sickness, results from infection by two subspecies of the protozoan flagellate parasite Trypanosoma brucei, termed Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, prevalent in western and eastern Africa respectively. These subspecies escape the trypanolytic potential of human serum, which efficiently acts against the prototype species Trypanosoma brucei brucei, responsible for the Nagana disease in cattle. We review the various strategies and components used by trypanosomes to counteract the immune defences of their host, highlighting the adaptive genomic evolution that occurred in both parasite and host to take the lead in this battle. The main parasite surface antigen, named Variant Surface Glycoprotein or VSG, appears to play a key role in different processes involved in the dialogue with the host.


Assuntos
Suscetibilidade a Doenças/imunologia , Predisposição Genética para Doença , Tripanossomíase Africana/etiologia , Imunidade Adaptativa , Apolipoproteína L1/genética , Apolipoproteína L1/metabolismo , Resistência à Doença/genética , Resistência à Doença/imunologia , Regulação da Expressão Gênica , Variação Genética , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/imunologia , Humanos , Imunidade Inata , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ligação Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Trypanosoma brucei gambiense/imunologia , Tripanossomíase Africana/metabolismo
7.
Nutrients ; 12(3)2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32213952

RESUMO

Queuine is a eukaryotic micronutrient, derived exclusively from eubacteria. It is incorporated into both cytosolic and mitochondrial transfer RNA to generate a queuosine nucleotide at position 34 of the anticodon loop. The transfer RNA of primary tumors has been shown to be hypomodified with respect to queuosine, with decreased levels correlating with disease progression and poor patient survival. Here, we assess the impact of queuine deficiency on mitochondrial bioenergetics and substrate metabolism in HeLa cells. Queuine depletion is shown to promote a Warburg type metabolism, characterized by increased aerobic glycolysis and glutaminolysis, concomitant with increased ammonia and lactate production and elevated levels of lactate dehydrogenase activity but in the absence of significant changes to proliferation. In intact cells, queuine deficiency caused an increased rate of mitochondrial proton leak and a decreased rate of ATP synthesis, correlating with an observed reduction in cellular ATP levels. Data from permeabilized cells demonstrated that the activity of individual complexes of the mitochondrial electron transport chain were not affected by the micronutrient. Notably, in queuine free cells that had been adapted to grow in galactose medium, the re-introduction of glucose permitted the mitochondrial F1FO-ATP synthase to operate in the reverse direction, acting to hyperpolarize the mitochondrial membrane potential; a commonly observed but poorly understood cancer trait. Together, our data suggest that queuosine hypomodification is a deliberate and advantageous adaptation of cancer cells to facilitate the metabolic switch between oxidative phosphorylation and aerobic glycolysis.


Assuntos
Metabolismo Energético , Guanina/análogos & derivados , Micronutrientes/deficiência , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Ativação Enzimática , Glutamina/metabolismo , Glicólise , Guanina/metabolismo , Células HeLa , Humanos , Mitocôndrias/ultraestrutura , Modelos Biológicos , RNA de Transferência/genética , RNA de Transferência/metabolismo
8.
iScience ; 23(9): 101476, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32889430

RESUMO

Human innate immunity to Trypanosoma brucei involves the trypanosome C-terminal kinesin TbKIFC1, which transports internalized trypanolytic factor apolipoprotein L1 (APOL1) within the parasite. We show that TbKIFC1 preferentially associates with cholesterol-containing membranes and is indispensable for mammalian infectivity. Knockdown of TbKIFC1 did not affect trypanosome growth in vitro but rendered the parasites unable to infect mice unless antibody synthesis was compromised. Surface clearance of Variant Surface Glycoprotein (VSG)-antibody complexes was far slower in these cells, which were more susceptible to capture by macrophages. This phenotype was not due to defects in VSG expression or trafficking but to decreased VSG mobility in a less fluid, stiffer surface membrane. This change can be attributed to increased cholesterol level in the surface membrane in TbKIFC1 knockdown cells. Clearance of surface-bound antibodies by T. brucei is therefore essential for infectivity and depends on high membrane fluidity maintained by the cholesterol-trafficking activity of TbKIFC1.

9.
Nat Rev Microbiol ; 4(6): 477-86, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16710327

RESUMO

African trypanosomes (the prototype of which is Trypanosoma brucei brucei) are protozoan parasites that infect a wide range of mammals. Human blood, unlike the blood of other mammals, has efficient trypanolytic activity, and this needs to be counteracted by these parasites. Resistance to this activity has arisen in two subspecies of Trypanosoma brucei - Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense - allowing these parasites to infect humans, and this results in sleeping sickness in East Africa and West Africa, respectively. Study of the mechanism by which T. b. rhodesiense escapes lysis by human serum led to the identification of an ionic-pore-forming apolipoprotein - known as apolipoprotein L1 - that is associated with high-density-lipoprotein particles in human blood. In this Opinion article, we argue that apolipoprotein L1 is the factor that is responsible for the trypanolytic activity of human serum.


Assuntos
Apolipoproteínas/fisiologia , Soros Imunes/imunologia , Lipoproteínas HDL/fisiologia , Trypanosoma brucei gambiense/fisiologia , Trypanosoma brucei rhodesiense/fisiologia , Tripanossomíase Africana/imunologia , Animais , Antígenos de Neoplasias/fisiologia , Apolipoproteína L1 , Proteínas Sanguíneas/fisiologia , Haptoglobinas/fisiologia , Humanos , Trypanosoma brucei gambiense/imunologia , Trypanosoma brucei rhodesiense/imunologia , Tripanossomíase Africana/parasitologia
10.
Front Immunol ; 10: 2137, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31572363

RESUMO

African trypanosomes, such as Trypanosoma brucei (T. brucei), are protozoan parasites of the mammalian vasculature and central nervous system that are best known for causing fatal human sleeping sickness. As exclusively extracellular parasites, trypanosomes are subject to constant challenge from host immune defenses but they have developed very effective strategies to evade and modulate these responses to maintain an infection while simultaneously prolonging host survival. Here we investigate host parasite interactions, especially within the CNS context, which are not well-understood. We demonstrate that T. brucei strongly upregulates the stress response protein, Heme Oxygenase 1 (HO-1), in primary murine glia and macrophages in vitro. Furthermore, using a novel AHADHinT. brucei cell line, we demonstrate that specific aromatic ketoacids secreted by bloodstream forms of T. brucei are potent drivers of HO-1 expression and are capable of inhibiting pro-IL1ß induction in both glia and macrophages. Additionally, we found that these ketoacids significantly reduced IL-6 and TNFα production by glia, but not macrophages. Finally, we present data to support Nrf2 activation as the mechanism of action by which these ketoacids upregulate HO-1 expression and mediate their anti-inflammatory activity. This study therefore reports a novel immune evasion mechanism, whereby T. brucei secretes amino-acid derived metabolites for the purpose of suppressing both the host CNS and peripheral immune response, potentially via induction of the Nrf2/HO-1 pathway.


Assuntos
Heme Oxigenase-1/imunologia , Macrófagos/imunologia , Proteínas de Membrana/imunologia , Fator 2 Relacionado a NF-E2/imunologia , Neuroglia/imunologia , Piruvatos/imunologia , Trypanosoma brucei brucei/imunologia , Animais , Inflamação/imunologia , Inflamação/patologia , Macrófagos/patologia , Camundongos , Neuroglia/patologia
11.
Mol Biochem Parasitol ; 157(2): 233-5, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18061288

RESUMO

Vesicle traffic to and from the surface is highly polarized in African trypanosomes. Actin is required for polarized endocytic traffic in bloodstream forms of African trypanosomes but its role in other pathways has remained equivocal. A combination of metabolic pulse chase labelling and surface biotinylation during the chase period along with the use of conditional RNA interference was employed to demonstrate that substantial loss of actin had no effect on the export of newly synthesized proteins to the surface of bloodstream and procyclic forms of Trypanosoma brucei. These results indicated that this trafficking pathway to the surface operates as normal even when actin levels are significantly lower than normal and endocytic activity is abolished. Taken together the data support the view that the secretory and endocytic pathways are not obligatorily coupled.


Assuntos
Actinas/fisiologia , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Actinas/genética , Animais , Inativação Gênica , Transporte Proteico , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/genética
12.
F1000Res ; 62017.
Artigo em Inglês | MEDLINE | ID: mdl-28620452

RESUMO

Cellular metabolic activity is a highly complex, dynamic, regulated process that is influenced by numerous factors, including extracellular environmental signals, nutrient availability and the physiological and developmental status of the cell. The causative agent of sleeping sickness, Trypanosoma brucei, is an exclusively extracellular protozoan parasite that encounters very different extracellular environments during its life cycle within the mammalian host and tsetse fly insect vector. In order to meet these challenges, there are significant alterations in the major energetic and metabolic pathways of these highly adaptable parasites. This review highlights some of these metabolic changes in this early divergent eukaryotic model organism.

13.
Nat Commun ; 6: 8078, 2015 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-26307671

RESUMO

Humans resist infection by the African parasite Trypanosoma brucei owing to the trypanolytic activity of the serum apolipoprotein L1 (APOL1). Following uptake by endocytosis in the parasite, APOL1 forms pores in endolysosomal membranes and triggers lysosome swelling. Here we show that APOL1 induces both lysosomal and mitochondrial membrane permeabilization (LMP and MMP). Trypanolysis coincides with MMP and consecutive release of the mitochondrial TbEndoG endonuclease to the nucleus. APOL1 is associated with the kinesin TbKIFC1, of which both the motor and vesicular trafficking VHS domains are required for MMP, but not for LMP. The presence of APOL1 in the mitochondrion is accompanied by mitochondrial membrane fenestration, which can be mimicked by knockdown of a mitochondrial mitofusin-like protein (TbMFNL). The BH3-like peptide of APOL1 is required for LMP, MMP and trypanolysis. Thus, trypanolysis by APOL1 is linked to apoptosis-like MMP occurring together with TbKIFC1-mediated transport of APOL1 from endolysosomal membranes to the mitochondrion.


Assuntos
Apolipoproteínas/metabolismo , Cinesinas/metabolismo , Lipoproteínas HDL/metabolismo , Lisossomos/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas de Protozoários/metabolismo , Apolipoproteína L1 , Apoptose , Transporte Biológico , Endocitose , Humanos , Membranas Intracelulares/metabolismo , Permeabilidade , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/patogenicidade , Trypanosoma brucei gambiense/metabolismo , Trypanosoma brucei gambiense/patogenicidade , Trypanosoma brucei rhodesiense/metabolismo , Trypanosoma brucei rhodesiense/patogenicidade
14.
PLoS One ; 8(1): e52846, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23335957

RESUMO

Flagellar attachment is a visibly striking morphological feature of African trypanosomes but little is known about the requirements for attachment at a molecular level. This study characterizes a previously undescribed membrane protein, FLA3, which plays an essential role in flagellar attachment in Trypanosoma brucei. FLA3 is heavily N-glycosylated, locates to the flagellar attachment zone and appears to be a bloodstream stage specific protein. Ablation of the FLA3 mRNA rapidly led to flagellar detachment and a concomitant failure of cytokinesis in the long slender bloodstream form but had no effect on the procyclic form. Flagellar detachment was obvious shortly after induction of the dsRNA and the newly synthesized flagellum was often completely detached after it emerged from the flagellar pocket. Within 12 h most cells possessed detached flagella alongside the existing attached flagellum. These results suggest that proteins involved in attachment are not shared between the new and old attachment zones. In other respects the detached flagella appear normal, they beat rapidly although directional motion was lost, and they possess an apparently normal axoneme and paraflagellar rod structure. The flagellar attachment zone appeared to be disrupted when FLA3 was depleted. Thus, while flagellar attachment is a constitutive feature of the life cycle of trypanosomes, attachment requires stage specific elements at the protein level.


Assuntos
Flagelos/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/fisiologia , Animais , Linhagem Celular , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Transporte Proteico , Interferência de RNA , Ratos
15.
Int J Oncol ; 43(3): 927-35, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23799546

RESUMO

The pyrrolo-1,5-benzoxazepines (PBOXs) are a novel group of selective apoptotic agents displaying promising therapeutic potential in both ex vivo chemotherapy-refractory patient samples and in vivo murine carcinoma models. In this report, we present novel data concerning the induction of autophagy by the PBOXs in adenocarcinoma-derived colon cancer cells. Autophagy is a lysosome-dependent degradative pathway recently associated with chemotherapy. However, whether autophagy facilitates cell survival in response to chemotherapy or contributes to chemotherapy-induced cell death is highly controversial. Autophagy was identified by enhanced expression of LC3B-II, an autophagosome marker, an increase in the formation of acridine orange-stained cells, indicative of increased vesicle formation and electron microscopic confirmation of autophagic structures. The vacuolar H+ ATPase inhibitor bafilomycin-A1 (BAF-A1) inhibited vesicle formation and enhanced the apoptotic potential of PBOX-6. These findings suggest a cytoprotective role of autophagy in these cells following prolonged exposure to PBOX-6. Furthermore, BAF-A1 and PBOX-6 interactions were determined to be synergistic and caspase-dependent. Potentiation of PBOX-6-induced apoptosis by BAF-A1 was associated with a decrease in the levels of the anti-apoptotic protein, Mcl-1. The data provide evidence that autophagy functions as a survival mechanism in colon cancer cells to PBOX-6-induced apoptosis and a rationale for the use of autophagy inhibitors to further enhance PBOX­6­induced apoptosis in colon cancer.


Assuntos
Adenocarcinoma/tratamento farmacológico , Autofagia/genética , Neoplasias do Colo/tratamento farmacológico , Macrolídeos/administração & dosagem , Oxazepinas/administração & dosagem , Pirróis/administração & dosagem , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Sinergismo Farmacológico , Humanos , Lisossomos/metabolismo , Lisossomos/patologia , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo
16.
Biochem Pharmacol ; 84(5): 612-24, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22705646

RESUMO

Recent clinical data demonstrated that the vascular targeting agent Combretastatin-A4 phosphate (CA-4P) prolonged survival of patients with advanced anaplastic thyroid cancer without any adverse side effects. However, as a single agent CA-4 failed to reduce tumour growth in the murine CT-26 adenocarcinoma colon cancer model. Furthermore, the molecular mechanism of the innate resistance of HT-29 human adenocarcinoma cells to CA-4 is largely unknown. In this report, we demonstrate for the first time that prolonged exposure to CA-4 and an azetidinone cis-restricted analogue, CA-432 (chemical name; 4-(3-Hydroxy-4-methoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)-azetidin-2-one) induced autophagy in adenocarcinoma-derived CT-26, Caco-2 and HT-29 cells but not in fibrosarcoma-derived HT-1080 cells. Autophagy is a fundamental self-catabolic process which can facilitate a prolonged cell survival in spite of adverse stress by generating energy via lysosomal degradation of cytoplasmic constituents. Autophagy was confirmed by acridine orange staining of vesicle formation, electron microscopy and increased expression of LC3-II. Combretastatin-induced autophagy was associated with a loss of mitochondrial membrane potential and elongation of the mitochondria. Furthermore, inhibition of autophagy by the vacuolar H(+)ATPase inhibitor Bafilomycin-A1 (BAF-A1) significantly enhanced CA-432 induced HT-29 cell death. Both CA-4 and its synthetic derivative, CA-432 induced the formation of large hyperdiploid cells in Caco-2 and CT-26 cells. The formation of these polyploid cells was significantly inhibited by autophagy inhibitor, BAF-A1. Results presented within demonstrate that autophagy is a novel response to combretastatin exposure and may be manipulated to enhance the therapeutic efficacy of this class of vascular targeting agents.


Assuntos
Adenocarcinoma/imunologia , Autofagia/efeitos dos fármacos , Neoplasias do Colo/imunologia , Estilbenos/farmacologia , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Western Blotting , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Citometria de Fluxo , Humanos , Potenciais da Membrana/efeitos dos fármacos , Microscopia Eletrônica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia
17.
PLoS One ; 5(8): e12282, 2010 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-20808867

RESUMO

Myosins are a multimember family of motor proteins with diverse functions in eukaryotic cells. African trypanosomes possess only two candidate myosins and thus represent a useful system for functional analysis of these motors. One of these candidates is an unusual class I myosin (TbMyo1) that is expressed at similar levels but organized differently during the life cycle of Trypanosoma brucei. This myosin localizes to the polarized endocytic pathway in bloodstream forms of the parasite. This organization is actin dependent. Knock down of TbMyo1 results in a significant reduction in endocytic activity, a cessation in cell division and eventually cell death. A striking morphological feature in these cells is an enlargement of the flagellar pocket, which is consistent with an imbalance in traffic to and from the surface. In contrast TbMyo1 is distributed throughout procyclic forms of the tsetse vector and a loss of approximately 90% of the protein has no obvious effects on growth or morphology. These results reveal a life cycle stage specific requirement for this myosin in essential endocytic traffic and represent the first description of the involvement of a motor protein in vesicle traffic in these parasites.


Assuntos
Miosina Tipo I/metabolismo , Vesículas Transportadoras/metabolismo , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Regulação da Expressão Gênica , Dados de Sequência Molecular , Miosina Tipo I/sangue , Miosina Tipo I/química , Miosina Tipo I/genética , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Trypanosoma brucei brucei/metabolismo
18.
FEBS J ; 276(23): 7187-99, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19895576

RESUMO

African trypanosomes possess high levels of alanine aminotransferase (EC 2.6.1.2), although the function of their activity remains enigmatic, especially in slender bloodstream forms where the metabolism of ketoacids does not occur. Therefore, the gene for alanine aminotransferase enzyme in Trypanosoma brucei (TbAAT) was characterized and its function assessed using a combination of RNA interference and gene knockout approaches. Surprisingly, as much as 95% or more of the activity appears to be unnecessary for growth of either bloodstream or procyclic forms respiring on glucose. A combination of RNA interference and NMR spectroscopy revealed an important role for the activity in procyclic forms respiring on proline. Under these conditions, the major end product of proline metabolism is alanine, and a reduction in TbAAT activity led to a proportionate decrease in the amount of alanine excreted along with an increase in the doubling time of the cells. These results provide evidence of a role for alanine aminotransferase in the metabolism of proline in African trypanosomes by linking glutamate produced by the initial oxidative steps of the pathway with pyruvate produced by the final oxidative step of the pathway. This step appears to be essential when proline is the primary carbon source, which is likely to be the physiological situation in the tsetse fly vector.


Assuntos
Alanina Transaminase/metabolismo , Prolina/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/enzimologia , Alanina Transaminase/genética , Animais , Sequência de Bases , Células Cultivadas , Cinética , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas de Protozoários/genética , Interferência de RNA , Trypanosoma brucei brucei/metabolismo
19.
J Biol Chem ; 280(11): 10410-8, 2005 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-15642735

RESUMO

Proteins from the endocytic pathway in bloodstream forms of Trypanosome brucei are modified by the addition of linear poly-N-acetyllactosamine side chains, which permits their isolation by tomato lectin affinity chromatography. Antibodies against this tomato lectin binding fraction were employed to screen a cDNA expression library from bloodstream forms of T. brucei. Two cDNAs were prominent among those selected. These cDNAs coded for two putative protein disulfide isomerases (PDIs) that respectively contained one and two double-cysteine redox-active sites and corresponded to a single domain PDI and a class 1 PDI. Assays of the purified recombinant proteins demonstrated that both proteins possess isomerase activity, but only the single domain PDI had a reducing activity. These PDIs possess a number of unusual features that distinguish them from previously characterized PDIs. The expression of both is developmentally regulated, they both co-localize with markers of the endocytic pathway, and both are modified by N-glycosylation. The larger PDI possesses N-glycans containing poly-N-acetyllactosamine, a modification that is indicative of processing in the Golgi and suggests the presence of a novel trafficking pathway for PDIs in trypanosomes. Although generally PDIs are considered essential, neither activity appeared to be essential for the growth of trypanosomes, at least in vitro.


Assuntos
Isomerases de Dissulfetos de Proteínas/química , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Western Blotting , Cromatografia , Clonagem Molecular , Cisteína/química , DNA/química , DNA Complementar/metabolismo , Dissulfetos , Endocitose , Genoma , Glicosídeo Hidrolases/metabolismo , Glicosilação , Immunoblotting , Imunoprecipitação , Lectinas/metabolismo , Solanum lycopersicum/metabolismo , Camundongos , Microscopia Confocal , Dados de Sequência Molecular , Oxirredução , Polissacarídeos/química , Isomerases de Dissulfetos de Proteínas/biossíntese , Estrutura Terciária de Proteína , Proteínas de Protozoários , RNA/química , Interferência de RNA , Proteínas Recombinantes/química , Relação Estrutura-Atividade , Frações Subcelulares , Fatores de Tempo
20.
Science ; 309(5733): 469-72, 2005 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-16020735

RESUMO

Apolipoprotein L-I is the trypanolytic factor of human serum. Here we show that this protein contains a membrane pore-forming domain functionally similar to that of bacterial colicins, flanked by a membrane-addressing domain. In lipid bilayer membranes, apolipoprotein L-I formed anion channels. In Trypanosoma brucei, apolipoprotein L-I was targeted to the lysosomal membrane and triggered depolarization of this membrane, continuous influx of chloride, and subsequent osmotic swelling of the lysosome until the trypanosome lysed.


Assuntos
Apolipoproteínas/química , Apolipoproteínas/metabolismo , Membranas Intracelulares/metabolismo , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Lisossomos/metabolismo , Trypanosoma brucei brucei/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Sequência de Aminoácidos , Animais , Ânions/metabolismo , Apolipoproteína L1 , Apolipoproteínas/genética , Apolipoproteínas/farmacologia , Células Imobilizadas , Cloretos/metabolismo , Colicinas/química , Colicinas/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/ultraestrutura , Canais Iônicos/metabolismo , Bicamadas Lipídicas/química , Lipoproteínas HDL/genética , Lipoproteínas HDL/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/ultraestrutura , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Permeabilidade , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/ultraestrutura
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA