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1.
Int J Mol Sci ; 25(2)2024 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-38255772

RESUMEN

Parkinson's disease (PD) is a complex disorder characterized by the impairment of the dopaminergic nigrostriatal system. PD has duplicated its global burden in the last few years, becoming the leading neurological disability worldwide. Therefore, there is an urgent need to develop innovative approaches that target multifactorial underlying causes to potentially prevent or limit disease progression. Accumulating evidence suggests that neuroinflammatory responses may play a pivotal role in the neurodegenerative processes that occur during the development of PD. Cortistatin is a neuropeptide that has shown potent anti-inflammatory and immunoregulatory effects in preclinical models of autoimmune and neuroinflammatory disorders. The goal of this study was to explore the therapeutic potential of cortistatin in a well-established preclinical mouse model of PD induced by acute exposure to the neurotoxin 1-methil-4-phenyl1-1,2,3,6-tetrahydropyridine (MPTP). We observed that treatment with cortistatin mitigated the MPTP-induced loss of dopaminergic neurons in the substantia nigra and their connections to the striatum. Consequently, cortistatin administration improved the locomotor activity of animals intoxicated with MPTP. In addition, cortistatin diminished the presence and activation of glial cells in the affected brain regions of MPTP-treated mice, reduced the production of immune mediators, and promoted the expression of neurotrophic factors in the striatum. In an in vitro model of PD, treatment with cortistatin also demonstrated a reduction in the cell death of dopaminergic neurons that were exposed to the neurotoxin. Taken together, these findings suggest that cortistatin could emerge as a promising new therapeutic agent that combines anti-inflammatory and neuroprotective properties to regulate the progression of PD at multiple levels.


Asunto(s)
Neuropéptidos , Enfermedad de Parkinson , Animales , Ratones , Enfermedad de Parkinson/tratamiento farmacológico , Neurotoxinas , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico
2.
Neuroendocrinology ; 112(8): 784-795, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-34649259

RESUMEN

INTRODUCTION: Scleroderma, or systemic sclerosis, is a complex connective tissue disorder characterized by autoimmunity, vasculopathy, and progressive fibrosis of the skin and internal organs. Because its aetiology is unknown, the identification of genes/factors involved in disease severity, differential clinical forms, and associated complications is critical for understanding its pathogenesis and designing novel treatments. Neuroendocrine mediators in the skin emerge as potential candidates. We investigated the role played by the neuropeptide cortistatin in a preclinical model of scleroderma. METHODS: Dermal fibrosis was induced by repetitive intradermal injections of bleomycin in wild-type and cortistatin-deficient mice. The histopathological signs and expression of fibrotic markers were evaluated in the skin and lungs. RESULTS: An inverse correlation between cortistatin levels and fibrogenic activation exists in the damaged skin and dermal fibroblasts. Bleomycin-challenged skin lesions of mice that are partially and totally deficient in cortistatin showed exacerbated histopathological signs of scleroderma, characterized by thicker and more fibrotic dermal layer, enlarged epidermis, and increased inflammatory infiltration in comparison to those of wild-type mice. Cortistatin deficiency enhanced dermal collagen deposits, connective tissue growth factor expression, loss of microvessels, and predisposition to suffer severe complications that co-occur with dermal exposition to bleomycin, including pulmonary fibrotic disease and increased mortality. Treatment with cortistatin mitigated these pathological processes. DISCUSSION/CONCLUSION: We identify cortistatin as an endogenous break of skin inflammation and fibrosis. Deficiency in cortistatin could be a marker of poor prognosis of scleroderma and associated complications. Cortistatin-based therapies emerge as attractive candidates to treat severe forms of systemic sclerosis and to manage fibrosis-related side effects of bleomycin chemotherapy in oncologic patients.


Asunto(s)
Neuropéptidos , Fibrosis Pulmonar , Esclerodermia Sistémica , Animales , Bleomicina/toxicidad , Modelos Animales de Enfermedad , Fibrosis , Ratones , Neuropéptidos/metabolismo , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Esclerodermia Sistémica/inducido químicamente , Esclerodermia Sistémica/metabolismo
3.
Cell Mol Life Sci ; 77(9): 1827-1845, 2020 May.
Artículo en Inglés | MEDLINE | ID: mdl-31372684

RESUMEN

Leishmaniasis comprises a group of neglected diseases caused by the protozoan parasite Leishmania spp. As is the case for other trypanosomatid parasites, Leishmania is auxotrophic for heme and must scavenge this essential compound from its human host. In mammals, the SLC transporter FLVCR2 mediates heme import across the plasma membrane. Herein we identify and characterize Leishmania major FLVCRb (LmFLVCRb), the first member of the FLVCR family studied in a non-metazoan organism. This protein localizes to the plasma membrane of the parasite and is able to bind heme. LmFLVCRb levels in Leishmania, which are modulated by overexpression thereof or the abrogation of an LmFLVCRb allele, correlate with the ability of the parasite to take up porphyrins. Moreover, injection of LmFLVCRb cRNA to Xenopus laevis oocytes provides these cells with the ability to take up heme. This process is temperature dependent, requires monovalent ions and is inhibited at basic pH, characteristics shared by the uptake of heme by Leishmania parasites. Interestingly, LmFLVCRb is essential as CRISPR/Cas9-mediated knockout parasites were only obtained in the presence of an episomal copy of the gene. In addition, deletion of just one of the alleles of the LmFLVCRb gene markedly impairs parasite replication as intracellular amastigotes as well as its virulence in an in vivo model of cutaneous leishmaniasis. Collectively, these results show that Leishmania parasites can rescue heme through plasma membrane transporter LFLVCRb, which could constitute a novel target for therapeutic intervention against Leishmania and probably other trypanosomatid parasites in which FLVCR genes are also present.


Asunto(s)
Hemo/metabolismo , Leishmania major/metabolismo , Leishmaniasis/parasitología , Macrófagos/parasitología , Proteínas de Transporte de Membrana/metabolismo , Porfirinas/metabolismo , Proteínas Protozoarias/metabolismo , Receptores Virales/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Leishmania major/patogenicidad , Leishmaniasis/metabolismo , Macrófagos/metabolismo , Proteínas de Transporte de Membrana/genética , Oocitos/metabolismo , Oocitos/parasitología , Proteínas Protozoarias/genética , Receptores Virales/genética , Homología de Secuencia , Virulencia , Xenopus laevis
4.
Mol Microbiol ; 101(6): 895-908, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27328668

RESUMEN

Pathogenic trypanosomatid parasites are auxotrophic for heme and they must scavenge it from their human host. Trypanosoma brucei (responsible for sleeping sickness) and Leishmania (leishmaniasis) can fulfill heme requirement by receptor-mediated endocytosis of host hemoglobin. However, the mechanism used to transfer hemoglobin-derived heme from the lysosome to the cytosol remains unknown. Here we provide strong evidence that HRG transporters mediate this essential step. In bloodstream T. brucei, TbHRG localizes to the endolysosomal compartment where endocytosed hemoglobin is known to be trafficked. TbHRG overexpression increases cytosolic heme levels whereas its downregulation is lethal for the parasites unless they express the Leishmania orthologue LmHR1. LmHR1, known to be an essential plasma membrane protein responsible for the uptake of free heme in Leishmania, is also present in its acidic compartments which colocalize with endocytosed hemoglobin. Moreover, LmHR1 levels modulated by its overexpression or the abrogation of an LmHR1 allele correlate with the mitochondrial bioavailability of heme from lysosomal hemoglobin. In addition, using heme auxotrophic yeasts we show that TbHRG and LmHR1 transport hemoglobin-derived heme from the digestive vacuole to the cytosol. Collectively, these results show that trypanosomatid parasites rescue heme from endocytosed hemoglobin through endolysosomal HRG transporters, which could constitute novel drug targets.


Asunto(s)
Hemo/metabolismo , Hemoglobinas/metabolismo , Leishmaniasis/metabolismo , Leishmaniasis/parasitología , Trypanosoma brucei brucei/metabolismo , Secuencia de Aminoácidos , Animales , Citosol/metabolismo , Endocitosis/fisiología , Leishmania/metabolismo , Leishmaniasis/sangre , Lisosomas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Transporte de Proteínas , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/parasitología
5.
Chemistry ; 23(9): 2157-2164, 2017 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-27925323

RESUMEN

The G-quadruplexes (G4s) are currently being explored as therapeutic targets in cancer and other pathologies. Six carbohydrate naphthalene diimide conjugates (carb-NDIs) have been synthesized as G4 ligands to investigate their potential selectivity in G4 binding and cell penetration. Carb-NDIs have shown certain selectivity for G4 structures against DNA duplexes, but different sugar moieties do not induce a preference for a specific G4 topology. Interestingly, when monosaccharides were attached through a short ethylene linker to the NDI scaffold, their cellular uptake was two- to threefold more efficient than that when the sugar was directly attached through its anomeric position. Moreover, a correlation between more efficient cell uptake of these carb-NDIs and their higher toxicity in cancerous cell lines has been observed. Carb-NDIs seem to be mainly translocated into cancer cells through glucose transporters (GLUT), of which GLUT4 plays a major role.

6.
J Biol Chem ; 289(21): 14583-99, 2014 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-24706753

RESUMEN

Vasoactive intestinal peptide (VIP) is an anti-inflammatory neuropeptide recently identified as a potential antimicrobial peptide. To overcome the metabolic limitations of VIP, we modified the native peptide sequence and generated two stable synthetic analogues (VIP51 and VIP51(6-30)) with better antimicrobial profiles. Herein we investigate the effects of both VIP analogues on cell viability, membrane integrity, and ultrastructure of various bacterial strains and Leishmania species. We found that the two VIP derivatives kill various non-pathogenic and pathogenic Gram-positive and Gram-negative bacteria as well as the parasite Leishmania major through a mechanism that depends on the interaction with certain components of the microbial surface, the formation of pores, and the disruption of the surface membrane. The cytotoxicity of the VIP derivatives is specific for pathogens, because they do not affect the viability of mammalian cells. Docking simulations indicate that the chemical changes made in the analogues are critical to increase their antimicrobial activities. Consequently, we found that the native VIP is less potent as an antibacterial and fails as a leishmanicidal. Noteworthy from a therapeutic point of view is that treatment with both derivatives increases the survival and reduces bacterial load and inflammation in mice with polymicrobial sepsis. Moreover, treatment with VIP51(6-30) is very effective at reducing lesion size and parasite burden in a model of cutaneous leishmaniasis. These results indicate that the VIP analogues emerge as attractive alternatives for treating drug-resistant infectious diseases and provide key insights into a rational design of novel agents against these pathogens.


Asunto(s)
Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Leishmania major/efectos de los fármacos , Péptido Intestinal Vasoactivo/farmacología , Secuencia de Aminoácidos , Animales , Endotoxemia/tratamiento farmacológico , Endotoxemia/microbiología , Femenino , Bacterias Gramnegativas/genética , Bacterias Grampositivas/genética , Enlace de Hidrógeno , Leishmania major/genética , Leishmania major/ultraestructura , Leishmaniasis Cutánea/tratamiento farmacológico , Leishmaniasis Cutánea/parasitología , Ratones , Ratones Endogámicos BALB C , Viabilidad Microbiana/efectos de los fármacos , Microscopía Electrónica , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Sepsis/tratamiento farmacológico , Sepsis/microbiología , Análisis de Supervivencia , Resultado del Tratamiento , Péptido Intestinal Vasoactivo/análogos & derivados , Péptido Intestinal Vasoactivo/química
7.
J Immunol ; 191(12): 6040-51, 2013 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-24249730

RESUMEN

We currently face an alarming resurgence in infectious diseases characterized by antimicrobial resistance and therapeutic failure. This has generated the urgent need of developing new therapeutic approaches that include agents with nontraditional modes of action. A recent interest focused on approaches based on our natural immune defenses, especially on peptides that combine innate antimicrobial activity against diverse pathogens and immunoregulatory functions. In this study, to our knowledge, we describe for the first time the antimicrobial activity of the neuropeptide urocortin II (UCNII) against a panel of Gram-positive and Gram-negative bacteria and tropical parasites of the genus Leishmania. Importantly, this cytotoxicity was selective for pathogens, because UCNII did not affect mammalian cell viability. Structurally, UCNII has a cationic and amphipathic design that resembles antimicrobial peptides. Using mutants and UCNII fragments, we determined the structural requirements for the interaction between the peptide and the surface of pathogen. Following its binding to pathogen, UCNII caused cell death through different membrane-disrupting mechanisms that involve aggregation and membrane depolarization in bacteria and pore formation in Leishmania. Noteworthily, UCNII killed the infective form of Leishmania major even inside the infected macrophages. Consequently, UCNII prevented mortality caused by polymicrobial sepsis and ameliorated pathological signs of cutaneous leishmaniasis. Besides its presence in body physical and mucosal barriers, we found that innate immune cells produce UCNII in response to infections. Therefore, UCNII could be considered as an ancient highly-conserved host peptide involved in the natural antimicrobial defense and emerge as an attractive alternative to current treatments for microbial disorders with associated drug resistances.


Asunto(s)
Hormona Liberadora de Corticotropina/fisiología , Leishmania/efectos de los fármacos , Leishmaniasis Cutánea/tratamiento farmacológico , Sepsis/tratamiento farmacológico , Urocortinas/fisiología , Secuencia de Aminoácidos , Animales , Membrana Celular/efectos de los fármacos , Hormona Liberadora de Corticotropina/química , Hormona Liberadora de Corticotropina/farmacología , Evaluación Preclínica de Medicamentos , Escherichia coli/efectos de los fármacos , Femenino , Humanos , Enlace de Hidrógeno , Inmunidad Innata , Perforación Intestinal/complicaciones , Perforación Intestinal/microbiología , Leishmania/ultraestructura , Leishmaniasis Cutánea/parasitología , Lipopolisacáridos/química , Macrófagos/parasitología , Potenciales de la Membrana/efectos de los fármacos , Ratones Endogámicos BALB C , Micrococcus luteus/efectos de los fármacos , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/uso terapéutico , Peritonitis/etiología , Peritonitis/microbiología , Unión Proteica , Conformación Proteica , Pseudomonas pseudoalcaligenes/efectos de los fármacos , Sepsis/etiología , Streptococcus mutans/efectos de los fármacos , Urocortinas/química , Urocortinas/farmacología
8.
Mol Microbiol ; 79(6): 1430-44, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21255121

RESUMEN

The characterization of LABCG5, a new intracellular ATP-binding cassette protein in Leishmania donovani, is described. Unlike other ABCG half-transporters, LABCG5 is not involved in either drug resistance or phospholipid efflux. However, we provide evidence suggesting that this protein is involved in intracellular haem trafficking. Thus, downregulation of LABCG5 function produced upon overexpression of an inactive version of the protein caused a dramatic growth arrest unless a haemin supplement was added or the mutated gene was eliminated. Supplementation with haemoglobin, an upstream metabolite normally sufficient to meet parasite haem requirements, was unable to rescue the growth defect phenotype. Haemoglobin endocytosis was not hampered in dominant-negative parasites and neither was haem uptake, a process that we show here to be dependent on a specific transporter. In contrast, LABCG5 function was required for the correct intracellular trafficking of haemoglobin-bound porphyrins to the mitochondria, not affecting the routing of free haem. Finally, LABCG5 binds haem through hydrophobic and electrostatic interactions. Altogether, these data suggest that LABCG5 is involved in the salvage of the haem released after the breakdown of internalized haemoglobin. As Leishmania is auxotrophic for haem, the pharmacological targeting of this route could represent a novel approach to control fatal visceral leishmaniasis.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Hemo/metabolismo , Leishmania donovani/metabolismo , Leishmaniasis Visceral/parasitología , Proteínas Protozoarias/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transporte Biológico , Línea Celular , Humanos , Leishmania donovani/genética , Leishmaniasis Visceral/metabolismo , Transporte de Proteínas , Proteínas Protozoarias/genética
9.
Pharmaceutics ; 14(12)2022 Dec 13.
Artículo en Inglés | MEDLINE | ID: mdl-36559278

RESUMEN

Cortistatin is a cyclic neuropeptide that recently emerged as an attractive therapeutic factor for treating inflammatory, autoimmune, fibrotic, and pain disorders. Despite of its efficiency and apparent safety in experimental preclinical models, its short half-life in body fluids and its potential pleiotropic effects, due to its promiscuity for several receptors expressed in various cells and tissues, represent two major drawbacks for the clinical translation of cortistatin-based therapies. Therefore, the design of new strategies focused on increasing the stability, bioavailability, and target specificity of cortistatin are lately demanded by the industry. Here, we generated by molecular engineering a new cortistatin-based prodrug formulation that includes, beside the bioactive cortistatin, a molecular-shield provided by the latency-associated protein of the transforming growth factor-ß1 and a cleavage site specifically recognized by metalloproteinases, which are abundant in inflammatory/fibrotic foci. Using different models of sepsis, inflammatory bowel disease, scleroderma, and pulmonary fibrosis, we demonstrated that this latent form of cortistatin was a highly effective protection against these severe disorders. Noteworthy, from a therapeutic point of view, is that latent cortistatin seems to require significantly lower doses and fewer administrations than naive cortistatin to reach the same efficacy. Finally, the metalloproteinase-cleavage site was essential for the latent molecule to exert its therapeutic action. In summary, latent cortistatin emerges as a promising innovative therapeutic tool for treating chronic diseases of different etiologies with difficult clinical solutions and as a starting point for a rational development of prodrugs based on the use of bioactive peptides.

10.
Neuroendocrinology ; 94(2): 89-100, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21734355

RESUMEN

Although necessary to eliminate pathogens, inflammation can lead to serious deleterious effects in the host if left unchecked. During the inflammatory response, further damage may arise from potential autoimmune responses occurring when the immune cells and molecules that respond to pathogen-derived antigens also react to self-antigens. In this sense, the identification of endogenous factors that control exacerbated immune responses is a key goal for the development of new therapeutic approaches for inflammatory and autoimmune diseases. Some neuropeptides that are produced during the ongoing inflammatory response have emerged as endogenous anti-inflammatory agents that could collaborate in tuning the balanced steady state of the immune system. These neuropeptides participate in maintaining immune tolerance through two distinct mechanisms: by regulating the balance between pro-inflammatory and anti-inflammatory factors, and by inducing the emergence of regulatory T cells with suppressive activity against autoreactive T cell effectors. Indeed, a functioning neuropeptide system contributes to general health, and alterations in the levels of these neuropeptides and/or their receptors lead to changes in susceptibility to inflammatory and autoimmune diseases. Recently, we found that some neuropeptides also have antimicrobial and antiparasitic actions, suggesting that they could act as primary mediators of innate defense, even in the most primitive organisms. In this review, we use the vasoactive intestinal peptide as example of an immunomodulatory neuropeptide to summarize the most relevant data found for other neuropeptides with similar characteristics, including adrenomedullin, urocortin, cortistatin and ghrelin.


Asunto(s)
Sistema Inmunológico/fisiología , Neuropéptidos/inmunología , Neuropéptidos/fisiología , Animales , Homeostasis , Humanos , Factores Inmunológicos/fisiología , Factores Inmunológicos/uso terapéutico , Inmunosupresores/farmacología , Inflamación/fisiopatología , Neuropéptidos/uso terapéutico , Sistemas Neurosecretores/fisiología
11.
Br J Pharmacol ; 178(21): 4368-4388, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34237151

RESUMEN

BACKGROUND AND PURPOSE: Acute lung injury (ALI), acute respiratory distress syndrome (ARDS) and pulmonary fibrosis remain major causes of morbidity, mortality and a healthcare burden in critically ill patient. There is an urgent need to identify factors causing susceptibility and for the design of new therapeutic agents. Here, we evaluate the effectiveness of the immunomodulatory neuropeptide cortistatin to regulate pulmonary inflammation and fibrosis in vivo. EXPERIMENTAL APPROACH: ALI/ARDS and pulmonary fibrosis were induced experimentally in wild-type and cortistatin-deficient mice by pulmonary infusion of the bacterial endotoxin LPS or the chemotherapeutic drug bleomycin, and the histopathological signs, pulmonary leukocyte infiltration and cytokines, and fibrotic markers were evaluated. KEY RESULTS: Partially deficient mice in cortistatin showed exacerbated pulmonary damage, pulmonary inflammation, alveolar oedema and fibrosis, and subsequent increased respiratory failure and mortality when challenged to LPS or bleomycin, even at low doses. Treatment with cortistatin reversed these aggravated phenotypes and protected from progression to severe ARDS and fibrosis, after high exposure to both injury agents. Moreover, cortistatin-deficient pulmonary macrophages and fibroblasts showed exaggerated ex vivo inflammatory and fibrotic responses, and treatment with cortistatin impaired their activation. Finally, the protective effects of cortistatin in ALI and pulmonary fibrosis were partially inhibited by specific antagonists for somatostatin and ghrelin receptors. CONCLUSION AND IMPLICATIONS: We identified cortistatin as an endogenous inhibitor of pulmonary inflammation and fibrosis. Deficiency in cortistatin could be a marker of poor prognosis in inflammatory/fibrotic pulmonary disorders. Cortistatin-based therapies could emerge as attractive candidates to treat severe ALI/ARDS, including SARS-CoV-2-associated ARDS.


Asunto(s)
Inflamación , Neuropéptidos , Neumonía , Animales , Modelos Animales de Enfermedad , Fibrosis , Inflamación/tratamiento farmacológico , Inflamación/patología , Lipopolisacáridos , Pulmón/patología , Ratones , Neuropéptidos/farmacología , Neumonía/inducido químicamente , Neumonía/tratamiento farmacológico
12.
Int J Parasitol Drugs Drug Resist ; 8(2): 203-212, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29649664

RESUMEN

The parasitic protozoan Trypanosoma brucei is the causative agent of human African trypanosomiasis (sleeping sickness) and nagana. Current drug therapies have limited efficacy, high toxicity and/or are continually hampered by the appearance of resistance. Antimicrobial peptides have recently attracted attention as potential parasiticidal compounds. Here, we explore circular bacteriocin AS-48's ability to kill clinically relevant bloodstream forms of T. brucei gambiense, T. brucei rhodesiense and T. brucei brucei. AS-48 exhibited excellent anti-trypanosomal activity in vitro (EC50 = 1-3 nM) against the three T. brucei subspecies, but it was innocuous to human cells at 104-fold higher concentrations. In contrast to its antibacterial action, AS-48 does not kill the parasite through plasma membrane permeabilization but by targeting intracellular compartments. This was evidenced by the fact that vital dye internalization-prohibiting concentrations of AS-48 could kill the parasite at 37 °C but not at 4 °C. Furthermore, AS-48 interacted with the surface of the parasite, at least in part via VSG, its uptake was temperature-dependent and clathrin-depleted cells were less permissive to the action of AS-48. The bacteriocin also caused the appearance of myelin-like structures and double-membrane autophagic vacuoles. These changes in the parasite's ultrastructure were confirmed by fluorescence microscopy as AS-48 induced the production of EGFP-ATG8.2-labeled autophagosomes. Collectively, these results indicate AS-48 kills the parasite through a mechanism involving clathrin-mediated endocytosis of VSG-bound AS-48 and the induction of autophagic-like cell death. As AS-48 has greater in vitro activity than the drugs currently used to treat T. brucei infection and does not present any signs of toxicity in mammalian cells, it could be an attractive lead compound for the treatment of sleeping sickness and nagana.


Asunto(s)
Autofagia/efectos de los fármacos , Bacteriocinas/farmacología , Muerte Celular/efectos de los fármacos , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Animales , Endocitosis/efectos de los fármacos , Humanos , Microscopía Fluorescente , Temperatura , Trypanosoma brucei brucei/ultraestructura , Trypanosoma brucei gambiense/efectos de los fármacos , Trypanosoma brucei rhodesiense/efectos de los fármacos , Tripanosomiasis Africana/sangre , Tripanosomiasis Africana/tratamiento farmacológico , Tripanosomiasis Africana/parasitología
13.
J Med Chem ; 61(3): 1231-1240, 2018 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-29323491

RESUMEN

G-quadruplexes (G4) are DNA secondary structures that take part in the regulation of gene expression. Putative G4 forming sequences (PQS) have been reported in mammals, yeast, bacteria, and viruses. Here, we present PQS searches on the genomes of T. brucei, L. major, and P. falciparum. We found telomeric sequences and new PQS motifs. Biophysical experiments showed that EBR1, a 29 nucleotide long highly repeated PQS in T. brucei, forms a stable G4 structure. G4 ligands based on carbohydrate conjugated naphthalene diimides (carb-NDIs) that bind G4's including hTel could bind EBR1 with selectivity versus dsDNA. These ligands showed important antiparasitic activity. IC50 values were in the nanomolar range against T. brucei with high selectivity against MRC-5 human cells. Confocal microscopy confirmed these ligands localize in the nucleus and kinetoplast of T. brucei suggesting they can reach their potential G4 targets. Cytotoxicity and zebrafish toxicity studies revealed sugar conjugation reduces intrinsic toxicity of NDIs.


Asunto(s)
Antiparasitarios/química , Antiparasitarios/farmacología , G-Cuádruplex/efectos de los fármacos , Genoma de Protozoos/genética , Imidas/química , Imidas/farmacología , Naftalenos/química , Naftalenos/farmacología , Animales , Antiparasitarios/toxicidad , Línea Celular , Humanos , Imidas/toxicidad , Ligandos , Naftalenos/toxicidad , Telómero/genética , Pez Cebra
14.
Parasit Vectors ; 10(1): 267, 2017 May 30.
Artículo en Inglés | MEDLINE | ID: mdl-28558770

RESUMEN

BACKGROUND: The G subfamily of ABC (ATP-binding cassette) transporters of Leishmania include 6 genes (ABCG1-G6), some with relevant biological functions associated with drug resistance and phospholipid transport. Several studies have shown that Leishmania LABCG2 transporter plays a role in the exposure of phosphatidylserine (PS), in virulence and in resistance to antimonials. However, the involvement of this transporter in other key biological processes has not been studied. METHODS: To better understand the biological function of LABCG2 and its nearly identical tandem-repeated transporter LABCG1, we have generated Leishmania major null mutant parasites for both genes (ΔLABCG1-2). NBD-PS uptake, infectivity, metacyclogenesis, autophagy and thiols were measured. RESULTS: Leishmania major ΔLABCG1-2 parasites present a reduction in NBD-PS uptake, infectivity and virulence. In addition, we have shown that ΔLABCG1-2 parasites in stationary phase growth underwent less metacyclogenesis and presented differences in the plasma membrane's lipophosphoglycan composition. Considering that autophagy is an important process in terms of parasite virulence and cell differentiation, we have shown an autophagy defect in ΔLABCG1-2 parasites, detected by monitoring expression of the autophagosome marker RFP-ATG8. This defect correlates with increased levels of reactive oxygen species and higher non-protein thiol content in ΔLABCG1-2 parasites. HPLC analysis revealed that trypanothione and glutathione were the main molecules accumulated in these ΔLABCG1-2 parasites. The decrease in non-protein thiol levels due to preincubation with buthionine sulphoximide (a γ-glutamylcysteine synthetase inhibitor) restored the autophagy process in ΔLABCG1-2 parasites, indicating a relationship between autophagy and thiol content. CONCLUSIONS: LABCG1-2 transporters from Leishmania could be considered as phosphatidylserine and non-protein thiol transporters. They probably accomplish transportation in conjunction with other molecules that are involved in oxidative stress, autophagy, metacyclogenesis and infectivity processes. The overall conclusion is that LABCG1-2 transporters could play a key role in Leishmania cell survival and infectivity.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Autofagia , Leishmania major/metabolismo , Leishmania major/patogenicidad , Leishmaniasis Cutánea/parasitología , Estrés Oxidativo , Proteínas Protozoarias/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Animales , Femenino , Humanos , Leishmania major/citología , Leishmania major/genética , Ratones , Ratones Endogámicos BALB C , Proteínas Protozoarias/genética , Virulencia
15.
Parasit Vectors ; 9: 7, 2016 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-26728034

RESUMEN

BACKGROUND: Mitochondria play essential biological functions including the synthesis and trafficking of porphyrins and iron/sulfur clusters (ISC), processes that in mammals involve the mitochondrial ATP-Binding Cassette (ABC) transporters ABCB6 and ABCB7, respectively. The mitochondrion of pathogenic protozoan parasites such as Leishmania is a promising goal for new therapeutic approaches. Leishmania infects human macrophages producing the neglected tropical disease known as leishmaniasis. Like most trypanosomatid parasites, Leishmania is auxotrophous for heme and must acquire porphyrins from the host. METHODS: LmABCB3, a new Leishmania major protein with significant sequence similarity to human ABCB6/ABCB7, was identified and characterized using bioinformatic tools. Fluorescent microscopy was used to determine its cellular localization, and its level of expression was modulated by molecular genetic techniques. Intracellular in vitro assays were used to demonstrate its role in amastigotes replication, and an in vivo mouse model was used to analyze its role in virulence. Functional characterization of LmABCB3 was carried out in Leishmania promastigotes and Saccharomyces cerevisiae. Structural analysis of LmABCB3 was performed using molecular modeling software. RESULTS: LmABCB3 is an atypical ABC half-transporter that has a unique N-terminal extension not found in any other known ABC protein. This extension is required to target LmABCB3 to the mitochondrion and includes a potential metal-binding domain. We have shown that LmABCB3 interacts with porphyrins and is required for the mitochondrial synthesis of heme from a host precursor. We also present data supporting a role for LmABCB3 in the biogenesis of cytosolic ISC, essential cofactors for cell viability in all three kingdoms of life. LmABCB3 fully complemented the severe growth defect shown in yeast lacking ATM1, an orthologue of human ABCB7 involved in exporting from the mitochondria a gluthatione-containing compound required for the generation of cytosolic ISC. Indeed, docking analyzes performed with a LmABCB3 structural model using trypanothione, the main thiol in this parasite, as a ligand showed how both, LmABCB3 and yeast ATM1, contain a similar thiol-binding pocket. Additionally, we show solid evidence suggesting that LmABCB3 is an essential gene as dominant negative inhibition of LmABCB3 is lethal for the parasite. Moreover, the abrogation of only one allele of the gene did not impede promastigote growth in axenic culture but prevented the replication of intracellular amastigotes and the virulence of the parasites in a mouse model of cutaneous leishmaniasis. CONCLUSIONS: Altogether our results present the previously undescribed LmABCB3 as an unusual mitochondrial ABC transporter essential for Leishmania survival through its role in the generation of heme and cytosolic ISC. Hence, LmABCB3 could represent a novel target to combat leishmaniasis.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Leishmania major/genética , Leishmaniasis/parasitología , Transportadoras de Casetes de Unión a ATP/genética , Animales , Hemo/metabolismo , Humanos , Hierro/metabolismo , Leishmania major/metabolismo , Leishmania major/patogenicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Modelos Moleculares , Transporte de Proteínas , Azufre/metabolismo , Virulencia
16.
PLoS Negl Trop Dis ; 7(4): e2179, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23638200

RESUMEN

Leishmaniasis is a neglected disease produced by the intracellular protozoan parasite Leishmania. In the present study, we show that LABCG2, a new ATP-binding cassette half-transporter (ABCG subfamily) from Leishmania, is involved in parasite virulence. Down-regulation of LABCG2 function upon expression of an inactive mutant version of this half-transporter (LABCG2(K/M)) is shown to reduce the translocation of short-chain analogues of phosphatidylserine (PS). This dominant-negative phenotype is specific for the headgroup of the phospholipid, as the movement of phospholipid analogues of phosphatidylcholine, phosphatidylethanolamine or sphingomyelin is not affected. In addition, promastigotes expressing LABCG2(K/M) expose less endogenous PS in the stationary phase than control parasites. Transient exposure of PS at the outer leaflet of the plasma membrane is known to be one of the mechanisms used by Leishmania to infect macrophages and to silence their immune response. Stationary phase/metacyclic promastigotes expressing LABCG2(K/M) are less infective for macrophages and show decreased pathogenesis in a mouse model of cutaneous leishmaniasis. Thus, mice infected with parasites expressing LABCG2(K/M) did not develop any lesion and showed significantly lower inflammation and parasite burden than mice infected with control parasites. Our results indicate that LABCG2 function is required for the externalization of PS in Leishmania promastigotes, a process that is involved in the virulence of the parasite.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Leishmania/metabolismo , Leishmania/patogenicidad , Fosfatidilserinas/metabolismo , Proteínas Protozoarias/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Animales , Femenino , Leishmania/genética , Leishmania major/genética , Leishmania major/metabolismo , Leishmania major/patogenicidad , Ratones , Ratones Endogámicos BALB C , Proteínas Protozoarias/genética
17.
Parasit Vectors ; 3: 116, 2010 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-21126352

RESUMEN

PCD in protozoan parasites has emerged as a fascinating field of parasite biology. This not only relates to the underlying mechanisms and their evolutionary implications but also to the impact on the parasite-host interactions within mammalian hosts and arthropod vectors. During recent years, common functions of apoptosis and autophagy in protozoa and during parasitic infections have emerged. Here, we review how distinct cell death pathways in Trypanosoma, Leishmania, Plasmodium or Toxoplasma may contribute to regulation of parasite cell densities in vectors and mammalian hosts, to differentiation of parasites, to stress responses, and to modulation of the host immunity. The examples provided indicate crucial roles of PCD in parasite biology. The existence of PCD pathways in these organisms and the identification as being critical for parasite biology and parasite-host interactions could serve as a basis for developing new anti-parasitic drugs that take advantage of these pathways.

18.
Autophagy ; 5(4): 551-4, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19337024

RESUMEN

We have recently demonstrated that some neuropeptides act as potent endogenous antiparasitic factors. These molecules kill trypanosomes through complex mechanisms that are difficult to escape by the parasite. Neuropeptides are endocytosed by the parasite, disrupt lysosome integrity, and alter the cellular compartmentalization of glycolytic enzymes. This promotes an energetic metabolism failure that initiates an autophagic-like cell death. The concept of autophagy is new for parasites and was mainly associated with differentiation or stress events. Here, we propose that this form of programmed cell death probably co-evolved with parasite-induced-neuropeptides after host infection, as a survival strategy favoring parasite transmission for a longer time by keeping the host alive.


Asunto(s)
Autofagia , Neuropéptidos/metabolismo , Parásitos/citología , Parásitos/metabolismo , Secuencia de Aminoácidos , Animales , Supervivencia Celular , Interacciones Huésped-Parásitos , Modelos Biológicos , Datos de Secuencia Molecular , Neuropéptidos/química , Trypanosoma/citología , Trypanosoma/metabolismo
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