Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 44
Filtrar
Más filtros













Intervalo de año de publicación
1.
Nat Commun ; 15(1): 1365, 2024 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-38355719

RESUMEN

Ribonucleoprotein complexes are composed of RNA, RNA-dependent proteins (RDPs) and RNA-binding proteins (RBPs), and play fundamental roles in RNA regulation. However, in the human malaria parasite, Plasmodium falciparum, identification and characterization of these proteins are particularly limited. In this study, we use an unbiased proteome-wide approach, called R-DeeP, a method based on sucrose density gradient ultracentrifugation, to identify RDPs. Quantitative analysis by mass spectrometry identifies 898 RDPs, including 545 proteins not yet associated with RNA. Results are further validated using a combination of computational and molecular approaches. Overall, this method provides the first snapshot of the Plasmodium protein-protein interaction network in the presence and absence of RNA. R-DeeP also helps to reconstruct Plasmodium multiprotein complexes based on co-segregation and deciphers their RNA-dependence. One RDP candidate, PF3D7_0823200, is functionally characterized and validated as a true RBP. Using enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP-seq), we demonstrate that this protein interacts with various Plasmodium non-coding transcripts, including the var genes and ap2 transcription factors.


Asunto(s)
Plasmodium , ARN , Humanos , ARN/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteoma/metabolismo , Proteínas de Unión al ARN/metabolismo , Plasmodium/genética
2.
bioRxiv ; 2024 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-38405848

RESUMEN

Over the last few decades, novel methods have been developed to study how chromosome positioning within the nucleus may play a role in gene regulation. Adaptation of these methods in the human malaria parasite, Plasmodium falciparum, has recently led to the discovery that the three-dimensional structure of chromatin within the nucleus may be critical in controlling expression of virulence genes (var genes). Recent work has implicated an unusual, highly conserved var gene called var2csa in contributing to coordinated transcriptional switching, however how this gene functions in this capacity is unknown. To further understand how var2csa influences var gene switching, targeted DNA double-strand breaks (DSBs) within the sub-telomeric region of chromosome 12 were used to delete the gene and the surrounding chromosomal region. To characterize the changes in chromatin architecture stemming from this deletion and how these changes could affect var gene expression, we used a combination of RNA-seq, Chip-seq and Hi-C to pinpoint epigenetic and chromatin structural modifications in regions of differential gene expression. We observed a net gain of interactions in sub-telomeric regions and internal var gene regions following var2csa knockout, indicating an increase of tightly controlled heterochromatin structures. Our results suggest that disruption of var2csa results not only in changes in var gene transcriptional regulation but also a significant tightening of heterochromatin clusters thereby disrupting coordinated activation of var genes throughout the genome. Altogether our result confirms a strong link between the var2csa locus, chromatin structure and var gene expression.

3.
Nat Commun ; 14(1): 5086, 2023 08 22.
Artículo en Inglés | MEDLINE | ID: mdl-37607941

RESUMEN

The complex life cycle of Plasmodium falciparum requires coordinated gene expression regulation to allow host cell invasion, transmission, and immune evasion. Increasing evidence now suggests a major role for epigenetic mechanisms in gene expression in the parasite. In eukaryotes, many lncRNAs have been identified to be pivotal regulators of genome structure and gene expression. To investigate the regulatory roles of lncRNAs in P. falciparum we explore the intergenic lncRNA distribution in nuclear and cytoplasmic subcellular locations. Using nascent RNA expression profiles, we identify a total of 1768 lncRNAs, of which 718 (~41%) are novels in P. falciparum. The subcellular localization and stage-specific expression of several putative lncRNAs are validated using RNA-FISH. Additionally, the genome-wide occupancy of several candidate nuclear lncRNAs is explored using ChIRP. The results reveal that lncRNA occupancy sites are focal and sequence-specific with a particular enrichment for several parasite-specific gene families, including those involved in pathogenesis and sexual differentiation. Genomic and phenotypic analysis of one specific lncRNA demonstrate its importance in sexual differentiation and reproduction. Our findings bring a new level of insight into the role of lncRNAs in pathogenicity, gene regulation and sexual differentiation, opening new avenues for targeted therapeutic strategies against the deadly malaria parasite.


Asunto(s)
Malaria Falciparum , Malaria , Parásitos , ARN Largo no Codificante , Humanos , Animales , Plasmodium falciparum/genética , ARN Largo no Codificante/genética , Malaria Falciparum/genética
4.
Nat Commun ; 13(1): 1275, 2022 03 11.
Artículo en Inglés | MEDLINE | ID: mdl-35277503

RESUMEN

The RAP (RNA-binding domain abundant in Apicomplexans) protein family has been identified in various organisms. Despite expansion of this protein family in apicomplexan parasites, their main biological functions remain unknown. In this study, we use inducible knockdown studies in the human malaria parasite, Plasmodium falciparum, to show that two RAP proteins, PF3D7_0105200 (PfRAP01) and PF3D7_1470600 (PfRAP21), are essential for parasite survival and localize to the mitochondrion. Using transcriptomics, metabolomics, and proteomics profiling experiments, we further demonstrate that these RAP proteins are involved in mitochondrial RNA metabolism. Using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (eCLIP-seq), we validate that PfRAP01 and PfRAP21 are true RNA-binding proteins and interact specifically with mitochondrial rRNAs. Finally, mitochondrial enrichment experiments followed by deep sequencing of small RNAs demonstrate that PfRAP21 controls mitochondrial rRNA expression. Collectively, our results establish the role of these RAP proteins in mitoribosome activity and contribute to further understanding this protein family in malaria parasites.


Asunto(s)
Malaria Falciparum , Ribosomas Mitocondriales , Plasmodium falciparum , Proteínas Protozoarias , Proteínas de Unión al ARN , Genómica , Humanos , Malaria Falciparum/parasitología , Ribosomas Mitocondriales/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo
5.
J Comput Aided Mol Des ; 34(11): 1117-1132, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32833084

RESUMEN

There is a pressing need to improve the efficiency of drug development, and nowhere is that need more clear than in the case of neglected diseases like malaria. The peculiarities of pyrimidine metabolism in Plasmodium species make inhibition of dihydroorotate dehydrogenase (DHODH) an attractive target for antimalarial drug design. By applying a pair of complementary quantitative structure-activity relationships derived for inhibition of a truncated, soluble form of the enzyme from Plasmodium falciparum (s-PfDHODH) to data from a large-scale phenotypic screen against cultured parasites, we were able to identify a class of antimalarial leads that inhibit the enzyme and abolish parasite growth in blood culture. Novel analogs extending that class were designed and synthesized with a goal of improving potency as well as the general pharmacokinetic and toxicological profiles. Their synthesis also represented an opportunity to prospectively validate our in silico property predictions. The seven analogs synthesized exhibited physicochemical properties in good agreement with prediction, and five of them were more active against P. falciparum growing in blood culture than any of the compounds in the published lead series. The particular analogs prepared did not inhibit s-PfDHODH in vitro, but advanced biological assays indicated that other examples from the class did inhibit intact PfDHODH bound to the mitochondrial membrane. The new analogs, however, killed the parasites by acting through some other, unidentified mechanism 24-48 h before PfDHODH inhibition would be expected to do so.


Asunto(s)
Antimaláricos/química , Inhibidores Enzimáticos/química , Malaria Falciparum/tratamiento farmacológico , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Plasmodium falciparum/efectos de los fármacos , Quinolonas/química , Antimaláricos/efectos adversos , Antimaláricos/farmacocinética , Dihidroorotato Deshidrogenasa , Diseño de Fármacos , Inhibidores Enzimáticos/efectos adversos , Inhibidores Enzimáticos/farmacocinética , Humanos , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Estructura Molecular , Relación Estructura-Actividad Cuantitativa , Quinolonas/efectos adversos , Quinolonas/farmacocinética
6.
Microb Genom ; 6(2)2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32017676

RESUMEN

Proteins interacting with DNA are fundamental for mediating processes such as gene expression, DNA replication and maintenance of genome integrity. Accumulating evidence suggests that the chromatin of apicomplexan parasites, such as Plasmodium falciparum, is highly organized, and this structure provides an epigenetic mechanism for transcriptional regulation. To investigate how parasite chromatin structure is being regulated, we undertook comparative genomics analysis using 12 distinct eukaryotic genomes. We identified conserved and parasite-specific chromatin-associated domains (CADs) and proteins (CAPs). We then used the chromatin enrichment for proteomics (ChEP) approach to experimentally capture CAPs in P. falciparum. A topological scoring analysis of the proteomics dataset revealed stage-specific enrichments of CADs and CAPs. Finally, we characterized, two candidate CAPs: a conserved homologue of the structural maintenance of chromosome 3 protein and a homologue of the crowded-like nuclei protein, a plant-like protein functionally analogous to animal nuclear lamina proteins. Collectively, our results provide a comprehensive overview of CAPs in apicomplexans, and contribute to our understanding of the complex molecular components regulating chromatin structure and genome architecture in these deadly parasites.


Asunto(s)
Cromatina/metabolismo , Malaria Falciparum/parasitología , Plasmodium falciparum/metabolismo , Proteoma/metabolismo , Proteínas Protozoarias/metabolismo , Cromatina/genética , Regulación de la Expresión Génica , Genoma de Protozoos , Humanos , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Unión Proteica , Proteoma/genética , Proteínas Protozoarias/genética
7.
Proc Natl Acad Sci U S A ; 116(8): 3183-3192, 2019 02 19.
Artículo en Inglés | MEDLINE | ID: mdl-30723152

RESUMEN

The positioning of chromosomes in the nucleus of a eukaryotic cell is highly organized and has a complex and dynamic relationship with gene expression. In the human malaria parasite Plasmodium falciparum, the clustering of a family of virulence genes correlates with their coordinated silencing and has a strong influence on the overall organization of the genome. To identify conserved and species-specific principles of genome organization, we performed Hi-C experiments and generated 3D genome models for five Plasmodium species and two related apicomplexan parasites. Plasmodium species mainly showed clustering of centromeres, telomeres, and virulence genes. In P. falciparum, the heterochromatic virulence gene cluster had a strong repressive effect on the surrounding nuclear space, while this was less pronounced in Plasmodium vivax and Plasmodium berghei, and absent in Plasmodium yoelii In Plasmodium knowlesi, telomeres and virulence genes were more dispersed throughout the nucleus, but its 3D genome showed a strong correlation with gene expression. The Babesia microti genome showed a classical Rabl organization with colocalization of subtelomeric virulence genes, while the Toxoplasma gondii genome was dominated by clustering of the centromeres and lacked virulence gene clustering. Collectively, our results demonstrate that spatial genome organization in most Plasmodium species is constrained by the colocalization of virulence genes. P. falciparum and P. knowlesi, the only two Plasmodium species with gene families involved in antigenic variation, are unique in the effect of these genes on chromosome folding, indicating a potential link between genome organization and gene expression in more virulent pathogens.


Asunto(s)
Genoma de Protozoos/genética , Heterocromatina/genética , Malaria Falciparum/genética , Plasmodium falciparum/genética , Animales , Centrómero/genética , Regulación de la Expresión Génica/genética , Genómica , Humanos , Malaria Falciparum/parasitología , Plasmodium berghei/genética , Plasmodium berghei/patogenicidad , Plasmodium falciparum/patogenicidad , Plasmodium knowlesi/genética , Plasmodium knowlesi/patogenicidad , Plasmodium vivax/genética , Plasmodium vivax/patogenicidad , Telómero/genética , Toxoplasma/genética , Toxoplasma/patogenicidad
8.
Nat Commun ; 9(1): 1910, 2018 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-29765020

RESUMEN

The development of malaria parasites throughout their various life cycle stages is coordinated by changes in gene expression. We previously showed that the three-dimensional organization of the Plasmodium falciparum genome is strongly associated with gene expression during its replication cycle inside red blood cells. Here, we analyze genome organization in the P. falciparum and P. vivax transmission stages. Major changes occur in the localization and interactions of genes involved in pathogenesis and immune evasion, host cell invasion, sexual differentiation, and master regulation of gene expression. Furthermore, we observe reorganization of subtelomeric heterochromatin around genes involved in host cell remodeling. Depletion of heterochromatin protein 1 (PfHP1) resulted in loss of interactions between virulence genes, confirming that PfHP1 is essential for maintenance of the repressive center. Our results suggest that the three-dimensional genome structure of human malaria parasites is strongly connected with transcriptional activity of specific gene families throughout the life cycle.


Asunto(s)
Genoma de Protozoos , Malaria Falciparum/parasitología , Familia de Multigenes , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Animales , Anopheles/parasitología , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Eritrocitos/parasitología , Femenino , Humanos , Estadios del Ciclo de Vida , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/fisiología , Proteínas Protozoarias/metabolismo
9.
Nucleic Acids Res ; 45(13): 7825-7840, 2017 Jul 27.
Artículo en Inglés | MEDLINE | ID: mdl-28531310

RESUMEN

Gene expression in Plasmodium falciparum is tightly regulated to ensure successful propagation of the parasite throughout its complex life cycle. The earliest transcriptomics studies in P. falciparum suggested a cascade of transcriptional activity over the course of the 48-hour intraerythrocytic developmental cycle (IDC); however, the just-in-time transcriptional model has recently been challenged by findings that show the importance of post-transcriptional regulation. To further explore the role of transcriptional regulation, we performed the first genome-wide nascent RNA profiling in P. falciparum. Our findings indicate that the majority of genes are transcribed simultaneously during the trophozoite stage of the IDC and that only a small subset of genes is subject to differential transcriptional timing. RNA polymerase II is engaged with promoter regions prior to this transcriptional burst, suggesting that Pol II pausing plays a dominant role in gene regulation. In addition, we found that the overall transcriptional program during gametocyte differentiation is surprisingly similar to the IDC, with the exception of relatively small subsets of genes. Results from this study suggest that further characterization of the molecular players that regulate stage-specific gene expression and Pol II pausing will contribute to our continuous search for novel antimalarial drug targets.


Asunto(s)
Genes Protozoarios , Plasmodium falciparum/genética , ARN Protozoario/genética , Animales , Epigénesis Genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Malaria Falciparum/sangre , Malaria Falciparum/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/patogenicidad , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Protozoario/metabolismo , Análisis de Secuencia de ARN , Transcripción Genética
10.
ACS Med Chem Lett ; 8(3): 355-360, 2017 Mar 09.
Artículo en Inglés | MEDLINE | ID: mdl-28337330

RESUMEN

Several kalihinol natural products, members of the broader isocyanoterpene family of antimalarial agents, are potent inhibitors of Plasmodium falciparum, the agent of the most severe form of human malaria. Our previous total synthesis of kalihinol B provided a blueprint to generate many analogues within this family, some as complex as the natural product and some much simplified and easier to access. Each analogue was tested for blood-stage antimalarial activity using both drug-sensitive and -resistant P. falciparum strains. Many considerably simpler analogues of the kalihinols retained potent activity, as did a compound with a different decalin scaffold made in only three steps from sclareolide. Finally, one representative compound showed reasonable stability toward microsomal metabolism, suggesting that the isonitrile functional group that is critical for activity is not an inherent liability in these compounds.

11.
Genome Biol ; 17(1): 147, 2016 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-27381095

RESUMEN

BACKGROUND: Gene expression is controlled at multiple levels, including transcription, stability, translation, and degradation. Over the years, it has become apparent that Plasmodium falciparum exerts limited transcriptional control of gene expression, while at least part of Plasmodium's genome is controlled by post-transcriptional mechanisms. To generate insights into the mechanisms that regulate gene expression at the post-transcriptional level, we undertook complementary computational, comparative genomics, and experimental approaches to identify and characterize mRNA-binding proteins (mRBPs) in P. falciparum. RESULTS: Close to 1000 RNA-binding proteins are identified by hidden Markov model searches, of which mRBPs encompass a relatively large proportion of the parasite proteome as compared to other eukaryotes. Several abundant mRNA-binding domains are enriched in apicomplexan parasites, while strong depletion of mRNA-binding domains involved in RNA degradation is observed. Next, we experimentally capture 199 proteins that interact with mRNA during the blood stages, 64 of which with high confidence. These captured mRBPs show a significant overlap with the in silico identified candidate RBPs (p < 0.0001). Among the experimentally validated mRBPs are many known translational regulators active in other stages of the parasite's life cycle, such as DOZI, CITH, PfCELF2, Musashi, and PfAlba1-4. Finally, we also detect several proteins with an RNA-binding domain abundant in Apicomplexans (RAP domain) that is almost exclusively found in apicomplexan parasites. CONCLUSIONS: Collectively, our results provide the most complete comparative genomics and experimental analysis of mRBPs in P. falciparum. A better understanding of these regulatory proteins will not only give insight into the intricate parasite life cycle but may also provide targets for novel therapeutic strategies.


Asunto(s)
Malaria Falciparum/genética , Plasmodium falciparum/genética , Biosíntesis de Proteínas , Proteoma/genética , Proteínas de Unión al ARN/biosíntesis , Regulación de la Expresión Génica , Genoma , Humanos , Malaria Falciparum/parasitología , Plasmodium falciparum/patogenicidad , Dominios Proteicos/genética , Procesamiento Postranscripcional del ARN/genética , Proteínas de Unión al ARN/genética
12.
J Proteome Res ; 15(8): 2787-801, 2016 08 05.
Artículo en Inglés | MEDLINE | ID: mdl-27291344

RESUMEN

A major obstacle in understanding the complex biology of the malaria parasite remains to discover how gene transcription is controlled during its life cycle. Accumulating evidence indicates that the parasite's epigenetic state plays a fundamental role in gene expression and virulence. Using a comprehensive and quantitative mass spectrometry approach, we determined the global and dynamic abundance of histones and their covalent post-transcriptional modifications throughout the intraerythrocytic developmental cycle of Plasmodium falciparum. We detected a total of 232 distinct modifications, of which 160 had never been detected in Plasmodium and 88 had never been identified in any other species. We further validated over 10% of the detected modifications and their expression patterns by multiple reaction monitoring assays. In addition, we uncovered an unusual chromatin organization with parasite-specific histone modifications and combinatorial dynamics that may be directly related to transcriptional activity, DNA replication, and cell cycle progression. Overall, our data suggest that the malaria parasite has a unique histone modification signature that correlates with parasite virulence.


Asunto(s)
Código de Histonas , Estadios del Ciclo de Vida/genética , Malaria/parasitología , Plasmodium falciparum/patogenicidad , Epigénesis Genética , Eritrocitos/parasitología , Histonas/metabolismo , Plasmodium falciparum/genética , Proteínas Protozoarias/efectos adversos , Proteínas Protozoarias/análisis , Transcripción Genética , Activación Transcripcional
13.
Bioorg Med Chem Lett ; 26(3): 854-857, 2016 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-26748697

RESUMEN

The marine natural product (-)-8,15-diisocyano-11(20)-amphilectene (1), isolated from the Caribbean sponge Svenzea flava, was used as scaffold to synthetize five new products, all of which were tested against laboratory strains of Plasmodium falciparum and Mycobacterium tuberculosis H37Rv. The scaffold contains two isocyanide units that are amenable to chemical manipulation, enabling them to be elaborated into a small library of sulfur and selenium compounds. Although most of the analogs prepared were less potent than the parent compound, 5 was nearly equipotent showing IC50 values of 0.0066 µM and 0.0025 µM, respectively, against two strains (Dd2 and 3D7) of the malaria parasite. On the other hand, when assayed against the tuberculosis bacterium, analogs 5 and 6 were found to be more potent than 1.


Asunto(s)
Antiinfecciosos/síntesis química , Productos Biológicos/química , Cianatos/química , Diterpenos/química , Isotiocianatos/química , Compuestos de Selenio/química , Animales , Antiinfecciosos/química , Antiinfecciosos/farmacología , Diterpenos/síntesis química , Diterpenos/farmacología , Concentración 50 Inhibidora , Mycobacterium tuberculosis/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Poríferos/química , Poríferos/metabolismo
14.
Bioorg Med Chem Lett ; 25(22): 5339-43, 2015 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-26421992

RESUMEN

A mixture-based combinatorial library of five Ugi adducts (4-8) incorporating known antitubercular and antimalarial pharmacophores was successfully synthesized, starting from the naturally occurring diisocyanide 3, via parallel Ugi four-center three-component reactions (U-4C-3CR). The novel α-acylamino amides obtained were evaluated for their antiinfective potential against laboratory strains of Mycobacterium tuberculosis H37Rv and chloroquine-susceptible 3D7 Plasmodium falciparum. Interestingly, compounds 4-8 displayed potent in vitro antiparasitic activity with higher cytotoxicity in comparison to their diisocyanide precursor 3, with the best compound exhibiting an IC50 value of 3.6 nM. Additionally, these natural product inspired hybrids potently inhibited in vitro thromboxane B2 (TXB2) and superoxide anion (O2(-)) generation from Escherichia coli lipopolysaccharide (LPS)-activated rat neonatal microglia, with concomitant low short-term toxicity.


Asunto(s)
Antiinflamatorios/síntesis química , Antiinflamatorios/farmacología , Cianuros/química , Cianuros/síntesis química , Mycobacterium tuberculosis/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacología , Aminas/química , Aminas/farmacología , Antiinflamatorios/química , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Quinolinas/química , Quinolinas/farmacología , Bibliotecas de Moléculas Pequeñas/química
15.
Tetrahedron ; 71(3): 487-494, 2015 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-26494928

RESUMEN

Bioassay-guided fractionation of the Caribbean sponge Svenzea flava collected near Mona Island, off the west coast of Puerto Rico, led to the isolation of two isocyanide amphilectane-type diterpenes named monamphilectines B and C (2 and 3). Attached to the backbone of each of these compounds is the first α-substituted monocyclic ß-lactam ring to be isolated from a marine organism. The molecular structures of 2 and 3 were established by spectroscopic methods and then confirmed unequivocally by chemical correlation and comparison of physical and chemical data with the natural products. The new ß-lactams were successfully synthesized in one step, starting from the known diisocyanide 4, via parallel Ugi four-center three-component reactions (U-4C-3CR) that also established their absolute stereostructures. Interestingly, compounds 2 and 3 exhibited activities in the low nanomolar range against the human malaria parasite Plasmodium falciparum.

16.
J Am Chem Soc ; 137(15): 4912-5, 2015 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-25815413

RESUMEN

Of the 50+ kalihinane diterpenoids reported to date, only five had been tested for antimalarial activity, in spite of the fact that kalihinol A is the most potent among the members of the larger family of antimalarial isocyanoterpenes. We have validated a strategy designed to access many of the kalihinanes with a 12-step enantioselective synthesis of kalihinol B, the tetrahydrofuran isomer of kalihinol A (a tetrahydropyran). Kalihinol B shows similarly high potency against chloroquine-resistant Plasmodium falciparum.


Asunto(s)
Antimaláricos/síntesis química , Antimaláricos/farmacología , Diterpenos/síntesis química , Diterpenos/farmacología , Plasmodium falciparum/efectos de los fármacos , Antimaláricos/química , Cloroquina/farmacología , Diterpenos/química , Resistencia a Medicamentos/efectos de los fármacos , Estructura Molecular , Pruebas de Sensibilidad Parasitaria
17.
J Nat Prod ; 77(11): 2418-22, 2014 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-25375026

RESUMEN

Metrodorea stipularis stem extracts were studied in the search for possible antichagastic, antimalarial, and antitumoral compounds using cruzain from Trypanosoma cruzi, Plasmodium falciparum, and cathepsins B and L, as molecular targets, respectively. Dihydrochalcones 1, 2, 3, and 4 showed significant inhibitory activity against all the targets. Compounds 1-4 displayed IC50 values ranging from 7.7 to 21.6 µM against cruzain; dihydrochalcones 2 and 4 inhibited the growth of three different strains of P. falciparum in low micromolar concentrations; and against cathepsins B and L these compounds presented good inhibitory activity with IC50 values ranging from 1.0 to 14.9 µM. The dihydrochalcones showed good selectivity in their inhibitory activities against the cysteine proteases.


Asunto(s)
Antiprotozoarios , Chalconas , Antimaláricos/química , Antimaláricos/aislamiento & purificación , Antimaláricos/farmacología , Antiprotozoarios/química , Antiprotozoarios/aislamiento & purificación , Antiprotozoarios/farmacología , Brasil , Catepsina B/antagonistas & inhibidores , Catepsina L/antagonistas & inhibidores , Chalconas/química , Chalconas/aislamiento & purificación , Chalconas/farmacología , Concentración 50 Inhibidora , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Tallos de la Planta/química , Plasmodium falciparum/efectos de los fármacos , Trypanosoma cruzi/efectos de los fármacos
18.
BMC Genomics ; 15: 347, 2014 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-24885191

RESUMEN

BACKGROUND: In eukaryotic organisms, packaging of DNA into nucleosomes controls gene expression by regulating access of the promoter to transcription factors. The human malaria parasite Plasmodium falciparum encodes relatively few transcription factors, while extensive nucleosome remodeling occurs during its replicative cycle in red blood cells. These observations point towards an important role of the nucleosome landscape in regulating gene expression. However, the relation between nucleosome positioning and transcriptional activity has thus far not been explored in detail in the parasite. RESULTS: Here, we analyzed nucleosome positioning in the asexual and sexual stages of the parasite's erythrocytic cycle using chromatin immunoprecipitation of MNase-digested chromatin, followed by next-generation sequencing. We observed a relatively open chromatin structure at the trophozoite and gametocyte stages, consistent with high levels of transcriptional activity in these stages. Nucleosome occupancy of genes and promoter regions were subsequently compared to steady-state mRNA expression levels. Transcript abundance showed a strong inverse correlation with nucleosome occupancy levels in promoter regions. In addition, AT-repeat sequences were strongly unfavorable for nucleosome binding in P. falciparum, and were overrepresented in promoters of highly expressed genes. CONCLUSIONS: The connection between chromatin structure and gene expression in P. falciparum shares similarities with other eukaryotes. However, the remarkable nucleosome dynamics during the erythrocytic stages and the absence of a large variety of transcription factors may indicate that nucleosome binding and remodeling are critical regulators of transcript levels. Moreover, the strong dependency between chromatin structure and DNA sequence suggests that the P. falciparum genome may have been shaped by nucleosome binding preferences. Nucleosome remodeling mechanisms in this deadly parasite could thus provide potent novel anti-malarial targets.


Asunto(s)
ADN/metabolismo , Malaria/parasitología , Nucleosomas/metabolismo , Plasmodium falciparum/genética , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Eritrocitos/parasitología , Secuenciación de Nucleótidos de Alto Rendimiento , Histonas/genética , Histonas/metabolismo , Humanos , Malaria/patología , Nucleasa Microcócica/metabolismo , Poli dA-dT/genética , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Transcripción Genética , Trofozoítos/metabolismo
19.
Genome Res ; 24(6): 974-88, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24671853

RESUMEN

The development of the human malaria parasite Plasmodium falciparum is controlled by coordinated changes in gene expression throughout its complex life cycle, but the corresponding regulatory mechanisms are incompletely understood. To study the relationship between genome architecture and gene regulation in Plasmodium, we assayed the genome architecture of P. falciparum at three time points during its erythrocytic (asexual) cycle. Using chromosome conformation capture coupled with next-generation sequencing technology (Hi-C), we obtained high-resolution chromosomal contact maps, which we then used to construct a consensus three-dimensional genome structure for each time point. We observed strong clustering of centromeres, telomeres, ribosomal DNA, and virulence genes, resulting in a complex architecture that cannot be explained by a simple volume exclusion model. Internal virulence gene clusters exhibit domain-like structures in contact maps, suggesting that they play an important role in the genome architecture. Midway during the erythrocytic cycle, at the highly transcriptionally active trophozoite stage, the genome adopts a more open chromatin structure with increased chromosomal intermingling. In addition, we observed reduced expression of genes located in spatial proximity to the repressive subtelomeric center, and colocalization of distinct groups of parasite-specific genes with coordinated expression profiles. Overall, our results are indicative of a strong association between the P. falciparum spatial genome organization and gene expression. Understanding the molecular processes involved in genome conformation dynamics could contribute to the discovery of novel antimalarial strategies.


Asunto(s)
Ensamble y Desensamble de Cromatina , Cromosomas/genética , Genoma de Protozoos , Modelos Genéticos , Plasmodium falciparum/genética , Regulación del Desarrollo de la Expresión Génica , Plasmodium falciparum/crecimiento & desarrollo , Esquizontes/metabolismo , Trofozoítos/metabolismo
20.
Autophagy ; 10(1): 80-92, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24275162

RESUMEN

Autophagy is a catabolic pathway typically induced by nutrient starvation to recycle amino acids, but can also function in removing damaged organelles. In addition, this pathway plays a key role in eukaryotic development. To date, not much is known about the role of autophagy in apicomplexan parasites and more specifically in the human malaria parasite Plasmodium falciparum. Comparative genomic analysis has uncovered some, but not all, orthologs of autophagy-related (ATG) genes in the malaria parasite genome. Here, using a genome-wide in silico analysis, we confirmed that ATG genes whose products are required for vesicle expansion and completion are present, while genes involved in induction of autophagy and cargo packaging are mostly absent. We subsequently focused on the molecular and cellular function of P. falciparum ATG8 (PfATG8), an autophagosome membrane marker and key component of the autophagy pathway, throughout the parasite asexual and sexual erythrocytic stages. In this context, we showed that PfATG8 has a distinct and atypical role in parasite development. PfATG8 localized in the apicoplast and in vesicles throughout the cytosol during parasite development. Immunofluorescence assays of PfATG8 in apicoplast-minus parasites suggest that PfATG8 is involved in apicoplast biogenesis. Furthermore, treatment of parasite cultures with bafilomycin A 1 and chloroquine, both lysosomotropic agents that inhibit autophagosome and lysosome fusion, resulted in dramatic morphological changes of the apicoplast, and parasite death. Furthermore, deep proteomic analysis of components associated with PfATG8 indicated that it may possibly be involved in ribophagy and piecemeal microautophagy of the nucleus. Collectively, our data revealed the importance and specificity of the autophagy pathway in the malaria parasite and offer potential novel therapeutic strategies.


Asunto(s)
Autofagia , Parásitos/citología , Plasmodium falciparum/citología , Transducción de Señal , Animales , Antimaláricos/farmacología , Apicoplastos/efectos de los fármacos , Apicoplastos/metabolismo , Autofagia/efectos de los fármacos , Autofagia/genética , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/metabolismo , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Técnica del Anticuerpo Fluorescente , Gametogénesis/efectos de los fármacos , Genes Protozoarios , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Estadios del Ciclo de Vida/efectos de los fármacos , Estadios del Ciclo de Vida/genética , Parásitos/efectos de los fármacos , Parásitos/genética , Parásitos/crecimiento & desarrollo , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Transporte de Proteínas/efectos de los fármacos , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA