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 TranscripcionalRESUMEN
Apicomplexan parasites are responsible for numerous important human diseases including toxoplasmosis, cryptosporidiosis, and most importantly malaria. There is a constant need for new antimalarials, and one of most keenly pursued drug targets is an ancient algal endosymbiont, the apicoplast. The apicoplast is essential for parasite survival, and several aspects of its metabolism and maintenance have been validated as targets of anti-parasitic drug treatment. Most apicoplast proteins are nuclear encoded and have to be imported into the organelle. Recently, a protein translocon typically required for endoplasmic reticulum associated protein degradation (ERAD) has been proposed to act in apicoplast protein import. Here, we show ubiquitylation to be a conserved and essential component of this process. We identify apicoplast localized ubiquitin activating, conjugating and ligating enzymes in Toxoplasma gondii and Plasmodium falciparum and observe biochemical activity by in vitro reconstitution. Using conditional gene ablation and complementation analysis we link this activity to apicoplast protein import and parasite survival. Our studies suggest ubiquitylation to be a mechanistic requirement of apicoplast protein import independent to the proteasomal degradation pathway.
Asunto(s)
Proteínas de Cloroplastos/metabolismo , Degradación Asociada con el Retículo Endoplásmico/fisiología , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Ubiquitinación/fisiología , Línea Celular , Proteínas de Cloroplastos/genética , Humanos , Plasmodium falciparum/genética , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Transporte de Proteínas/fisiología , Proteínas Protozoarias/genética , Toxoplasma/genéticaRESUMEN
PURPOSE: To report potentially pathogenic mutations in the keratin 3 (KRT3) and keratin 12 (KRT12) genes in two individuals with clinically diagnosed Meesmann corneal dystrophy (MECD). METHODS: Slit-lamp examination was performed on the probands and available family members to identify characteristic features of MECD. After informed consent was obtained, saliva samples were obtained as a source of genomic DNA, and screening of KRT3 and KRT12 was performed. Potentially pathogenic variants were screened for in 200 control chromosomes. PolyPhen-2, SIFT, and PANTHER were used to predict the functional impact of identified variants. Short tandem repeat genotyping was performed to confirm paternity. RESULTS: Slit-lamp examination of the first proband demonstrated bilateral, diffusely distributed, clear epithelial microcysts, consistent with MECD. Screening of KRT3 revealed a heterozygous missense variant in exon 1, c.250C>T (p.(Arg84Trp)), which has a minor allele frequency of 0.0076 and was not identified in 200 control chromosomes. In silico analysis with PolyPhen-2 and PANTHER predicted the variant to be damaging to protein function; however, SIFT analysis predicted tolerance of the variant. The second proband demonstrated bilateral, diffusely distributed epithelial opacities that appeared gray-white on direct illumination and translucent on retroillumination. Neither parent demonstrated corneal opacities. Screening of KRT12 revealed a novel heterozygous insertion/deletion variant in exon 6, c.1288_1293delinsAGCCCT (p.(Arg430_Arg431delinsSerPro)). This variant was not present in either of the proband's parents or in 200 control chromosomes and was predicted to be damaging by PolyPhen-2, PANTHER, and SIFT. Haplotype analysis confirmed paternity of the second proband, indicating that the variant arose de novo. CONCLUSIONS: We present a novel KRT12 mutation, representing the first de novo mutation and the first indel in KRT12 associated with MECD. In addition, we report a variant of uncertain significance in KRT3 in an individual with MECD. Although the potential pathogenicity of this variant is unknown, it is the first variant affecting the head domain of K3 to be reported in an individual with MECD and suggests that disease-causing variants associated with MECD may not be restricted to primary sequence alterations of either the helix-initiation or helix-termination motifs of K3 and K12.
Asunto(s)
Distrofia Corneal Epitelial Juvenil de Meesmann/genética , Queratina-12/genética , Queratina-3/genética , Mutación , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Niño , Distrofia Corneal Epitelial Juvenil de Meesmann/patología , Análisis Mutacional de ADN , Femenino , Heterocigoto , Humanos , Mutación INDEL , Queratina-12/química , Queratina-3/química , Masculino , Persona de Mediana Edad , Mutación Missense , Linaje , Polimorfismo de Nucleótido SimpleRESUMEN
PURPOSE: To report the identification of a novel frameshift mutation and copy number variation (CNV) in PIKFYVE in two probands with fleck corneal dystrophy (FCD). METHODS: Slit-lamp examination was performed to identify characteristic features of FCD. After genomic DNA was collected, PCR amplification and automated sequencing of all 41 exons of PIKFYVE was performed. Using genomic DNA, quantitative PCR (qPCR) was performed to detect CNVs within PIKFYVE. RESULTS: In the first FCD proband, numerous panstromal punctate opacities were observed in each of the proband's corneas, consistent with the diagnosis of FCD. Screening of PIKFYVE demonstrated a novel heterozygous frameshift mutation in exon 19, c.3151dupA, which is predicted to encode for a truncated PIKFYVE protein, p.(Asp1052Argfs*18). This variant was identified in an affected sister but not in the proband's unaffected mother or brother or 200 control chromosomes. The second FCD proband presented with bilateral, discrete, punctate, grayish-white stromal opacities. Exonic screening of PIKFYVE revealed no causative variant. However, CNV analysis demonstrated the hemizygous deletion of exons 15 and 16. CONCLUSIONS: We report a novel heterozygous frameshift mutation (c.3151dupA) and a CNV in PIKFYVE, representing the first CNV and the fifth frameshift mutation associated with FCD.
Asunto(s)
Secuencia de Bases , Distrofias Hereditarias de la Córnea/genética , Variaciones en el Número de Copia de ADN , Mutación del Sistema de Lectura , Fosfatidilinositol 3-Quinasas/genética , Eliminación de Secuencia , Adulto , Córnea/metabolismo , Córnea/patología , Distrofias Hereditarias de la Córnea/diagnóstico , Distrofias Hereditarias de la Córnea/patología , Análisis Mutacional de ADN , Exones , Femenino , Expresión Génica , Heterocigoto , Humanos , Datos de Secuencia MolecularRESUMEN
Ubiquitination is a posttranslational modification that regulates protein degradation and signaling in eukaryotes. Although it is acknowledged that pathogens exploit ubiquitination to infect mammalian cells, it remains unknown how microbes interact with the ubiquitination machinery in medically relevant arthropods. Here, we show that the ubiquitination machinery is present in the tick Ixodes scapularis and demonstrate that the E3 ubiquitin ligase named x-linked inhibitor of apoptosis protein (XIAP) restricts bacterial colonization of this arthropod vector. We provide evidence that xiap silencing significantly increases tick colonization by the bacterium Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis. We also demonstrate that (i) XIAP polyubiquitination is dependent on the really interesting new gene (RING) catalytic domain, (ii) XIAP polyubiquitination occurs via lysine (K)-63 but not K-48 residues, and (iii) XIAP-dependent K-63 polyubiquitination requires zinc for catalysis. Taken together, our data define a role for ubiquitination during bacterial colonization of disease vectors.
Asunto(s)
Anaplasma phagocytophilum/fisiología , Vectores Arácnidos/enzimología , Ehrlichiosis/microbiología , Ixodes/enzimología , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Animales , Vectores Arácnidos/microbiología , Dominio Catalítico , Humanos , Ixodes/microbiología , Interferencia de ARN , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Proteína Inhibidora de la Apoptosis Ligada a X/genéticaRESUMEN
Malaria is one of the deadliest infectious diseases worldwide. The most severe form is caused by the eukaryotic protozoan parasite Plasmodium falciparum. Recent studies have highlighted the importance of post-translational regulations for the parasite's progression throughout its life cycle, protein ubiquitylation being certainly one of the most abundant. The specificity of its components and the wide range of biological processes in which it is involved make the ubiquitylation pathway a promising source of suitable targets for anti-malarial drug development. Here, we combined immunofluorescent microscopy, biochemical assays, in silico prediction, and mass spectrometry analysis using the multidimensional protein identification technology, or MudPIT, to describe the P. falciparum ubiquitome. We found that ubiquitin conjugates are detected at every morphological stage of the parasite erythrocytic cycle. Furthermore, we detected that more than half of the parasite's proteome represents possible targets for ubiquitylation, especially proteins found to be present at the most replicative stage of the asexual cycle, the trophozoite stage. A large proportion of ubiquitin conjugates were also detected at the schizont stage, consistent with a cell activity slowdown to prepare for merozoite differentiation and invasion. Finally, for the first time in the human malaria parasite, our results strongly indicate the presence of heterologous mixed conjugations, SUMO/UB. This discovery suggests that sumoylated proteins may be regulated by ubiquitylation in P. falciparum. Altogether, our results present the first stepping stone toward a better understanding of ubiquitylation and its role(s) in the biology of the human malaria parasite.
Asunto(s)
Malaria Falciparum/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Proteína SUMO-1/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/fisiología , Humanos , Malaria Falciparum/tratamiento farmacológicoRESUMEN
BACKGROUND: In recent years, a major increase in the occurrence of drug resistant falciparum malaria has been reported. Choline analogs, such as the bisthiazolium T4, represent a novel class of compounds with strong potency against drug sensitive and resistant P. falciparum clones. Although T4 and its analogs are presumed to target the parasite's lipid metabolism, their exact mechanism of action remains unknown. Here we have employed transcriptome and proteome profiling analyses to characterize the global response of P. falciparum to T4 during the intraerythrocytic cycle of this parasite. RESULTS: No significant transcriptional changes were detected immediately after addition of T4 despite the drug's effect on the parasite metabolism. Using the Ontology-based Pattern Identification (OPI) algorithm with an increased T4 incubation time, we demonstrated cell cycle arrest and a general induction of genes involved in gametocytogenesis. Proteomic analysis revealed a significant decrease in the level of the choline/ethanolamine-phosphotransferase (PfCEPT), a key enzyme involved in the final step of synthesis of phosphatidylcholine (PC). This effect was further supported by metabolic studies, which showed a major alteration in the synthesis of PC from choline and ethanolamine by the compound. CONCLUSION: Our studies demonstrate that the bisthiazolium compound T4 inhibits the pathways of synthesis of phosphatidylcholine from choline and ethanolamine in P. falciparum, and provide evidence for post-transcriptional regulations of parasite metabolism in response to external stimuli.
Asunto(s)
Antimaláricos/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Tiazoles/farmacología , Algoritmos , Animales , Ciclo Celular/efectos de los fármacos , Células Cultivadas , Colina/metabolismo , Eritrocitos/parasitología , Etanolaminas/metabolismo , Perfilación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatidilcolinas/biosíntesis , Plasmodium falciparum/enzimología , Proteoma/genética , ARN Protozoario/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masas en Tándem , Transcripción Genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismoRESUMEN
Posterior polymorphous corneal dystrophy 1 (PPCD1) is a genetic disorder that affects corneal endothelial cell function and leads to loss of visual acuity. PPCD1 has been linked to a locus on chromosome 20 in multiple families; however, Sanger sequencing of protein-coding genes in the consensus region failed to identify any causative missense mutations. In this study, custom capture probes were utilized for targeted next-generation sequencing of the linked region in a previously reported family with PPCD1. Variants were detected through two bioinformatics pipelines and filtered according to multiple criteria. Additionally, a high-resolution microarray was used to detect copy number variations. No non-synonymous variants in the protein-coding region of annotated genes were identified. However, 12 single nucleotide variants in 10 genes, and 9 indels in 7 genes met the filtering criteria and were considered candidate variants for PPCD1. Eleven single nucleotide variants were confirmed by Sanger sequencing, including 2 synonymous variants and 9 non-coding variants, in 9 genes. One microdeletion was detected in an intron of OVOL2 by microarray but was subsequently not identified by PCR. Using a comprehensive next-generation sequencing approach, a total of 16 genes containing single nucleotide variants or indels that segregated with the affected phenotype in an affected family previously mapped to the PPCD1 locus were identified. Screening of these candidate genes in other families previously mapped to the PPCD1 locus will likely result in the identification of the genetic basis of PPCD1.
Asunto(s)
Distrofias Hereditarias de la Córnea/genética , Polimorfismo de Nucleótido Simple , Algoritmos , Biología Computacional , Variaciones en el Número de Copia de ADN , Salud de la Familia , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Proteínas de Microfilamentos/genética , Proteínas Asociadas a Microtúbulos/genética , Mutación Missense , Regiones Promotoras Genéticas , Trombomodulina/genética , Factores de Transcripción/genéticaRESUMEN
PURPOSE: To investigate the role of the zinc finger e-box binding homeobox 1 (ZEB1) transcription factor in posterior polymorphous corneal dystrophy 3 by demonstrating its ability to regulate type IV collagen gene transcription via binding to putative E2 box motifs. METHODS: Putative E2 box motifs were identified by in silico analysis within the promoter region of collagen, type IV, alpha3 (COL4A3) and collagen, type IV, alpha4 (COL4A4). To test the ability of ZEB1 to bind to each identified E2 box, electrophoretic mobility shift assays were performed by incubating ZEB1-enriched nuclear extracts with DIG-labeled probes containing one of each of the identified E2 box motifs. Dual-luciferase reporter assays were performed to test the effects of ZEB1 on the luciferase activity of COL4A3 and cadherin 1 (CDH1) promoter constructs, and to determine the effect of a ZEB1 truncating mutation on CDH1 promoter activity. RESULTS: ZEB1 exhibited binding to six of the nine COL4A3 E2 box probes, whereas no binding was observed for either of the two COL4A4 E2 box probes. ZEB1 overexpression resulted in reduced activity of the COL4A3 promoter construct containing all identified E2 box motifs, whereas a truncating ZEB1 mutation led to the loss of ZEB1-dependent repression of the CDH1 promoter. CONCLUSIONS: COL4A3 gene expression is negatively regulated by ZEB1 binding to E2 box motifs in the COL4A3 promoter region. Therefore, the altered expression of type IV collagens, particularly COL4A3, in the corneal endothelium in individuals with PPCD3 is likely due to reduced transcriptional repression in the setting of a single functional ZEB1 allele.
Asunto(s)
Autoantígenos/genética , Colágeno Tipo IV/genética , Distrofias Hereditarias de la Córnea/genética , ADN/genética , Endotelio Corneal/metabolismo , Regulación de la Expresión Génica , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Autoantígenos/biosíntesis , Células Cultivadas , Colágeno Tipo IV/biosíntesis , Distrofias Hereditarias de la Córnea/metabolismo , Distrofias Hereditarias de la Córnea/patología , Ensayo de Cambio de Movilidad Electroforética , Endotelio Corneal/patología , Epítopos , Humanos , Immunoblotting , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/biosíntesis , Dedos de ZincRESUMEN
PURPOSE: To assess the impact of zinc finger E-box binding homeobox 1 (ZEB1) gene mutations associated with posterior polymorphous corneal dystrophy 3 (PPCD3) and Fuchs' endothelial corneal dystrophy (FECD). METHODS: Thirteen of the 27 previously reported ZEB1 truncating mutations associated with PPCD3 and the six previously reported ZEB1 missense mutations associated with FECD were generated and transiently transfected into a corneal endothelial cell line. Protein abundance was determined by immunoblotting, while intracellular localization was determined by fluorescence confocal microscopy. RESULTS: Three of the 13 ZEB1 truncated mutants, and none of the missense mutants, showed significant decrease in mutant ZEB1 protein levels. Predominant nuclear localization was observed for truncated ZEB1 mutant proteins with a predicted molecular weight of less than 92 kilodaltons. The two largest mutant proteins that lacked a putative nuclear localization signal (NLS), p.(Ser638Cysfs*5) and p.(Gln884Argfs*37), primarily localized to the cytoplasm, while the NLS-containing mutant proteins, p.(Glu997Alafs*7) and p.(Glu1039Glyfs*6), primarily localized to the nucleus. All the missense ZEB1 mutant proteins were exclusively present in the nucleus. CONCLUSIONS: ZEB1 truncating mutations result in a significant decrease and/or impaired nuclear localization of the encoded protein, indicating that ZEB1 haploinsufficiency in PPCD3 may result from decreased protein production and/or impaired cellular localization. Conversely, as the reported ZEB1 missense mutations do not significantly impact protein abundance or nuclear localization, the effect of these mutations on ZEB1 function and their relationship to FECD, if any, remain to be elucidated.
Asunto(s)
ADN/genética , Endotelio Corneal/metabolismo , Distrofia Endotelial de Fuchs/genética , Proteínas de Homeodominio/genética , Mutación , Factores de Transcripción/genética , Línea Celular , Análisis Mutacional de ADN , Endotelio Corneal/patología , Distrofia Endotelial de Fuchs/metabolismo , Distrofia Endotelial de Fuchs/patología , Proteínas de Homeodominio/metabolismo , Humanos , Immunoblotting , Microscopía Confocal , Transducción de Señal , Factores de Transcripción/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
Cytosine DNA methylation is an epigenetic mark in most eukaryotic cells that regulates numerous processes, including gene expression and stress responses. We performed a genome-wide analysis of DNA methylation in the human malaria parasite Plasmodium falciparum. We mapped the positions of methylated cytosines and identified a single functional DNA methyltransferase (Plasmodium falciparum DNA methyltransferase; PfDNMT) that may mediate these genomic modifications. These analyses revealed that the malaria genome is asymmetrically methylated and shares common features with undifferentiated plant and mammalian cells. Notably, core promoters are hypomethylated, and transcript levels correlate with intraexonic methylation. Additionally, there are sharp methylation transitions at nucleosome and exon-intron boundaries. These data suggest that DNA methylation could regulate virulence gene expression and transcription elongation. Furthermore, the broad range of action of DNA methylation and the uniqueness of PfDNMT suggest that the methylation pathway is a potential target for antimalarial strategies.
Asunto(s)
Metilación de ADN , ADN Protozoario/química , Genoma de Protozoos , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Cromatografía Liquida , ADN Protozoario/metabolismo , ADN-Citosina Metilasas/metabolismo , Epigénesis Genética , Eritrocitos/parasitología , Humanos , Plasmodium falciparum/enzimología , Espectrometría de Masas en TándemRESUMEN
Over the past few years only, next-generation sequencing technologies became accessible and many applications were rapidly derived, such as the development of RNA-seq, a technique that uses deep sequencing to profile whole transcriptomes. RNA-seq has the power to discover new transcripts and splicing variants, single-nucleotide variations, fusion genes, and mRNA level-based expression profiles. Preparing RNA-seq libraries can be delicate and usually obligates buying expensive kits that require large amounts of stating materials. The method presented here is flexible and cost-effective. Using this method, we prepared high-quality strand-specific RNA-seq libraries from RNA extracted from the human malaria parasite Plasmodium falciparum. The libraries are compatible with Illumina(®)'s sequencers Genome Analyzer and Hi-Seq. The method can, however, be easily adapted to other platforms.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Plasmodium falciparum/genética , Análisis de Secuencia de ARN/métodos , Adenosina/aislamiento & purificación , Tampones (Química) , División del ADN , ADN Complementario/síntesis química , Desoxirribonucleasas/química , Biblioteca de Genes , Polímeros/aislamiento & purificación , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , ARN Protozoario/genética , ARN Protozoario/aislamiento & purificaciónRESUMEN
Ubiquitin-dependent protein degradation within malarial parasites is a burgeoning field of interest due to several encouraging reports of proteasome inhibitors that were able to confer antimalarial activity. Despite the growing interest in the Plasmodium proteasome system, relatively little investigation has been done to actually characterize the parasite degradation machinery. In this report, we provide an initial biological investigation of the ubiquitylating components of the endoplasmic reticulum-associated degradation (ERAD) system, which is a major pathway in targeting misfolded proteins from the ER to the cytosol for proteasome degradation. We are able to show that the ERAD system is essential for parasite survival and that the putative Plasmodium HRD1 (E3 ubiquitin ligase), UBC (E2 ubiquitin conjugating enzyme) and UBA1 (E1 ubiquitin activating enzyme) are able to mediate in vitro ubiquitylation. Furthermore, by using immunofluorescence, we report that Plasmodium HRD1 localizes to the ER membranes, while the Plasmodium UBC and UBA1 localize to the cytosol. In addition, our gene disruption experiments indicate that the Plasmodium HRD1 is likely essential. We have conducted an initial characterization of the ubiquitylating components of the Plasmodium ERAD system, a major pathway for protein degradation and parasite maintenance. In conjunction with promising proteasome inhibitor studies, we explore the possibility of targeting the Plasmodium ERAD system for future bottom-up drug development approaches.