RESUMO
Artemisinin and its derivatives (ARTs) are the frontline drugs against malaria, but resistance is jeopardizing their effectiveness. ART resistance is mediated by mutations in the parasite's Kelch13 protein, but Kelch13 function and its role in resistance remain unclear. In this study, we identified proteins located at a Kelch13-defined compartment. Inactivation of eight of these proteins, including Kelch13, rendered parasites resistant to ART, revealing a pathway critical for resistance. Functional analysis showed that these proteins are required for endocytosis of hemoglobin from the host cell. Parasites with inactivated Kelch13 or a resistance-conferring Kelch13 mutation displayed reduced hemoglobin endocytosis. ARTs are activated by degradation products of hemoglobin. Hence, reduced activity of Kelch13 and its interactors diminishes hemoglobin endocytosis and thereby ART activation, resulting in parasite resistance.
Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Resistência a Medicamentos/genética , Endocitose/genética , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Antimaláricos/uso terapêutico , Artemisininas/uso terapêutico , Hemoglobinas/metabolismo , Humanos , Malária Falciparum/tratamento farmacológico , MutaçãoRESUMO
Epigenetic regulatory mechanisms are central to the development and survival of all eukaryotic organisms. These mechanisms critically depend on the marking of chromatin domains with distinctive histone tail modifications (PTMs) and their recognition by effector protein complexes. Here we used quantitative proteomic approaches to unveil interactions between PTMs and associated reader protein complexes of Plasmodium falciparum, a unicellular parasite causing malaria. Histone peptide pull-downs with the most prominent and/or parasite-specific PTMs revealed the binding preference for 14 putative and novel reader proteins. Amongst others, they highlighted the acetylation-level-dependent recruitment of the BDP1/BDP2 complex and identified an PhD-finger protein (PHD 1, PF3D7_1008100) that could mediate a cross-talk between H3K4me2/3 and H3K9ac marks. Tagging and interaction proteomics of 12 identified proteins unveiled the composition of 5 major epigenetic complexes, including the elusive TBP-associated-factor complex as well as two distinct GCN5/ADA2 complexes. Furthermore, it has highlighted a remarkable degree of interaction between these five (sub)complexes. Collectively, this study provides an extensive inventory of PTM-reader interactions and composition of epigenetic complexes. It will not only fuel further explorations of gene regulation amongst ancient eukaryotes, but also provides a stepping stone for exploration of PTM-reader interactions for antimalarial drug development.
Assuntos
Epigênese Genética/genética , Regulação da Expressão Gênica/genética , Histonas/metabolismo , Plasmodium falciparum/genética , Processamento de Proteína Pós-Traducional/genética , Cromatina/metabolismo , Humanos , Malária Falciparum/genética , Malária Falciparum/parasitologia , MetilaçãoRESUMO
MNase-seq allows the genome-wide examination of the nucleosome landscape by determination of nucleosome positioning and occupancy. Typically, native or formaldehyde fixed chromatin is subjected to digestion by micrococcal nuclease (MNase), which degrades linker DNA and yields mainly mono-nucleosomes. The resulting material can be processed directly or can be subjected to an optional chromatin immunoprecipitation step (MNase-ChIP-seq). De-crosslinked and purified DNA is then subjected to next-generation sequencing. The protocol presented here has been tailored for the analysis of nucleosome landscape in the malaria parasite, Plasmodium falciparum, but most steps are directly applicable to other cell types. We also discuss general considerations for experimental design and computational analysis, which are crucial for accurate investigation of the nucleosome landscape.
Assuntos
Imunoprecipitação da Cromatina , Sequenciamento de Nucleotídeos em Larga Escala , Nuclease do Micrococo/metabolismo , Nucleossomos/metabolismo , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina/métodos , DNA/genética , DNA/metabolismo , Clivagem do DNA , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Controle de QualidadeRESUMO
In eukaryotes, the chromatin architecture has a pivotal role in regulating all DNA-associated processes and it is central to the control of gene expression. For Plasmodium falciparum, a causative agent of human malaria, the nucleosome positioning profile of regulatory regions deserves particular attention because of their extreme AT-content. With the aid of a highly controlled MNase-seq procedure we reveal how positioning of nucleosomes provides a structural and regulatory framework to the transcriptional unit by demarcating landmark sites (transcription/translation start and end sites). In addition, our analysis provides strong indications for the function of positioned nucleosomes in splice site recognition. Transcription start sites (TSSs) are bordered by a small nucleosome-depleted region, but lack the stereotypic downstream nucleosome arrays, highlighting a key difference in chromatin organization compared to model organisms. Furthermore, we observe transcription-coupled eviction of nucleosomes on strong TSSs during intraerythrocytic development and demonstrate that nucleosome positioning and dynamics can be predictive for the functionality of regulatory DNA elements. Collectively, the strong nucleosome positioning over splice sites and surrounding putative transcription factor binding sites highlights the regulatory capacity of the nucleosome landscape in this deadly human pathogen.
