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1.
Nucleic Acids Res ; 48(17): 9660-9680, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32890403

RESUMO

Maintenance of genome integrity is critical to guarantee transfer of an intact genome from parent to offspring during cell division. DNA polymerases (Pols) provide roles in both replication of the genome and the repair of a wide range of lesions. Amongst replicative DNA Pols, translesion DNA Pols play a particular role: replication to bypass DNA damage. All cells express a range of translesion Pols, but little work has examined their function in parasites, including whether the enzymes might contribute to host-parasite interactions. Here, we describe a dual function of one putative translesion Pol in African trypanosomes, which we now name TbPolIE. Previously, we demonstrated that TbPolIE is associated with telomeric sequences and here we show that RNAi-mediated depletion of TbPolIE transcripts results in slowed growth, altered DNA content, changes in cell morphology, and increased sensitivity to DNA damaging agents. We also show that TbPolIE displays pronounced localization at the nuclear periphery, and that its depletion leads to chromosome segregation defects and increased levels of endogenous DNA damage. Finally, we demonstrate that TbPolIE depletion leads to deregulation of telomeric variant surface glycoprotein genes, linking the function of this putative translesion DNA polymerase to host immune evasion by antigenic variation.


Assuntos
Variação Antigênica , DNA Polimerase Dirigida por DNA/metabolismo , Telômero/genética , Trypanosoma brucei brucei/genética , Linhagem Celular , Núcleo Celular/enzimologia , Núcleo Celular/genética , Segregação de Cromossomos , Replicação do DNA , DNA Polimerase Dirigida por DNA/genética , Regulação da Expressão Gênica , Genoma de Protozoário , Humanos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Interferência de RNA , Telômero/metabolismo , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/patogenicidade , Glicoproteínas Variantes de Superfície de Trypanosoma/genética
2.
PLoS Negl Trop Dis ; 14(9): e0008568, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32936798

RESUMO

Trypanosoma brucei is a single celled eukaryotic parasite and the causative agent of human African trypanosomiasis and nagana in cattle. Aside from its medical relevance, T. brucei has also been key to the discovery of several general biological principles including GPI-anchoring, RNA-editing and trans-splicing. The parasite contains a single mitochondrion with a singular genome. Recent studies have identified several molecular components of the mitochondrial genome segregation machinery (tripartite attachment complex, TAC), which connects the basal body of the flagellum to the mitochondrial DNA of T. brucei. The TAC component in closest proximity to the mitochondrial DNA is TAC102. Here we apply and compare three different approaches (proximity labelling, immunoprecipitation and yeast two-hybrid) to identify novel interactors of TAC102 and subsequently verify their localisation. Furthermore, we establish the direct interaction of TAC102 and p166 in the unilateral filaments of the TAC.


Assuntos
DNA Mitocondrial/genética , Imunoprecipitação , Proteínas de Protozoários/genética , Técnicas do Sistema de Duplo-Híbrido , Animais , Bovinos , Doenças dos Bovinos/parasitologia , DNA de Cinetoplasto/metabolismo , Flagelos/metabolismo , Genoma Mitocondrial/genética , Humanos , Mitocôndrias/genética , Mitocôndrias/fisiologia , Trypanosoma brucei brucei/genética , Tripanossomíase Africana/veterinária
3.
Nucleic Acids Res ; 48(15): 8704-8723, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32738044

RESUMO

Trypanosoma brucei is a parasitic protozoan that undergoes a complex life cycle involving insect and mammalian hosts that present dramatically different nutritional environments. Mitochondrial metabolism and gene expression are highly regulated to accommodate these environmental changes, including regulation of mRNAs that require extensive uridine insertion/deletion (U-indel) editing for their maturation. Here, we use high throughput sequencing and a method for promoting life cycle changes in vitro to assess the mechanisms and timing of developmentally regulated edited mRNA expression. We show that edited CYb mRNA is downregulated in mammalian bloodstream forms (BSF) at the level of editing initiation and/or edited mRNA stability. In contrast, edited COIII mRNAs are depleted in BSF by inhibition of editing progression. We identify cell line-specific differences in the mechanisms abrogating COIII mRNA editing, including the possible utilization of terminator gRNAs that preclude the 3' to 5' progression of editing. By examining the developmental timing of altered mitochondrial mRNA levels, we also reveal transcript-specific developmental checkpoints in epimastigote (EMF), metacyclic (MCF), and BSF. These studies represent the first analysis of the mechanisms governing edited mRNA levels during T. brucei development and the first to interrogate U-indel editing in EMF and MCF life cycle stages.


Assuntos
Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mitocondrial/genética , RNA de Protozoário/genética , Trypanosoma brucei brucei/genética , Mitocôndrias/genética , Proteínas de Protozoários/genética , Edição de RNA/genética , RNA Guia/genética , Trypanosoma brucei brucei/metabolismo
4.
PLoS One ; 15(8): e0237187, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833981

RESUMO

INTRODUCTION: Infection of equids with Trypanosoma brucei (T. brucei) ssp. is of socioeconomic importance across sub-Saharan Africa as the disease often progresses to cause fatal meningoencephalitis. Loop-mediated isothermal amplification (LAMP) has been developed as a cost-effective molecular diagnostic test and is potentially applicable for use in field-based laboratories. PART I: Threshold levels for T. brucei ssp. detection by LAMP were determined using whole equine blood specimens spiked with known concentrations of parasites. Results were compared to OIE antemortem gold standard of T. brucei-PCR (TBR-PCR). RESULTS I: Threshold for detection of T. brucei ssp. on extracted DNA from whole blood was 1 parasite/ml blood for LAMP and TBR-PCR, and there was excellent agreement (14/15) between tests at high (1 x 103/ml) concentrations of parasites. Detection threshold was 100 parasites/ml using LAMP on whole blood (LWB). Threshold for LWB improved to 10 parasites/ml with detergent included. Performance was excellent for LAMP at high (1 x 103/ml) concentrations of parasites (15/15, 100%) but was variable at lower concentrations. Agreement between tests was weak to moderate, with the highest for TBR-PCR and LAMP on DNA extracted from whole blood (Cohen's kappa 0.95, 95% CI 0.64-1.00). PART II: A prospective cross-sectional study of working equids meeting clinical criteria for trypanosomiasis was undertaken in The Gambia. LAMP was evaluated against subsequent TBR-PCR. RESULTS II: Whole blood samples from 321 equids in The Gambia were processed under field conditions. There was weak agreement between LWB and TBR-PCR (Cohen's kappa 0.34, 95% CI 0.19-0.49) but excellent agreement when testing CSF (100% agreement on 6 samples). CONCLUSIONS: Findings support that LAMP is comparable to PCR when used on CSF samples in the field, an important tool for clinical decision making. Results suggest repeatability is low in animals with low parasitaemia. Negative samples should be interpreted in the context of clinical presentation.


Assuntos
Doenças dos Cavalos/parasitologia , Cavalos/parasitologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Trypanosoma brucei brucei/genética , Tripanossomíase Africana/diagnóstico , Tripanossomíase Africana/veterinária , Animais , Estudos Transversais , DNA de Protozoário/sangue , DNA de Protozoário/genética , Feminino , Gâmbia , Masculino , Técnicas de Diagnóstico Molecular/economia , Técnicas de Amplificação de Ácido Nucleico/economia , Reação em Cadeia da Polimerase/economia , Estudos Prospectivos , Sensibilidade e Especificidade , Tripanossomíase Africana/parasitologia
5.
Nucleic Acids Res ; 48(17): e99, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32756897

RESUMO

Mitochondrial gene expression in African trypanosomes and other trypanosomatid pathogens requires a U-nucleotide specific insertion/deletion-type RNA-editing reaction. The process is catalyzed by a macromolecular protein complex known as the editosome. Editosomes are restricted to the trypanosomatid clade and since editing is essential for the parasites, the protein complex represents a near perfect target for drug intervention strategies. Here, we report the development of an improved in vitro assay to monitor editosome function. The test system utilizes fluorophore-labeled substrate RNAs to analyze the processing reaction by automated, high-throughput capillary electrophoresis (CE) in combination with a laser-induced fluorescence (LIF) readout. We optimized the assay for high-throughput screening (HTS)-experiments and devised a multiplex fluorophore-labeling regime to scrutinize the U-insertion/U-deletion reaction simultaneously. The assay is robust, it requires only nanogram amounts of materials and it meets all performance criteria for HTS-methods. As such the test system should be helpful in the search for trypanosome-specific pharmaceuticals.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Edição de RNA , Trypanosoma brucei brucei/genética , Fluoresceína/química , Corantes Fluorescentes/química , Genoma Mitocondrial , Reação em Cadeia da Polimerase Multiplex/métodos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Guia/química , RNA Guia/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Uridina Trifosfato/química
6.
Nucleic Acids Res ; 48(15): 8645-8662, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32614436

RESUMO

In Trypanosoma brucei, mitochondrial pre-mRNAs undergo 3'-5' exonucleolytic processing, 3' adenylation and uridylation, 5' pyrophosphate removal, and, often, U-insertion/deletion editing. The 3' modifications are modulated by pentatricopeptide repeat (PPR) Kinetoplast Polyadenylation Factors (KPAFs). We have shown that KPAF3 binding to the 3' region stabilizes properly trimmed transcripts and stimulates their A-tailing by KPAP1 poly(A) polymerase. Conversely, poly(A) binding KPAF4 shields the nascent A-tail from uridylation and decay thereby protecting pre-mRNA upon KPAF3 displacement by editing. While editing concludes in the 5' region, KPAF1/2 dimer induces A/U-tailing to activate translation. Remarkably, 5' end recognition and pyrophosphate hydrolysis by the PPsome complex also contribute to mRNA stabilization. Here, we demonstrate that KPAF4 functions as a heterodimer with KPAF5, a protein lacking discernable motifs. We show that KPAF5 stabilizes KPAF4 to enable poly(A) tail recognition, which likely leads to mRNA stabilization during the editing process and impedes spontaneous translational activation of partially-edited transcripts. Thus, KPAF4/5 represents a poly(A) binding element of the mitochondrial polyadenylation complex. We present evidence that RNA editing substrate binding complex bridges the 5' end-bound PPsome and 3' end-bound polyadenylation complexes. This interaction may enable mRNA circularization, an apparently critical element of mitochondrial mRNA stability and quality control.


Assuntos
Polinucleotídeo Adenililtransferase/genética , Proteínas de Protozoários/genética , RNA de Protozoário/genética , Trypanosoma brucei brucei/genética , Mitocôndrias/genética , Poliadenilação/genética , Proteínas de Protozoários/química , Edição de RNA/genética , Precursores de RNA/genética , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA de Protozoário/química , Fatores de Poliadenilação e Clivagem de mRNA/genética
7.
Mol Cell ; 79(4): 629-644.e4, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32679035

RESUMO

In contrast to the bacterial translation machinery, mitoribosomes and mitochondrial translation factors are highly divergent in terms of composition and architecture. There is increasing evidence that the biogenesis of mitoribosomes is an intricate pathway, involving many assembly factors. To better understand this process, we investigated native assembly intermediates of the mitoribosomal large subunit from the human parasite Trypanosoma brucei using cryo-electron microscopy. We identify 28 assembly factors, 6 of which are homologous to bacterial and eukaryotic ribosome assembly factors. They interact with the partially folded rRNA by specifically recognizing functionally important regions such as the peptidyltransferase center. The architectural and compositional comparison of the assembly intermediates indicates a stepwise modular assembly process, during which the rRNA folds toward its mature state. During the process, several conserved GTPases and a helicase form highly intertwined interaction networks that stabilize distinct assembly intermediates. The presented structures provide general insights into mitoribosomal maturation.


Assuntos
Ribossomos Mitocondriais/química , RNA Ribossômico/metabolismo , Subunidades Ribossômicas Maiores/química , Trypanosoma brucei brucei/metabolismo , Microscopia Crioeletrônica , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Ribossomos Mitocondriais/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Ribossômico/química , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores/metabolismo , Trypanosoma brucei brucei/genética
8.
PLoS Biol ; 18(6): e3000741, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32520929

RESUMO

Mitochondrial metabolic remodeling is a hallmark of the Trypanosoma brucei digenetic life cycle because the insect stage utilizes a cost-effective oxidative phosphorylation (OxPhos) to generate ATP, while bloodstream cells switch to aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector, the dynamics of the parasite's metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro-induced differentiation to follow changes at the RNA, protein, and metabolite levels. This multi-omics and cell-based profiling showed an immediate redirection of electron flow from the cytochrome-mediated pathway to an alternative oxidase (AOX), an increase in proline consumption, elevated activity of complex II, and certain tricarboxylic acid (TCA) cycle enzymes, which led to mitochondrial membrane hyperpolarization and increased reactive oxygen species (ROS) levels. Interestingly, these ROS molecules appear to act as signaling molecules driving developmental progression because ectopic expression of catalase, a ROS scavenger, halted the in vitro-induced differentiation. Our results provide insights into the mechanisms of the parasite's mitochondrial rewiring and reinforce the emerging concept that mitochondria act as signaling organelles through release of ROS to drive cellular differentiation.


Assuntos
Metabolômica , Mitocôndrias/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/metabolismo , Trifosfato de Adenosina/biossíntese , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Respiração Celular/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Elétrons , Glucose/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Oxirredução , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Prolina/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Transcriptoma/genética , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/genética
9.
Nat Commun ; 11(1): 1498, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198348

RESUMO

Despite histone H2A variants and acetylation of histones occurring in almost every eukaryotic organism, it has been difficult to establish direct functional links between canonical histones or H2A variant acetylation, deposition of H2A variants and transcription. To disentangle these complex interdependent processes, we devised a highly sensitive strategy for quantifying histone acetylation levels at specific genomic loci. Taking advantage of the unusual genome organization in Trypanosoma brucei, we identified 58 histone modifications enriched at transcription start sites (TSSs). Furthermore, we found TSS-associated H4 and H2A.Z acetylation to be mediated by two different histone acetyltransferases, HAT2 and HAT1, respectively. Whereas depletion of HAT2 decreases H2A.Z deposition and shifts the site of transcription initiation, depletion of HAT1 does not affect H2A.Z deposition but reduces total mRNA levels by 50%. Thus, specifically reducing H4 or H2A.Z acetylation levels enabled us to reveal distinct roles for these modifications in H2A.Z deposition and RNA transcription.


Assuntos
Histonas/metabolismo , Processamento de Proteína Pós-Traducional , RNA/metabolismo , Trypanosoma brucei brucei/metabolismo , Acetilação , Linhagem Celular , Genômica , Histona Acetiltransferases/metabolismo , Código das Histonas , Nucleossomos , RNA Mensageiro , Sítio de Iniciação de Transcrição , Transcriptoma , Trypanosoma brucei brucei/genética
10.
Trends Parasitol ; 36(4): 337-355, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32191849

RESUMO

Trypanosoma brucei spp. cause African human and animal trypanosomiasis, a burden on health and economy in Africa. These hemoflagellates are distinguished by a kinetoplast nucleoid containing mitochondrial DNAs of two kinds: maxicircles encoding ribosomal RNAs (rRNAs) and proteins and minicircles bearing guide RNAs (gRNAs) for mRNA editing. All RNAs are produced by a phage-type RNA polymerase as 3' extended precursors, which undergo exonucleolytic trimming. Most pre-mRNAs proceed through 3' adenylation, uridine insertion/deletion editing, and 3' A/U-tailing. The rRNAs and gRNAs are 3' uridylated. Historically, RNA editing has attracted major research effort, and recently essential pre- and postediting processing events have been discovered. Here, we classify the key players that transform primary transcripts into mature molecules and regulate their function and turnover.


Assuntos
Edição de RNA/fisiologia , RNA Mitocondrial/metabolismo , RNA de Protozoário/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , RNA Mitocondrial/genética , RNA de Protozoário/genética , Trypanosoma brucei brucei/genética
11.
Nat Commun ; 11(1): 620, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001697

RESUMO

Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5'-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 Å resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds.


Assuntos
Coenzimas/química , Cristalização , IMP Desidrogenase/química , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico , Clonagem Molecular , Guanosina Monofosfato , Modelos Moleculares , Conformação Proteica , Células Sf9 , Trypanosoma brucei brucei/genética
12.
mSphere ; 5(1)2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32075879

RESUMO

Kinetoplastid parasites, including Trypanosoma brucei, Trypanosoma cruzi, and Leishmania, harbor unique organelles known as glycosomes, which are evolutionarily related to peroxisomes. Glycosome/peroxisome biogenesis is mediated by proteins called peroxins that facilitate organelle formation, proliferation, and degradation and import of proteins housed therein. Import of matrix proteins occurs via one of two pathways that are dictated by their peroxisome targeting sequence (PTS). In PTS1 import, a C-terminal tripeptide sequence, most commonly SKL, is recognized by the soluble receptor Pex5. In PTS2 import, a less conserved N-terminal sequence is recognized by Pex7. The soluble receptors deliver their cargo to the import channel consisting minimally of Pex13 and Pex14. While much of the import process is conserved, kinetoplastids are the only organisms to have two Pex13s, Pex13.1 and Pex13.2. It is unclear why trypanosomes require two Pex13s when one is sufficient for most eukaryotes. To interrogate the role of Pex13.2, we have employed biochemical approaches to partially resolve the composition of the Pex13/Pex14 import complexes in T. brucei and characterized glycosome morphology and protein import in Pex13.2-deficient parasites. Here, we show that Pex13.2 is an integral glycosome membrane protein that interacts with Pex13.1 and Pex14. The N terminus of Pex13.2 faces the cytoplasmic side of the membrane, where it can facilitate interactions required for protein import. Two-dimensional gel electrophoresis revealed three glycosome membrane complexes containing combinations of Pex13.1, Pex13.2, and Pex14. The silencing of Pex13.2 resulted in parasites with fewer, larger glycosomes and disrupted glycosome protein import, suggesting the protein is involved in glycosome biogenesis as well as protein import. Furthermore, superresolution microscopy demonstrated that Pex13.2 localizes to discrete foci in the glycosome periphery, indicating that the glycosome periphery is not homogenous.IMPORTANCE Trypanosoma brucei causes human African trypanosomiasis and a wasting disease called Nagana in livestock. Current treatments are expensive, toxic, and difficult to administer. Because of this, the search for new drug targets is essential. T. brucei has glycosomes that are essential to parasite survival; however, our ability to target them in drug development is hindered by our lack of understanding about how these organelles are formed and maintained. This work forwards our understanding of how the parasite-specific protein Pex13.2 functions in glycosome protein import and lays the foundation for future studies focused on blocking Pex13.2 function, which would be lethal to bloodstream-form parasites that reside in the mammalian bloodstream.


Assuntos
Microcorpos/metabolismo , Peroxinas/metabolismo , Peroxissomos/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/genética , Citosol/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Peroxinas/genética , Peroxissomos/genética , Transporte Proteico , Proteínas de Protozoários/genética
13.
Nat Commun ; 11(1): 844, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-32051413

RESUMO

African trypanosomes (Trypanosoma) are vector-borne haemoparasites that survive in the vertebrate bloodstream through antigenic variation of their Variant Surface Glycoprotein (VSG). Recombination, or rather segmented gene conversion, is fundamental in Trypanosoma brucei for both VSG gene switching and for generating antigenic diversity during infections. Trypanosoma vivax is a related, livestock pathogen whose VSG lack structures that facilitate gene conversion in T. brucei and mechanisms underlying its antigenic diversity are poorly understood. Here we show that species-wide VSG repertoire is broadly conserved across diverse T. vivax clinical strains and has limited antigenic repertoire. We use variant antigen profiling, coalescent approaches and experimental infections to show that recombination plays little role in diversifying T. vivax VSG sequences. These results have immediate consequences for both the current mechanistic model of antigenic variation in African trypanosomes and species differences in virulence and transmission, requiring reconsideration of the wider epidemiology of animal African trypanosomiasis.


Assuntos
Variação Antigênica/genética , Variação Antigênica/imunologia , Recombinação Genética/genética , Trypanosoma vivax/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Glicoproteínas Variantes de Superfície de Trypanosoma/imunologia , DNA de Protozoário , Evolução Molecular , Genoma de Protozoário , Interações Hospedeiro-Parasita/imunologia , Evasão da Resposta Imune , Filogenia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Homologia de Sequência , Especificidade da Espécie , Transcriptoma , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/imunologia , Tripanossomíase Africana/imunologia , Tripanossomíase Africana/parasitologia , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo
14.
mSphere ; 5(1)2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32102938

RESUMO

RAP1 is a telomere protein that is well conserved from protozoa to mammals. It plays important roles in chromosome end protection, telomere length control, and gene expression/silencing at both telomeric and nontelomeric loci. Interaction with different partners is an important mechanism by which RAP1 executes its different functions in yeast. The RAP1 ortholog in Trypanosoma brucei is essential for variant surface glycoprotein (VSG) monoallelic expression, an important aspect of antigenic variation, where T. brucei regularly switches its major surface antigen, VSG, to evade the host immune response. Like other RAP1 orthologs, T. brucei RAP1 (TbRAP1) has conserved functional domains, including BRCA1 C terminus (BRCT), Myb, MybLike, and RAP1 C terminus (RCT). To study functions of various TbRAP1 domains, we established a strain in which one endogenous allele of TbRAP1 is flanked by loxP repeats, enabling its conditional deletion by Cre-mediated recombination. We replaced the other TbRAP1 allele with various mutant alleles lacking individual functional domains and examined their nuclear localization and protein interaction abilities. The N terminus, BRCT, and RCT of TbRAP1 are required for normal protein levels, while the Myb and MybLike domains are essential for normal cell growth. Additionally, the Myb domain of TbRAP1 is required for its interaction with T. brucei TTAGGG repeat-binding factor (TbTRF), while the BRCT domain is required for its self-interaction. Furthermore, the TbRAP1 MybLike domain contains a bipartite nuclear localization signal that is required for its interaction with importin α and its nuclear localization. Interestingly, RAP1's self-interaction and the interaction between RAP1 and TRF are conserved from kinetoplastids to mammals. However, details of the interaction interfaces have changed throughout evolution.IMPORTANCE Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, to evade the host immune response. VSGs are expressed from subtelomeres in a monoallelic fashion. TbRAP1, a telomere protein, is essential for cell viability and VSG monoallelic expression and suppresses VSG switching. Although TbRAP1 has conserved functional domains in common with its orthologs from yeasts to mammals, the domain functions are unknown. RAP1 orthologs have pleiotropic functions, and interaction with different partners is an important means by which RAP1 executes its different roles. We have established a Cre-loxP-mediated conditional knockout system for TbRAP1 and examined the roles of various functional domains in protein expression, nuclear localization, and protein-protein interactions. This system enables further studies of TbRAP1 point mutation phenotypes. We have also determined functional domains of TbRAP1 that are required for several different protein interactions, shedding light on the underlying mechanisms of TbRAP1-mediated VSG silencing.


Assuntos
Variação Antigênica , Inativação Gênica , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/genética , Proteínas de Ligação a Telômeros/genética , Trypanosoma brucei brucei/genética , Alelos , Glicoproteínas de Membrana/genética , Mutação Puntual , Telômero/genética , Proteínas de Ligação a Telômeros/metabolismo
15.
Proc Natl Acad Sci U S A ; 117(5): 2613-2621, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31964820

RESUMO

Tsetse-transmitted African trypanosomes must develop into mammalian-infectious metacyclic cells in the fly's salivary glands (SGs) before transmission to a new host. The molecular mechanisms that underlie this developmental process, known as metacyclogenesis, are poorly understood. Blocking the few metacyclic parasites deposited in saliva from further development in the mammal could prevent disease. To obtain an in-depth perspective of metacyclogenesis, we performed single-cell RNA sequencing (scRNA-seq) from a pool of 2,045 parasites collected from infected tsetse SGs. Our data revealed three major cell clusters that represent the epimastigote, and pre- and mature metacyclic trypanosome developmental stages. Individual cell level data also confirm that the metacyclic pool is diverse, and that each parasite expresses only one of the unique metacyclic variant surface glycoprotein (mVSG) coat protein transcripts identified. Further clustering of cells revealed a dynamic transcriptomic and metabolic landscape reflective of a developmental program leading to infectious metacyclic forms preadapted to survive in the mammalian host environment. We describe the expression profile of proteins that regulate gene expression and that potentially play a role in metacyclogenesis. We also report on a family of nonvariant surface proteins (Fam10) and demonstrate surface localization of one member (named SGM1.7) on mature metacyclic parasites. Vaccination of mice with recombinant SGM1.7 reduced parasitemia early in the infection. Future studies are warranted to investigate Fam10 family proteins as potential trypanosome transmission blocking vaccine antigens. Our experimental approach is translationally relevant for developing strategies to prevent other insect saliva-transmitted parasites from infecting and causing disease in mammalian hosts.


Assuntos
Insetos Vetores/parasitologia , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/genética , Moscas Tsé-Tsé/parasitologia , Animais , Feminino , Humanos , Estágios do Ciclo de Vida , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/imunologia , RNA de Protozoário/genética , Glândulas Salivares/parasitologia , Análise de Sequência de RNA , Análise de Célula Única , Transcriptoma , Trypanosoma brucei brucei/imunologia , Tripanossomíase Africana/imunologia , Tripanossomíase Africana/parasitologia
16.
mSphere ; 5(1)2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31969475

RESUMO

Lucy Glover's research focuses on the role of DNA repair and recombination in antigenic variation in the parasite Trypanosoma brucei, the causative agent of both human and animal African trypanosomiasis. In this mSphere of Influence article, she reflects on how "A CRISPR-based approach for proteomic analysis of a single genomic locus" by Z. J. Waldrip, S. D. Byrum, A. J. Storey, J. Gao, et al. (Epigenetics 9:1207-1211, 2014, https://doi.org/10.4161/epi.29919) made an impact on her research by taking the precision of CRISPR-Cas9 and repurposing it to look at single-locus proteomics. By using this technology in trypanosomes, Dr. Glover and her colleagues could study the dynamic accumulation of repair proteins after specific damage and gain insight into how the location of a double-strand break (DSB) dictates repair pathway choice and how this may influence immune evasion in these parasites.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Reparo do DNA , Proteômica/métodos , Variação Antigênica , Edição de Genes , Recombinação Genética , Trypanosoma brucei brucei/genética
18.
RNA ; 26(1): 69-82, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31704716

RESUMO

Most mitochondrial mRNAs are transcribed as polycistronic precursors that are cleaved by endonucleases to produce mature mRNA transcripts. However, recent studies have shown that mitochondrial transcripts in the kinetoplastid protozoan, Trypanosoma brucei, are transcribed individually. Also unlike most mitochondrial mRNAs, the 5' end of these transcripts harbor a triphosphate that is hydrolyzed. This modification is carried out by a putative Nudix hydrolase called MERS1. The Nudix motif in MERS1 is degenerate, lacking a conserved glutamic acid, thus it is unclear how it may bind its substrates and whether it contains a Nudix fold. To obtain insight into this unusual hydrolase, we determined structures of apo, GTP-bound and RNA-bound T. brucei MERS1 to 2.30 Å, 2.45 Å, and 2.60 Å, respectively. The MERS1 structure has a unique fold that indeed contains a Nudix motif. The nucleotide bound structures combined with binding studies reveal that MERS1 shows preference for RNA sequences with a central guanine repeat which it binds in a single-stranded conformation. The apo MERS1 structure indicates that a significant portion of its nucleotide binding site folds upon substrate binding. Finally, a potential interaction region for a binding partner, MERS2, that activates MERS1 was identified. The MERS2-like peptide inserts a glutamate near the missing Nudix acidic residue in the RNA binding pocket, suggesting how the enzyme may be activated. Thus, the combined studies reveal insight into the structure and enzyme properties of MERS1 and its substrate-binding activities.


Assuntos
RNA Mensageiro/química , RNA Mitocondrial/química , Trypanosoma brucei brucei/enzimologia , Modelos Moleculares , Conformação de Ácido Nucleico , RNA/metabolismo , RNA Mensageiro/genética , RNA Mitocondrial/genética , RNA de Protozoário/química , RNA de Protozoário/genética , Alinhamento de Sequência , Trypanosoma brucei brucei/genética
19.
Mol Microbiol ; 113(2): 430-451, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31743541

RESUMO

ZC3H20 and ZC3H21 are related trypanosome proteins with two C(x)8 C(x)5 C(x)3 H zinc finger motifs. ZC3H20 is present at a low level in replicating mammalian-infective bloodstream forms, but becomes more abundant when they undergo growth arrest at high density; ZC3H21 appears only in the procyclic form of the parasite, which infects Tsetse flies. Each protein binds to several hundred mRNAs, with overlapping but not identical specificities. Both increase expression of bound mRNAs, probably through recruitment of the MKT1-PBP1 complex. At least 28 of the bound mRNAs decrease after depletion of ZC3H20, or of ZC3H20 and ZC3H21 together; their products include procyclic-specific proteins of the plasma membrane and energy metabolism. Simultaneous depletion of ZC3H20 and ZC3H21 causes procyclic forms to shrink and stop growing; in addition to decreases in target mRNAs, there are other changes suggestive of loss of developmental regulation. The bloodstream-form-specific protein RBP10 controls ZC3H20 and ZC3H21 expression. Interestingly, some ZC3H20/21 target mRNAs also bind to and are repressed by RBP10, allowing for dynamic regulation as RBP10 decreases and ZC3H20 and ZC3H21 increase during differentiation.


Assuntos
RNA Mensageiro/metabolismo , Trypanosoma brucei brucei , Dedos de Zinco/genética , Animais , Regulação da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Protozoários/genética , RNA de Protozoário , Proteínas de Ligação a RNA/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Moscas Tsé-Tsé/parasitologia
20.
J Mol Biol ; 432(2): 410-426, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31726063

RESUMO

Prozymes are pseudoenzymes that stimulate the function of weakly active enzymes through complex formation. The major Trypanosoma brucei protein arginine methyltransferase, TbPRMT1 enzyme (ENZ), requires TbPRMT1 prozyme (PRO) to form an active heterotetrameric complex. Here, we present the X-ray crystal structure of the TbPRMT1 ENZ-Δ52PRO tetrameric complex with the cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.4 Å resolution. The individual ENZ and PRO units adopt the highly-conserved PRMT domain architecture and form an antiparallel heterodimer that corresponds to the canonical homodimer observed in all previously reported PRMTs. In turn, two such heterodimers assemble into a tetramer both in the crystal and in solution with twofold rotational symmetry. ENZ is unstable in absence of PRO and incapable of forming a homodimer due to a steric clash of an ENZ-specific tyrosine within the dimerization arm, rationalizing why PRO is required to complement ENZ to form a PRMT dimer that is necessary, but not sufficient for PRMT activity. The PRO structure deviates from other, active PRMTs in that it lacks the conserved η2 310-helix within the Rossmann fold, abolishing cofactor binding. In addition to its chaperone function for ENZ, PRO substantially contributes to substrate binding. Heterotetramerization is required for catalysis, as heterodimeric ENZ-PRO mutants lack binding affinity and methyltransferase activity toward the substrate protein TbRGG1. Together, we provide a structural basis for TbPRMT1 ENZ activation by PRO heterotetramer formation, which is conserved across all kinetoplastids, and describe a chaperone function of the TbPRMT1 prozyme, which represents a novel mode of PRMT regulation.


Assuntos
Complexos Multiproteicos/ultraestrutura , Conformação Proteica , Proteína-Arginina N-Metiltransferases/ultraestrutura , S-Adenosil-Homocisteína/química , Trypanosoma brucei brucei/ultraestrutura , Sequência de Aminoácidos/genética , Catálise , Cristalografia por Raios X , Dimerização , Metilação , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/genética , Especificidade por Substrato/genética , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética
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