Estudio de factores proteicos asociados a la regulación de los genes HSP70 en Leishmania braziliensis

La leishmaniasis constituye un grupo de enfermedades producidas por parásitos protozoos pertenecientes al género Leishmania. Durante su ciclo de vida, el parásito alterna entre un vector invertebrado y un hospedero vertebrado, lo que le exige desarrollar cambios morfológicos y bioquímicos que le permitan sobrevivir y adaptarse al estrés celular que debe enfrentar en ambos hospederos. Adaptación que depende de la regulación de la expresión de una amplia variedad de genes, dentro de los cuales se encuentran los genes HSP70. Se ha demostrado que estos parásitos, tanto del subgénero Leishmania como del subgénero Viannia, poseen dos tipos de genes HSP70 (HSP70-I y II), los cuales se diferencian en su región 3´ no traducida (3´ UTR); diferencias relacionadas con los mecanismos moleculares de adaptación del parásito para tolerar el estrés térmico al que es sometido durante su ciclo de vida. Ensayos recientes, en L. braziliensis, han identificado proteínas con afinidad a las regiones UTR de los ARNm de estos genes, hallazgo de gran importancia teniendo en cuenta que la regulación de su expresión génica ocurre principalmente a nivel postranscripcional, y está mediada por la interacción entre elementos reguladores cis y trans del parásito. Entre las proteínas identificadas en dicho estudio se encuentran las proteínas LbrM.25.2210 (SCD6) y LbrM.30.3080 (RBP42), para las cuales, en esta Tesis, se estudiaron sus características moleculares y funcionales como factores proteicos implicados en la regulación génica en Leishmania. Inicialmente, se confirmó la capacidad de estas proteínas para interactuar de forma directa con moléculas de ARN. Observándose que estas proteínas son capaces de interactuar con un motivo con características de elemento ARE. Por otra parte, la interacción de estas proteínas con moléculas de ARN también fue demostrada in vivo, encontrándose asociados varios transcritos codificantes para proteínas relacionadas con el metabolismo y degradación celular. De forma particularmente destacable, asociada a la proteína RBP42 se identificó el transcrito correspondiente al gen HSP70-II. Así mismo, se encontraron asociaciones de las proteínas de estudio con transcritos codificantes para proteínas involucradas en la diferenciación y sobrevivencia del parásito en condiciones de choque térmico. Con el propósito de dilucidar los posibles procesos biológicos en los cuales podrían estar involucradas estas proteínas se determinó el interactoma de cada una de ellas. Cabe destacar que los interactomas comparten una alta proporción de proteínas, lo que sugiere algún tipo de relación funcional; de hecho, las dos proteínas forman parte de los interactomas recíprocos. Ambos interactomas están enriquecidos en proteínas asociadas a los procesos de traducción, metabolismo de ARN y regulación de la expresión génica. Como apoyo a un posible papel de estas proteínas en la regulación postranscripcional, cabe indicar que se encontraron varias proteínas cuyos homólogos han sido reportados como constituyentes de gránulos de ARN, principalmente cuerpos de procesamiento y gránulos de estrés. Finalmente, para analizar la función de SCD6 y RBP42 se generaron líneas mutantes de parásitos que sobreexpresan las proteínas fusión SCD6-mCherry y RBP42-mCherry. La línea sobreexpresante control (expresión de la proteína mCherry) de L. braziliensis pudo ser establecida, pero, por el contrario, las líneas de sobreexpresión para cada una de estas proteínas, luego de una semana de crecimiento, perdieron viabilidad. Estos resultados, si bien no concluyentes, sugieren que la sobreexpresión de SCD6 y RBP42 en L. braziliensis afecta negativamente al desarrollo y sobrevivencia del parásito. Como alternativa, se logró, generar estas líneas de sobreexpresión utilizando la especie L. major, lo que permitió determinar importantes implicaciones de estas proteínas en el desarrollo de los parásitos; observándose alteraciones morfológicas significativas y el retardo del crecimiento de los parásitos en condiciones fisiológicas de crecimiento, siendo el efecto mucho más pronunciado en condiciones de estrés térmico. Por otra parte, la capacidad infectiva in vitro de estos parásitos mutantes resulta significativamente afectada en relación con la cepa silvestre. En conjunto, este estudio aporta la caracterización de las proteínas SCD6 y RBP42 como proteínas de unión a ARN que, junto al resto de análisis complementarios sugieren su relevante implicación en la regulación de la estabilidad o degradación de un amplio grupo de transcritos en Leishmania.Pontificia Universidad JaverianaLeishmaniasis is a disease caused by protozoan parasites of the Leishmania genus. These parasites present a complex digenetic life cycle alternating between invertebrate and vertebrate hosts. Therefore, these parasites must develop morphological and biochemical changes that allow them to survive the host defense mechanisms and adapt to the intracellular environment within the human phagocytic cells. This adaptation depends on the regulation of a wide variety of genes, and the HSP70 genes are found among them. Parasites of Leishmania and Viannia subgenera present two types of HSP70 (HSP70-I and II) genes, which differ in their 3’ untranslated regions (3’ UTR); these differences would be related to the molecular mechanisms regulating the adaptation to the thermal stress that the parasite suffers through its life cycle. Recent assays in L. braziliensis have identified different proteins with affinity to the UTRs of these genes, results of great importance considering that the regulation of gene expression in these parasites occurs mainly at the posttranscriptional level, mediated by the interaction between cis and trans regulatory elements. Among the proteins identified, the LbrM.25.2210 (SCD6) and LbrM.30.3080 (RBP42) were the focus of this Thesis, in which their molecular and functional characterization was undertaken, considering their putative function as protein factors involved in the gene regulation in Leishmania. Initially, we confirmed the capacity of SCD6 and RBP42 proteins to directly interact with RNA molecules, particularly, with a RNA motif related to the ARE elements. On the other hand, the binding capacity of these proteins in vivo was also demonstrated, finding several transcripts associated to SCD6 and RBP42 proteins. Transcripts involved mainly in metabolism pathways and cellular proteolysis. Remarkably, the transcript corresponds to the HSP70-II gene was identified associated with the RBP42 protein. Likewise, transcripts involved in the differentiation and survival of the parasites in thermal stress conditions, were found associated both to SCD6 and RBP42 proteins. In order to elucidate the possible biological processes in which these proteins could be involved, the interactome of each protein was determined. Notably, the interactomes share a high proportion of proteins, suggesting some type of functional relationship; in fact, both proteins were identified in the reciprocal interactomes. The interactomes are enriched in proteins associated with translation, RNA metabolism and regulation of gene expression processes. In support of a possible role of SCD6 and RBP42 proteins in the post-transcriptional regulation was the finding of several proteins, whose homologues have been reported as constituents of RNA granules, mainly processing bodies and stress granules. Finally, to analyze the SCD6 and RBP42 function, overexpressing lines of parasites were generated. Initially, transfection assays using constructions expressing either SCD6 or RBP42 proteins, fused to the mCherry protein, were done in L. braziliensis parasites. Whereas the control parasite line (parasites that express the mCherry protein) was obtained, the parasites overexpressing SCD6 or RBP42, however, after one week of growth, lost their viability. These results, while not conclusive, could indicate that SCD6 and RBP42 overexpression, negatively affects the development and survival of the parasites. As an alternative, it was tried, with successful results, to generate the overexpression lines in L. major parasites; these lines allowed us to determine important implications of these proteins in the developmental of the parasites. Significant morphological alterations were observed together with a lower rate of growth, whose more drastic effect was better observed when the parasites were grown at 37ºC. On the other hand, the infective in vitro capacity of these mutant parasites was significantly affected, as well as their intracellular proliferation rate, regarding the wild type strain. Overall, this study describes the structural features of SCD6 and RBP42 proteins, and their in vitro and in vivo capacities to bind RNA. Proteomics data and cellular analysis point to a relevant implication of SCD6 and RBP42 proteins as regulators for a wide group of transcripts in Leishmania.Doctor en Ciencias BiológicasDoctorad

Trypanosoma brucei parasites occupy and functionally adapt to the adipose tissue in mice

This work was supported by 55007419 (HHMI) and 2151 (EMBO) to L.M.F., D.P.-N., F.B., and F.G.; FCT fellowships to S.T., F.R.-F., and F.A.-B. (SFRH/BPD/89833/2012, SFRH/BD/51286/2010, and SFRH/BD/80718/2011, respectively); Wellcome Trust grant (093228), MRC MR/M020118/1, and European Community Seventh Framework Programme under grant agreement No. 602773 (Project KINDRED) to S.A.Y. and T.K.S.; and PAI 7/41 (Belspo) and ERC-NANOSYM to J.V.D.A.Trypanosoma brucei is an extracellular parasite that causes sleeping sickness. In mammalian hosts, trypanosomes are thought to exist in two major niches: early in infection, they populate the blood; later, they breach the blood-brain barrier. Working with a well-established mouse model, we discovered that adipose tissue constitutes a third major reservoir for T. brucei. Parasites from adipose tissue, here termed adipose tissue forms (ATFs), can replicate and were capable of infecting a naive animal. ATFs were transcriptionally distinct from bloodstream forms, and the genes upregulated included putative fatty acid β-oxidation enzymes. Consistent with this, ATFs were able to utilize exogenous myristate and form β-oxidation intermediates, suggesting that ATF parasites can use fatty acids as an external carbon source. These findings identify the adipose tissue as a niche for T. brucei during its mammalian life cycle and could potentially explain the weight loss associated with sleeping sickness.Publisher PDFPeer reviewe

Functional insights from a surface antigen mRNA-bound proteome

Trypanosoma brucei is the causative agent of human sleeping sickness. The parasites' Variant Surface Glycoprotein (VSG) enables them to evade adaptive immunity via anti-genic variation. VSG comprises 10% of total cell protein and the high stability of VSG mRNA is essential for trypanosome survival. To determine how VSG mRNA stability is maintained, we used mRNA affinity purification to identify all its associated proteins. CFB2, an unconventional RNA-binding protein with an F-box domain, was specifically enriched with VSG mRNA. We demonstrate that CFB2 is essential for VSG mRNA stabil-ity, describe cis acting elements within the VSG 3'-untranslated region that regulate the interaction, identify trans-acting factors that are present in the VSG messenger ribonu-cleoprotein particle and mechanistically explain how CFB2 stabilizes the mRNA of this key pathogenicity factor. Beyond T. brucei, the mRNP purification approach has the potential to supply detailed biological insight into metabolism of relatively abundant mRNAs in any eukaryote.Fil: do Nascimento, Larissa Melo. Ruprecht Karls Universitat Heidelberg; AlemaniaFil: Egler, Franziska. Ruprecht Karls Universitat Heidelberg; AlemaniaFil: Arnold, Katharina. Ruprecht Karls Universitat Heidelberg; AlemaniaFil: Papavasiliou, Nina. Ruprecht Karls Universitat Heidelberg; Alemania. Deutsche Krebsforschungszentrum; AlemaniaFil: Clayton, Christine. Ruprecht Karls Universitat Heidelberg; AlemaniaFil: Erben, Esteban Daniel. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina. Ruprecht Karls Universitat Heidelberg; Alemania. Deutsche Krebsforschungszentrum; Alemani

Genome-wide analysis of 30 -untranslated regions supports the existence of post-transcriptional regulons controlling gene expression in trypanosomes

In eukaryotic cells, a group of messenger ribonucleic acids (mRNAs) encoding functionally interrelated proteins together with the trans-acting factors that coordinately modulate their expression is termed a post-transcriptional regulon, due to their partial analogy to a prokaryotic polycistron. This mRNA clustering is organized by sequence-specific RNA-binding proteins (RBPs) that bind cis-regulatory elements in the noncoding regions of genes, and mediates the synchronized control of their fate. These recognition motifs are often characterized by conserved sequences and/or RNA structures, and it is likely that various classes of cis-elements remain undiscovered. Current evidence suggests that RNA regulons govern gene expression in trypanosomes, unicellular parasites which mainly use post-transcriptional mechanisms to control protein synthesis. In this study, we used motif discovery tools to test whether groups of functionally related trypanosomatid genes contain a common cis-regulatory element. We obtained conserved structured RNA motifs statistically enriched in the noncoding region of 38 out of 53 groups of metabolically related transcripts in comparison with a random control. These motifs have a hairpin loop structure, a preferred sense orientation and are located in close proximity to the open reading frames. We found that 15 out of these 38 groups represent unique motifs in which most 30 -UTR signature elements were group-specific. Two extensively studied Trypanosoma cruzi RBPs, TcUBP1 and TcRBP3 were found associated with a few candidate RNA regulons. Interestingly, 13 motifs showed a strong correlation with clusters of developmentally co-expressed genes and six RNA elements were enriched in gene clusters affected after hyperosmotic stress. Here we report a systematic genome-wide in silico screen to search for novel RNA-binding sites in transcripts, and describe an organized network of several coordinately regulated cohorts of mRNAs in T. cruzi. Moreover, we found that structured RNA elements are also conserved in other human pathogens. These results support a model of regulation of gene expression by multiple post-transcriptional regulons in trypanosomes.Fil: de Gaudenzi, Javier Gerardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Carmona, Santiago Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Agüero, Fernan Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Frasch, Alberto Carlos C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentin

Codon choice directs constitutive mRNA levels in trypanosomes.

Selective transcription of individual protein coding genes does not occur in trypanosomes and the cellular copy number of each mRNA must be determined post-transcriptionally. Here, we provide evidence that codon choice directs the levels of constitutively expressed mRNAs. First, a novel codon usage metric, the gene expression codon adaptation index (geCAI), was developed that maximised the relationship between codon choice and the measured abundance for a transcriptome. Second, geCAI predictions of mRNA levels were tested using differently coded GFP transgenes and were successful over a 25-fold range, similar to the variation in endogenous mRNAs. Third, translation was necessary for the accelerated mRNA turnover resulting from codon choice. Thus, in trypanosomes, the information determining the levels of most mRNAs resides in the open reading frame and translation is required to access this information

Genomic and Proteomic Studies on the Mode of Action of Oxaboroles against the African Trypanosome

SCYX-7158, an oxaborole, is currently in Phase I clinical trials for the treatment of human African trypanosomiasis. Here we investigate possible modes of action against Trypanosoma brucei using orthogonal chemo-proteomic and genomic approaches. SILAC-based proteomic studies using an oxaborole analogue immobilised onto a resin was used either in competition with a soluble oxaborole or an immobilised inactive control to identify thirteen proteins common to both strategies. Cell-cycle analysis of cells incubated with sub-lethal concentrations of an oxaborole identified a subtle but significant accumulation of G2 and >G2 cells. Given the possibility of compromised DNA fidelity, we investigated long-term exposure of T. brucei to oxaboroles by generating resistant cell lines in vitro. Resistance proved more difficult to generate than for drugs currently used in the field, and in one of our three cell lines was unstable. Whole-genome sequencing of the resistant cell lines revealed single nucleotide polymorphisms in 66 genes and several large-scale genomic aberrations. The absence of a simple consistent mechanism among resistant cell lines and the diverse list of binding partners from the proteomic studies suggest a degree of polypharmacology that should reduce the risk of resistance to this compound class emerging in the field. The combined genetic and chemical biology approaches have provided lists of candidates to be investigated for more detailed information on the mode of action of this promising new drug clas

Regulation of RNA binding proteins in trypanosomatid protozoan parasites

Posttranscriptional mechanisms have a critical role in the overall outcome of gene expression. These mechanisms are especially relevant in protozoa from the genus Trypanosoma, which is composed by death threatening parasites affecting people in Sub-saharan Africa or in the Americas. In these parasites the classic view of regulation of transcription initiation to modulate the products of a given gene cannot be applied. This is due to the presence of transcription start sites that give rise to long polycistronic units that need to be processed costranscriptionally by trans-splicing and polyadenylation to give mature monocistronic mRNAs. Posttranscriptional mechanisms such as mRNA degradation and translational repression are responsible for the final synthesis of the required protein products. In this context, RNA-binding proteins (RBPs) in trypanosomes have a relevant role as modulators of mRNA abundance and translational repression by associating to the 3’ untranslated regions in mRNA. Many different RBPs have been proposed to modulate cohorts of mRNAs in trypanosomes. However, the current understanding of their functions lacks a dynamic view on the different steps at which these RBPs are regulated. Here, we discuss different evidences to propose regulatory events for different RBPs in these parasites. These events vary from regulated developmental expression, to biogenesis of cytoplasmic ribonucleoprotein complexes in the nucleus, and condensation of RBPs and mRNA into large cytoplasmic granules. Finally, we discuss how newly identified posttranslational modifications of RBPs and mRNA metabolism-related proteins could have an enormous impact on the modulation of mRNA abundance. To understand these modifications is especially relevant in these parasites due to the fact that the enzymes involved could be interesting targets for drug therapy.Fil: Romaniuk, María Albertina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Cervini Bohm, Gabriela Marta . Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Cassola, Alejandro Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentin