11 research outputs found

    The MB2 gene family of Plasmodium species has a unique combination of S1 and GTP-binding domains

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    BACKGROUND: Identification and characterization of novel Plasmodium gene families is necessary for developing new anti-malarial therapeutics. The products of the Plasmodium falciparum gene, MB2, were shown previously to have a stage-specific pattern of subcellular localization and proteolytic processing. RESULTS: Genes homologous to MB2 were identified in five additional parasite species, P. knowlesi, P. gallinaceum, P. berghei, P. yoelii, and P. chabaudi. Sequence comparisons among the MB2 gene products reveal amino acid conservation of structural features, including putative S1 and GTP-binding domains, and putative signal peptides and nuclear localization signals. CONCLUSIONS: The combination of domains is unique to this gene family and indicates that MB2 genes comprise a novel family and therefore may be a good target for drug development

    Characterization of immunoglobulin G antibodies to Plasmodium falciparum sporozoite surface antigen MB2 in malaria exposed individuals

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    <p>Abstract</p> <p>Background</p> <p>MB2 protein is a sporozoite surface antigen on the human malaria parasite <it>Plasmodium falciparum</it>. MB2 was identified by screening a <it>P. falciparum </it>sporozoite cDNA expression library using immune sera from a protected donor immunized via the bites of <it>P. falciparum</it>-infected irradiated mosquitoes. It is not known whether natural exposure to <it>P. falciparum </it>also induces the anti-MB2 response and if this response differs from that in protected individuals immunized via the bites of <it>P. falciparum </it>infected irradiated mosquitoes. The anti-MB2 antibody response may be part of a robust protective response against the sporozoite.</p> <p>Methods</p> <p>Fragments of polypeptide regions of MB2 were constructed as recombinant fusions sandwiched between glutathione S-transferase and a hexa histidine tag for bacterial expression. The hexa histidine tag affinity purified proteins were used to immunize rabbits and the polyclonal sera evaluated in an <it>in vitro </it>inhibition of sporozoite invasion assay. The proteins were also used in immunoblots with sera from a limited number of donors immunized via the bites of <it>P. falciparum </it>infected irradiated mosquitoes and plasma and serum obtained from naturally exposed individuals in Kenya.</p> <p>Results</p> <p>Rabbit polyclonal antibodies targeting the non-repeat region of the basic domain of MB2 inhibited sporozoites entry into HepG2-A16 cells <it>in vitro</it>. Analysis of serum from five human volunteers that were immunized via the bites of <it>P. falciparum </it>infected irradiated mosquitoes that developed immunity and were completely protected against subsequent challenge with non-irradiated parasite also had detectable levels of antibody against MB2 basic domain. In contrast, in three volunteers not protected, anti-MB2 antibodies were below the level of detection. Sera from protected volunteers preferentially recognized a non-repeat region of the basic domain of MB2, whereas plasma from naturally-infected individuals also had antibodies that recognize regions of MB2 that contain a repeat motif in immunoblots. Sequence analysis of eleven field isolates and four laboratory strains showed that these antigenic regions of the basic domain of the <it>MB2 </it>gene are highly conserved in parasites obtained from different parts of the world. Moreover, anti-MB2 antibodies also were detected in the plasma of 83% of the individuals living in a malaria endemic area of Kenya (n = 41).</p> <p>Conclusion</p> <p>A preliminary analysis of the human humoral response against MB2 indicates that it may be an additional highly conserved target for immune intervention at the pre-erythrocytic stage of <it>P. falciparum </it>life cycle.</p

    Molecular Biology

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    Plasmodium falciparum translational machinery condones polyadenosine repeats

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    Plasmodium falciparum is a causative agent of human malaria. Sixty percent of mRNAs from its extremely AT-rich (81%) genome harbor long polyadenosine (polyA) runs within their ORFs, distinguishing the parasite from its hosts and other sequenced organisms. Recent studies indicate polyA runs cause ribosome stalling and frameshifting, triggering mRNA surveillance pathways and attenuating protein synthesis. Here, we show that P. falciparum is an exception to this rule. We demonstrate that both endogenous genes and reporter sequences containing long polyA runs are efficiently and accurately translated in P. falciparum cells. We show that polyA runs do not elicit any response from No Go Decay (NGD) or result in the production of frameshifted proteins. This is in stark contrast to what we observe in human cells or T. thermophila, an organism with similar AT-content. Finally, using stalling reporters we show that Plasmodium cells evolved not to have a fully functional NGD pathway

    Protein Synthesis Adaptation to the AU-Rich Transcriptome of Plasmodium falciparum

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    The process of protein synthesis whereby a messenger RNA is decoded into an amino acid chainis conserved among the domains. Fastidious protein synthesis is necessary for organism survival. However, exceptions negatively affecting the mRNA translation cycle – inadvertently or by design – may occur. Polyadenosine tracts are one such motif causing ribosomal stalling and frameshifting in almost all organisms tested thus far; save Plasmodium spp. Thus, with ~60% of their protein-coding genome harboring polyadenosine tracts, the elucidation of such paradigm-breaking adaptations enabling Plasmodium spp. to translate this typically problematic motif without issue is salient from both basic science and clinical perspectives. Using biochemical and structural approaches, I report on the parasite ability to express polyA motifs and ribosome alterations enabling polylysine synthesis. The developed PP7-mRIP assay reveals RBP differences among varying mRNA substrates, revealing a previously uncharacterized, parasite-specific AU-rich binding protein bound to polyA tract reporter mRNA. Finally, the parasite exhibits altered binding of the essential ribosomal protein RACK1, vital for translation cap-dependent initiation and quality control activation, that would invariably alter ribosome- associated quality control pathway signaling, ostensibly aiding polyA translation

    Isolation and Characterisation of the GPI:Protein Transamidase From Leishmania mexicana

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    Many eukaryotic cell surface proteins are attached to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. Trypanosomatid parasitic protozoa such as Leishmania and Trypanosoma brucei make extensive use of this method of protein surface attachment. The perceived importance of GPI-anchored proteins makes the GPI biosynthetic pathway a good target for anti-parasite chemotherapy. The terminal step in the pathway is the addition of complete pre-formed GPI anchors to the carboxyl-termini of proteins. This involves replacing a GPI signal sequence with a GPI anchor in a transamidation reaction. Two of the proteins involved in this step have been identified in yeast and mammals. One of these components, GPI8, has significant homology to a family of plant cysteine proteinases, the legumains, and GPI8 is therefore believed to be the catalytic subunit of the transamidase. The work described in this thesis focussed on the cloning of the GPI8 gene from Leishmania mexicana and its characterisation. The predicted protein shares 31% identity with yeast and human homologues. The nucleotide sequence of a fragment of the T. brucei GPI8 gene has also been obtained. Targeted gene replacement of the single copy L. mexicana GPI8 produced GPI8 null mutants. The loss of GPI8 was confirmed by Southern blotting. The phenotype of the GPI8 null mutants was analysed with the following findings: (1) Mutant promastigotes grow well in culture. (2) GP63 is not detected on the promastigote surface and is greatly reduced in promastigote lysates. (3) Episomal re-expression of GPI8 restored GP63 to the cell surface. (4) GPI8 null mutants are able to infect macrophages in vitro to approximately wild type levels, and replicate within macrophages. (5) GPI8 null mutants are capable of forming lesions in mice. These data show that GPI-anchored proteins of L. mexicana are not essential for growth of promastigotes, invasion of macrophages by promastigotes, or infection of mice. It remains to be established if GPI-anchored proteins are required for survival in the sandfly. Sequence comparison of L. mexicana, yeast and human GPI8 proteins identified two potential active site cysteine residues. Mutation in which Cys216 was converted to a glycine led to loss of GPI8 activity, as assessed by GP63 surface expression, indicating that this may be the active site cysteine. Recombinant GPI8 was produced in E. coli and used to inoculate rabbits for the production of anti-serum. These antibodies detected recombinant protein but failed to detect protein in L. mexicana cell lysates. Antibodies raised against a peptide of GPI8 also recognised recombinant GPI8, but not GPI8 in L. mexicana lysates. A protein of 38-40 kDa, however, was detected in cell lysates of T. brucei with the antirecombinant GPI8 antibodies. This antiserum gave a similar immunofluorescence pattern in T. brucei to those of an epitope-tagged ribosomal protein, QM. There is some overlap in fluorescence patterns between tagged QM and the endoplasmic reticulum marker protein BiP. This suggests that GPI8 is located in the ER of T. brucei, the same subcellular location that has been identified in yeast. A fusion protein of GPI8 and GFP was expressed in L. mexicana to attempt to localise GPI8 in this organism. The fusion protein, however, was unable to restore GPI8 function in the null mutant, therefore data on its localisation cannot be inferred

    NMR insights into molecular recognition: structure and interactions of peptides and proteins

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    Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Departamento de Bioquímica y Biología Molecular I, leída el 16-12-2016La investigación de las bases moleculares que gobiernan los procesos biológicos es esencial para comprenderlos, así como para entender sus implicaciones y el impacto que tienen en el organismo de los seres vivos. En dichos procesos, los eventos de reconocimiento biomolecular son cruciales y es necesario lograr una descripción detallada a nivel atómico para conocer las particularidades de los mecanismos, su regulación, selectividad, especificidad… En este contexto, la espectroscopía de RMN es una técnica que proporciona gran cantidad de información fiable que permite estudiar las características de los eventos de reconocimiento biomolecular con resolución atómica, lo que es clave para deducir y clarificar los fundamentos de los relevantes procesos biológicos. En este trabajo se ha empleado la espectroscopía de RMN, en combinación con diversas técnicas bioquímicas, fisicoquímicas y computacionales, para estudiar varios sistemas biológicos de interés en los que están presentes algunos eventos de reconocimiento biomolecular representativos, tales como las interacciones péptido–membrana, las interacciones carbohidrato–proteína, o las interacciones proteína–proteína. El objeto de esta tesis es profundizar en el conocimiento sobre varios casos de los citados eventos de reconocimiento biomolecular y reforzar el papel de la espectroscopía de RMN como una potente herramienta para abordar la caracterización de las interacciones biomoleculares. Esta tesis está estructurada en seis capítulos. El capítulo 2 describe el estudio de una serie de péptidos derivados del dominio de unión a colina de la autolisina de pneumococo, LytA. Este estudio está dirigido a averiguar si dichos péptidos mantienen su estructura nativa y su capacidad de unir colina cuando se encuentran aislados de la proteína completa. Utilizando espectroscopía de RMN en disolución, dicroísmo circular (CD) y técnicas de fluorescencia, se han descubierto tres péptidos que adoptan conformaciones de horquilla β nativas en disolución acuosa, tal y como se pretendía, y que sufren una inesperada transición reversible de estructura β a estructura α en presencia de micelas de detergente. Se ha propuesto una explicación para la interacción que tiene lugar entre estos péptidos y las micelas de detergente, y se han caracterizado las bases fisicoquímicas de la transición estructural observada mediante el estudio de variantes de uno de estos péptidos...The investigation of the molecular bases governing biological processes is essential to understand them, as well as their implications and impact in the organism of living creatures. In these processes, biomolecular recognition events are pivotal and the search for a comprehensive description at atomic level is necessary to know the details of mechanisms, regulation, selectivity, specificity, etc. In this context, NMR spectroscopy is a technique that provides a vast diversity of reliable information allowing the study of the characteristics of biomolecular recognition events at atomic resolution, which is the key to infer and clarify the fundamentals of relevant biological processes. In this work, NMR spectroscopy combined with diverse biochemical, physicochemical, and computational techniques have been utilised to study several interesting biological systems involving some representative biomolecular recognition events, such as peptide–membrane interactions, carbohydrate–protein interactions, and protein–protein interactions. The aim of this thesis is to go deeper in the knowledge of some biomolecular recognition events, and reinforce that NMR spectroscopy as a powerful tool to address the characterization of biomolecular interactions. This thesis has been structured in six chapters. Chapter 2 describes the study of a series of peptides derived from the choline–binding domain of pneumococcal autolysin LytA, aimed to find out whether they maintain their native structure and the ability to bind choline when isolated from the full–length protein. Using solution NMR, CD and fluorescence techniques, three peptides were found to show native–like, β–hairpin conformations in aqueous solution, as intended, and undergo an unexpected, reversible β–to–α transition in the presence of detergent micelles. An explanation to the interaction between these peptides and detergent micelle has been proposed, and the physicochemical bases of the observed structural transition were characterised by studying variants of one of these peptides...Depto. de Bioquímica y Biología MolecularFac. de Ciencias QuímicasTRUEunpu

    Drug Discovery and Structural Studies on Mycobacterium tuberculosis Proteins Related to Drug Resistance and Persistence

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    Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), which is one of the leading infectious diseases worldwide. The current therapy for drug-sensitive TB is complex and lasts for at least six months. Improper use of antibiotics during this regimen has led to the emergence of drug resistance, which represents a grave threat to human health. This problem is further exacerbated by the ability of the bacterium to persist in the host in a non-replicating state despite the use of antibiotics. The majority of antibiotics currently used to treat tuberculosis only affect replicating bacteria. Therefore, it is critical to develop new antitubercular drugs that can shorten the current therapy while maintaining activity against persistent bacteria as well as the drug-resistant strains. In this dissertation, structural and drug discovery studies on Mtb proteins related to drug resistance and persistence are presented. InhA, the enoyl-ACP-reductase enzyme of the mycolic acid biosynthesis pathway, is the molecular target of the antitubercular prodrugs isoniazid and ethionamide, and it is one of the best validated targets for Mtb drug discovery. A target-based high throughput screening and a structure-based drug design were performed to identify potent activation-free InhA inhibitors that were effective against drug-resistant Mtb strains. The molecular basis of InhA inhibition by these inhibitors was revealed by X-ray crystallography. In addition, the mode of action for ethionamide was revealed by using a cell-based activation system and X-ray crystallography. Furthermore, it was demonstrated that InhA is the clinically relevant primary target of isoniazid. The regulation of InhA function was also studied, which revealed that phosphorylation of InhA occurs at its C-terminal. Phosphomimetic mutants showed that phosphorylation decreases InhA activity by decreasing the affinity toward cofactor NADH. The results of these studies are presented in Chapters II, III, IV, and V. It is essential to understand the physiology of the bacterium to target the persistent state of Mtb infection. In Chapter VI, I report our studies on CarD, an essential Mtb transcription regulator that is required for persistent infection. The structure of the CarD/RNAP complex was determined by X-ray crystallography and the CarD-DNA interactions were investigated
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