A destabilisation domain approach to define the in vivo functional importance of PfHsp70-1 and PfHsp40 in the intraerythrocytic life cycle of Plasmodium falciparum

The apicomplexan malaria parasite, Plasmodium falciparum is capable of invading red blood cells and causes the most virulent form of malaria. The life cycle of P. falciparum involves the migration from the poikilothermic mosquito vector to warm-blooded human host and vice versa. Such transition introduces radical differences between the cellular environments where the parasite resides, imposing physiological stress. The diverse environmental insults in addition to the febrile fever episodes imparts challenge on the proteostasis, resulting in the evolutionary selection of a diverse network of molecular chaperones. In fact, some molecular chaperones are essential for the survival of Plasmodium. Due to the developing resistance of Plasmodium against currently available drugs, heat shock proteins have received extensive research attention as antimalarial targets in recent years. Plasmodium codes for one Hsp90 homologue and a constitutively expressed heat inducible cytosolic Hsp70 known as PfHsp70-1. In general, Hsp70 interacts with co-chaperone Hsp40 initiating the protein folding machinery that finally interacts with Hsp90 to maintain proteostasis in a cell. PfHsp90 has been found to be essential for the intraerythrocytic development of P. falciparum. Although there have been some in vitro studies on the biology of PfHsp70-1, the information on the in vivo essential function of PfHsp70-1 and its interaction with PfHsp40 is limited. In this study, we wanted to identify the in vivo biological importance of PfHsp70-1 and one of its predicted co-chaperones, PfHsp40 by the overexpression of the dominant negative alleles tagged to recently characterised destabilisation domain (dd) to regulate protein level. We expressed a dominant negative PfHsp70-1 possessing a point mutation (E187K), severely affecting normal domain movement important for its function. PfHsp40 was mutated in the conserved HPD motif (D34N) necessary for establishing interaction with PfHsp70-1. Unfortunately, we could not obtain sufficient overexpression of the episomal dominant negative versions to override the function of the endogenous proteins in a competitive manner. The cellular levels of endogenous proteins were higher by several folds compared to that of the episomally expressed dominant negative alleles. The destabilisation strategy has been reported to be successful for studying certain plasmodial proteins. But in contrast, during our work the level of almost none of the candidate chaperones could be controlled by either FKBP or E. coli DHFR derived destabilisation domains in a ligand dependent fashion. Although, the level of wild type PfHsp70-1 could be regulated by this strategy, the dominant negative version with only one amino acid substitution made it non responsive to dd tagging and further ligand treatment. At the same time, the level of control proteins could be efficiently regulated by stabilising ligands. Recently, success of destabilization domain strategy for conditional knockdown of several genes has been reported. But in contrast, our observations in this study unravel the possible drawbacks. We assume that the success of such an approach is greatly protein dependent. Based on the several reports initially this approach appeared to be the most beneficial system. But, the failure to successfully implement this strategy demands careful consideration in selecting an alternative future approach to study the function of essential genes in Plasmodium

Proteomic analysis of Plasmodium falciparum histone deacetylase 1 complex proteins

Plasmodium falciparum histone deacetylases (PfHDACs) are an important class of epigenetic regulators that alter protein lysine acetylation, contributing to regulation of gene expression and normal parasite growth and development. PfHDACs are therefore under investigation as drug targets for malaria. Despite this, our understanding of the biological roles of these enzymes is only just beginning to emerge. In higher eukaryotes, HDACs function as part of multi-protein complexes and act on both histone and non-histone substrates. Here, we present a proteomics analysis of PfHDAC1 immunoprecipitates, identifying 26 putative P. falciparum complex proteins in trophozoite-stage asexual intraerythrocytic parasites. The co-migration of two of these (P. falciparum heat shock proteins 70-1 and 90) with PfHDAC1 was validated using Blue Native PAGE combined with Western blot. These data provide a snapshot of possible PfHDAC1 interactions and a starting point for future studies focused on elucidating the broader function of PfHDACs in Plasmodium parasites

Detection of the in vitro modulation of Plasmodium falciparum Arf1 by Sec7 and ArfGAP domains using a colorimetric plate-based assay:

The regulation of human Arf1 GTPase activity by ArfGEFs that stimulate GDP/GTP exchange and ArfGAPs that mediate GTP hydrolysis has attracted attention for the discovery of Arf1 inhibitors as potential anti-cancer agents. The malaria parasite Plasmodium falciparum encodes a Sec7 domain-containing protein - presumably an ArfGEF - and two putative ArfGAPs, as well as an Arf1 homologue (PfArf1) that is essential for blood-stage parasite viability. However, ArfGEF and ArfGAP-mediated activation/deactivation of PfArf1 has not been demonstrated

Portuguese history storyboard

This paper intends to present relevant facts about the Portuguese culture and history, so as to enable a better understanding of who the Portuguese are and provide an overall perspective of the course of history in this westernmost part of Europe. Although the choice of historical facts was subjective by nature, it is believed it achieves the aim of presenting information in a critical but blithesome way, with a view to also deconstructing national stereotypes, such as that Portuguese people are always late or are crazy about football. Finally, it focuses on some information about the Portuguese language mainly to serve as a term of comparison with other European languages

The Plasmodium Export Element Revisited

We performed a bioinformatical analysis of protein export elements (PEXEL) in the putative proteome of the malaria parasite Plasmodium falciparum. A protein family-specific conservation of physicochemical residue profiles was found for PEXEL-flanking sequence regions. We demonstrate that the family members can be clustered based on the flanking regions only and display characteristic hydrophobicity patterns. This raises the possibility that the flanking regions may contain additional information for a family-specific role of PEXEL. We further show that signal peptide cleavage results in a positional alignment of PEXEL from both proteins with, and without, a signal peptide

Compartmentation of Redox Metabolism in Malaria Parasites

Malaria, caused by the apicomplexan parasite Plasmodium, still represents a major threat to human health and welfare and leads to about one million human deaths annually. Plasmodium is a rapidly multiplying unicellular organism undergoing a complex developmental cycle in man and mosquito – a life style that requires rapid adaptation to various environments. In order to deal with high fluxes of reactive oxygen species and maintain redox regulatory processes and pathogenicity, Plasmodium depends upon an adequate redox balance. By systematically studying the subcellular localization of the major antioxidant and redox regulatory proteins, we obtained the first complete map of redox compartmentation in Plasmodium falciparum. We demonstrate the targeting of two plasmodial peroxiredoxins and a putative glyoxalase system to the apicoplast, a non-photosynthetic plastid. We furthermore obtained a complete picture of the compartmentation of thioredoxin- and glutaredoxin-like proteins. Notably, for the two major antioxidant redox-enzymes – glutathione reductase and thioredoxin reductase – Plasmodium makes use of alternative-translation-initiation (ATI) to achieve differential targeting. Dual localization of proteins effected by ATI is likely to occur also in other Apicomplexa and might open new avenues for therapeutic intervention

Analysis of protein trafficking signals of a member of the P. falciparum stevor multi-gene family

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Intracellular protozoan parasites of humans: the role of molecular chaperones in development and pathogenesis

Certain kinetoplastid (Leishmania spp. and Trypanosoma cruzi) and apicomplexan parasites (Plasmodium falciparum and Toxoplasma gondii) are capable of invading human cells as part of their pathology. These parasites appear to have evolved a relatively expanded or diverse complement of genes encoding molecular chaperones. The gene families encoding heat shock protein 90 (Hsp90) and heat shock protein 70 (Hsp70) chaperones show significant expansion and diversity (especially for Leishmania spp. and T. cruzi), and in particular the Hsp40 family appears to be an extreme example of phylogenetic radiation. In general, Hsp40 proteins act as co-chaperones of Hsp70 chaperones, forming protein folding pathways that integrate with Hsp90 to ensure proteostasis in the cell. It is tempting to speculate that the diverse environmental insults that these parasites endure have resulted in the evolutionary selection of a diverse and expanded chaperone network. Hsp90 is involved in development and growth of all of these intracellular parasites, and so far represents the strongest candidate as a target for chemotherapeutic interventions. While there have been some excellent studies on the molecular and cell biology of Hsp70 proteins, relatively little is known about the biological function of Hsp70-Hsp40 in teractions in these intracellular parasites. This review focuses on intracellular protozoan parasites of humans, and provides a critique of the role of heat shock proteins in development and pathogenesis, especially the molecular chaperones Hsp90, Hsp70 and Hsp40

Der Nachweis eines Mitglieds der OMP85-Proteinfamilie im Apikoplasten von Toxoplasma gondii

Der intrazelluläre Parasit Toxoplasma gondii zeichnet sich wie die meisten Mitglieder der Chromalveolaten durch den Besitz einer sekundären Plastide aus, die man als Apikoplasten bezeichnet. Diese Art von Plastide ist durch einen Vorgang der sekundären Endosymbiose entstanden, bei der ein Rhodophyt von einer eukaryotischen Zelle aufgenommen und im Laufe der Evolution als Organell etabliert wurde. Aufgrund des horizontalen Gentransfers zwischen dem Nukleus und der Plastide werden die meisten Proteine des Apikoplasten im Wirtsgenom kodiert und im ER synthetisiert. Proteine des Apikoplastenstromas müssen daher aus dem ER, über die insgesamt vier Membranen der Plastide mit Hilfe einer Proteinimportmaschinerie zu ihrem Bestimmungsort befördert werden. Wie dieser Import im Detail erfolgt, ist zum jetzigen Zeitpunkt noch unklar. Jedoch konnten einige Komponenten dieser Maschinerie, wie SELMA in der PPM und ein Tic20-Homolog in der innersten Membran des Apikoplasten identifiziert werden. Basierend auf bioinformatischen Analysen konnten zwei Proteine der OMP85-Familie im Genom von T. gondii identifiziert werden. Die Mitglieder dieser Familie können aufgrund ihrer Funktionalität zwei Subtypen (Toc75- und Sam50-Subtyp) zugeordnet werden. Während Proteine des Toc75-Subtyps am Transport von Proteinen über Membranen beteiligt sind, sind die des Sam50-Subtyps in der Integration von Proteinen in Membranen involviert. Im Rahmen dieser Arbeit wurde für eines der beiden in T. gondii identifizierten OMP85 Proteine (TgOMP85) eine Funktion im Proteinimport in die Plastide postuliert. Nach Überprüfung des Genmodells konnte TgOMP85 eindeutig der OMP85-Familie zugeordnet und im Apikoplasten des genannten Parasiten lokalisiert werden. Des Weiteren wurde der Nachweis erbracht, dass TgOMP85 eine funktionelle BTS-Sequenz aufweist, welche den Transport und die Lokalisation in den Apikoplasten vermittelt. Um die Vermutung, dass es sich bei TgOMP85 um eine, in der dritten Apikoplastenmembran lokalisierte, Komponente der Proteinimportmaschinerie handelt, bestätigen zu können, sind weiterführende Analysen nötig. Das zweite identifizierte Protein (TgSam50) könnte aufgrund seiner Gensequenz und mitochondrialen Lokalisation dem Sam50-Subtyp zugeordnet werden. Ob es jedoch tatsächlich an der Assemblierung und Integration von Proteinen in die äußere mitochondriale Membran involviert ist, kann zum jetzigen Zeitpunkt nicht beantwortet werden