Biochemistry and Pharmacology - Research Publications
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ItemAn FtsH Protease Is Recruited to the Mitochondrion of Plasmodium falciparum(PUBLIC LIBRARY SCIENCE, 2013-09-13)The two organelles, apicoplast and mitochondrion, of the malaria parasite Plasmodium falciparum have unique morphology in liver and blood stages; they undergo complex branching and looping prior to division and segregation into daughter merozoites. Little is known about the molecular processes and proteins involved in organelle biogenesis in the parasite. We report the identification of an AAA+/FtsH protease homolog (PfFtsH1) that exhibits ATP- and Zn(2+)-dependent protease activity. PfFtsH1 undergoes processing, forms oligomeric assemblies, and is associated with the membrane fraction of the parasite cell. Generation of a transfectant parasite line with hemagglutinin-tagged PfFtsH1, and immunofluorescence assay with anti-PfFtsH1 Ab demonstrated that the protein localises to P. falciparum mitochondria. Phylogenetic analysis and the single transmembrane region identifiable in PfFtsH1 suggest that it is an i-AAA like inner mitochondrial membrane protein. Expression of PfFtsH1 in Escherichia coli converted a fraction of bacterial cells into division-defective filamentous forms implying a sequestering effect of the Plasmodium factor on the bacterial homolog, indicative of functional conservation with EcFtsH. These results identify a membrane-associated mitochondrial AAA+/FtsH protease as a candidate regulatory protein for organelle biogenesis in P. falciparum.
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ItemNo Preview AvailableTargeting and function of proteins mediating translation initiation in organelles of Plasmodium falciparum(WILEY, 2015-05)The malaria parasite Plasmodium falciparum has two translationally active organelles - the apicoplast and mitochondrion, which import nuclear-encoded translation factors to mediate protein synthesis. Initiation of translation is a complex step wherein initiation factors (IFs) act in a regulated manner to form an initiation complex. We identified putative organellar IFs and investigated the targeting, structure and function of IF1, IF2 and IF3 homologues encoded by the parasite nuclear genome. A single PfIF1 is targeted to the apicoplast. Apart from its critical ribosomal interactions, PfIF1 also exhibited nucleic-acid binding and melting activities and mediated transcription anti-termination. This suggests a prominent ancillary function for PfIF1 in destabilisation of DNA and RNA hairpin loops encountered during transcription and translation of the A+T rich apicoplast genome. Of the three putative IF2 homologues, only one (PfIF2a) was an organellar protein with mitochondrial localisation. We additionally identified an IF3 (PfIF3a) that localised exclusively to the mitochondrion and another protein, PfIF3b, that was apicoplast targeted. PfIF3a exhibited ribosome anti-association activity, and monosome splitting by PfIF3a was enhanced by ribosome recycling factor (PfRRF2) and PfEF-G(Mit). These results fill a gap in our understanding of organellar translation in Plasmodium, which is the site of action of several anti-malarial compounds.
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ItemPromiscuous thymic expression of an autoantigen gene does not result in negative selection of pathogenic T cells(AMER ASSOC IMMUNOLOGISTS, 2005-11-01)"Promiscuous" thymic expression of peripheral autoantigens can contribute to immunological tolerance in some cases. However, in this study we show that thymic mRNA expression alone cannot predict a contribution to thymic tolerance. Autoimmune gastritis is caused by CD4+ T cells directed to the alpha (H/Kalpha) and beta (H/Kbeta) subunits of the gastric membrane protein the H+/K+ ATPase. H/Kalpha mRNA is expressed in the thymus, but H/Kbeta expression is barely detectable. In this study, we demonstrate that thymic H/Kalpha in wild-type mice or mice that overexpressed H/Kalpha did not result in negative selection of pathogenic anti-H/Kalpha T cells. However, negative selection of anti-H/Kalpha T cells did occur if H/Kbeta was artificially overexpressed in the thymus. Given that H/Kalpha cannot be exported from the endoplasmic reticulum and is rapidly degraded in the absence of H/Kbeta, we conclude that H/Kalpha epitopes are unable to access MHC class II loading compartments in cells of the normal thymus. This work, taken together with our previous studies, highlights that thymic autoantigen expression does not necessarily result in the induction of tolerance.