Housekeeping and other metabolic functions of the Plasmodium plastid

The malaria parasite carries a plastid called the apicoplast that has been the subject of intense study in the last 15 years. Having originated from red-algal plastids, the apicoplast has lost its ability to photosynthesize, but carries out other essential functions such as type-II fatty acid synthesis, biosynthesis of haem and isoprenoid synthesis; the DOXP pathway for isoprenoid synthesis has recently been demonstrated to be the only pathway critical for parasite survival in the erythrocytic stage. The apicoplast also has a functional Suf system for assembly of (Fe–S) complexes on target proteins. The organelle has a 35 kb, double-stranded DNA genome that encodes a set of RNAs and proteins, the latter being translated from organellar mRNA by an active translation machinery, a major component of which is encoded by the nucleus. This article reviews current knowledge of housekeeping functions of the Plasmodium apicoplast and its (Fe–S) assembly system and discusses these components as sites for drug intervention against malaria

Anticancer Activity of Cissus quadrangularis: An in vitro 2D Model Based Study

Cissus quadrangularis (CQ) is a perennial rambling shrub of the grape family commonly known a

Ssd1 Is Required for Thermotolerance and Hsp104-Mediated Protein Disaggregation in Saccharomyces cerevisiae▿

In the budding yeast Saccharomyces cerevisiae, the Hsp104-mediated disaggregation of protein aggregates is essential for thermotolerance and to facilitate the maintenance of prions. In humans, protein aggregation is associated with neuronal death and dysfunction in many neurodegenerative diseases. Mechanisms of aggregation surveillance that regulate protein disaggregation are likely to play a major role in cell survival after acute stress. However, such mechanisms have not been studied. In a screen using the yeast gene deletion library for mutants unable to survive an aggregation-inducing heat stress, we find that SSD1 is required for Hsp104-mediated protein disaggregation. SSD1 is a polymorphic gene that plays a role in cellular integrity, longevity, and pathogenicity in yeast. Allelic variants of SSD1 regulate the level of thermotolerance and cell wall remodeling. We have shown that Ssd1 influences the ability of Hsp104 to hexamerize, to interact with the cochaperone Sti1, and to bind protein aggregates. These results provide a paradigm for linking Ssd1-mediated cellular integrity and Hsp104-mediated disaggregation to ensure the survival of cells with fewer aggregates

The effect of fusidic acid on Plasmodium falciparum elongation factor G (EF-G)

Inhibition of growth of the malaria parasite Plasmodium falciparum by known translation-inhibitory antibiotics has generated interest in understanding their action on the translation apparatus of the two genome containing organelles of the malaria parasite: the mitochondrion and the relic plastid (apicoplast). We report GTPase activity of recombinant EF-G proteins that are targeted to the organelles and further use these to test the effect of the EF-G inhibitor fusidic acid (FA) on the factor–ribosome interface. Our results monitoring locking of EF-G·GDP onto surrogate Escherichia coli ribosomes as well as multi-turnover GTP hydrolysis by the factor indicate that FA has a greater effect on apicoplast EF-G compared to the mitochondrial counterpart. Deletion of a three amino acid (GVG) sequence in the switch I loop that is conserved in proteins of the mitochondrial EF-G1 family and the Plasmodium mitochondrial factor, but is absent in apicoplast EF-G, demonstrated that this motif contributes to differential inhibition of the two EF-Gs by FA. Additionally, the drug thiostrepton, that is known to target the apicoplast and proteasome, enhanced retention of only mitochondrial EF-G on ribosomes providing support for the reported effect of the drug on parasite mitochondrial translation

Interaction of apicoplast-encoded elongation factor (EF) EF-Tu with nuclear-encoded EF-Ts mediates translation in the Plasmodium falciparum plastid

Protein translation in the plastid (apicoplast) of Plasmodium spp. is of immense interest as a target for potential anti-malarial drugs. However, the molecular data on apicoplast translation needed for optimisation and development of novel inhibitors is lacking. We report characterisation of two key translation elongation factors in Plasmodium falciparum, apicoplast-encoded elongation factor PfEF-Tu and nuclear-encoded PfEF-Ts. Recombinant PfEF-Tu hydrolysed GTP and interacted with its presumed nuclear-encoded partner PfEF-Ts. The EF-Tu inhibitor kirromycin affected PfEF-Tu activity in vitro, indicating that apicoplast EF-Tu is indeed the target of this drug. The predicted PfEF-Ts leader sequence targeted GFP to the apicoplast, confirming that PfEF-Ts functions in this organelle. Recombinant PfEF-Ts mediated nucleotide exchange on PfEF-Tu and homology modeling of the PfEF-Tu:PfEF-Ts complex revealed PfEF-Ts-induced structural alterations that would expedite GDP release from PfEF-Tu. Our results establish functional interaction between two apicoplast translation factors encoded by genes residing in different cellular compartments and highlight the significance of their sequence/structural differences from bacterial elongation factors in relation to inhibitor activity. These data provide an experimental system to study the effects of novel inhibitors targeting PfEF-Tu and PfEF-Tu.PfEF-Ts interaction. Our finding that apicoplast EF-Tu possesses chaperone-related disulphide reductase activity also provides a rationale for retention of the tufA gene on the plastid genome

Rohitukine promoted ROS production and loss Mitochondrial content in gene knockout strains of yeast.

<p>(a) DCFDA staining (b) Graphical representation of Relative formation of reactive oxygen species (ROS) measured by H2DCFDA staining in WT and gene knockout strains of yeast as quantified using Image J software ***p < 0.001.(c) Mitotracker Deep Red staining (d) Graphical representation for fluorescence intensity of mitochondrial content of the budding yeast as quantified using Image J software ***p <0.001.</p

Recycling factors for ribosome disassembly in the apicoplast and mitochondrion of Plasmodium falciparum

The reduced genomes of the apicoplast and mitochondrion of the malaria parasite Plasmodium falciparum are actively translated and antibiotic-mediated translation inhibition is detrimental to parasite survival. In order to understand recycling of organellar ribosomes, a critical step in protein translation, we identified ribosome recycling factors (RRF) encoded by the parasite nuclear genome. Targeting of PfRRF1 and PfRRF2 to the apicoplast and mitochondrion respectively was established by localization of leader sequence–GFP fusions. Unlike any RRF characterized thus far, PfRRF2 formed dimers with disulphide interaction(s) and additionally localized in the cytoplasm, thus suggesting adjunct functions for the factor. PfRRF1 carries a large 108-amino-acid insertion in the functionally critical hinge region between the head and tail domains of the protein, yet complemented Escherichia coli RRF in the LJ14frr<SUP>ts</SUP> mutant and disassembled surrogate E. coli 70S ribosomes in the presence of apicoplast-targeted EF-G. Recombinant PfRRF2 bound E. coli ribosomes and could split monosomes in the presence of the relevant mitochondrial EF-G but failed to complement the LJ14frr<SUP>ts</SUP> mutant. Although proteins comprising subunits of P. falciparum organellar ribosomes are predicted to differ from bacterial and mitoribosomal counterpar<SUP>ts</SUP>, our results indicate that the essential interactions required for recycling are conserved in parasite organelles

(a) RT-PCR analysis of <i>Slt2</i> and <i>Hog1</i> gene in budding yeast after drug treatment (i:Untreated control, ii: drug treated) (b) The expression of Slt2 and Hog1 mRNA, expressed as the ratio of densitometric measurement of the sample to the corresponding internal control (β-actin) (i: Untreated control, ii: drug treated) (c) Docking studies of Rohitukine with human two different type of member of MAPK pathway. (i) p38 (Hog1 in <i>S</i>. <i>cerevisiae</i>) and (ii) ERK5 (Slt2 in <i>S</i>. <i>cerevisiae</i>).

<p>(a) RT-PCR analysis of <i>Slt2</i> and <i>Hog1</i> gene in budding yeast after drug treatment (i:Untreated control, ii: drug treated) (b) The expression of Slt2 and Hog1 mRNA, expressed as the ratio of densitometric measurement of the sample to the corresponding internal control (β-actin) (i: Untreated control, ii: drug treated) (c) Docking studies of Rohitukine with human two different type of member of MAPK pathway. (i) p38 (Hog1 in <i>S</i>. <i>cerevisiae</i>) and (ii) ERK5 (Slt2 in <i>S</i>. <i>cerevisiae</i>).</p

Rohitukine affected the apoptosis-associated protein levels in A549 cells.

<p><b>Cells were treated with Rohitukine at 30</b> μ<b>M for 24hrs, and then the total proteins were prepared and determined as described in methods.</b> (a) The levels of proteins expression of p53 and Bcl-2 (b) proteins expression of caspase9 were estimated by Western blotting. Band intensities were calculated by densitometry and change in protein expression after Rohitukine treatment was calculated with respect to controls and expressed as fold change in graph. (c), (d) & (e) densitometry for p53, Bcl-2, and caspase9 blot respectively. Results were normalized to β-actin. The data are represented as means ±SD of three independent experiments (** <i>P</i> < 0.01 versus control).</p

Rohitukine causes DNA damage and induction of apoptosis.

<p>(a) A.O. staining (b) Graphical representation for fluorescence intensity of apoptotic death of the Yeast cells as quantified using Image J software ***p < 0.001 (c) DNA damage revealed by Nuc Blue Live Cell Stain (d) Graphical representation for fluorescence intensity of nucleic acid of the yeast cells as quantified using Image J software ***p < 0.001.</p