Lack of an efficient endoplasmic reticulum-localized recycling system protects peroxiredoxin IV from hyperoxidation

Typical 2-cys peroxiredoxins are required to remove hydrogen peroxide from several different cellular compartments. Their activity can be regulated by hyperoxidation and consequent inactivation of the active site peroxidatic cysteine. Here we have developed a simple assay to quantify the hyperoxidation of peroxiredoxins. Hyperoxidation of peroxiredoxins can only occur efficiently in the presence of a recycling system usually based on thioredoxin and thioredoxin reductase. We demonstrate that there is a marked difference in the sensitivity of the endoplasmic reticulum-localized peroxiredoxin to hyperoxidation compared to either the cytosolic or mitochondrial enzymes. Each enzyme is equally sensitive to hyperoxidation in the presence of a robust recycling system. Our results demonstrate that the peroxiredoxin IV recycling in the ER is much less efficient than in the cytosol or mitochondria leading to the protection of peroxiredoxin IV from hyperoxidation

A Communication Library Using Active Messages to Improve Performance of PVM

We present a communication library to improve performance of PVM. The new library introduces communication primitives based on Active Messages. We propose a hybrid scheme that includes a signal driven message notification scheme plus controlled polling. The new communication library is tested along with the normal PVM library to assess the improvement in performance

A disrupted transsulphuration pathway results in accumulation of redox metabolites and induction of gametocytogenesis in malaria

Intra-erythrocytic growth of malaria parasite is known to induce redox stress. In addition to haem degradation which generates reactive oxygen species (ROS), the parasite is also thought to efflux redox active homocysteine. To understand the basis underlying accumulation of homocysteine, we have examined the transsulphuration (TS) pathway in the parasite, which is known to convert homocysteine to cysteine in higher eukaryotes. Our bioinformatic analysis revealed absence of key enzymes in the biosynthesis of cysteine namely cystathionine-beta-synthase and cystathionine-gamma-lyase in the parasite. Using mass spectrometry, we confirmed the absence of cystathionine, which is formed by enzymatic conversion of homocysteine thereby confirming truncation of TS pathway. We also quantitated levels of glutathione and homocysteine in infected erythrocytes and its spent medium. Our results showed increase in levels of these metabolites intracellularly and in culture supernatants. Our results provide a mechanistic basis for the long-known occurrence of hyperhomocysteinemia in malaria. Most importantly we find that homocysteine induces the transcription factor implicated in gametocytogenesis namely AP2-G and consequently triggers sexual stage conversion. We confirmed this observation both in vitro using Plasmodium falciparum cultures, and in vivo in the mouse model of malaria. Our study implicates homocysteine as a potential physiological trigger of gametocytogenesis

A secreted Heat shock protein 90 of <i>Trichomonas vaginalis</i>

<div><p><i>Trichomonas vaginalis</i> is a causative agent of Trichomoniasis, a leading non-viral sexually transmitted disease worldwide. In the current study, we show Heat shock protein 90 is essential for its growth. Upon genomic analysis of the parasite, it was found to possess seven ORFs which could potentially encode Hsp90 isoforms. We identified a cytosolic Hsp90 homolog, four homologs which can align to truncated cytosolic Hsp90 gene products along with two Grp94 homologs (ER isoform of Hsp90). However, both Grp94 orthologs lacked an ER retention motif. In cancer cells, it is very well established that Hsp90 is secreted and regulates key clients involved in metastases, migration, and invasion. Since <i>Trichomonas</i> Grp94 lacks ER retention motif, we examined the possibility of its secretion. By using cell biology and biochemical approaches we show that the Grp94 isoform of Hsp90 is secreted by the parasite by the classical ER-Golgi pathway. This is the first report of a genome encoded secreted Hsp90 in a clinically important parasitic protozoan.</p></div

TV910 is secreted via a BFA-sensitive secretory pathway.

<p>(A) Immunoblot shows inhibition of TV910 secretion into medium upon BFA treatment. Ponceau profile is shown as loading control. (B) The graph shows quantitation of signal. (C) Autoradiograph of IP for secreted TV910 at 0 and 3 H of the chase after 2 H pulse of S³⁵ labeling with and without BFA treatment. Upon BFA treatment no signal for secreted TV910 was observed at 3 H. (D) Autoradiograph of total labeled proteins of lysate shows a time-dependent decrease from 0 to 3 H. Upon BFA treatment higher signal was observed in cells due to inhibition of signal. (E) Autoradiograph of total labeled secreted proteins show a time-dependent increase in signal and upon BFA treatment a clear decrease was seen in signal due to inhibition of secretion.</p

TV910 is secreted by <i>Trichomonas</i>.

<p><b>(</b>A) Immunoblot shows TV910 in the cellular lysate and spent medium consistent with the secretion of TV910. No signal for alpha-tubulin was observed in spent media confirming cellular integrity. (B) Immunoblot for lysate and spent medium of <i>Giardia</i> trophozoites shows a prominent signal in the lysate and no signal in spent medium. (C) Coomassie profile of total secretome of <i>Trichomonas vaginalis</i>. (D) MS/MS spectra of the peptide ‘VTEDPRGNTLGR’ of TV910 detected in the tryptic digest corresponding to 75–100 kDa region. (E) TV910 sequence showing the peptides identified by MS/MS analysis. Peptides in green were identified with a confidence of 95% or more. (F) TV910 is secreted in a time-dependent manner. (a) Autoradiograph of IP of TV910 in the spent medium for different time points following the 2 H pulse of labeling. Protein A lane does not show any signal. A time-dependent increase is seen in the TV910 signal. (b) Autoradiograph of total labeled proteins of lysate shows a time-dependent decrease. (c) Autoradiograph of total labeled secreted proteins shows a time-dependent increase in signal. (d) Autoradiograph of IP for GlHsp90 after pulse-chase shows signal only in the lysate and no signal in spent media.</p

<i>Trichomonas</i> expresses all three full-length Hsp90 isoforms and depends on Hsp90 for its growth.

<p>(A) Sequence analysis upstream of the three full-length Hsp90 isoforms in <i>T</i>. <i>vaginalis</i> shows the presence of conserved M5-like and Inr elements. (B) Agarose gel shows RT- PCR amplicon for full-length transcripts of TV560, TV810, and TV910, thus confirming their expression at the transcript level. Lane-1: PCR using respective primers from cDNA, Lane 2: no Reverse transcription control to control for genomic DNA contamination, Lane 3: no template control. (C) GI₅₀ for growth arrest by 17-AAG was determined by counting viable cells after 24 h treatment with the drug. The graph shows growth inhibition of <i>Trichomonas</i> by 17-AAG for with a GI₅₀ of 708 nM.</p

Both cytosolic Hsp90 TV560 and the Grp94 isoform TV910 are functional ATPase <i>in vitro</i>.

<p>(A) The binding affinity of the natural ligand ATP to Hsp90s was determined using tryptophan fluorescence quenching. Changes in intrinsic fluorescence intensity upon ligand binding was plotted against ligand concentration. The dissociation constant, k_d, for ATP binding, was found to be 538.3 μM for TV560 and 722 μM for TV910. (B) & (C) The rate of ATP hydrolysis was measured by the hydrolysis of ATP to ADP where γ³²P- labeled ATP was used as a tracer. A Michaelis–Menten plot shows the rate of ATP hydrolysis plotted against ATP concentration. Inset graph is the representative Lineweaver–Burke plot for each of the Hsp90’s ATPase activities. (B) ATPase activity of TV560 and (C) ATPase activity of TV910. (D) The binding affinities of pharmacological inhibitor 17-AAG to Hsp90s were determined using tryptophan fluorescence quenching. The dissociation constant, k_d, for 17-AAG binding, was found to be 11.45 μM for TV560 and 15.73 μM for TV910. (E) 17-AAG potently inhibits ATPase activity of cytosolic parasitic Hsp90s. ATPase assay was performed in the presence of increasing concentrations of 17-AAG and percent remaining activity was calculated. Graphs show TV560 inhibition by 17-AAG with an IC₅₀ of 22.98 μM, and TV910 inhibition by 17-AAG with an IC₅₀ of 87.05 μM. (F) The table shows a comparison of biochemical parameters of TV910 and TV560 with human Hsp90 ^a[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006493#pntd.0006493.ref027" target="_blank">27</a>], ^b[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006493#pntd.0006493.ref028" target="_blank">28</a>], ^c[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006493#pntd.0006493.ref008" target="_blank">8</a>].</p