Proteomics of Trypanosoma evansi Infection in Rodents

infection using mass spectrometry (MS). in mice infected with camel isolate. Homology driven searches for protein identification from MS/MS data led to most of the matches arising from related Trypanosoma species. Proteins identified belonged to various functional categories including metabolic enzymes; DNA metabolism; transcription; translation as well as cell-cell communication and signal transduction. TCA cycle enzymes were strikingly missing, possibly suggesting their low abundances. The clinical proteome revealed the presence of known and potential drug targets such as oligopeptidases, kinases, cysteine proteases and more. infections

Rif1 Regulates the Fate of DNA Entanglements during Mitosis

Clearance of entangled DNA from the anaphase mid-region must accurately proceed in order for chromosomes to segregate with high fidelity. Loss of Taz1 (fission yeast ortholog of human TRF1/TRF2) leads to stalled telomeric replication forks that trigger telomeric entanglements; the resolution of these entanglements fails at ≤20°C. Here, we investigate these entanglements and their promotion by the conserved replication/repair protein Rif1. Rif1 plays no role in taz1Δ fork stalling. Rather, Rif1 localizes to the anaphase mid-region and regulates the resolution of persisting DNA structures. This anaphase role for Rif1 is genetically separate from the role of Rif1 in S/G2, though both roles require binding to PP1 phosphatase, implying spatially and temporally distinct Rif1-regulated phosphatase substrates. Rif1 thus acts as a double-edged sword. Although it inhibits the resolution of taz1Δ telomere entanglements, it promotes the resolution of non-telomeric ultrafine anaphase bridges at ≤20°C. We suggest a unifying model for Rif1’s seemingly diverse roles in chromosome segregation in eukaryotes

Heat shock protein 90 from neglected protozoan parasites

Significant advances have been made in our understanding of heat shock protein 90 (Hsp90) in terms of its structure, biochemical characteristics, post-translational modifications, interactomes, regulation and functions. In addition to yeast as a model several new systems have now been examined including flies, worms, plants as well as mammalian cells. This review discusses themes emerging out of studies reported on Hsp90 from infectious disease causing protozoa. A common theme of sensing and responding to host cell microenvironment emerges out of analysis of Hsp90 in Malaria, Trypanosmiasis as well as Leishmaniasis. In addition to their functional roles, the potential of Hsp90 from these infectious disease causing organisms to serve as drug targets and the current status of this drug development endeavor are discussed. Finally, a unique and the only known example of a split Hsp90 gene from another disease causing protozoan Giardia lamblia and its evolutionary significance are discussed. Clearly studies on Hsp90 from protozoan parasites promise to reveal important new paradigms in Hsp90 biology while exploring its potential as an anti-infective drug target. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90). (C) 2011 Elsevier B.V. All rights reserved

<i>Trans</i>-spliced Heat Shock Protein 90 Modulates Encystation in <i>Giardia lamblia</i>

<div><p>Background</p><p>Hsp90 from <i>Giardia lamblia</i> is expressed by splicing of two independently transcribed RNA molecules, coded by genes named HspN and HspC located 777 kb apart. The reasons underlying such unique <i>trans</i>-splicing based generation of GlHsp90 remain unclear.</p><p>Principle Finding</p><p>In this study using mass-spectrometry we identify the sequence of the unique, junctional peptide contributed by the 5′ UTR of HspC ORF. This peptide is critical for the catalytic function of Hsp90 as it harbours an essential “Arg” in its sequence. We also show that full length GlHsp90 possesses all the functional hall marks of a canonical Hsp90 including its ability to bind and hydrolyze ATP. Using qRT-PCR as well as western blotting approach we find the reconstructed Hsp90 to be induced in response to heat shock. On the contrary we find GlHsp90 to be down regulated during transition from proliferative trophozoites to environmentally resistant cysts. This down regulation of GlHsp90 appears to be mechanistically linked to the encystation process as we find pharmacological inhibition of GlHsp90 function to specifically induce encystation.</p><p>Significance</p><p>Our results implicate the <i>trans</i>-spliced GlHsp90 from <i>Giardia lamblia</i> to regulate an essential stage transition in the life cycle of this important human parasite.</p></div

Comparative table, representing kinetic parameters of Hsp90 from diverse biological systems.

<p>* - adopted from Pallavi <i>et al.</i>, 2010 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002829#pntd.0002829-Pallavi1" target="_blank">[12]</a>.</p

GlHsp90 junctional peptide is contributed by the 5′ UTR of HspC gene.

<p><b><i>A</i></b>, Total protein profile of <i>G. lamblia</i> trophozoite. The band corresponding to Hsp90 was analyzed by mass spectrometry. <b><i>B</i></b> and <b><i>D</i></b>, Representation of the <i>trans</i>-spliced full-length mature Hsp90 mRNA and its product respectively. The region highlighted in yellow represents the signature peptide for <i>trans</i>-splicing reaction unique to full length Hsp90 which is contributed from 5′ UTR of HspC ORF and also covers the non-coding strand of the hypothetical ORF (GL50803_31962). <b><i>C</i></b>, MS/MS spectrum of the signature peptide (a part of junctional peptide, GVVDCDDLPLNISRE).</p

Inhibition of GlHsp90 induces encystation.

<p><b><i>A</i></b>, Encystation efficiency of trophozoites as a function of Hsp90 inhibition by 17AAG. Step1 shows an increase in cyst formation upon Hsp90 inhibition at sub-lethal level. Trophozoites treated with varying concentration of 17AAG show transformation to be dose dependent (with 9, 27 and 62 fold cyst formation at 0.1, 0.5 and 1 µM of inhibitor). <b><i>B</i></b>, the cysts formed from the treated cultures showed all the characteristic features of a mature cyst as shown in upper panel with specific staining of CWP1 of cyst wall component and 4 nuclei, representing the features of mature cysts. <b><i>C</i></b>, Determination of encystation efficiency of cells treated with metranidazole before (blue bar) and during (red bar) encystation do not produce any better cysts than the normal trophozoites. <b><i>D</i></b>, Encystation efficiency did not change in response to heat shock. Varying duration of heat shock followed by 90 mins recovered cells encyst similarly as the normal cells maintained at 37°C.</p

<i>Trans</i>-spliced GlHsp90 is an active ATPase.

<p><b><i>A</i></b>, <b><i>B</i></b>, and <b><i>C</i></b>, Binding curve of ATP, GA and 17AAG to full length Hsp90 respectively. The Kd values determined for ATP, GA and 17AAG binding were 629 µM, 1.5 µM and 17 µM respectively. <b><i>D</i></b>, Michaelis-Menten plot showing the rate of ATP hydrolysis plotted against ATP concentration. The determined biochemical parameters are tabulated and presented in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002829#pntd-0002829-t001" target="_blank">Table 1</a>. <b><i>E</i></b>, 17AAG can inhibit the ATPase activity of Hsp90. EC₅₀ value was obtained upon plotting % activity remaining against the Log concentration of 17AAG. EC₅₀ value was found to be 11.96 µM.</p

GlHsp90 is induced upon heat shock.

<p><b><i>A</i></b>, HspN and HspC promoter elements in blue box (Genbank accession number for HspN and HspC ORFs are AB561868.1 and AB561869.1 respectively), marked in yellow represents the 5′ UTR of HspC, the arrow along HspC ORF is the direction of transcription for HspC ORF. The opposite arrow represents the direction of transcription of the hypothetical ORF (GL50803_31692) which spans the 5′ UTR sequence of HspC in the opposite strand. <b><i>B</i></b>, Real-time PCR analysis to study transcriptional expression of full-length Hsp90 and precursor RNAs using specific primers show abundance of full length Hsp90. <b><i>C</i></b>, Graph showing the relative levels of induction of full-length, HspN and HspC transcripts from <i>Giardia</i> trophozoites as response to heat stress. Inset shows the relative increase in the levels of HspN upon heat shock. Both the precursors and the product were observed to be induced about 2 times at the transcript level. <b><i>D</i></b>, Equal proteins from heat shocked and control cells were resolved and probed with anti-Hsp90 antibody. The upper panel represents the signal corresponding to full length Hsp90 whereas lower panel shows the tubulin control blot. The bar graph represents the quantification of the corresponding blot in which GlHsp90 shows 2.3 fold increase (normalized to Tubulin control) as compared to control cells.</p

GlHsp90 is down-regulated during encystation.

<p><b><i>A</i></b>, The mature cysts show characteristic cyst marker, CWP1 in upper panel with four nuclei which are used for qRT-PCR and western blot analysis. <b><i>B</i></b>, Quantitative RT-PCR results show that the level of Hsp90 and its gene components get modulated in cyst stage in comparison with trophozoites. The values are normalized to internal standard, GAPDH, and represented. <b><i>C</i></b>, Western blot analysis of Hsp90 during the course of encystation show steady decrease in Hsp90 levels. Quantification of the western blot show that cysts produce 50% less Hsp90 than the active trophozoites, at the beginning of the encystation.</p