Dataset Information

Quantitative proteomics of Giardia exposures to HKMT inhibitors

ABSTRACT: We explored differential protein expression (DEPs) during sublethal exposures of HKMT inhibitors (BIX-01294 = 10uM (B), Chaetocin = 20uM (C)) in log-phase (growth; TY) and encystation (differentiation; EC) at 8 and 24 hours relative to DMSO controls (D). In total, 18,527 peptides from 1,981 proteins in log-phase, and 19,852 peptides from 2,097 proteins were analysed in encysting cultures. In total, 56 proteins were up- and 36 down-regulated in log-phase trophozoites and 32 up- and 12 down during encystation upon BIX-01294-treatment relative to the DMSO controls. Following Chaetocin-treatment, 195 proteins were up- and 121 down-regulated in log-phase trophozoites, and 63 up- and 12 down during encystation. We have presented results by cell-phase given positive correlations were driven by culture condition and not between either Chaetocin or BIX-01294 inhibitor).

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Giardia Lamblia Atcc 50803

TISSUE(S): Trophozoite, Binucleate Cell

DISEASE(S): Giardiasis

SUBMITTER: Samantha Emery

LAB HEAD: Aaron Jex

PROVIDER: PXD016747 | Pride | 2020-08-04

REPOSITORIES: Pride

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Publications

Eukaryote-Conserved Methylarginine Is Absent in Diplomonads and Functionally Compensated in Giardia.

Emery-Corbin Samantha J SJ Hamey Joshua J JJ Ansell Brendan R E BRE Balan Balu B Tichkule Swapnil S Stroehlein Andreas J AJ Cooper Crystal C McInerney Bernie V BV Hediyeh-Zadeh Soroor S Vuong Daniel D Crombie Andrew A Lacey Ernest E Davis Melissa J MJ Wilkins Marc R MR Bahlo Melanie M Svärd Staffan G SG Gasser Robin B RB Jex Aaron R AR

Molecular biology and evolution 20201201 12

Methylation is a common posttranslational modification of arginine and lysine in eukaryotic proteins. Methylproteomes are best characterized for higher eukaryotes, where they are functionally expanded and evolved complex regulation. However, this is not the case for protist species evolved from the earliest eukaryotic lineages. Here, we integrated bioinformatic, proteomic, and drug-screening data sets to comprehensively explore the methylproteome of Giardia duodenalis-a deeply branching parasiti ...[more]

PMID: 32702104

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Project description:Background: Cyst formation in parasitic protist Giardia duodenalis is critical to its transmission. Proteomic data is essential for the system-level understanding of the molecular mechanisms managing stage transition from a flagellated, binucleate trophozoite to a tetra-nucleate cyst. The current encystation proteome quantifies 16% of predicted protein coding genes and lacks complete coverage of the developmental transition. Given recent gains in mass spectrometry instrumentation, we have generated significantly improved proteomes for Giardia across encystation, providing framework for transcriptome-proteome correlation drawing insights into molecular networks modulating encystation in Giardia. Results: We have reproducibly identified 3,796 (64.6% of Giardia proteins) and quantified 3,402 proteins (56.12 % of Giardia proteins), tripling the currently published encystation proteome coverage. This includes timepoints from standard trophozoite growth (TY), during low-bile encystation priming (LB), mid-encystation (EC), and enriched, mature cysts (C), making this the first proteome study across the entire encystation process. This is also the first proteome analysis of trophozoites during low bile priming prior to in vitro induction of encystation, and analysis of mature cyst proteomes. Almost 28% (1,102 proteins) of the proteome is differentially changed during encystation including within proteasomal machinery, metabolic pathways and lipid metabolism, indicating a multimodal regulation in encystation. To further understand novel observations in lipid metabolism, we used hidden markov model (HMM) based domain prediction strategies and i-Tasser based structural modelling to provide evidence for the presence of three families of lipid transporters (LTP’s) StARkin (steroidogenic acute regulatory protein–related lipid transfer), ORP/Osh (Oxysterol Binding protein related protein), GLTP (Glycosphingolipid transfer protein) in Giardia. Finally, we performed functional interaction studies using in silico docking of newly identified LTP’s with corresponding lipid ligands to validate their interaction. This identified a shift in lipid species dependency in Giardia as the parasite progress towards cyst formation.

Project description:We previously showed that epigenetic transcriptional silencing of the amoebapore gene (Ehap-a) in E. histolytica G3 trophozoites was accompanied by the down-regulation of two other members of the saposin-like gene family (Ehap-b, and EhSaplip1). Comparison of the transcriptomes of G3 trophozoites with those of the parent HM-1:IMSS strain revealed 72 additional transcripts that were either down- or up-regulated at least 7 fold. One of the polyA+, up-regulated (>200) transcripts (459.m00030), had the potential to encode a 66 amino acid protein. Antibodies raised against a synthetic N-terminal peptide of the gene did not recognize any such protein in trophozoite lysates. Trophozoite cultures grown with the proteasome inhibitor MG-132 to prevent protein degradation, also did not have any detectable protein. Transfections with a plasmid in which the Ehactin gene promoter elements were used to flank the 459.m gene, significantly increased their level transcript but again, no protein was detected. Transfectants in which the 459.m ORF was placed downstream to the GST gene, expressed a fused protein which reacted with the antibody against the m.459 peptide. Constructs with the CAT reporter gene placed under the 459.m gene promoter element or fused to the C-terminal sequence of the 459.m ORF were prepared to determine if the higher levels of 459.m transcript found in the G3 trophozoites were due to changes in the stability of the transcript or increased transcription. No difference was observed in the very weak expressions seen in both G3 and HM-1:IMSS transfectants. Similarly, transfectants with a plasmid construct in which the 459.m gene was placed under its regulatory elements, did not induce an increase in the transcript levels. Our conclusions are that the transcript of the 459.m gene is an mRNA-like ncRNA and that modulations of transcript levels, especially of hypothetical genes, may not correlate with changes in the proteome. Samples include two replicates of the HM-1:IMSS strain and the amoebapore a silenced G3 strain (G3(A))

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