Oral Presentation 24th Annual Lorne Proteomics Symposium 2019

The reduced protein methylation network in the early-branching protozoan parasite, Giardia duodenalis (#21)

Samantha J Emery-Corbin 1 , Swapnil Tichkule 1 , Balu Balan 1 2 , Brendan RE Ansell 1 , Louise Baker 1 , Daniel Vuong 3 , Ernest Lacey 3 4 , Staffan G Svärd 5 , Aaron R Jex 1 2
  1. Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia
  2. Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Melbourne, Victoria, Australia
  3. Microbial Screening Technologies Pty. Ltd., Smithfield, NSW, Australia
  4. Macquarie University, North Ryde, NSW, Australia
  5. Cell and Molecular Biology, Uppsala University, Uppsala, Sweden

Giardia duodenalis causes ~300 million cases of gastroenteritis (giardiasis) annually, with its unique cell biology shaped by early-branching origins in the eukaryotic phylogeny. In particular, protein methylation enzymes in Giardia are minimised relative to higher eukaryotes, with no annotated demethylase or protein arginine methyltransferase (PRMT) domain-containing proteins, and only six SET-domain-containing proteins in support of reduced lysine methylation (K-Me) machinery. However, despite a lack of methylation data, there is sufficient evidence for K-Me regulation in surface antigen switching, differentiation and drug resistance.

We have comprehensively demonstrated a conserved, functionally essential K-Me network in Giardia. Using in silico structural modelling, domain homology and inhibitor auto-docking, we have consolidated known Class V SET-domain methyltransferases, and annotated new Class I seven-beta-strand lysine methyltransferases.  We have detected no PRMTs as per previous studies, which we further verified by no detectable arginine methylation via immunoblotting, and the lack of PRMT inhibitor activity in chemical screens.  Immunoaffinity enrichment (IAP) of lysine methylation in the infective (trophozoite) and transmission (cyst) life-stages has identified 524 methylation sites on 322 proteins. Cytoskeletal proteins are significantly enriched, and we detected methylated RNA helicases and ribosomal/ribonucleoproteins in support of a role of K-Me in gene regulation, as well as on histone H2 and H3 variants. Additional mass spectrometry of histone-enriched fractions has allowed us to detect over 50 acetylation, methylation and phosphorylation sites on Giardia histone variants for the first time, including conserved, canonical H3 methyl marks. Indeed, only inhibitors of histone lysine methyltransferases (HKMTs) have detectable activity in Giardia, in particular inhibitors for H3 methyl marks. Indeed, inhibitor exposure assays during in vitro log-phase and encysting cultures, complimented by quantitative proteomics of over 2000 proteins, provides first links between gene regulation, phenotype and inhibitor pharmacology, and confirms K-Me plays a dynamic, essential role in Giardia biology.