Eukaryote-Conserved Methylarginine Is Absent in Diplomonads and Functionally Compensated in Giardia Samantha J. Emery-Corbin ,*,1,2 Joshua J. Hamey,3 Brendan R.E. Ansell,1,2 Balu Balan,1,2,4 Swapnil Tichkule,1,2 Andreas J. Stroehlein,4 Crystal Cooper,5 Bernie V. McInerney,6 Soroor Hediyeh-Zadeh,2,7 Daniel Vuong,8 Andrew Crombie,8 Ernest Lacey,8,9 Melissa J. Davis,2,7 Marc R. Wilkins,3 Melanie Bahlo,1,2 Staffan G. Sv€ard,10 Robin B. Gasser,4 and Aaron R. Jex1,2,4 1 Population Health and Immunity Division, The Walter and Eliza Hall Institute of Medical Research,Melbourne,VIC,Australia Downloaded from https://academic.oup.com/mbe/article/37/12/3525/5875618 by Uppsala Universitetsbibliotek user on 12 February 2021 2Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia 3School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia 4Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC, Australia 5Central Analytical Research Facility (CARF), Institute for Future Environments, Queensland University of Technology, Brisbane, QLD, Australia 6Australian Proteome Analysis Facility (APAF), Macquarie University, North Ryde, NSW, Australia 7Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia 8Microbial Screening Technologies, Smithfield, NSW, Australia 9Chemistry and Biomolecular Sciences, Faculty of Science, Macquarie University, North Ryde, NSW, Australia 10Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden *Corresponding author: E-mail: [email protected]. Associate editor: Claudia Russo PRIDE Data Reviewer Login Details Project Name: Quantitative proteomics of Giardia exposures to HKMT inhibitors Project accession: PXD016747 Username: [email protected] Password: ytC0i80t Project Name: Arginine and lysine methylproteomes of Giardia duodenalis Project accession: PXD016813 Username: [email protected] Password: dOkSWMPD Abstract Article 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 parasitic protist. We demonstrate that Giardia and related diplomonads lack arginine-methyltransferases and have remodeled conserved RGG/RG motifs targeted by these enzymes. We also provide experimental evidence for methylarginine absence in proteomes of Giardia but readily detect methyllysine. We bio- informatically infer 11 lysine-methyltransferases in Giardia, including highly diverged Su(var)3-9, Enhancer-of-zeste and Trithorax proteins with reduced domain architectures, and novel annotations demonstrating conserved methyllysine regulation of eukaryotic elongation factor 1 alpha. Using mass spectrometry, we identify more than 200 methyllysine sites in Giardia, including in species-specific gene families involved in cytoskeletal regulation, enriched in coiled-coil features. Finally, we use known methylation inhibitors to show that methylation plays key roles in replication and cyst formation in this parasite. This study highlights reduced methylation enzymes, sites, and functions early in eukaryote evolution, including absent methylarginine networks in the Diplomonadida. These results challenge the view that arginine meth- ylation is eukaryote conserved and demonstrate that functional compensation of methylarginine was possible preceding expansion and diversification of these key networks in higher eukaryotes. Key words: methylproteome, methylarginine, methyllysine, Diplomonadida, Metamonada, Giardia. ß The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Open Access Mol. Biol. Evol. 37(12):3525–3549 doi:10.1093/molbev/msaa186 Advance Access publication July 23, 2020 3525 Emery-Corbin et al. doi:10.1093/molbev/msaa186 MBE Introduction Metamonads have many reduced or eliminated Posttranslational modifications (PTMs) of proteins coordi- eukaryote-conserved gene families (Morrison et al. 2007; nate cell development, signaling, and gene regulation Leger et al. 2017), including protein MTases (Iyer et al. 2008; (Larsen et al. 2016; Blanc and Richard 2017; Murn and Shi Fisk and Read 2011). There are five MTase Classes (I–V), and 2017). Methylations (Me) of lysine (Lys/K/K-Me) and arginine those targeting proteins are usually Class I (seven-stranded b- (Arg/R/R-Me) are common PTMs of eukaryotic proteins, cat- sheet [7bS]) and Class V (“Su(var)3-9, Enhancer-of-zeste and alyzed by methyltransferases (MTases) enzyme classes that Trithorax,” SET-domain) (Schubert et al. 2003). Early investi- arose during the evolution of eukaryotes and are best known gations of Giardia protein MTases suggest an absence of ar- Downloaded from https://academic.oup.com/mbe/article/37/12/3525/5875618 by Uppsala Universitetsbibliotek user on 12 February 2021 for epigenetic regulation (Lanouette et al. 2014; Murn and Shi ginine methyltransferase (PRMT) classes (Iyer et al. 2008; Fisk 2017). Improved technologies have identified abundant andRead2011) and divergent Class V n-lysine methyltrans- methylated substrates in higher eukaryotes (Lanouette et al. ferases (K-MTases) (Sonda et al. 2010). These findings high- 2014; Moore and Gozani 2014) and roles for methylation in light reduced methylproteomes in Giardia accordant with its metabolism, translation, and the modulation of RNA-binding deep-branching origins but which have been implicated in proteins (Erce et al. 2012; Castello, Horos, et al. 2016; Murn regulating parasite development, antimicrobial resistance, and Shi 2017; Hamey and Wilkins 2018). These discoveries and virulence (Sonda et al. 2010; Carranza et al. 2016; have driven identification of new protein MTase classes Salusso et al. 2017; Emery et al. 2018). Giardia MTases are (Cloutieret al. 2013; Hamey and Wilkins 2018)andtheir essentially unexplored but could represent novel targets to extrachromatin specificities (Dillonet al. 2005; Moore and disrupt parasites and their life cycles. Gozani 2014; Hamey et al. 2018), with relevance for cell de- Giardia is a protist whose evolution precedes the ex- velopment in all eukaryotes, and clinical relevance for neuro- panded methylproteome of yeast and vertebrates, with an- logical disorders and cancer in humans (Guccione and ticipated unique adaptions to its host gastrointestinal niches. Richard 2019; Han et al. 2019). It is an ideal lineage to contrast with model organisms, in- Eukaryotic methylproteomes are best characterized in cluding humans, to better understand the evolved complex- mammals and yeast. These methylproteomes exhibit numer- ity of higher eukaryotic methylproteomes. Here, we ous evolutionary expansions of methylated substrates (Larsen characterize the methylproteome of G. duodenalis and ex- et al. 2016)andMTases(Bachand 2007; Lanouette et al. 2014), plore its evolution within the Metamonada. Notably, we lo- with vertebrate methylproteomes further featuring multisite calize a lack of PRMT enzymes and their preferred motifs in and state (mono-, di-, and tri-) methyl-regulation (e.g., histo- Diplomonadida species and do not detect methylarginine in nes [Lanouette et al. 2014]). Saccharomyces cerevisiae (bud- the Giardia proteome by multiple methods, confirming ab- ding yeast) represents the most complete eukaryotic sence of arginine methylation networks—a first among methylation model with simpler, conserved methylpro- eukaryotes. Using mass spectrometry (MS) and novel teomes (Low and Wilkins 2012). However, yeasts have already methyl-site filtering strategies, we identified over 200 high- accumulated paralogous MTases with expanded architec- confidence K-Me sites in Giardia. These provide the evolu- tures and new regulators (Iyer et al. 2008), and diverged tionarily earliest evidence of conserved eukaryotic elongation from the methylation networks which evolved with factor 1 alpha (eEF1a) lysine methylation and abundant eukaryote-specific protein methylation (R-Me, K-Me). This species-specific methyllysine adaptations, enriched in coiled- complexity presents obstacles in identifying regulators of coils within cytoskeletal proteins. Lastly, we disrupt in vitro nonhistone and nonnuclear protein methylation (Murn models of parasite transmission and virulence using methyl- and Shi 2017), deciphering functional significance between ation inhibitors, demonstrating functional roles for K-Me in methylated and nonmethylated protein interactomes Giardia cytoskeletal regulation and encystation. This compre- (Cornettetal.2019), and understanding PTM coregulation hensive functional and evolutionary exploration of the (e.g., phosphorylation [Larsen et al. 2016]). Identifying a sim- Giardia methylproteome