Mem Inst Oswaldo Cruz, Rio de Janeiro, VOLUME 115 | JANUARY 2020
Original Article

A new gene inventory of the ubiquitin and ubiquitin-like conjugation pathways in Giardia intestinalis

Isabel Cristina Castellanos1, Eliana Patricia Calvo2,+, Moisés Wasserman3

1Universidad Escuela de Administración de Negocios, Departamento de Ciencias Básicas, Bogotá, Colombia
2Universidad El Bosque, Laboratorio de Virología, Bogotá, Colombia
3Universidad Nacional de Colombia, Laboratorio de Investigaciones Básicas en Bioquímica, Bogotá, Colombia

DOI: 10.1590/0074-02760190242
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BACKGROUND Ubiquitin (Ub) and Ub-like proteins (Ub-L) are critical regulators of complex cellular processes such as the cell cycle, DNA repair, transcription, chromatin remodeling, signal translation, and protein degradation. Giardia intestinalis possesses an experimentally proven Ub-conjugation system; however, a limited number of enzymes involved in this process were identified using basic local alignment search tool (BLAST). This is due to the limitations of BLAST’s ability to identify homologous functional regions when similarity between the sequences dips to < 30%. In addition Ub-Ls and their conjugating enzymes have not been fully elucidated in Giardia.

OBJETIVE To identify the enzymes involved in the Ub and Ub-Ls conjugation processes using intelligent systems based on the hidden Markov models (HMMs).

METHODS We performed an HMM search of functional Pfam domains found in the key enzymes of these pathways in Giardia’s proteome. Each open reading frame identified was analysed by sequence homology, domain architecture, and transcription levels.

FINDINGS We identified 118 genes, 106 of which corresponded to the ubiquitination process (Ub, E1, E2, E3, and DUB enzymes). The E3 ligase group was the largest group with 82 members; 71 of which harbored a characteristic RING domain. Four Ub-Ls were identified and the conjugation enzymes for NEDD8 and URM1 were described for first time. The 3D model for Ub-Ls displayed the β-grasp fold typical. Furthermore, our sequence analysis for the corresponding activating enzymes detected the essential motifs required for conjugation.

MAIN CONCLUSIONS Our findings highlight the complexity of Giardia’s Ub-conjugation system, which is drastically different from that previously reported, and provides evidence for the presence of NEDDylation and URMylation enzymes in the genome and transcriptome of G. intestinalis.

01. Ciechanover A. The unravelling of the ubiquitin system. Nat Rev Mol Cell Biol. 2015; 16(5): 322-24.
02. Cappadocia L, Lima CD. Ubiquitin-like protein conjugation: structures, chemistry, and mechanism. Chem Rev. 2018; 118(3): 889-918.
03. Metzger MB, Hristova VA, Weissman AM. HECT and RING finger families of E3 ubiquitin ligases at a glance. J Cell Sci. 2012; 125(3): 531-7.
04. Clague MJ, Urbé S, Komander D. Breaking the chains: deubiquitylating enzyme specificity begets function. Nat Rev Mol Cell Biol. 2019; 20: 338-52.
05. Carranza PG, Lujan HD. New insights regarding the biology of Giardia lamblia. Microbes Infect. 2010; 12(1): 71-80.
06. Niño CA, Chaparro J, Soffientini P, Polo S, Wasserman M. Ubiquitination dynamics in the early-branching eukaryote Giardia intestinalis. Microbiol Open. 2013; 2(3): 525-39.
07. Ray A, Sarkar S. The proteasome of the differently-diverged eukaryote Giardia lamblia and its role in remodeling of the microtubule-based cytoskeleton. Crit Rev Microbiol. 2017; 43(4): 481-92.
08. Krebber H, Wöstmann C, Bakker-Grunwald T. Evidence for the existence of a single ubiquitin gene in Giardia lamblia. FEBS Lett. 1994; 343(3): 234-6.
09. Catic A, Sun Z, Ratner D, Misaghi S, Spooner E, Samuelson J, et al. Sequence and structure evolved separately in a ribosomal ubiquitin variant. EMBO J. 2007; 26(14): 3474-83.
10. Gallego E, Alvarado M, Wasserman M. Identification and expression of the protein ubiquitination system in Giardia intestinalis. Parasitol Res. 2007; 101(1): 1-7.
11. Ponder E, Bogyo M. Ubiquitin-like modifiers and their deconjugating enzymes in medically important parasitic protozoa. Eukaryot Cell. 2007; 6(11): 1943-52.
12. Iyer LM, Burroughs AM, Aravind L. The prokaryotic antecedents of the ubiquitin-signaling system and the early evolution of ubiquitin-like β-grasp domains. Genome Biol. 2006; 7(7): R60.
13. Vranych CV, Merino MC, Zamponi N, Touz MC, Rópolo AS. SUMOylation in Giardia lamblia: a conserved post-translational modification in one of the earliest divergent eukaryotes. Biomolecules. 2012; 2(3): 312-30.
14. Jüdes A, Bruch A, Klassen R, Helm M, Schaffrath R. Sulfur transfer and activation by ubiquitin-like modifier system Uba4•URM1 link protein urmylation and tRNA thiolation in yeast. Microb Cell. 2016; 3(11): 554-64.
15. Uchida C, Kitagawa M. RING-, HECT-, and RBR-type E3 ubiquitin ligases: involvement in human cancer. Current Cancer Drug Targets. 2016; 16(2): 157-74.
16. Yuan X, Zhang S, Liu S, Yu M, Su H, Shu H, et al. Global analysis of ankyrin repeat domain C3HC4-type RING finger gene family in plants. PLoS One. 2013; 8(3): e58003.
17. Guo B, McMillan BJ, Blacklow SC. Structure and function of the Mind bomb E3 ligase in the context of Notch signal transduction. Curr Opin Struct Biol. 2016; 41: 38-45.
18. Ponts N, Yang J, Chung DW, Prudhomme J, Girke T, Horrocks P, et al. Deciphering the ubiquitin-mediated pathway in apicomplexan parasites: a potential strategy to interfere with parasite virulence. PLoS One. 2008; 3(6): e2386.
19. Wang P, Gong P, Wang W, Li J, Ai Y, Zhang X. An Eimeria acervulina OTU protease exhibits linkage-specific deubiquitinase activity. Parasitol Res. 2019; 118(1): 47-55.
20. Franzen O, Jerlström-Hultqvist J, Einarsson E, Ankarklev J, Ferella M, Andersson B, et al. Transcriptome profiling of Giardia intestinalis using strand-specific RNASeq. PLoS Comput Biol. 2013; 93: e1003000.
21. Morf L, Spycher C, Rehrauer H, Fournier CA, Morrison HG, Hehl AB. The transcriptional response to encystation stimuli in Giardia lamblia is restricted to a small set of genes. Eukaryot Cell. 2010; 9(10): 1566-76.
22. Arya S, Sharma G, Gupta P, Tiwari S. In silico analysis of ubiquitin / ubiquitin-like modifiers and their conjugating enzymes in Entamoeba species. Parasitol Res. 2012; 111(1): 37-51.
23. Mishra SK, Ammon T, Popowicz GM, Krajewski M, Nagel RJ, Ares M, et al. Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing. Nature. 2011; 474(7350): 173-8.
24. Gómez V, Wasserman M. Interactions between Giardia duodenalis Sm proteins and their association with spliceosomal snRNAs. Parasitol Res. 2017; 116(2): 617-26.
25. Bagchi S, Oniku AE, Topping K, Mamhoud ZN, Paget TA. Programmed cell death in Giardia. Parasitology. 2012; 139(7): 894-903.
26. Gannavaram S, Connelly PS, Daniels MP, Duncan R, Salotra P, Nakhasi HL. Deletion of mitochondrial associated ubiquitin fold modifier protein UFM1 in Leishmania donovani results in loss of β-oxidation of fatty acids and blocks cell division in the amastigote stage. Mol Microbiol. 2012; 86(1): 187-98.
27. Liao S, Hu H, Wang T, Tu X, Li Z. The protein neddylation pathway in Trypanosoma brucei functional characterization and substrate identification. J Biol Chem. 2017; 292(3): 1081-91.
28. Di Genova BM, da Silva RC, da Cunha JPC, Gargantini PR, Mortara RA, Tonelli RR. Protein SUMOylation is involved in cell-cycle progression and cell morphology in Giardia lamblia. J Eukaryot Microbiol. 2017; 64(4): 491-503.
29. Kumari R, Gupta P, Tiwari S. Ubc7/Ube2g2 ortholog in Entamoeba histolytica: connection with the plasma membrane and phagocytosis. Parasitol Res. 2018; 117(5): 1599-611.
30. Nakayama KI, Nakayama K. Ubiquitin ligases: cell-cycle control and cancer. Nat Rev Cancer. 2006; 6(5): 369-81.
31. Eme L, Trilles A, Moreira D, Brochier-Armanet C. The phylogenomic analysis of the anaphase promoting complex and its targets points to complex and modern-like control of the cell cycle in the last common ancestor of eukaryotes. BMC Evol Biol. 2011; 11(1): 265.
32. Gourguechon S, Holt LJ, Cande WZ. The Giardia cell cycle progresses independently of the anaphase-promoting complex. J Cell Sci. 2013; 126(10): 2246-55.

+ Corresponding author:
Received 10 July 2019
Accepted 02 January 2020

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