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Calcium-binding proteins that are type B″ regulatory subunits of phosphatase 2A in Giardia intestinalis

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Abstract

Giardia intestinalis is a protozoan parasite that colonizes the upper part of the small intestine of its mammalian hosts. The trophozoite, which is the replicative stage, has a complex cytoskeleton that allows it to move and adhere to intestinal cells. It has been proposed that protein phosphatase 2A (PP2A) participates in the regulation of changes to the parasite cytoskeleton during its life cycle. However, how PP2A is involved in this regulation remains unclear since its substrates and regulators have not been characterized. In this work, we report the bioinformatic and experimental analysis of two potential regulatory B″ subunits of PP2A in Giardia, both of which are calcium-binding proteins. In this work, in silico and experimental evidence of the binding of both proteins to calcium is presented; the proteins are shown to interact with the catalytic PP2A subunit in the trophozoite stage, and they exhibit different subcellular localization patterns. Because PP2A is a heterotrimer, homology analysis of the different subunits of PP2A indicates that fewer holoenzyme combinations can be formed in this parasite than in other organisms. Our results suggest that the localization of PP2A may be associated with calcium-dependent signaling through its B″ type regulatory subunits.

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References

  • Adam RD (2001) Biology of Giardia lamblia. Clin Microbiol Rev. 14:447–475

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Alvarado ME, Rubiano C, Velandia D, Wasserman M (2018) In silico analysis of the EF-hand proteins in the genome of Giardia intestinalis assembly A. Parasitol Res. 117:1035–1041

    Article  PubMed  Google Scholar 

  • Aurrecoechea C, Brestelli J, Brunk BP, Carlton JM, Dommer J, Fischer S et al (2009) GiardiaDB and TrichDB: integrated genomic resources for the eukaryotic protist pathogens Giardia lamblia and Trichomonas vaginalis. Nucleic Acids Res. 37(Database issue):D526–D530

    Article  CAS  PubMed  Google Scholar 

  • Basu S (2011) PP2A in the regulation of cell motility and invasion. Curr Protein Pept Sci. 12:3–11

    Article  CAS  PubMed  Google Scholar 

  • Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T et al (2014) SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 42(W1):W252–W258

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Davis AJ, Yan Z, Martinez B, Mumby MC (2008) Protein phosphatase 2A is targeted to cell division control protein 6 by a calcium-binding regulatory subunit. J Biol Chem. 283:16104–16114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dobson S, Kumar R, Bracchi-Ricard V, Freeman S, Al-Murrani SW, Johnson C et al (2003) Characterization of a unique aspartate-rich protein of the SET/TAF-family in the human malaria parasite, Plasmodium falciparum, which inhibits protein phosphatase 2A. Mol Biochem Parasitol. 126:239–250

    Article  CAS  PubMed  Google Scholar 

  • Dovega R, Tsutakawa S, Quistgaard EM, Anandapadamanaban M, Löw C et al (2014) Structural and biochemical characterization of human PR70 in isolation and in complex with the scaffolding subunit of protein phosphatase 2A. PLoS ONE 9(7):e101846

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Elam CA, Wirschell M, Yamamoto R, Fox LA, York K, Kamiya R et al (2011) An axonemal PP2A B-subunit is required for PP2A localization and flagellar motility. Cytoskeleton (Hoboken) 68:363–372

    Article  CAS  Google Scholar 

  • Elmendorf HG, Dawson SC, McCaffery JM (2003) The cytoskeleton of Giardia lamblia. Int J Parasitol. 33:3–28

    Article  PubMed  Google Scholar 

  • Gillin FD, Diamond LS (1980) Clonal growth of Giardia lamblia trophozoites in a semisolid agarose medium. J Parasitol. 66:350–352

    Article  CAS  PubMed  Google Scholar 

  • Groves MR, Hanlon N, Turowski P, Hemmings BA, Barford D (1999) The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs. Cell. 96:99–110

    Article  CAS  PubMed  Google Scholar 

  • Hardin WR, Li R, Xu J, Shelton AM, Alas GCM, Minin VN et al (2017) Myosin-independent cytokinesis in Giardia utilizes flagella to coordinate force generation and direct membrane trafficking. Proc Natl Acad Sci U S A. 114:E5854–E5863

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hoffman A, Taleski G, Sontag E (2017) The protein serine/threonine phosphatases PP2A, PP1 and calcineurin: a triple threat in the regulation of the neuronal cytoskeleton. Mol Cell Neurosci. 84:119–131

    Article  CAS  PubMed  Google Scholar 

  • Janssens V, Longin S, Goris J (2008) PP2A holoenzyme assembly: in cauda venenum (the sting is in the tail). Trends Biochem Sci. 33:113–121

    Article  CAS  PubMed  Google Scholar 

  • Keister DB (1983) Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans R Soc Trop Med Hyg. 77:487–488

    Article  CAS  PubMed  Google Scholar 

  • Kiely M, Kiely PA (2015) PP2A: the wolf in sheep’s clothing? Cancers (Basel). 7:648–669

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kono Y, Maeda K, Kuwahara K, Yamamoto H, Miyamoto E, Yonezawa K et al (2002) MCM3-binding GANP DNA-primase is associated with a novel phosphatase component G5PR. Genes Cells. 7:821–834

    Article  CAS  PubMed  Google Scholar 

  • Lauwaet T, Davids BJ, Torres-Escobar A, Birkeland SR, Cipriano MJ, Preheim SP et al (2007) Protein phosphatase 2A plays a crucial role in Giardia lamblia differentiation. Mol Biochem Parasitol. 152:80–89

    Article  CAS  PubMed  Google Scholar 

  • Magenta A, Fasanaro P, Romani S, Di Stefano V, Capogrossi MC, Martelli F (2008) Protein phosphatase 2A subunit PR70 interacts with pRb and mediates its dephosphorylation. Mol Cell Biol. 28:873–882

    Article  CAS  PubMed  Google Scholar 

  • Manning G, Reiner DS, Lauwaet T, Dacre M, Smith A, Zhai Y et al (2011) The minimal kinome of Giardia lamblia illuminates early kinase evolution and unique parasite biology. Genome Biol. 12:R66

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Maruyama K, Mikawa T, Ebashi S (1984) Detection of calcium binding proteins by 45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis. J Biochem. 95:511–519

    Article  CAS  PubMed  Google Scholar 

  • McGuffin L, Buenavista MT, Roche DB (2013) The ModFOLD4 server for the quality assessment of 3D protein models. Nucleic Acids Res. 41:W368–W372

    Article  PubMed  PubMed Central  Google Scholar 

  • Mi H, Muruganujan A, Casagrande JT, Thomas PD (2013) Large-scale gene function analysis with the PANTHER classification system. Nat Protoc. 8:1551–1566

    Article  CAS  PubMed  Google Scholar 

  • Mitchell A, Chang H-Y, Daugherty L, Fraser M, Hunter S, Lopez R et al (2015) The InterPro protein families database: the classification resource after 15 years. Nucleic Acid Res 43(Database issue):D213–D221

    Article  PubMed  Google Scholar 

  • Morrison HG, McArthur AG, Gillin FD, Aley SB, Adam RD et al (2007) Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science. 317(5846):1921–1926

    Article  CAS  PubMed  Google Scholar 

  • Nohynková E, Tumová P, Kulda J (2006) Cell division of Giardia intestinalis: flagellar developmental cycle involves transformation and exchange of flagella between mastigonts of a diplomonad cell. Eukaryot Cell. 5:753–761

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nosala C, Dawson SC (2015) The critical role of the cytoskeleton in the pathogenesis of Giardia. Curr Clin Microbiol Rep. 2:155–162

    Article  PubMed  PubMed Central  Google Scholar 

  • Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem. 2004; 25: 1605–1612.

    Article  CAS  PubMed  Google Scholar 

  • Piovesan D, Tabaro F, Paladin L, Necci M, Micetic I, Camilloni C et al (2018 Jan 4) MobiDB 3.0: more annotations for intrinsic disorder, conformational diversity and interactions in proteins. Nucleic Acids Res. 46(D1):D471–D476. https://doi.org/10.1093/nar/gkx1071

    Article  PubMed  Google Scholar 

  • Rothberg KG, Jetton N, Hubbard JG, Powell DA, Pandarinath V, Ruben L (2014) Identification of a protein phosphatase 2A family member that regulates cell cycle progression in Trypanosoma brucei. Mol Biochem Parasitol. 194:48–52

    Article  CAS  PubMed  Google Scholar 

  • Sangodkar J, Farrington CC, McClinch K, Galsky MD, Kastrinsky DB, Narla G (2016) All roads lead to PP2A: exploiting the therapeutic potential of this phosphatase. FEBS J. 283:1004–1024

    Article  CAS  PubMed  Google Scholar 

  • Sents W, Ivanova E, Lambrecht C, Haesen D, Janssens V (2013) The biogenesis of active protein phosphatase 2A holoenzymes: a tightly regulated process creating phosphatase specificity. FEBS J. 280:644–661

    Article  CAS  PubMed  Google Scholar 

  • Shi Y (2009) Serine/threonine phosphatases: mechanism through structure. Cell. 139:468–484

    Article  CAS  PubMed  Google Scholar 

  • Touz MC, Lujan HD, Hayes SF, Nash TE (2003) Sorting of encystation-specific cysteine protease to lysosome-like peripheral vacuoles in Giardia lamblia requires a conserved tyrosine-based motif. J Biol Chem. 278:6420–6426

    Article  CAS  PubMed  Google Scholar 

  • Vandomme A, Fréville A, Cailliau K, Kalamou H, Bodart JF, Khalife J et al (2014) PhosphoTyrosyl phosphatase activator of Plasmodium falciparum: identification of its residues involved in binding to and activation of PP2A. Int J Mol Sci. 15:2431–2453

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wlodarchak N, Guo F, Satyshur KA, Jiang L, Jeffrey PD, Sun T et al (2013) Structure of the Ca2+-dependent PP2A heterotrimer and insights into Cdc6 dephosphorylation. Cell Res. 23:931–946

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wu CG, Zheng A, Jiang L, Rowse M, Stanevich V, Chen H et al (2017) Methylation-regulated decommissioning of multimeric PP2A complexes. Nat Commun. 8:2272

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yee J, Nash TE (1995) Transient transfection and expression of firefly luciferase in Giardia lamblia. Proc Natl Acad Sci U S A. 92:5615–5619

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9:40

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors would like to thank Dra. Myriam Velandia (Universidad El Bosque, Bogotá) for her kind collaboration.

Funding

This work was supported by COLCIENCIAS (Fondo Nacional de Financiamiento, Contrato de Recuperación Contingente-FP44842-334-2014—M.W.).

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Correspondence to Magda E. Alvarado.

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Section Editor: Panagiotis Karanis

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Alvarado, M.E., Rubiano, C., Sánchez, W. et al. Calcium-binding proteins that are type B″ regulatory subunits of phosphatase 2A in Giardia intestinalis. Parasitol Res 117, 3205–3214 (2018). https://doi.org/10.1007/s00436-018-6019-z

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  • DOI: https://doi.org/10.1007/s00436-018-6019-z

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