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Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation

Nature volume 520, pages 378–382 (2015)Cite this article

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Abstract

Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack their genetic and epigenetic machinery to change host cell phenotypic states. Among the Apicomplexa phylum of obligate intracellular parasites, which cause veterinary and human diseases, Theileria is the only genus that transforms its mammalian host cells1. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-1 (ref. 2). The transformed phenotypes are reversed by treatment with the theilericidal drug buparvaquone3. We used comparative genomics to identify a homologue of the peptidyl-prolyl isomerase PIN1 in T. annulata (TaPIN1) that is secreted into the host cell and modulates oncogenic signalling pathways. Here we show that TaPIN1 is a bona fide prolyl isomerase and that it interacts with the host ubiquitin ligase FBW7, leading to its degradation and subsequent stabilization of c-JUN, which promotes transformation. We performed in vitro and in silico analysis and in vivo zebrafish xenograft experiments to demonstrate that TaPIN1 is directly inhibited by the anti-parasite drug buparvaquone (and other known PIN1 inhibitors) and is mutated in a drug-resistant strain. Prolyl isomerization is thus a conserved mechanism that is important in cancer and is used by Theileria parasites to manipulate host oncogenic signalling.

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Figure 1: Theileria parasites secrete a conserved PIN1 PPIase protein.
Figure 2: TaPIN1 is a functional homologue of hPIN1 involved in transformation.
Figure 3: Inhibition of TaPIN1 activity blocks transformation in vitro and in vivo.
Figure 4: TaPIN1 activates the oncogenic c-JUN pathway via FBW7 ubiquitination.

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Acknowledgements

We thank G. Langsley, M. A. Darghouth and M. Weitzman for critical reading of the manuscript and advice on this study. We thank members of the UMR7216 for discussions. J.B.W. thanks C. Gawer for advice and support. We thank the following for providing reagents: G. Langsley for TBL3-, BL3- and TpMD409-infected cells and for a Theileria complementary DNA library; C. Francastel for NIH/3T3 cells; G. Del Sal for Pin1−/− murine immortalized fibroblasts; B. E. Clurman for FBW7 plasmids; J. Baum for the rabbit anti-Apicomplexa actin antibody; and T. Uchida for DTM. Measurements of the PPIase activities were performed at the Flexstation III facility of the Biologie Fonctionnelle et Adaptative laboratory. Confocal analysis was performed at the microscopy facility of the ImagoSeine platform (Jacques Monod Institute). This work was supported by National Institutes of Health grant R01CA167677 to K.P.L., the Association for International Cancer Research (#08-0111), the French National Research Agency (ANR) (Blanc 11-BSV3-016-01), and the “Who Am I?” Laboratory of Excellence #ANR-11-LABX-0071 funded by the French Government through its “Investments for the Future” program operated by the ANR under grant #ANR-11-IDEX-0005-01.

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Author notes

  1. S. Medjkane and J. B. Weitzman: These authors contributed equally to this work.

Authors and Affiliations

  1. Université Paris Diderot, Sorbonne Paris Cité, Epigenetics and Cell Fate, UMR 7216 CNRS, 75013 Paris, France ,

    J. Marsolier, M. Perichon, L. Fritsch, S. Ait-Si-Ali, S. Medjkane & J. B. Weitzman

  2. Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, 30602, Georgia, USA

    J. D. DeBarry

  3. Université Paris Diderot, Sorbonne Paris Cité, Molécules Thérapeutiques in silico, INSERM UMR-S 973, 75013 Paris, France ,

    B. O. Villoutreix

  4. INSERM, UMR 866, Equipe labellisée Ligue contre le Cancer and Laboratoire d’Excellence LipSTIC, 21000 Dijon, France ,

    J. Chluba, T. Lopez & C. Garrido

  5. University of Burgundy, Faculty of Medicine and Pharmacy, 21000 Dijon, France ,

    J. Chluba, T. Lopez & C. Garrido

  6. Centre anticancéreux George François Leclerc, CGFL, 21000 Dijon, France ,

    C. Garrido

  7. Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, 02215, Massachusetts, USA

    X. Z. Zhou & K. P. Lu

  8. Laboratoire de Parasitologie, Ecole Nationale de Médecine Vétérinaire, Université de la Manouba, 2020 Sidi Thabet, Tunisia ,

    M. Mhadhbi

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Contributions

J.M. and M.P. performed the experiments; J.D.D. performed the comparative genomics bioinformatics screen; B.O.V. performed the structural three-dimensional modelling; J.C., T.L. and C.G. designed and executed the zebrafish experiments; L.F., S.A.-S.-A., X.Z.Z. and K.P.L. provided critical reagents and advice; M.M. provided genomic DNA from buparvaquone-resistant parasites; J.M., S.M. and J.B.W. conceived the study and designed experiments, analysed the data and wrote the paper. All authors read the final version of the manuscript.

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Correspondence to J. B. Weitzman.

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Extended data figures and tables

Extended Data Figure 1 Bioinformatic screen and in silico predicted signal peptide for TaPIN1.

a, On 10 September 2011, search strategies at EuPathDB were used to search for all T. annulata protein-encoding genes with predicted signal peptides (SignalP 2.0 HMM). Six-hundred and eighty-nine genes were returned. One-hundred and thirty-eight of these were found to have a predicted signal peptide only in T. annulata and not in their T. gondii orthologues. Among these proteins, we excluded (1) hypothetical proteins, (2) proteins that are not expressed at the macroschizont stage, as the macroschizont is the stage of the parasite associated with host cell transformation, and (3) proteins that are predicted to be targeted to the apicoplast of the parasite. We obtained 33 proteins (right panel). b, Sequence alignment of PIN1 proteins in H. sapiens (human), B. taurus (cow), M. musculus (mouse) and T. annulata revealed a predicted signal peptide (underlined) and conserved PPIase domain in TaPIN1. Stars indicate TaPIN1 residues mutated in the study. c, Example of signal peptide predicted with SignaIP 3.0 Server.

Extended Data Figure 2 TaPIN1 is conserved between T. annulata and T. parva.

a, As the T. parva TpPIN1 sequence did not have an annotated signal peptide, we reanalysed sequence alignment of pin1 genes in T. annulata (Tapin1) and T. parva (Tppin1) (National Center for Biotechnology Information (NCBI) BLAST). The annotated T. annulata intron is indicated in grey and the annotated methionine codon (ATG) in T. parva is in black (underlined). Tapin1 and Tppin1 are well conserved, including in the intronic sequence. We conclude that the T. parva Tppin1 annotation should be modified in light of our polymerase chain reaction (PCR) and western blot analysis (see Fig. 1b and Extended Data Fig. 5c). b, Sequence alignment of pin1 genes in T. annulata (Tapin1) and T. orientalis (Topin1) (NCBI BLAST). The annotated T. annulata intron is indicated in grey and the annotated methionine codon in T. orientalis is in black (underlined). Tapin1 and Topin1 are less conserved than Tapin1 and Tppin1.

Extended Data Figure 3 BtPin1 is not regulated by the parasite.

a, b, Buparvaquone (Bup) treatment had no effect on the mRNA levels (a) of bovine Pin1 in infected (TBL3) or non-infected (BL3) cells as assessed by qPCR analysis, or on the protein level (b) as assessed by immunoblot analysis. Con, control. Average ± s.d., n = 3. b, The result is representative of three independent experiments.

Extended Data Figure 4 Characterization of the TaPIN1 antibody and secretion of TpPIN1 in T. parva.

a, The antibody raised against TaPIN1 specifically recognizes the Theileria protein in NIH/3T3 cells stably transfected with TaPIN1 but not the empty vector in NIH/3T3 cells stably transfected with control or the human protein in NIH/3T3 cells stably transfected with hPIN1. Both Theileria and human versions of PIN1 are present in NIH/3T3 cells stably transfected with TaPIN1 and NIH/3T3 cells stably transfected with hPIN1, respectively. b, The antibody raised against TaPIN1 specifically recognizes GST–TaPIN1 but not GST and GST–hPIN1 beads. c, TpPIN1 protein was detected in the nuclear and cytoplasmic fractions of T. parva-infected TpMD409 cells and decreased upon buparvaquone (Bup) treatment. Con, control. Antibodies recognizing apicomplexan actin, bovine tubulin or bovine histone H3 were used as controls. d, TaPIN1 expression in BL3 or parasite-infected TBL3 cells was examined by confocal microscopy analysis using an antibody raised specifically against Theileria TaPIN1, counterstaining with DAPI. Three-dimensional reconstructions in BL3 and TBL3 cells are shown. e, Immunostaining of TaPIN1 and haemagglutin tag (HA) in NIH/3T3 cells stably expressing TaPIN1 and NIH/3T3 cells stably expressing hPIN1; counterstaining with DAPI. All the results are representatives of three independent experiments. Objective used, 60×.

Extended Data Figure 5 TaPIN1 functionally replaces hPIN1.

a, The HA-tagged version of TaPIN1 and hPIN1 can rescue cell spreading defects in Pin1−/− knockout murine immortalized fibroblasts. Cell spreading was assessed by Phalloidin-TRITC staining. Con, control (transfection with the appropriate empty vector). b, TaPIN1 causes centrosome amplification. NIH/3T3 fibroblasts stably expressing TaPIN1 or hPIN1 were arrested at the G1/S transition by aphidicolin, stained with anti-γ-tubulin antibody (arrow) and counterstained with DAPI. Photographs are representative of one cell in three independent experiments. Objective used, 60×.

Extended Data Figure 6 The PPIase domain of PIN1 is well conserved.

a, Sequence alignment of PIN1 genes in H. sapiens, A. thaliana, T. brucei and T. annulata revealed the presence or the absence of a conserved WW domain. Percentages of identity are indicated. The magenta box indicates the predicted signal peptide of TaPIN1. b, Homology models for TaPIN1 wild type (WT) and the A53P mutant based on sequence identity/similarity with hPIN1. The Ala to Pro mutation in TaPIN1 appears to induce a conformational change within and nearby the catalytic loop. c, The buparvaquone molecule can be docked in the active site of hPIN1 or in the active site of the wild-type TaPIN1 predicted structure. Here, the second lowest docked energy pose is shown (the best predicted energy pose is shown Fig. 3a). These two predicted binding poses are fully consistent with the different binding modes of some inhibitors co-crystallized with hPIN1. Yet, considering the computations shown in e, we suggest that the most likely pose for buparvaquone corresponds to pose 1, as reported in Fig. 3a. However, independently of the selected poses, we expect that buparvaquone would not fit well in the catalytic pocket due to structural changes induced by the A53P mutation. d, Three-dimensional structure of the experimental hPIN1 structure and the predicted TaPIN1 wild-type and A53P mutant structures, alongside Trypanosome TbPIN and Arabidopsis PIN1At models (ribbon diagram). The three-dimensional structures are well conserved among these proteins with some differences, for instance, in the catalytic loops. The TaPIN1, TbPIN1 and PIN1At enzymes lack the WW domain present in hPIN1 yet the overall fold in the catalytic area is well conserved, suggesting that accurate homology models for TaPIN1 can be built using the approach used here. e, The experimental structure of hPIN1 is represented as a solid surface with a view down the active site, showing a small co-crystallized ligand next to the catalytic site residue Cys 113. In the same orientation, small chemical fragments are predicted to bind in the catalytic site region with FTmap. Using this information, we propose that the most likely binding pose for buparvaquone is the one shown in Fig. 3.

Extended Data Figure 7 Buparvaquone-resistant parasites are mutated in the TaPIN1 PPIase domain.

a, Sequence alignment of the Tapin1 gene in T. annulata wild-type (WT) or buparvaquone-resistant genomes (NCBI BLAST). The annotated T. annulata intron is indicated in grey. Buparvaquone-resistant parasites have three mutations indicated in black. b, Sequence alignment of the TaPIN1 protein in T. annulata wild-type or buparvaquone-resistant strains. Buparvaquone-resistant parasites have a mutation in the PPIase domain (residue 53): Ala to Pro mutation. c, Signal peptide predicted of TaPIN1(A53P) with SignaIP 3.0 Server. d, The measure of TaPIN1–GST and TaPIN1(A53P)–GST catalytic activity were determined using PPIase assay (chymotrypsin-coupled assay) (average ± s.d., n = 4).

Extended Data Figure 8 Inhibition of BtPin1 does not affect Theileria-associated cell transformation.

a, Buparvaquone (Bup), juglone (Jug) and DTM decreased the viability of cells infected with T. parva (TpMD409). Host cell viability was assessed using the XTT assay after 72 h. b, Colony formation of parasite-infected TpMD409 cells in soft agar was lost after 72 h treatment with buparvaquone, juglone or DTM. Numbers of macroscopic colonies per plate were counted after 10 days. c, siBtPin1 has no effect on TBL3 transformed phenotypes. TBL3 cells were transiently transfected with siControl or siBtPin1. The average number of colonies per plate is shown (average ± s.d., n = 3). Indicated protein levels were detected by western blot analysis using specific antibodies. Actin was used as loading control (results are representative of three independent experiments). d–f, Overexpression of the TaPIN1 A53P mutant rescued the buparvaquone but not juglone effects on Theileria-infected cells. WT, wild type. All data represent three independent experiments (average ± s.d., n = 3). The SPSS 19.0 program was used for statistics. P < 0.05, P < 0.01. NS, not significant.

Extended Data Figure 9 TaPIN1 changes c-JUN stability through the regulation of FBW7.

a, TaPIN1 interacts with endogenous mouse FBW7. Protein extracts from NIH/3T3 cells stably expressing HA–Flag–TaPIN1 or HA–Flag–Control (Con) were used for HA immunoprecipitation, followed by immunoblot analysis using FBW7 and HA antibodies. b, TaPIN1 interacts with endogenous bovine FBW7 protein. Parasite-infected TBL3 and TpMD409 cell lysates were incubated with the indicated GST-coupled beads, followed by immunoblot analysis using a FBW7 antibody. c, d, Analysis of indicated bovine gene expression by qPCR in TBL3 cells following buparvaquone (Bup) or juglone (Jug) treatment with or without TaPIN1 wild type (WT) or A53P mutant. β-Actin and H2A mRNAs were used for normalization (data represent three independent experiments; average ± s.d., n = 3). e, siBtPin1 does not affect FBW7α or c-JUN protein levels. TBL3 were transiently transfected by siControl (siCon) or siBtPin1. Indicated protein levels were detected by western blot analysis using specific antibodies. Actin was used as loading control. f, Inhibition of TaPIN1 by buparvaquone/juglone increased c-JUN ubiquitination and decreased FBW7 ubiquitination. Parasite-infected TBL3 cells (± buparvaquone or juglone) were incubated with MG132, followed by immunoprecipitation of endogenous c-JUN or FBW7 and immunoblot analysis with the indicated antibodies. Con, control. g, The effect of buparvaquone on c-JUN ubiquitination was rescued by overexpression of TaPIN1(A53P). Parasite-infected TBL3 cells (± buparvaquone or juglone) were transfected with TaPIN1 wild type, TaPIN1(A53P) or empty vector (Con) and then treated with MG132, followed by immunoprecipitation of endogenous c-JUN and immunoblot analysis with the indicated antibodies. h, Measure of c-MYC, KLF5 and activated NOTCH1 protein levels in TBL3 cells upon buparvaquone treatment. Tubulin was used as loading control. i, Measure of c-MYC, KLF5 and activated NOTCH1 protein levels in TBL3 cells upon FBW7α ectopic expression. Con, control (transfection with the appropriate empty vector). Tubulin was used as loading control. a, b, e–h, All the results are representative of three independent experiments. NS, not significant.

Extended Data Figure 10 Original blot.

a, From Fig. 1b: TaPIN1 protein was detected in the host cytoplasm and nucleus, in contrast with apicomplexan actin (TaActin). Bovine histone H3 (nuclear) and tubulin (cytoplasmic) proteins were controls. b, From Fig. 4a: endogenous TaPIN1 interacts with FBW7α isoform. Protein extracts from TBL3 expressing Flag–hFBW7 isoforms or Flag–control (Con) were immunoprecipitated and immunoblotted with TaPIN1 or Flag antibodies. c, From Fig. 4b: inhibition of TaPIN1 by buparvaquone (Bup), juglone (Jug) or DTM increased FBW7α protein levels and decreased c-JUN expression in TBL3 cells. Actin/tubulin were loading controls. d, From Fig. 4c: Inhibition of Fbw7 increases c-JUN protein levels in TBL3 cells, whereas ectopic FBW7α expression reduced c-JUN protein levels. Bovine actin/tubulin were loading controls. Con, empty vector. e, From Fig. 4h: efficiency of two independent c-Jun-targeting siRNAs. Bovine tubulin loading control.

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Marsolier, J., Perichon, M., DeBarry, J. et al. Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation. Nature 520, 378–382 (2015). https://doi.org/10.1038/nature14044

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