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Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation.

Marsolier J, Perichon M, DeBarry JD, Villoutreix BO, Chluba J, Lopez T, Garrido C, Zhou XZ, Lu KP, Fritsch L, Ait-Si-Ali S, Mhadhbi M, Medjkane S, Weitzman JB - Nature (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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

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 cells. 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 buparvaquone. 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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TaPin1 is a functional homologue of hPin1 involved in transformationa. hPin1 and TaPin1 catalytic PPIase activities measured by in vitro chymotrypsin-coupled using a Pin1 substrate peptide (Suc-Ala-Glu-Pro-Phe-pNA). No activity was detected for GST alone or control substrate peptide (Suc-Ala-Ala-Pro-Phe-pNA).b. TaPin1 and hPin1 increased CyclinD1-Luciferase promoter activity when transfected in TBL3 cells.c. C92A and K38A TaPin1 mutants showed reduced activation of cyclinD1 promoter when transfected in TBL3 cells.d. TaPin1 or hPin1 induced CyclinD1-Luciferase promoter activity in pin1−/− immortalized fibroblasts.e. TaPin1 causes centrosome amplification. NIH3T3 fibroblasts stably expressing TaPin1 or hPin1 were arrested at the G1/S transition by aphidicolin, stained with anti-γ-tubulin antibody. 300 cells were scored.f. TaPin1 or hPin1 transfection increased colony foci formation in Pin1 knockdown MCF10A-Ras/Neu cells. Expression of transfected TaPin1 and hPin1 in MCF10A-Ras/Neu-shPin1 detected with an anti-FLAG antibody. Actin was a loading control.Note: “Con” indicates Control empty vector transfection.Data represent 3 independent experiments (average ± sd). The p-values were calculated using the Dunnett method for multiple comparisons with the TaPin1WT for Figure 2c. For Figures 2b / 2d / 2e / 2f: the p-values were corrected using Dunnett multiple comparisons with the control. *p<0.05, **p<0.01, ***p<0.001.
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Figure 2: TaPin1 is a functional homologue of hPin1 involved in transformationa. hPin1 and TaPin1 catalytic PPIase activities measured by in vitro chymotrypsin-coupled using a Pin1 substrate peptide (Suc-Ala-Glu-Pro-Phe-pNA). No activity was detected for GST alone or control substrate peptide (Suc-Ala-Ala-Pro-Phe-pNA).b. TaPin1 and hPin1 increased CyclinD1-Luciferase promoter activity when transfected in TBL3 cells.c. C92A and K38A TaPin1 mutants showed reduced activation of cyclinD1 promoter when transfected in TBL3 cells.d. TaPin1 or hPin1 induced CyclinD1-Luciferase promoter activity in pin1−/− immortalized fibroblasts.e. TaPin1 causes centrosome amplification. NIH3T3 fibroblasts stably expressing TaPin1 or hPin1 were arrested at the G1/S transition by aphidicolin, stained with anti-γ-tubulin antibody. 300 cells were scored.f. TaPin1 or hPin1 transfection increased colony foci formation in Pin1 knockdown MCF10A-Ras/Neu cells. Expression of transfected TaPin1 and hPin1 in MCF10A-Ras/Neu-shPin1 detected with an anti-FLAG antibody. Actin was a loading control.Note: “Con” indicates Control empty vector transfection.Data represent 3 independent experiments (average ± sd). The p-values were calculated using the Dunnett method for multiple comparisons with the TaPin1WT for Figure 2c. For Figures 2b / 2d / 2e / 2f: the p-values were corrected using Dunnett multiple comparisons with the control. *p<0.05, **p<0.01, ***p<0.001.

Mentions: To explore the functional PPIase activity of the secreted TaPin1 protein, we developed a chymotrypsin-coupled in vitro assay and found that TaPin1 and hPin1 catalytic activities were comparable (Fig. 2a). TaPin1 and hPin1 were also equivalent in activation of the cyclinD1-Luciferase reporter in bovine B cells (Fig. 2b), an established readout for Pin1 activity9. We mutated key C92 and K38 residues in TaPin1 and showed loss of the PPIase activity (Fig. 2c). Furthermore, TaPin1 rescued cyclinD1 promoter activity and cell spreading defects in pin1−/− mouse fibroblasts (Fig. 2d, Extended Data Fig. 5a). Mammalian Pin1 overexpression disrupts cell cycle regulation causing centrosome amplification and cell transformation17. TaPin1 also induced centrosome duplication when overexpressed in mouse fibroblasts (Fig. 2e, Extended Data Fig. 5b). Furthermore, TaPin1 functionally replaced mammalian Pin1 and rescued colony formation as effectively as hPin1 in human breast cancer cells with knocked-down Pin1 (Fig. 2f). These combined results show that Theileria secretes a bona fide phosphorylation-dependent PPIase which could contribute to host cell transformation.


Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation.

Marsolier J, Perichon M, DeBarry JD, Villoutreix BO, Chluba J, Lopez T, Garrido C, Zhou XZ, Lu KP, Fritsch L, Ait-Si-Ali S, Mhadhbi M, Medjkane S, Weitzman JB - Nature (2015)

TaPin1 is a functional homologue of hPin1 involved in transformationa. hPin1 and TaPin1 catalytic PPIase activities measured by in vitro chymotrypsin-coupled using a Pin1 substrate peptide (Suc-Ala-Glu-Pro-Phe-pNA). No activity was detected for GST alone or control substrate peptide (Suc-Ala-Ala-Pro-Phe-pNA).b. TaPin1 and hPin1 increased CyclinD1-Luciferase promoter activity when transfected in TBL3 cells.c. C92A and K38A TaPin1 mutants showed reduced activation of cyclinD1 promoter when transfected in TBL3 cells.d. TaPin1 or hPin1 induced CyclinD1-Luciferase promoter activity in pin1−/− immortalized fibroblasts.e. TaPin1 causes centrosome amplification. NIH3T3 fibroblasts stably expressing TaPin1 or hPin1 were arrested at the G1/S transition by aphidicolin, stained with anti-γ-tubulin antibody. 300 cells were scored.f. TaPin1 or hPin1 transfection increased colony foci formation in Pin1 knockdown MCF10A-Ras/Neu cells. Expression of transfected TaPin1 and hPin1 in MCF10A-Ras/Neu-shPin1 detected with an anti-FLAG antibody. Actin was a loading control.Note: “Con” indicates Control empty vector transfection.Data represent 3 independent experiments (average ± sd). The p-values were calculated using the Dunnett method for multiple comparisons with the TaPin1WT for Figure 2c. For Figures 2b / 2d / 2e / 2f: the p-values were corrected using Dunnett multiple comparisons with the control. *p<0.05, **p<0.01, ***p<0.001.
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Figure 2: TaPin1 is a functional homologue of hPin1 involved in transformationa. hPin1 and TaPin1 catalytic PPIase activities measured by in vitro chymotrypsin-coupled using a Pin1 substrate peptide (Suc-Ala-Glu-Pro-Phe-pNA). No activity was detected for GST alone or control substrate peptide (Suc-Ala-Ala-Pro-Phe-pNA).b. TaPin1 and hPin1 increased CyclinD1-Luciferase promoter activity when transfected in TBL3 cells.c. C92A and K38A TaPin1 mutants showed reduced activation of cyclinD1 promoter when transfected in TBL3 cells.d. TaPin1 or hPin1 induced CyclinD1-Luciferase promoter activity in pin1−/− immortalized fibroblasts.e. TaPin1 causes centrosome amplification. NIH3T3 fibroblasts stably expressing TaPin1 or hPin1 were arrested at the G1/S transition by aphidicolin, stained with anti-γ-tubulin antibody. 300 cells were scored.f. TaPin1 or hPin1 transfection increased colony foci formation in Pin1 knockdown MCF10A-Ras/Neu cells. Expression of transfected TaPin1 and hPin1 in MCF10A-Ras/Neu-shPin1 detected with an anti-FLAG antibody. Actin was a loading control.Note: “Con” indicates Control empty vector transfection.Data represent 3 independent experiments (average ± sd). The p-values were calculated using the Dunnett method for multiple comparisons with the TaPin1WT for Figure 2c. For Figures 2b / 2d / 2e / 2f: the p-values were corrected using Dunnett multiple comparisons with the control. *p<0.05, **p<0.01, ***p<0.001.
Mentions: To explore the functional PPIase activity of the secreted TaPin1 protein, we developed a chymotrypsin-coupled in vitro assay and found that TaPin1 and hPin1 catalytic activities were comparable (Fig. 2a). TaPin1 and hPin1 were also equivalent in activation of the cyclinD1-Luciferase reporter in bovine B cells (Fig. 2b), an established readout for Pin1 activity9. We mutated key C92 and K38 residues in TaPin1 and showed loss of the PPIase activity (Fig. 2c). Furthermore, TaPin1 rescued cyclinD1 promoter activity and cell spreading defects in pin1−/− mouse fibroblasts (Fig. 2d, Extended Data Fig. 5a). Mammalian Pin1 overexpression disrupts cell cycle regulation causing centrosome amplification and cell transformation17. TaPin1 also induced centrosome duplication when overexpressed in mouse fibroblasts (Fig. 2e, Extended Data Fig. 5b). Furthermore, TaPin1 functionally replaced mammalian Pin1 and rescued colony formation as effectively as hPin1 in human breast cancer cells with knocked-down Pin1 (Fig. 2f). These combined results show that Theileria secretes a bona fide phosphorylation-dependent PPIase which could contribute to host cell transformation.

Bottom Line: 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.

View Article: PubMed Central - PubMed

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

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 cells. 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 buparvaquone. 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.

Show MeSH
Related in: MedlinePlus