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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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Inhibition of bovine Pin1 does not affect Theileria-associated cell transformationa. 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 72h.b. Colony formation of parasite-infected TpMD409 cells in soft-agar was lost after 72h treatment with Buparvaquone (Bup), Juglone (Jug) or DTM. Number of macroscopic colonies per plate were counted after 10 days.c. siPin1 has no effect on TBL3 transformed phenotypes. TBL3 were transiently transfected with siControl or siPin1. The average number of colonies per plate is shown. (average ± sd, n=3). Indicated protein levels were detected by Western blot analysis using specific antibodies. Actin was used as loading control (Results representative of 3 independent experiments).d/f. Overexpression of the TaPin1 Mutant A53P rescued the Buparvaquone but not Juglone effects on Theileria infected cells.All data represent 3 independent experiments (average ± sd, n=3). The SPSS 19.0 program (SPSS Inc. Chicago, IL, USA) was used for statistics. *p<0.05, **p<0.01.
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Figure 12: Inhibition of bovine Pin1 does not affect Theileria-associated cell transformationa. 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 72h.b. Colony formation of parasite-infected TpMD409 cells in soft-agar was lost after 72h treatment with Buparvaquone (Bup), Juglone (Jug) or DTM. Number of macroscopic colonies per plate were counted after 10 days.c. siPin1 has no effect on TBL3 transformed phenotypes. TBL3 were transiently transfected with siControl or siPin1. The average number of colonies per plate is shown. (average ± sd, n=3). Indicated protein levels were detected by Western blot analysis using specific antibodies. Actin was used as loading control (Results representative of 3 independent experiments).d/f. Overexpression of the TaPin1 Mutant A53P rescued the Buparvaquone but not Juglone effects on Theileria infected cells.All data represent 3 independent experiments (average ± sd, n=3). The SPSS 19.0 program (SPSS Inc. Chicago, IL, USA) was used for statistics. *p<0.05, **p<0.01.

Mentions: We predicted that Buparvaquone might target TaPin1 directly and that Juglone (or other Pin1 inhibitors) could functionally replace Buparvaquone to block parasite transformation. Both Buparvaquone and Juglone inhibited TaPin1 PPIase activity in vitro, as did the unrelated non-quinone inhibitor DTM14, albeit to a lesser degree (Fig. 3b). Buparvaquone-resistant Theileria strains are an emerging clinical concern for cattle in infected areas23 and mutations in the cytochrome B gene were recently reported24. But mitochondrial and non-mitochondrial pathways might cooperate in transformation and participate in drug resistance. We sequenced the TaPin1 gene in genomic DNA from a drug-resistant isolate and identified a mutation (A53>P substitution) in the catalytic loop of TaPin1 (Extended Data Fig. 7). Structural modeling suggested that this mutation could affect the nearby catalytic region and disturb ligand binding; computational docking indicated that the small Juglone molecule could react with the thiolate group of C113 or C92 in hPin1, TaPin1 or mutant TaPin1-A53P. However, the A53P mutation might impede interaction with the bulky, hydrophobic moiety of the larger Buparvaquone compound (MW=326, cf. Juglone MW=176), creating steric clashes between the inhibitor and residues in the modified structure (Fig. 3a, Extended Data Fig. 6c). Mutant TaPin1-A53P was catalytically active on the Pin1 substrate and was inhibited by Juglone and DTM, but not by Buparvaquone (Fig. 3b). The Pin1 inhibitors (Buparvaquone, Juglone and DTM) all reduced parasite load and viability of host cells infected with T. annulata or T. parva (Fig. 3c-d, Extended Data Fig. 8a) and blocked colony growth of parasitized cells in soft-agar assays in vitro (Fig. 3e, Extended Data Fig. 8b). In contrast, knocking down the endogenous bovine BtPin1 did not affect colony formation (Extended Data Fig. 8c). Transfection with mutant TaPin1-A53P rendered TBL3 cells resistant to Buparvaquone, but not Juglone, treatment (Extended Data Fig. 8d). Similarly, Juglone inhibited both WT and mutant TaPin1 activity in the cyclinD1-luciferase assay, but only the mutant was resistant to Buparvaquone (Extended Data Fig. 8e). Fish xenograft models are effective for monitoring in vivo tumor formation and for drug testing25 and are emerging as important experimental models to study cancer26. We used a zebrafish xenograft experimental system to test drug effects on tumor growth in vivo and observed a two-fold increase in tumor growth of infected cells which was efficiently inhibited by the anti-Pin1 drugs (Fig. 3f-g). Thus, our modelling predictions, biochemical analysis in vitro, transformation assays and tumor growth in vivo all support the targeting of TaPin1 by Buparvaquone and the role of TaPin1 in Theileria-induced 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)

Inhibition of bovine Pin1 does not affect Theileria-associated cell transformationa. 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 72h.b. Colony formation of parasite-infected TpMD409 cells in soft-agar was lost after 72h treatment with Buparvaquone (Bup), Juglone (Jug) or DTM. Number of macroscopic colonies per plate were counted after 10 days.c. siPin1 has no effect on TBL3 transformed phenotypes. TBL3 were transiently transfected with siControl or siPin1. The average number of colonies per plate is shown. (average ± sd, n=3). Indicated protein levels were detected by Western blot analysis using specific antibodies. Actin was used as loading control (Results representative of 3 independent experiments).d/f. Overexpression of the TaPin1 Mutant A53P rescued the Buparvaquone but not Juglone effects on Theileria infected cells.All data represent 3 independent experiments (average ± sd, n=3). The SPSS 19.0 program (SPSS Inc. Chicago, IL, USA) was used for statistics. *p<0.05, **p<0.01.
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Figure 12: Inhibition of bovine Pin1 does not affect Theileria-associated cell transformationa. 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 72h.b. Colony formation of parasite-infected TpMD409 cells in soft-agar was lost after 72h treatment with Buparvaquone (Bup), Juglone (Jug) or DTM. Number of macroscopic colonies per plate were counted after 10 days.c. siPin1 has no effect on TBL3 transformed phenotypes. TBL3 were transiently transfected with siControl or siPin1. The average number of colonies per plate is shown. (average ± sd, n=3). Indicated protein levels were detected by Western blot analysis using specific antibodies. Actin was used as loading control (Results representative of 3 independent experiments).d/f. Overexpression of the TaPin1 Mutant A53P rescued the Buparvaquone but not Juglone effects on Theileria infected cells.All data represent 3 independent experiments (average ± sd, n=3). The SPSS 19.0 program (SPSS Inc. Chicago, IL, USA) was used for statistics. *p<0.05, **p<0.01.
Mentions: We predicted that Buparvaquone might target TaPin1 directly and that Juglone (or other Pin1 inhibitors) could functionally replace Buparvaquone to block parasite transformation. Both Buparvaquone and Juglone inhibited TaPin1 PPIase activity in vitro, as did the unrelated non-quinone inhibitor DTM14, albeit to a lesser degree (Fig. 3b). Buparvaquone-resistant Theileria strains are an emerging clinical concern for cattle in infected areas23 and mutations in the cytochrome B gene were recently reported24. But mitochondrial and non-mitochondrial pathways might cooperate in transformation and participate in drug resistance. We sequenced the TaPin1 gene in genomic DNA from a drug-resistant isolate and identified a mutation (A53>P substitution) in the catalytic loop of TaPin1 (Extended Data Fig. 7). Structural modeling suggested that this mutation could affect the nearby catalytic region and disturb ligand binding; computational docking indicated that the small Juglone molecule could react with the thiolate group of C113 or C92 in hPin1, TaPin1 or mutant TaPin1-A53P. However, the A53P mutation might impede interaction with the bulky, hydrophobic moiety of the larger Buparvaquone compound (MW=326, cf. Juglone MW=176), creating steric clashes between the inhibitor and residues in the modified structure (Fig. 3a, Extended Data Fig. 6c). Mutant TaPin1-A53P was catalytically active on the Pin1 substrate and was inhibited by Juglone and DTM, but not by Buparvaquone (Fig. 3b). The Pin1 inhibitors (Buparvaquone, Juglone and DTM) all reduced parasite load and viability of host cells infected with T. annulata or T. parva (Fig. 3c-d, Extended Data Fig. 8a) and blocked colony growth of parasitized cells in soft-agar assays in vitro (Fig. 3e, Extended Data Fig. 8b). In contrast, knocking down the endogenous bovine BtPin1 did not affect colony formation (Extended Data Fig. 8c). Transfection with mutant TaPin1-A53P rendered TBL3 cells resistant to Buparvaquone, but not Juglone, treatment (Extended Data Fig. 8d). Similarly, Juglone inhibited both WT and mutant TaPin1 activity in the cyclinD1-luciferase assay, but only the mutant was resistant to Buparvaquone (Extended Data Fig. 8e). Fish xenograft models are effective for monitoring in vivo tumor formation and for drug testing25 and are emerging as important experimental models to study cancer26. We used a zebrafish xenograft experimental system to test drug effects on tumor growth in vivo and observed a two-fold increase in tumor growth of infected cells which was efficiently inhibited by the anti-Pin1 drugs (Fig. 3f-g). Thus, our modelling predictions, biochemical analysis in vitro, transformation assays and tumor growth in vivo all support the targeting of TaPin1 by Buparvaquone and the role of TaPin1 in Theileria-induced 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