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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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Bovine Pin1 is not regulated by the parasiteBuparvaquone 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 and on the protein level (b) as assessed by immunoblot analysis. “Con” indicates Control and “Bup” indicates Buparvaquone treatment. (average ± sd, n=3). The result in 3b is representative of 3 independent experiments.
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Figure 7: Bovine Pin1 is not regulated by the parasiteBuparvaquone 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 and on the protein level (b) as assessed by immunoblot analysis. “Con” indicates Control and “Bup” indicates Buparvaquone treatment. (average ± sd, n=3). The result in 3b is representative of 3 independent experiments.

Mentions: To identify proteins secreted by Theileria into the host cell which could contribute to transformation4-6, we conducted an in silico screen of parasite genomes; we identified 689 proteins in the T. annulata genome with a predicted signal peptide. Comparison with T. gondii (a non-transforming apicomplexan parasite) proteome, narrowed the candidate list to 33 proteins with a Theileria-specific signal peptide (Extended Data Fig. 1a). We focused on the TA18945 gene encoding a homologue of the human parvulin Pin1 (hPin1) Peptidyl Prolyl Isomerase (PPIase) as mammalian Pin1 regulates cell proliferation, pluripotency and survival7,8 and contributes to tumorigenesis9,10. hPin1 catalyzes the cis/trans isomerization of peptidyl-prolyl bonds in phosphorylated Ser/Thr-Pro motifs inducing conformational changes that affect substrate stability and activity11,12 and there are several small-molecule inhibitors of hPin113-15. The TA18945-encoded protein has a signal peptide and a highly conserved PPIase domain (Extended Data Fig. 1b-c), but lacks the WW domain important for substrate recognition of mammalian Pin111. A gene in the T. parva genome, also associated with transformation, encodes a conserved TpPin1 predicted protein, whereas the signal peptide is not conserved in the related T. orientalis genome which does not transform host cells16 (Extended Data Fig. 2a-b). We detected Theileria Pin1 transcripts in B cells infected with T. annulata or T. parva and they decreased upon Buparvaquone treatment (Fig. 1a). The levels of host bovine BtPin1 transcripts were unaffected by Theileria infection or Buparvaquone treatment (Extended Data Fig. 3). An antibody generated against a TaPin1-specific peptide (NPVNRNTGMAVTR) recognized parasite Pin1 protein or transfected TaPin1 in mouse fibroblasts, but not mammalian Pin1 (Fig. 1b, Extended Data Fig. 4a-e). Confocal microscopy and immunoblot analysis located the parasite Pin1 protein to both the host cell cytoplasm and nucleus (Fig. 1b-c, Extended Data Fig. 4c-d). The host nuclear signal in the confocal images was 10-fold over background in parasitized cells (205.0 ± 15.48 nuclear fluorescence intensity/pixel compared to 21.45 ± 8.50 in controls p<0.0001, n=31). Thus, comparative parasite genomics identified TaPin1 which is secreted into the host cytoplasm and nucleus.


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)

Bovine Pin1 is not regulated by the parasiteBuparvaquone 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 and on the protein level (b) as assessed by immunoblot analysis. “Con” indicates Control and “Bup” indicates Buparvaquone treatment. (average ± sd, n=3). The result in 3b is representative of 3 independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401560&req=5

Figure 7: Bovine Pin1 is not regulated by the parasiteBuparvaquone 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 and on the protein level (b) as assessed by immunoblot analysis. “Con” indicates Control and “Bup” indicates Buparvaquone treatment. (average ± sd, n=3). The result in 3b is representative of 3 independent experiments.
Mentions: To identify proteins secreted by Theileria into the host cell which could contribute to transformation4-6, we conducted an in silico screen of parasite genomes; we identified 689 proteins in the T. annulata genome with a predicted signal peptide. Comparison with T. gondii (a non-transforming apicomplexan parasite) proteome, narrowed the candidate list to 33 proteins with a Theileria-specific signal peptide (Extended Data Fig. 1a). We focused on the TA18945 gene encoding a homologue of the human parvulin Pin1 (hPin1) Peptidyl Prolyl Isomerase (PPIase) as mammalian Pin1 regulates cell proliferation, pluripotency and survival7,8 and contributes to tumorigenesis9,10. hPin1 catalyzes the cis/trans isomerization of peptidyl-prolyl bonds in phosphorylated Ser/Thr-Pro motifs inducing conformational changes that affect substrate stability and activity11,12 and there are several small-molecule inhibitors of hPin113-15. The TA18945-encoded protein has a signal peptide and a highly conserved PPIase domain (Extended Data Fig. 1b-c), but lacks the WW domain important for substrate recognition of mammalian Pin111. A gene in the T. parva genome, also associated with transformation, encodes a conserved TpPin1 predicted protein, whereas the signal peptide is not conserved in the related T. orientalis genome which does not transform host cells16 (Extended Data Fig. 2a-b). We detected Theileria Pin1 transcripts in B cells infected with T. annulata or T. parva and they decreased upon Buparvaquone treatment (Fig. 1a). The levels of host bovine BtPin1 transcripts were unaffected by Theileria infection or Buparvaquone treatment (Extended Data Fig. 3). An antibody generated against a TaPin1-specific peptide (NPVNRNTGMAVTR) recognized parasite Pin1 protein or transfected TaPin1 in mouse fibroblasts, but not mammalian Pin1 (Fig. 1b, Extended Data Fig. 4a-e). Confocal microscopy and immunoblot analysis located the parasite Pin1 protein to both the host cell cytoplasm and nucleus (Fig. 1b-c, Extended Data Fig. 4c-d). The host nuclear signal in the confocal images was 10-fold over background in parasitized cells (205.0 ± 15.48 nuclear fluorescence intensity/pixel compared to 21.45 ± 8.50 in controls p<0.0001, n=31). Thus, comparative parasite genomics identified TaPin1 which is secreted into the host cytoplasm and nucleus.

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