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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 changes c-Jun stability through the regulation of FBW7a. TaPin1 interacts with endogenous mouse FBW7. Protein extracts from NIH3T3 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 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 WT or Mutant A53P. β-actin and H2A mRNAs were used for normalization (data represent 3 independent experiments - average ± sd, n=3).e. siPin1 does not affect FBW7a or c-Jun protein levels. TBL3 were transiently transfected by siControl “siCon” or si-bovine-Pin1. 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 (Bup) or Juglone (Jug) - “Con” indicates Control) were incubated with MG132, followed by immunoprecipitation of endogenous c-Jun or FBW7 and immunoblot analysis with the indicated antibodies.g. The effect of Buparvaquone on c-Jun ubiquitination was rescued by overexpression of TaPin1 Mutant A53P. Parasite-infected TBL3 cells (+/− Buparvaquone (Bup) or Juglone (Jug) - “Con” indicates Control) were transfected with TaPin1 WT, TaPin1 Mutant 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 Notch 1 protein levels in TBL3 upon Buparvaquone (Bup) treatment. “Con” indicates Control. Tubulin was used as loading control.i. Measure of c-Myc, KLF5 and activated Notch 1 protein levels in TBL3 upon FBW7α ectopic expression. “Con” indicates Control = transfection with the appropriate empty vector. Tubulin was used as loading control.All the results in a, b, e, f, g, h and i are representatives of 3 independent experiments.
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Figure 13: TaPin1 changes c-Jun stability through the regulation of FBW7a. TaPin1 interacts with endogenous mouse FBW7. Protein extracts from NIH3T3 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 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 WT or Mutant A53P. β-actin and H2A mRNAs were used for normalization (data represent 3 independent experiments - average ± sd, n=3).e. siPin1 does not affect FBW7a or c-Jun protein levels. TBL3 were transiently transfected by siControl “siCon” or si-bovine-Pin1. 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 (Bup) or Juglone (Jug) - “Con” indicates Control) were incubated with MG132, followed by immunoprecipitation of endogenous c-Jun or FBW7 and immunoblot analysis with the indicated antibodies.g. The effect of Buparvaquone on c-Jun ubiquitination was rescued by overexpression of TaPin1 Mutant A53P. Parasite-infected TBL3 cells (+/− Buparvaquone (Bup) or Juglone (Jug) - “Con” indicates Control) were transfected with TaPin1 WT, TaPin1 Mutant 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 Notch 1 protein levels in TBL3 upon Buparvaquone (Bup) treatment. “Con” indicates Control. Tubulin was used as loading control.i. Measure of c-Myc, KLF5 and activated Notch 1 protein levels in TBL3 upon FBW7α ectopic expression. “Con” indicates Control = transfection with the appropriate empty vector. Tubulin was used as loading control.All the results in a, b, e, f, g, h and i are representatives of 3 independent experiments.

Mentions: To investigate how TaPin1 affects host signaling pathways, we studied relevant substrates targeted by TaPin1. hPin1 targets many proteins, including the ubiquitin ligase FBW727, which exerts anti-tumor function by degrading oncoproteins required for cellular proliferation, such as c-Jun28,29. Since c-Jun is induced (and critical) during Theileria-induced transformation2, we examined whether TaPin1 targets the conserved bovine FBW7 protein. We found that TaPin1 interacts with host FBW7 in Theileria-infected cells or murine fibroblasts (Extended Data Fig. 9a-b). Conversely, FBW7α in particular, but not other isoforms, co-immunoprecipitated TaPin1 from parasitized cells (Fig. 4a). FBW7α protein levels were reduced in parasitized cells compared with uninfected cells and correlated with elevated c-Jun levels (Fig. 4b). Pharmacological TaPin1 inhibition restored FBW7 protein expression and reduced c-Jun levels, without affecting mRNA expression (Fig. 4b, Extended Data Fig. 9c-d). But knocking-down bovine Pin1 did not affect FBW7 or c-Jun protein levels (Extended Data Fig. 9e). siRNA FBW7 knockdown caused accumulation of c-Jun protein, whereas exogenous FBW7α transfection decreased c-Jun protein levels (Fig. 4c). Furthermore, knocking-down FBW7 increased AP-1 activity, measured by a Luciferase reporter assay, and rescued the inhibition of AP-1 by Buparvaquone or Juglone (Fig. 4d-e). TaPin1 inhibition caused increased c-Jun ubiquitination in TBL3 cells and decreased FBW7 (auto)-ubiquitination (Extended Data Fig. 9f). c-Jun ubiquitination was FBW7-dependent, as this effect was abolished by siRNA targeting bovine FBW7 (Fig. 4f). Half-life analysis using cycloheximide showed that siFBW7 increased c-Jun stability and rescued c-Jun levels following TaPin1 inhibition (Fig. 4g). In addition, the TaPin1-A53P mutant rescued the effect of Buparvaquone, but not Juglone, on c-Jun ubiquitination and transcriptional activity (reflected by the MMP-9 AP-1 target gene) (Extended Data Fig. 8f, Fig. 9g). As mammalian FBW7 targets many protein substrates, we examined the effects of Buparvaquone treatment or FBW7α transfection, but noted no changes in levels of endogenous c-Myc or activated Notch 1 proteins, while there was a modest effect on the KLF5 transcription factor (Extended Data Fig. 9h, i). These combined results suggest that c-Jun is the major target of TaPin1-FBW7α in our cells. Finally, FBW7α overexpression or c-Jun knockdown both caused significant reduction of colony growth in soft-agar proliferation assays of parasitized TBL3 cells (Fig. 4h).


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 changes c-Jun stability through the regulation of FBW7a. TaPin1 interacts with endogenous mouse FBW7. Protein extracts from NIH3T3 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 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 WT or Mutant A53P. β-actin and H2A mRNAs were used for normalization (data represent 3 independent experiments - average ± sd, n=3).e. siPin1 does not affect FBW7a or c-Jun protein levels. TBL3 were transiently transfected by siControl “siCon” or si-bovine-Pin1. 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 (Bup) or Juglone (Jug) - “Con” indicates Control) were incubated with MG132, followed by immunoprecipitation of endogenous c-Jun or FBW7 and immunoblot analysis with the indicated antibodies.g. The effect of Buparvaquone on c-Jun ubiquitination was rescued by overexpression of TaPin1 Mutant A53P. Parasite-infected TBL3 cells (+/− Buparvaquone (Bup) or Juglone (Jug) - “Con” indicates Control) were transfected with TaPin1 WT, TaPin1 Mutant 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 Notch 1 protein levels in TBL3 upon Buparvaquone (Bup) treatment. “Con” indicates Control. Tubulin was used as loading control.i. Measure of c-Myc, KLF5 and activated Notch 1 protein levels in TBL3 upon FBW7α ectopic expression. “Con” indicates Control = transfection with the appropriate empty vector. Tubulin was used as loading control.All the results in a, b, e, f, g, h and i are representatives of 3 independent experiments.
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Figure 13: TaPin1 changes c-Jun stability through the regulation of FBW7a. TaPin1 interacts with endogenous mouse FBW7. Protein extracts from NIH3T3 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 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 WT or Mutant A53P. β-actin and H2A mRNAs were used for normalization (data represent 3 independent experiments - average ± sd, n=3).e. siPin1 does not affect FBW7a or c-Jun protein levels. TBL3 were transiently transfected by siControl “siCon” or si-bovine-Pin1. 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 (Bup) or Juglone (Jug) - “Con” indicates Control) were incubated with MG132, followed by immunoprecipitation of endogenous c-Jun or FBW7 and immunoblot analysis with the indicated antibodies.g. The effect of Buparvaquone on c-Jun ubiquitination was rescued by overexpression of TaPin1 Mutant A53P. Parasite-infected TBL3 cells (+/− Buparvaquone (Bup) or Juglone (Jug) - “Con” indicates Control) were transfected with TaPin1 WT, TaPin1 Mutant 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 Notch 1 protein levels in TBL3 upon Buparvaquone (Bup) treatment. “Con” indicates Control. Tubulin was used as loading control.i. Measure of c-Myc, KLF5 and activated Notch 1 protein levels in TBL3 upon FBW7α ectopic expression. “Con” indicates Control = transfection with the appropriate empty vector. Tubulin was used as loading control.All the results in a, b, e, f, g, h and i are representatives of 3 independent experiments.
Mentions: To investigate how TaPin1 affects host signaling pathways, we studied relevant substrates targeted by TaPin1. hPin1 targets many proteins, including the ubiquitin ligase FBW727, which exerts anti-tumor function by degrading oncoproteins required for cellular proliferation, such as c-Jun28,29. Since c-Jun is induced (and critical) during Theileria-induced transformation2, we examined whether TaPin1 targets the conserved bovine FBW7 protein. We found that TaPin1 interacts with host FBW7 in Theileria-infected cells or murine fibroblasts (Extended Data Fig. 9a-b). Conversely, FBW7α in particular, but not other isoforms, co-immunoprecipitated TaPin1 from parasitized cells (Fig. 4a). FBW7α protein levels were reduced in parasitized cells compared with uninfected cells and correlated with elevated c-Jun levels (Fig. 4b). Pharmacological TaPin1 inhibition restored FBW7 protein expression and reduced c-Jun levels, without affecting mRNA expression (Fig. 4b, Extended Data Fig. 9c-d). But knocking-down bovine Pin1 did not affect FBW7 or c-Jun protein levels (Extended Data Fig. 9e). siRNA FBW7 knockdown caused accumulation of c-Jun protein, whereas exogenous FBW7α transfection decreased c-Jun protein levels (Fig. 4c). Furthermore, knocking-down FBW7 increased AP-1 activity, measured by a Luciferase reporter assay, and rescued the inhibition of AP-1 by Buparvaquone or Juglone (Fig. 4d-e). TaPin1 inhibition caused increased c-Jun ubiquitination in TBL3 cells and decreased FBW7 (auto)-ubiquitination (Extended Data Fig. 9f). c-Jun ubiquitination was FBW7-dependent, as this effect was abolished by siRNA targeting bovine FBW7 (Fig. 4f). Half-life analysis using cycloheximide showed that siFBW7 increased c-Jun stability and rescued c-Jun levels following TaPin1 inhibition (Fig. 4g). In addition, the TaPin1-A53P mutant rescued the effect of Buparvaquone, but not Juglone, on c-Jun ubiquitination and transcriptional activity (reflected by the MMP-9 AP-1 target gene) (Extended Data Fig. 8f, Fig. 9g). As mammalian FBW7 targets many protein substrates, we examined the effects of Buparvaquone treatment or FBW7α transfection, but noted no changes in levels of endogenous c-Myc or activated Notch 1 proteins, while there was a modest effect on the KLF5 transcription factor (Extended Data Fig. 9h, i). These combined results suggest that c-Jun is the major target of TaPin1-FBW7α in our cells. Finally, FBW7α overexpression or c-Jun knockdown both caused significant reduction of colony growth in soft-agar proliferation assays of parasitized TBL3 cells (Fig. 4h).

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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Related in: MedlinePlus