Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition of TaPin1 activity blocks transformation in vitro and in vivoa. Homology prediction models for TaPin1 and TaPin1-A53P Mutant based on similarity with hPin1. The TaPin1-A53P mutation induces a conformational change near the catalytic loop (green arrow). Computational analysis predicted docking of Juglone, Buparvaquone or DTM molecules in the Pin1 active sites (see Extended Data Fig. 6c for alternative). Bup/Jug: red=polar and negatively-charged residues; blue=polar and positively charged residues; yellow= S atoms; white=remaining residues. DTM: blue = N atoms; amber = S atoms. Colours in small molecule: O atoms in red; other atoms in yellow.b. TaPin1 and TaPin1 A53P catalytic PPIase activity, measured in vitro chymotrypsin-coupled assay, upon treatment with Buparvaquone (Bup), Juglone (Jug) or DTM. “Con” indicates Control solutions.c. Drug treatment (72h) eliminated Theileria parasites in infected cells (parasite nuclei counted following DAPI staining).d. Pin1 inhibitors decreased the viability of infected TBL3 cells (XTT assay 72h).e. Drug treatment decreased colony formation of parasitized cells in soft-agar (72h treatment with Buparvaquone (Bup), Juglone (Jug) or DTM): macroscopic colonies/plate after 10 days.f. TaPin1 inhibition reduced Xenograft tumor growth in zebrafish embryos (with a Zeiss AxioZoom V16 Macroscope at day 1 and day 4 post-injection). The median tumor development Day4:Day1 is shown. “n” = number of embryos.g. Representative images from individual zebrafish embryos photographed with a Zeiss AxioZoom V16 Macroscope. Scalebar = 200 μm.“Con” = vector solutions alone.Data represent 3 independent experiments (average ± sd, n=3). For Figures 3c / 3d / 3e: the p-values were corrected using the Dunnett test multiple comparisons with the control. An unpaired Mann-Whitney test was performed for the zebrafish experiments to analyze the significant difference between the control and treatment groups. *p<0.05, **p<0.01, ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4401560&req=5

Figure 3: Inhibition of TaPin1 activity blocks transformation in vitro and in vivoa. Homology prediction models for TaPin1 and TaPin1-A53P Mutant based on similarity with hPin1. The TaPin1-A53P mutation induces a conformational change near the catalytic loop (green arrow). Computational analysis predicted docking of Juglone, Buparvaquone or DTM molecules in the Pin1 active sites (see Extended Data Fig. 6c for alternative). Bup/Jug: red=polar and negatively-charged residues; blue=polar and positively charged residues; yellow= S atoms; white=remaining residues. DTM: blue = N atoms; amber = S atoms. Colours in small molecule: O atoms in red; other atoms in yellow.b. TaPin1 and TaPin1 A53P catalytic PPIase activity, measured in vitro chymotrypsin-coupled assay, upon treatment with Buparvaquone (Bup), Juglone (Jug) or DTM. “Con” indicates Control solutions.c. Drug treatment (72h) eliminated Theileria parasites in infected cells (parasite nuclei counted following DAPI staining).d. Pin1 inhibitors decreased the viability of infected TBL3 cells (XTT assay 72h).e. Drug treatment decreased colony formation of parasitized cells in soft-agar (72h treatment with Buparvaquone (Bup), Juglone (Jug) or DTM): macroscopic colonies/plate after 10 days.f. TaPin1 inhibition reduced Xenograft tumor growth in zebrafish embryos (with a Zeiss AxioZoom V16 Macroscope at day 1 and day 4 post-injection). The median tumor development Day4:Day1 is shown. “n” = number of embryos.g. Representative images from individual zebrafish embryos photographed with a Zeiss AxioZoom V16 Macroscope. Scalebar = 200 μm.“Con” = vector solutions alone.Data represent 3 independent experiments (average ± sd, n=3). For Figures 3c / 3d / 3e: the p-values were corrected using the Dunnett test multiple comparisons with the control. An unpaired Mann-Whitney test was performed for the zebrafish experiments to analyze the significant difference between the control and treatment groups. *p<0.05, **p<0.01, ***p<0.001.

Mentions: In a search for potential inhibitors, we noted that the chemical structure of Buparvaquone is similar to Juglone, a well-characterized inhibitor of mammalian Pin113. The TaPin1 sequence exhibits over 47% identity with hPin1 in the PPIase domain (Extended Data Fig. 6a). Our homology models of TaPin1 protein based on published hPin1 experimental data suggest a similar structure with a conserved catalytic pocket (Fig. 3a, Extended Data Fig. 6b). Notably, several Pin1 homologues also lack the WW domain, including Arabidopsis thaliana Pin1At18-20, MdPin1 in Malus domestica and the parasite Trypanosoma brucei TbPin1 homologue20-22, and the predicted TaPin1 model closely resembles these structures (Extended Data Fig. 6d). We investigated the hPin1 experimental structure and the TaPin1 predicted model with the binding pocket and hot-spot detection algorithm FTMap, using the server FTFlex. Notably, we found key hot-spot regions in the catalytic site area, matching the substrate binding region of hPin1 (Extended Data Fig. 6). Juglone and Buparvaquone molecules could be docked into the active site of both TaPin1 and hPin1 by in silico analysis (Fig. 3a, Extended Data Fig. 6c).


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 TaPin1 activity blocks transformation in vitro and in vivoa. Homology prediction models for TaPin1 and TaPin1-A53P Mutant based on similarity with hPin1. The TaPin1-A53P mutation induces a conformational change near the catalytic loop (green arrow). Computational analysis predicted docking of Juglone, Buparvaquone or DTM molecules in the Pin1 active sites (see Extended Data Fig. 6c for alternative). Bup/Jug: red=polar and negatively-charged residues; blue=polar and positively charged residues; yellow= S atoms; white=remaining residues. DTM: blue = N atoms; amber = S atoms. Colours in small molecule: O atoms in red; other atoms in yellow.b. TaPin1 and TaPin1 A53P catalytic PPIase activity, measured in vitro chymotrypsin-coupled assay, upon treatment with Buparvaquone (Bup), Juglone (Jug) or DTM. “Con” indicates Control solutions.c. Drug treatment (72h) eliminated Theileria parasites in infected cells (parasite nuclei counted following DAPI staining).d. Pin1 inhibitors decreased the viability of infected TBL3 cells (XTT assay 72h).e. Drug treatment decreased colony formation of parasitized cells in soft-agar (72h treatment with Buparvaquone (Bup), Juglone (Jug) or DTM): macroscopic colonies/plate after 10 days.f. TaPin1 inhibition reduced Xenograft tumor growth in zebrafish embryos (with a Zeiss AxioZoom V16 Macroscope at day 1 and day 4 post-injection). The median tumor development Day4:Day1 is shown. “n” = number of embryos.g. Representative images from individual zebrafish embryos photographed with a Zeiss AxioZoom V16 Macroscope. Scalebar = 200 μm.“Con” = vector solutions alone.Data represent 3 independent experiments (average ± sd, n=3). For Figures 3c / 3d / 3e: the p-values were corrected using the Dunnett test multiple comparisons with the control. An unpaired Mann-Whitney test was performed for the zebrafish experiments to analyze the significant difference between the control and treatment groups. *p<0.05, **p<0.01, ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401560&req=5

Figure 3: Inhibition of TaPin1 activity blocks transformation in vitro and in vivoa. Homology prediction models for TaPin1 and TaPin1-A53P Mutant based on similarity with hPin1. The TaPin1-A53P mutation induces a conformational change near the catalytic loop (green arrow). Computational analysis predicted docking of Juglone, Buparvaquone or DTM molecules in the Pin1 active sites (see Extended Data Fig. 6c for alternative). Bup/Jug: red=polar and negatively-charged residues; blue=polar and positively charged residues; yellow= S atoms; white=remaining residues. DTM: blue = N atoms; amber = S atoms. Colours in small molecule: O atoms in red; other atoms in yellow.b. TaPin1 and TaPin1 A53P catalytic PPIase activity, measured in vitro chymotrypsin-coupled assay, upon treatment with Buparvaquone (Bup), Juglone (Jug) or DTM. “Con” indicates Control solutions.c. Drug treatment (72h) eliminated Theileria parasites in infected cells (parasite nuclei counted following DAPI staining).d. Pin1 inhibitors decreased the viability of infected TBL3 cells (XTT assay 72h).e. Drug treatment decreased colony formation of parasitized cells in soft-agar (72h treatment with Buparvaquone (Bup), Juglone (Jug) or DTM): macroscopic colonies/plate after 10 days.f. TaPin1 inhibition reduced Xenograft tumor growth in zebrafish embryos (with a Zeiss AxioZoom V16 Macroscope at day 1 and day 4 post-injection). The median tumor development Day4:Day1 is shown. “n” = number of embryos.g. Representative images from individual zebrafish embryos photographed with a Zeiss AxioZoom V16 Macroscope. Scalebar = 200 μm.“Con” = vector solutions alone.Data represent 3 independent experiments (average ± sd, n=3). For Figures 3c / 3d / 3e: the p-values were corrected using the Dunnett test multiple comparisons with the control. An unpaired Mann-Whitney test was performed for the zebrafish experiments to analyze the significant difference between the control and treatment groups. *p<0.05, **p<0.01, ***p<0.001.
Mentions: In a search for potential inhibitors, we noted that the chemical structure of Buparvaquone is similar to Juglone, a well-characterized inhibitor of mammalian Pin113. The TaPin1 sequence exhibits over 47% identity with hPin1 in the PPIase domain (Extended Data Fig. 6a). Our homology models of TaPin1 protein based on published hPin1 experimental data suggest a similar structure with a conserved catalytic pocket (Fig. 3a, Extended Data Fig. 6b). Notably, several Pin1 homologues also lack the WW domain, including Arabidopsis thaliana Pin1At18-20, MdPin1 in Malus domestica and the parasite Trypanosoma brucei TbPin1 homologue20-22, and the predicted TaPin1 model closely resembles these structures (Extended Data Fig. 6d). We investigated the hPin1 experimental structure and the TaPin1 predicted model with the binding pocket and hot-spot detection algorithm FTMap, using the server FTFlex. Notably, we found key hot-spot regions in the catalytic site area, matching the substrate binding region of hPin1 (Extended Data Fig. 6). Juglone and Buparvaquone molecules could be docked into the active site of both TaPin1 and hPin1 by in silico analysis (Fig. 3a, Extended Data Fig. 6c).

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