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The structure of bradyzoite-specific enolase from Toxoplasma gondii reveals insights into its dual cytoplasmic and nuclear functions.

Ruan J, Mouveaux T, Light SH, Minasov G, Anderson WF, Tomavo S, Ngô HM - Acta Crystallogr. D Biol. Crystallogr. (2015)

Bottom Line: Furthermore, direct physical interactions of both nuclear TgENO1 and TgENO2 with the TgMAG1 gene promoter are demonstrated in vivo using chromatin immunoprecipitation (ChIP) assays.Structural and biochemical studies reveal that T. gondii enolase functions are multifaceted, including the coordination of gene regulation in parasitic stage development.Enolase 1 provides a potential lead in the design of drugs against Toxoplasma brain cysts.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Structural Genomics of Infectious Diseases, Northwestern University, 320 E. Superior Street, Morton 7-601, Chicago, IL 60611, USA.

ABSTRACT
In addition to catalyzing a central step in glycolysis, enolase assumes a remarkably diverse set of secondary functions in different organisms, including transcription regulation as documented for the oncogene c-Myc promoter-binding protein 1. The apicomplexan parasite Toxoplasma gondii differentially expresses two nuclear-localized, plant-like enolases: enolase 1 (TgENO1) in the latent bradyzoite cyst stage and enolase 2 (TgENO2) in the rapidly replicative tachyzoite stage. A 2.75 Å resolution crystal structure of bradyzoite enolase 1, the second structure to be reported of a bradyzoite-specific protein in Toxoplasma, captures an open conformational state and reveals that distinctive plant-like insertions are located on surface loops. The enolase 1 structure reveals that a unique residue, Glu164, in catalytic loop 2 may account for the lower activity of this cyst-stage isozyme. Recombinant TgENO1 specifically binds to a TTTTCT DNA motif present in the cyst matrix antigen 1 (TgMAG1) gene promoter as demonstrated by gel retardation. Furthermore, direct physical interactions of both nuclear TgENO1 and TgENO2 with the TgMAG1 gene promoter are demonstrated in vivo using chromatin immunoprecipitation (ChIP) assays. Structural and biochemical studies reveal that T. gondii enolase functions are multifaceted, including the coordination of gene regulation in parasitic stage development. Enolase 1 provides a potential lead in the design of drugs against Toxoplasma brain cysts.

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In vitro interaction of TgENO1 and TgENO2 with the bradyzoite TgMAG1 promoter as shown by chromatin immunoprecipitation (ChIP) analyses. (a) Upstream and downstream primers of the TTTTCT motif in TgMAG1 for PCR of the ChIP products. (b) Polyclonal antibodies to bradyzoite-specific TgENO1 precipitated ChIP fragments from the TgMAG1 promoter. (c) Polyclonal antibodies to tachyzoite-specific TgENO2 also pulled down ChIP fragments from TgMAG1 promoter.
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fig7: In vitro interaction of TgENO1 and TgENO2 with the bradyzoite TgMAG1 promoter as shown by chromatin immunoprecipitation (ChIP) analyses. (a) Upstream and downstream primers of the TTTTCT motif in TgMAG1 for PCR of the ChIP products. (b) Polyclonal antibodies to bradyzoite-specific TgENO1 precipitated ChIP fragments from the TgMAG1 promoter. (c) Polyclonal antibodies to tachyzoite-specific TgENO2 also pulled down ChIP fragments from TgMAG1 promoter.

Mentions: Having shown that TgENO1 binds to a specific DNA motif by gel retardation in vitro, we focused on validating its binding to the MAG1 gene promoter in vivo. We showed that the rabbit polyclonal antibodies specific to TgENO1 pulled down the MAG1 promoter from chromatin extracts of intracellular parasites (Fig. 7 ▶b) using two distinct pair of primers that span the TTTTCT motif (Fig. 7 ▶a). In addition, the MAG1 promoter was also similarly pulled down with the polyclonal anti-ENO2 antibodies and the same pair of primers (Fig. 7 ▶c). These results indicate that both bradyzoite-specific TgENO1 and tachyzoite-specific TgENO2 are capable of binding to the TgMAG1 gene promoter, suggesting a possible role of nuclear enolases in the control of gene expression in T. gondii. This proposed role is further confirmed by targeted disruption of TgENO1, which resulted in changes in the transcripts of nuclear genes and a reduction in the brain-cyst burden in chronically infected mice (Mouveaux et al., 2014 ▶). This moonlighting enzyme is thus proposed to play a transcriptional regulatory role in brain-cyst development.


The structure of bradyzoite-specific enolase from Toxoplasma gondii reveals insights into its dual cytoplasmic and nuclear functions.

Ruan J, Mouveaux T, Light SH, Minasov G, Anderson WF, Tomavo S, Ngô HM - Acta Crystallogr. D Biol. Crystallogr. (2015)

In vitro interaction of TgENO1 and TgENO2 with the bradyzoite TgMAG1 promoter as shown by chromatin immunoprecipitation (ChIP) analyses. (a) Upstream and downstream primers of the TTTTCT motif in TgMAG1 for PCR of the ChIP products. (b) Polyclonal antibodies to bradyzoite-specific TgENO1 precipitated ChIP fragments from the TgMAG1 promoter. (c) Polyclonal antibodies to tachyzoite-specific TgENO2 also pulled down ChIP fragments from TgMAG1 promoter.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356359&req=5

fig7: In vitro interaction of TgENO1 and TgENO2 with the bradyzoite TgMAG1 promoter as shown by chromatin immunoprecipitation (ChIP) analyses. (a) Upstream and downstream primers of the TTTTCT motif in TgMAG1 for PCR of the ChIP products. (b) Polyclonal antibodies to bradyzoite-specific TgENO1 precipitated ChIP fragments from the TgMAG1 promoter. (c) Polyclonal antibodies to tachyzoite-specific TgENO2 also pulled down ChIP fragments from TgMAG1 promoter.
Mentions: Having shown that TgENO1 binds to a specific DNA motif by gel retardation in vitro, we focused on validating its binding to the MAG1 gene promoter in vivo. We showed that the rabbit polyclonal antibodies specific to TgENO1 pulled down the MAG1 promoter from chromatin extracts of intracellular parasites (Fig. 7 ▶b) using two distinct pair of primers that span the TTTTCT motif (Fig. 7 ▶a). In addition, the MAG1 promoter was also similarly pulled down with the polyclonal anti-ENO2 antibodies and the same pair of primers (Fig. 7 ▶c). These results indicate that both bradyzoite-specific TgENO1 and tachyzoite-specific TgENO2 are capable of binding to the TgMAG1 gene promoter, suggesting a possible role of nuclear enolases in the control of gene expression in T. gondii. This proposed role is further confirmed by targeted disruption of TgENO1, which resulted in changes in the transcripts of nuclear genes and a reduction in the brain-cyst burden in chronically infected mice (Mouveaux et al., 2014 ▶). This moonlighting enzyme is thus proposed to play a transcriptional regulatory role in brain-cyst development.

Bottom Line: Furthermore, direct physical interactions of both nuclear TgENO1 and TgENO2 with the TgMAG1 gene promoter are demonstrated in vivo using chromatin immunoprecipitation (ChIP) assays.Structural and biochemical studies reveal that T. gondii enolase functions are multifaceted, including the coordination of gene regulation in parasitic stage development.Enolase 1 provides a potential lead in the design of drugs against Toxoplasma brain cysts.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Structural Genomics of Infectious Diseases, Northwestern University, 320 E. Superior Street, Morton 7-601, Chicago, IL 60611, USA.

ABSTRACT
In addition to catalyzing a central step in glycolysis, enolase assumes a remarkably diverse set of secondary functions in different organisms, including transcription regulation as documented for the oncogene c-Myc promoter-binding protein 1. The apicomplexan parasite Toxoplasma gondii differentially expresses two nuclear-localized, plant-like enolases: enolase 1 (TgENO1) in the latent bradyzoite cyst stage and enolase 2 (TgENO2) in the rapidly replicative tachyzoite stage. A 2.75 Å resolution crystal structure of bradyzoite enolase 1, the second structure to be reported of a bradyzoite-specific protein in Toxoplasma, captures an open conformational state and reveals that distinctive plant-like insertions are located on surface loops. The enolase 1 structure reveals that a unique residue, Glu164, in catalytic loop 2 may account for the lower activity of this cyst-stage isozyme. Recombinant TgENO1 specifically binds to a TTTTCT DNA motif present in the cyst matrix antigen 1 (TgMAG1) gene promoter as demonstrated by gel retardation. Furthermore, direct physical interactions of both nuclear TgENO1 and TgENO2 with the TgMAG1 gene promoter are demonstrated in vivo using chromatin immunoprecipitation (ChIP) assays. Structural and biochemical studies reveal that T. gondii enolase functions are multifaceted, including the coordination of gene regulation in parasitic stage development. Enolase 1 provides a potential lead in the design of drugs against Toxoplasma brain cysts.

Show MeSH
Related in: MedlinePlus