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Proteomics for the discovery of nuclear bile acid receptor FXR targets.

Gardmo C, Tamburro A, Modica S, Moschetta A - Biochim. Biophys. Acta (2011)

Bottom Line: Nineteen spots with a more than two-fold difference in protein amounts were found by 2D-DIGE and 20 proteins were identified by MALDI-TOF MS as putative novel FXR targets.In conclusion, our study provides the impetus to include proteomic analysis for the identification of novel targets of transcription factors, such as NRs.This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Pharmacology, Consorzio Mario Negri Sud, Santa Maria Imbaro (CH), 66030, Italy. gardmo@negrisud.it

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Identification of potential FXR targets. A) Hepatic cytosolic protein samples from wild type mice treated with INT-747 or vehicle were compared in a 2D-DIGE. Four examples of spots differentially expressed are shown and the proteins were identified by MALDI-TOF MS. The pH and molecular weights scales are indicated in the figure. B) Western blot on four of the proteins identified as potential FXR targets in the proteomics analysis. Pooled hepatic cytosolic protein samples from wild type mice treated with INT-747 or vehicle were compared (n = 5). The quantified results were normalized against β-actin. C) RT-qPCR on four genes corresponding to proteins identified as potential FXR targets in the proteomics analysis. Five individual samples were analyzed for each treatment and the error bars represent the standard deviation. *p < 0.05, Student's t-test.
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f0005: Identification of potential FXR targets. A) Hepatic cytosolic protein samples from wild type mice treated with INT-747 or vehicle were compared in a 2D-DIGE. Four examples of spots differentially expressed are shown and the proteins were identified by MALDI-TOF MS. The pH and molecular weights scales are indicated in the figure. B) Western blot on four of the proteins identified as potential FXR targets in the proteomics analysis. Pooled hepatic cytosolic protein samples from wild type mice treated with INT-747 or vehicle were compared (n = 5). The quantified results were normalized against β-actin. C) RT-qPCR on four genes corresponding to proteins identified as potential FXR targets in the proteomics analysis. Five individual samples were analyzed for each treatment and the error bars represent the standard deviation. *p < 0.05, Student's t-test.

Mentions: In a 2D-DIGE assay, comparing hepatic protein samples from INT-747 with vehicle treated mice, 19 spots were more than two-fold different in protein levels (Fig. 1A, Tables S1 and S2). For seven spots the amount of protein was higher in the INT-747 samples compared to vehicle, while in the rest it was reduced by the FXR ligand treatment. The proteins in the spots were identified by MALDI-TOF MS (Table 1). In spot 8302 two different proteins were found, argininosuccinate synthase and 3-ketoacylCoA thiolase. It is therefore unclear which protein, or if possibly both were down regulated in response to the FXR activation. In this study we also compared hepatic samples from FXR−/− mice, treated with the semi-synthetic bile acid INT-747 or vehicle, and no differences in protein amounts could be detected for any of the proteins in Table 1 (data not shown). This comparison was made as an objective control for non-FXR mediated effects that could relate to the weak agonistic effect of INT-747 on the membrane TGR5 receptor [28].


Proteomics for the discovery of nuclear bile acid receptor FXR targets.

Gardmo C, Tamburro A, Modica S, Moschetta A - Biochim. Biophys. Acta (2011)

Identification of potential FXR targets. A) Hepatic cytosolic protein samples from wild type mice treated with INT-747 or vehicle were compared in a 2D-DIGE. Four examples of spots differentially expressed are shown and the proteins were identified by MALDI-TOF MS. The pH and molecular weights scales are indicated in the figure. B) Western blot on four of the proteins identified as potential FXR targets in the proteomics analysis. Pooled hepatic cytosolic protein samples from wild type mice treated with INT-747 or vehicle were compared (n = 5). The quantified results were normalized against β-actin. C) RT-qPCR on four genes corresponding to proteins identified as potential FXR targets in the proteomics analysis. Five individual samples were analyzed for each treatment and the error bars represent the standard deviation. *p < 0.05, Student's t-test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3117992&req=5

f0005: Identification of potential FXR targets. A) Hepatic cytosolic protein samples from wild type mice treated with INT-747 or vehicle were compared in a 2D-DIGE. Four examples of spots differentially expressed are shown and the proteins were identified by MALDI-TOF MS. The pH and molecular weights scales are indicated in the figure. B) Western blot on four of the proteins identified as potential FXR targets in the proteomics analysis. Pooled hepatic cytosolic protein samples from wild type mice treated with INT-747 or vehicle were compared (n = 5). The quantified results were normalized against β-actin. C) RT-qPCR on four genes corresponding to proteins identified as potential FXR targets in the proteomics analysis. Five individual samples were analyzed for each treatment and the error bars represent the standard deviation. *p < 0.05, Student's t-test.
Mentions: In a 2D-DIGE assay, comparing hepatic protein samples from INT-747 with vehicle treated mice, 19 spots were more than two-fold different in protein levels (Fig. 1A, Tables S1 and S2). For seven spots the amount of protein was higher in the INT-747 samples compared to vehicle, while in the rest it was reduced by the FXR ligand treatment. The proteins in the spots were identified by MALDI-TOF MS (Table 1). In spot 8302 two different proteins were found, argininosuccinate synthase and 3-ketoacylCoA thiolase. It is therefore unclear which protein, or if possibly both were down regulated in response to the FXR activation. In this study we also compared hepatic samples from FXR−/− mice, treated with the semi-synthetic bile acid INT-747 or vehicle, and no differences in protein amounts could be detected for any of the proteins in Table 1 (data not shown). This comparison was made as an objective control for non-FXR mediated effects that could relate to the weak agonistic effect of INT-747 on the membrane TGR5 receptor [28].

Bottom Line: Nineteen spots with a more than two-fold difference in protein amounts were found by 2D-DIGE and 20 proteins were identified by MALDI-TOF MS as putative novel FXR targets.In conclusion, our study provides the impetus to include proteomic analysis for the identification of novel targets of transcription factors, such as NRs.This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Pharmacology, Consorzio Mario Negri Sud, Santa Maria Imbaro (CH), 66030, Italy. gardmo@negrisud.it

Show MeSH
Related in: MedlinePlus