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PGE1 and PGA1 bind to Nurr1 and activate its transcriptional function

Nature Chemical Biology volume 16, pages 876–886 (2020)Cite this article

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Abstract

The orphan nuclear receptor Nurr1 is critical for the development, maintenance and protection of midbrain dopaminergic (mDA) neurons. Here we show that prostaglandin E1 (PGE1) and its dehydrated metabolite, PGA1, directly interact with the ligand-binding domain (LBD) of Nurr1 and stimulate its transcriptional function. We also report the crystallographic structure of Nurr1-LBD bound to PGA1 at 2.05 Å resolution. PGA1 couples covalently to Nurr1-LBD by forming a Michael adduct with Cys566, and induces notable conformational changes, including a 21° shift of the activation function-2 helix (H12) away from the protein core. Furthermore, PGE1/PGA1 exhibit neuroprotective effects in a Nurr1-dependent manner, prominently enhance expression of Nurr1 target genes in mDA neurons and improve motor deficits in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mouse models of Parkinson’s disease. Based on these results, we propose that PGE1/PGA1 represent native ligands of Nurr1 and can exert neuroprotective effects on mDA neurons, via activation of Nurr1’s transcriptional function.

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Fig. 1: Identification of PGE1 and 8-iso-PGE1 as Nurr1 activators from brain tissue extracts.
Fig. 2: Crystal structure of PGA1-bound Nurr1-LBD and its molecular and functional analyses.
Fig. 3: Structural comparison of apo and PGA1-bound Nurr1-LBD.
Fig. 4: Direct binding of PGE1/PGA1 to Nurr1-LBD or EP2.
Fig. 5: Neuroprotective effect of PGE1/PGA1 is dependent on Nurr1.
Fig. 6: Neuroprotective effects of PGE1 and PGA1 in the MPTP-induced PD mouse model.

Data availability

The PDB accession code for the coordinates and structure factors of Nurr1-LBD in complex with PGA1 is PDB 5Y41. Source data for Figs. 1–3 and Extended Data Figs. 1–9 are presented with the paper.

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Acknowledgements

We thank various members of the molecular neurobiology laboratory past and present who participated in the project. In particular, we thank B.-S. Han, H.-Y. Jung, J. Lee and J. Sung Koh for technical assistance. We also acknowledge the contribution of the scientists and staff on the PXII and PXIII (Paul Scherrer Institute, Switzerland) beamlines for their expert assistance during crystal data collection. This work was supported by NIH grant nos. NS070577 and NS084869 (to K.-S.K.), NRF-2018M3A9B5023055 grant (to C.-H.K.), Ministry of Education Singapore AcRF Tier 2 Grant (no. ARC55/16) and Tang Tieng See Advancement Fund (to H.S.Y.), and National Medical Research Council, Singapore (grant no. TCR/013-NNI/2014; to K.L.L. and H.S.Y.).

Author information

Author notes

  1. These authors contributed equally: Sreekanth Rajan, Yongwoo Jang, Chun-Hyung Kim, Woori Kim.

Authors and Affiliations

  1. School of Biological Sciences, Nanyang Technological University, Singapore, Singapore

    Sreekanth Rajan, Hui Ting Toh, Aida Serra, Julien Lescar, Jun Yeob Yoo, Serap Beldar, Hong Ye & Ho Sup Yoon

  2. Molecular Neurobiology Laboratory, Department of Psychiatry, McLean Hospital, Harvard Medical School, Belmont, MA, USA

    Yongwoo Jang, Chun-Hyung Kim, Woori Kim, Jeha Jeon, Bin Song, Melissa Feitosa, Yeahan Kim, Dabin Hwang & Kwang-Soo Kim

  3. Department of Biomedical Engineering, Hanyang University, Seoul, Korea

    Yongwoo Jang

  4. Paean Biotechnology, Daejeon, Korea

    Chun-Hyung Kim

  5. Nanyang Institute of Technology in Health and Medicine, Interdisciplinary Graduate School, Nanyang Technological University, Singapore, Singapore

    Hui Ting Toh

  6. NTU Institute of Structural Biology, Nanyang Technological University, Singapore, Singapore

    Julien Lescar & Ho Sup Yoon

  7. Experimental Drug Development Centre, Agency for Science, Technology and Research, Nanos, Singapore, Singapore

    Congbao Kang

  8. Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore, Singapore

    Xue-Wei Liu

  9. National Neuroscience Institute, Singapore, Singapore

    Geraldine Goh & Kah-Leong Lim

  10. Lee Kong Chian School of Medicine, Singapore, Singapore

    Kah-Leong Lim

  11. Department of Bioscience and Biotechnology, Konkuk University, Gwangjin-gu, Seoul, Republic of Korea

    Hye Min Park & Choong Hwan Lee

  12. Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Sungwhan F. Oh

  13. Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

    Gregory A. Petsko

  14. Program in Neuroscience and Harvard Stem Cell Institute, McLean Hospital, Harvard Medical School, Belmont, MA, USA

    Kwang-Soo Kim

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  1. Sreekanth Rajan
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Contributions

K.-S.K. and H.S.Y. initiated and supervised the project. S.R., Y.J., C.H.K. and W.K. were responsible for the overall design and performance of experiments. S.R., H.T.T., H.Y., C.K., A.S., J.L., J.Y.Y., S.B., H.Y., C.K., X.L., G.G. and K.L.L. performed and analyzed structural studies. Y.J., C.H.K., W.K, J.J., B.S., M.F., Y.K., D.H., H.M.P., S.F.O. and C.H.L. performed and analyzed functional and biological studies. K.-S.K., H.S.Y., S.R., Y.J., C.H.K., W.K. and G.A.P. analyzed the data and wrote the paper. All authors contributed to the discussion and final approval of the paper.

Corresponding authors

Correspondence to Ho Sup Yoon or Kwang-Soo Kim.

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H.S.Y. is a nonexecutive director of Lifex Biolabs. The remaining authors have no competing interests to disclose.

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Supplementary information

Extended Data Fig. 1 Identification of PGE1 from brain tissue extract.

(a) Isolation of endogenous ligand candidates using a combination of boiling, acetone precipitation, and ultrafiltration (3,000 molecular weight cut-off). Fractions were monitored for their Nurr1-enhancing activity using a cell-based luciferase assay system. Nurr1-enhancing activity was unaffected by boiling and acetone precipitation. n = 3 independent experiments, Data are presented as mean ± s.d. (b) Following ultrafiltration, filtrates were fractionated by HPLC column C-18 and each fraction was assayed for Nurr1-activating activities. Fraction 5 contained the most activity and thus was used for the mass spectrometry (MS) analysis. n = 3 independent experiments, Data are presented as mean ± s.d. (c) Candidate compounds that are tested after identification by an ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC–qTOF-MS/MS).

Extended Data Fig. 2 Direct binding of PGE1 and PGA1 to Nurr1-LBD.

(a–f) Molecular interaction of Nurr1-LBD with PGE1 (a and b) and PGA1 (c and d) studied using 2D HSQC NMR titration experiments with uniformly 15N-labeled Nurr1-LBD. (a) Close-up view of a section of the overlay of free Nurr1-LBD (red) and Nurr1-LBD with PGE1 (1:4, green; 1:10, blue), with residues showing chemical shift perturbations (with arrows) or intensity changes (boxed in red) labelled. (b) Selected residues were mapped on the crystal structure of Nurr1-LBD (PDB: 1OVL) in surface representation, with a close-up section (as inset) showing affected helices H4, H11 and H12, with amino acid residues indicated. (c) Close-up view of a section of the overlay of free Nurr1-LBD (red) and Nurr1-LBD with PGA1 (1:5, green; 1:10, blue), with residues showing chemical shift perturbations (with arrows) or intensity changes (boxed in red) labelled. Residues (Leu559, Gln571 and Thr595) showing additional peaks upon PGA1 incubation are marked with asterisks (*). This indicates that the PGE1 (a) and PGA1 (c) interaction with Nurr1-LBD matches the typical two-state binding model (P + L ⇄ PL) and an induced-fit binding model (P + L ⇄ PLopen → PLclosed), respectively. (d) Mapping of Nurr1-LBD residues perturbed in the presence of PGA1 reveals that both PGE1 (a and b) and PGA1 (c and d) recognize the same binding region on Nurr1-LBD, with maximum perturbation observed in helices H11 and H12. Residues showing chemical shifts and line broadening are coloured in purple while L410 is coloured in red (b and d), as its peak disappeared upon PGA1 binding. (e, f) PGA1 increases the transcriptional activity of Nurr1-based reporter constructs: Nurr1-LBD-dependent (e) and full-length Nurr1-dependent (f) transcriptional activities in SK-N-BE(2)C cells. n = 3 independent experiments, Data are presented as mean ± s.e.m.

Extended Data Fig. 3 Chemical shift perturbation plot of Nurr1-LBD upon PGE1 and PGA1 binding.

Chemical shift perturbation plot of Nurr1-LBD upon PGE1 (a) / PGA1 (b) binding (1:10 ratio) and their corresponding peak intensity plots (PGE1 (c) / PGA1 (d)) revealing residues with perturbed resonances and/or line broadening upon ligand binding. (*) denotes the peak belonging to L410 which disappeared upon PGA1 binding.

Extended Data Fig. 4 PGE1 conversion to PGA1 under crystal condition.

(a) The overlaid 2Fo-Fc (blue) and composite omit (pink) electron density maps contoured at 1σ cut-off confirming the conversion of PGE1 to PGA1, evident from the covalent bonding density with Cys566. (b) Mass spectrometry data of PGE1 incubated with Nurr1-LBD under crystallization buffer condition (100 mM MES, pH 5.5 and 200 mM MgCl2) confirming the conversion of PGE1 to PGA1, as revealed by the covalent complex molecular mass of 30,862 Da (Nurr1-LBD328–598 is 30,525 Da and PGA1 is 336.5 Da).

Extended Data Fig. 5 Crystal structure of PGA1-bound Nurr1-LBD and its molecular and functional analyses.

(a) Cartoon representation of Nurr1-LBD (blue) with PGA1 shown in sphere mode. (b) Interactions between PGA1 and Nurr1 residues (labelled) through hydrophobic contacts (grey broken lines) and hydrogen bonds (blue broken lines). Only chain B in the asymmetric unit are shown here, as the electron density for the PGA1 attached to this chain was complete. (c) PGJ2 and 15d-PGJ2 show no effect on the transcriptional activity of Nurr1-LBD. n = 3 independent experiments, Data are presented as mean ± s.e.m. (d) 15d-PGJ2 (3 μM), but not PGE1 (1 μM) or PGA1 (10 μM), induces the transcriptional activity of PPARγ-LBD.

Extended Data Fig. 6 Effects of mutations at Nurr1 residues interacting with the chain B (Arg515, His516, Arg563, Thr567).

(a), with the chain A (Phe443, Leu570, Ile573, Leu591) (b), and effects of mutations at the residue Cys566 (c) on PGA1 (10 μM)-induced transcriptional activation of Nurr1-LBD in SK-N-BE(2)C cells. n = 3 independent experiments, Data are presented as mean ± s.e.m.

Extended Data Fig. 7 Effects of EP2 agonists and antagonists on the transcriptional activity of Nurr1-LBD.

(a) The EP2 agonist, AH13205 activates Nurr1’s transcriptional activity, whereas EP3/EP4 agonists (Sulprostone and CAY10598) do not. (b) EP2 antagonist, PF-04418948 suppresses PGE1-induced transcriptional activation of Nurr1, whereas EP1/EP3/EP4 antagonists (SC-19220, L-798106, and L-161982) do not. (c) The synthetic PGE1 analogue misoprostol, activates Nurr1’s transcriptional activity in SK-N-BE(2)C cells. n = 3 independent experiments, Data are presented as mean ± s.e.m.

Extended Data Fig. 8 Protective effects of PGE1 and PGA1 against MPP+ in MN9D cells.

(a, b) Determination of protective effects of PGE1 and PGA1 in MN9D cells under MPP+-induced oxidative stress measured by MTT reduction. (a) Cells were treated with MPP+ (0–1000 µM) for 24 hrs. Cell viabilities assessed by MTT reduction assay show that treatment with 500 µM of MPP+ significantly induces 50% of cell death. (b) Pre-treatments with PGE1/PGA1 (24 hrs prior to MPP+ treatment) increase cell viability against the MPP+ induced oxidative stress in MN9D cells. *P < 0.05, **P < 0.01 compared to 0 µM; ###P < 0.001 compared to the absence of MPP+, unpaired two-tailed t-test; n = 3 independent samples per group. Data are mean ± s.e.m. (c, d) Protective effects of PGE1 and PGA1 measured by LDH release. (c) Cytotoxicity determined by LDH release assay also reveals that treatment with 500 µM of MPP+ significantly induces 50% of cell death in MN9D cells. (d) Similar to MTT reduction assay, pre-treatments with PGE1/PGA1 reduce cytotoxicity under the MPP+-induced oxidative stress. **P < 0.01, ***P < 0.001 compared to 0 µM; ###P < 0.001 compared to the absence of MPP+, unpaired two-tailed t-test; n = 3 independent samples per group. Data are mean ± s.e.m.

Extended Data Fig. 9 Effects of PGE1/PGA1 in the MPTP-induced reduction of DA levels.

The administration of PGE1/PGA1 significantly restores the MPTP-induced reduction of DA levels in the SN (a) and in the striatum (b). One-way ANOVA, Tukey’s post-hoc test; n = 5 per group. Data are mean ± s.e.m.

Extended Data Fig. 10 Mass spectrometry data between PGA1 and Nurr1-LBD under NMR condition.

Mass spectrometry data confirming the formation of the covalent bond between PGA1 (red line) with Nurr1-LBD356–598 (28.035 kDa), while PGE1 (blue dotted line) does not form such a covalent attachment under the NMR buffer conditions (20 mM sodium phosphate (pH 7.5) buffer containing 50 mM NaCl, 0.01% NaN3 in 90% H2O/10% D2O). The apo Nurr1-LBD356–598 (black line) (27.698 kDa) is shown for reference. The molecular weight of PGA1 is 336.5 Da. This also corroborates with the two-state binding and induced-fit model observed from NMR data (Extended Data Fig. 2a, c).

Supplementary information

Supplementary Information

Supplementary Figs. 1–18 and Tables 1–4.

Reporting Summary

Supplementary Video 1

PGA1 binding on Nurr1-LBD

Supplementary Video 2

Superposition of unbound over PGA1 bound Nurr1

Supplementary Video 3

Conformational changes induced by PGA1 binding

Source data

Source Data Fig. 1

Uncut Gel for Fig. 1g

Source Data Fig. 2

Uncut Gel for Fig. 2d

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Rajan, S., Jang, Y., Kim, CH. et al. PGE1 and PGA1 bind to Nurr1 and activate its transcriptional function. Nat Chem Biol 16, 876–886 (2020). https://doi.org/10.1038/s41589-020-0553-6

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