Molecular basis of ligand-dependent Nurr1-RXRα activation
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eLife assessment
This is a fundamental study of the activation process of Nurr1, an orphan nuclear receptor that may be a significant target for the treatment of neurodegenerative disorders. Nurr1 functions as a monomer, but may also heterodimerize with RXR which represses Nurr1 transcriptional activation. The authors provide compelling evidence for Nurr1 activation through ligand-induced dissociation of an inactive Nurr1-RXRa heterodimer. These data will be important for biochemists and cell biologists working on regulatory / activation mechanisms of nuclear hormone receptors.
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Abstract
Small molecule compounds that activate transcription of Nurr1-retinoid X receptor alpha (RXRα) (NR4A2-NR2B1) nuclear receptor heterodimers are implicated in the treatment of neurodegenerative disorders, but function through poorly understood mechanisms. Here, we show that RXRα ligands activate Nurr1-RXRα through a mechanism that involves ligand-binding domain (LBD) heterodimer protein-protein interaction (PPI) inhibition, a paradigm distinct from classical pharmacological mechanisms of ligand-dependent nuclear receptor modulation. NMR spectroscopy, PPI, and cellular transcription assays show that Nurr1-RXRα transcriptional activation by RXRα ligands is not correlated with classical RXRα agonism but instead correlated with weakening Nurr1-RXRα LBD heterodimer affinity and heterodimer dissociation. Our data inform a model by which pharmacologically distinct RXRα ligands (RXRα homodimer agonists and Nurr1-RXRα heterodimer selective agonists that function as RXRα homodimer antagonists) operate as allosteric PPI inhibitors that release a transcriptionally active Nurr1 monomer from a repressive Nurr1-RXRα heterodimeric complex. These findings provide a molecular blueprint for ligand activation of Nurr1 transcription via small molecule targeting of Nurr1-RXRα.
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eLife assessment
This is a fundamental study of the activation process of Nurr1, an orphan nuclear receptor that may be a significant target for the treatment of neurodegenerative disorders. Nurr1 functions as a monomer, but may also heterodimerize with RXR which represses Nurr1 transcriptional activation. The authors provide compelling evidence for Nurr1 activation through ligand-induced dissociation of an inactive Nurr1-RXRa heterodimer. These data will be important for biochemists and cell biologists working on regulatory / activation mechanisms of nuclear hormone receptors.
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Reviewer #1 (Public Review):
The nuclear receptor Nurr1 is a target of interest in neurodegenerative diseases like Parkinson's and Alzheimer's, but its mechanism of activation on NBRE-containing promoters and potential druggability is unknown. A heterodimer of Nurr1 with RXRa can be activated by a subset of ligands that bind to the RXRa ligand binding domain (LBD). Here, the authors provide evidence that transcriptional activation occurs through ligand-induced dissociation of the heterodimer, leading to an active Nurr1 monomer.
NMR spectroscopy and other biophysical, biochemical, and cell-based assays provide a strong foundation for the work. The manuscript is well-written and easy to follow, and for the most part, it thoughtfully addresses experimental results and data interpretation with reasonable caveats. However, a reliance on …
Reviewer #1 (Public Review):
The nuclear receptor Nurr1 is a target of interest in neurodegenerative diseases like Parkinson's and Alzheimer's, but its mechanism of activation on NBRE-containing promoters and potential druggability is unknown. A heterodimer of Nurr1 with RXRa can be activated by a subset of ligands that bind to the RXRa ligand binding domain (LBD). Here, the authors provide evidence that transcriptional activation occurs through ligand-induced dissociation of the heterodimer, leading to an active Nurr1 monomer.
NMR spectroscopy and other biophysical, biochemical, and cell-based assays provide a strong foundation for the work. The manuscript is well-written and easy to follow, and for the most part, it thoughtfully addresses experimental results and data interpretation with reasonable caveats. However, a reliance on simple correlative analyses, including some with rather modest correlations (R2 values {less than or equal to} 0.5), may fail to account for some potentially interesting outlier ligands and oversimplify conclusions. Despite this possible oversimplification, this manuscript provides solid evidence of their discovery of an interesting mechanism by which a subset of RXRɑ ligands leads to transcriptional activation of Nurr1 at NBRE promoters--this is an exciting finding that could be potentially relevant in the development of neuroprotective therapies.
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Reviewer #2 (Public Review):
Nurr1 is a nuclear receptor and is important for mammalian brain development and homeostasis. Dysfunctional Nurr1 transcriptional activities are implicated in neurodegenerative diseases like Parkinson's. This exquisite ligand-dependent and specific transcriptional reprogramming make nuclear receptors ideal drug targets. However, the design of Nurr1-selective ligands has been confounded by the fact that Nurr1's ligand binding pocket appears to collapse in x-ray crystal structures. Interestingly, RXRalpha-targeted ligands, Nurr1's obligate heterodimer binding partner, show differential effects on Nurr1's transcriptional activities. In this study, the authors aimed to address how RXRalpha ligands lead to Nurr1 transcriptional activation. By combining biochemical approaches, NMR spectroscopy, and transcriptional …
Reviewer #2 (Public Review):
Nurr1 is a nuclear receptor and is important for mammalian brain development and homeostasis. Dysfunctional Nurr1 transcriptional activities are implicated in neurodegenerative diseases like Parkinson's. This exquisite ligand-dependent and specific transcriptional reprogramming make nuclear receptors ideal drug targets. However, the design of Nurr1-selective ligands has been confounded by the fact that Nurr1's ligand binding pocket appears to collapse in x-ray crystal structures. Interestingly, RXRalpha-targeted ligands, Nurr1's obligate heterodimer binding partner, show differential effects on Nurr1's transcriptional activities. In this study, the authors aimed to address how RXRalpha ligands lead to Nurr1 transcriptional activation. By combining biochemical approaches, NMR spectroscopy, and transcriptional reporter gene assays in neuronal cells, the authors convincingly show that these select RXRalpha ligands elicit an allosteric effect that reduces Nurr1 binding affinity. They further show that monomeric Nurr1 is a highly effective enhancer of the promoter that is repressed in the presence of RXRalpha. Overall, this is a well-presented and robust study as presented and the conclusions are supported by their evidence. This study should have a profound impact on the field as it provides a clear structural mechanism for ligand-dependent Nurr1 activation in neuronal cells.
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Reviewer #3 (Public Review):
Nurr1 is an orphan nuclear receptor that may be a significant target for the treatment of neurodegenerative disorders. Targeting Nurr1 with small molecule ligands has been challenging, but there has been some progress in the identification of synthetic ligands that appear to increase Nurr1 activity. Nurr1 functions as a monomer, but may also heterodimerize with RXR. Heterodimerization appears to repress Nurr1 transcriptional activation via NBRE-driven reporters. Importantly, small molecule ligands that appear to selectively activate the Nurr1/RXR heterodimer complex (and not the Nurr1 or RXR homodimers, individually) have been identified. Exactly how these ligands function in this manner is unclear.
Here, the authors demonstrate that Nurr-1/RXR agonists actually function by perturbing the heterodimer …
Reviewer #3 (Public Review):
Nurr1 is an orphan nuclear receptor that may be a significant target for the treatment of neurodegenerative disorders. Targeting Nurr1 with small molecule ligands has been challenging, but there has been some progress in the identification of synthetic ligands that appear to increase Nurr1 activity. Nurr1 functions as a monomer, but may also heterodimerize with RXR. Heterodimerization appears to repress Nurr1 transcriptional activation via NBRE-driven reporters. Importantly, small molecule ligands that appear to selectively activate the Nurr1/RXR heterodimer complex (and not the Nurr1 or RXR homodimers, individually) have been identified. Exactly how these ligands function in this manner is unclear.
Here, the authors demonstrate that Nurr-1/RXR agonists actually function by perturbing the heterodimer formation providing Nurr1 monomers that are much more active in driving transcription. The authors demonstrate this with a range of biochemical, biophysical, and cell-based methodologies. Cotransfection assays examining the activity of Nurr1 on an NBRE reporter illustrate that RXRalpha is a repressor of Nurr1 transcriptional activity and that this is mediated by the RXR LBD. Using this experimental model as well as RXR coactivator interaction assays (biochemical) and RXR/DR1 reporter cotransfection assays, the authors examined multiple classes of RXR ligands (RXR agonists, modulators, antagonists, and Nurr1/RXRa selective agonists) to compare their activities. The Nurr1/RXR heterodimer agonists were quite effective at inducing transcription in the Nurr1/RXR assay but relatively ineffective in the RXR - coactivator binding assay or the RXR cotransfection assay. Using the array of ligands, the authors show that the Nurr1/RXR activity does not correlate to the ability of compounds to induce RXR to recruit a coactivator or activate RXR-mediated transcription. This suggests that Nurr1/RXR heterodimer agonists may not be mediating transcriptional activation via the "standard" mechanism. One weakness here is that some compounds used in the Nurr1/RXR transcription assay are not included in the other assays and may not have been included in the correlation studies. Assessment of RXR/Nurr1 dimerization in the presence of the ligands was assessed by ITC and demonstrated a correlation between the weakening of heterodimer formation and Nurr1/transcriptional activity suggesting that modulation of dimerization may be a mechanism by which the Nurr1/RXR heterodimer specific ligands function. NMR and analytical SEC data support this hypothesis as well. With regard to the physiological significance of these observations, no studies were completed on actual Nurr1 target genes addressing this type of mechanism offering limitations on the applicability of the hypothesis. However, the mechanism proposed is strongly supported by the data and offers a novel paradigm for the development of drugs targeting this receptor and possibly other nuclear receptors as well.
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