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Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins.

ABSTRACT: B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of cross-talk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins.

SUBMITTER: Godoi PHC

PROVIDER: S-EPMC4933167 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins.

Godoi Paulo H C PHC Wilkie-Grantham Rachel P RP Hishiki Asami A Sano Renata R Matsuzawa Yasuko Y Yanagi Hiroko H Munte Claudia E CE Chen Ya Y Yao Yong Y Marassi Francesca M FM Kalbitzer Hans R HR Matsuzawa Shu-Ichi SI Reed John C JC

The Journal of biological chemistry 20160419 27

B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), mod ...[more]

PMID: 27129202

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Project description:Resveratrol is a polyphenolic phytochemical found in fruits, nuts and vegetables that contributes to the remarkable dietary effects of polyphenolic as inhibitors aging and multiple aging related diseases. In addition, resveratrol has been extensively investigated as an inhibitor of inflammatory diseases including cancer, however, the underlying mechanisms of these chemotherapeutic effects of resveratrol are not completely understood. In cancer cells resveratrol inhibits cell growth, survival, migration and invasion, and many of the effects of resveratrol resemble those observed for bis-indole derived (CDIM) compounds that bind the pro-oncogenic nuclear receptor 4A1 (NR4A1, Nur77) and act as receptor antagonists. Using an isothermal titration calorimetry binding assay, we observed that resveratrol bound to the ligand binding domain of NR4A1 with a KD value of 2.4 µM and a ΔG of -32.2 kJ/mol. Resveratrol also inhibited NR4A1-dependent transactivation in H460 and H1299 lung cancer cells suggesting that resveratrol is an NR4A1 antagonist. This observation was confirmed in a series of functional (cell proliferation, survival, migration and invasion) and gene expression assays in H460 and H1299 cells showing that treatment with resveratrol mimicked the effects of NR4A1 knockdown and were similar to results of previous studies using CDIM/NR4A1 antagonists. These data indicate that applications of resveratrol may be more effective in patients that overexpress NR4A1 which is a negative prognostic factor for patients with some solid tumor-derived cancers. Significance Statement We have examined the mechanism of action of resveratrol and show binding to NR4A1 (KD = 2.4 µM) and inhibition of NR4A1-dependent transactivation in lung cancer cells. Treatment of H460 and H1299 lung cancer cells with resveratrol inhibits cell growth, survival, migration/invasion and related genes, and acts as an NR4A1 antagonist. Resveratrol can now be used more effectively in cancer chemotherapy by targeting patients that overexpress NR4A1 in lung cancer.

Project description:The nuclear receptor subfamily 4 group A member 1 (NR4A1) gene plays a crucial role in both osteoporosis and adipogenesis. The present study investigated the mechanisms by which NR4A1 influences osteoblastogenesis and adipogenesis in human bone marrow‑derived mesenchymal stem cells (BMD‑MSCs). NR4A1 was overexpressed or knocked down in mouse MC3T3‑E1 osteoblast cells and 3T3‑L1 adipocyte cells, as well as in PCS‑500‑012, a BMD‑MSC line. The alkaline phosphatase (ALP) assay and Alizarin Red S staining were performed using MC3T3‑E1 and BMD‑MSCs to assess ALP activity and mineralization, while Oil Red O staining was used to assess the lipid content in 3T3‑L1 cells and BMD‑MSCs. Total RNA was isolated from control, NR4A1‑overexpressing and NR4A1 small interfering RNA (siRNA; siNR4A1)‑treated BMD‑MSCs. RNA sequencing (RNA‑seq) was performed to identify differentially expressed genes, followed by ingenuity pathway analysis (IPA) to determine the role of NR4A1 in osteoblastogenesis and adipogenesis. NR4A1 or Nr4a1 knockdown tended to increase ALP activity and significantly increased calcification in BMD‑MSCs (P<0.005) and MC3T3‑E1 cells (P<0.005), respectively. By contrast, NR4A1 or Nr4a1 overexpression significantly decreased ALP activity and calcification. NR4A1 or Nr4a1 knockdown and overexpression significantly decreased and increased adipogenesis, respectively, in BMD‑MSCs (P<0.005 and <0.05, respectively) and 3T3‑L1 cells (P<0.005 in both). Treatments of BMD‑MSCs with an NR4A1 antagonist, 1,1‑bis(3'‑indolyl)‑1‑(p‑hydroxyphenyl) methane and siNR4A1 showed similar results. RNA‑seq and IPA in control, NR4A1 knockdown and NR4A1 overexpressing cells indicated that Notch signaling mediated the effects of NR4A1 in osteoblastogenesis and adipogenesis. Expression of mastermind‑like transcriptional coactivator 3 was reduced in the Notch signaling pathway in cells treated with siNR4A1. In conclusion, NR4A1 suppressed osteoblastogenesis and promotes adipogenesis in human BMD‑MSCs. The present study also suggested that NR4A1 plays a role in the progression of osteoporosis and adipogenesis by modulating the Notch signaling cascade.

Dataset Information

Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins.

Publications

Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins.

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