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 crosstalk 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.In metazoans, Bcl-2 5 family proteins regulate several cellular processes, most prominently apoptosis (programmed cell death) and also autophagy (1, 2). Based on primary amino acid sequence similarity, members of the Bcl-2 protein family share at least one of four modular components, termed Bcl-2 homology (BH) domains. Of these, BH3 has an amphipathic ␣-helical structure and plays a pivotal role in both the activation of proapoptotic and the suppression of anti-apoptotic members of the family. For example, the BH3 domains of Bim, Bid, and Puma trigger conformational changes in the executioners Bax and Bak, leading to their oligomerization and permeation of the outer mitochondrial membrane. Downstream consequences of Bax and Bak activation include release of cytochrome c and, ultimately, the activation of caspase-9 and other cell death proteases. Conversely, the BH3 domains of these and other BH3-carrying proteins bind anti-apoptotic Bcl-2 family proteins and inhibit their ability to prevent Bax/Bak oligomerization in the outer mitochondrial membrane.The BH3 domain of pro-apoptotic proteins operates analogous to a ligand to interact with a predominantly hydrophobic receptor-like binding crevice on the surfaces of anti-apoptotic Bcl-2 family proteins. In addition to regulating apoptosis, the BH3-binding pocket on anti-apoptotic Bcl-2 family proteins has been reported to bind a BH3-like domain in the autophagy protein Beclin-1, and this interaction has been shown to play an important role in regulating autophagy (3). Prior work has dissected the binding affinity of various BH3 peptides (ligands) to anti-apoptotic Bcl-2 proteins (receptors), and three-dimensional structural data are available for several of these pairs (reviewed in Ref. 4).Targeting the BH3-binding cleft of anti-apo...

The nuclear receptor NR4A1 is regulated by SUMO modification to induce autophagic cell death

“…Furthermore, Dendrogenin A, a mammalian cholesterol metabolite ligand of liver-X-receptors (LCRs) induces Nr4a1 expression in association with excessive autophagic cell death both in vitro and in vivo [15,16], displaying anticancer and chemopreventive properties in mice [17]. Interestingly, NR4A1 interaction with anti-apoptotic BCL2 family members outside the BH3 domain induces autophagic cell death [18], which seems to be mediated by releasing BECN1 in a model of cigarette smokeinduced autophagic cell death [19]. Taken together, these findings indicate that NR4A1 could function as a broad inducer of autophagic cell death.…”

The nuclear receptor NR4A1 is regulated by SUMO modification to induce autophagic cell death

“…Furthermore, Dendrogenin A, a mammalian cholesterol metabolite ligand of liver-X-receptors (LCRs) induces Nr4a1 expression in association with excessive autophagic cell death both in vitro and in vivo (15, 16), displaying anticancer and chemopreventive properties in mice(17). Interestingly, NR4A1 interaction with anti-apoptotic BCL2 family members outside the BH3 domain induces autophagic cell death (18), which seems to be mediated by releasing BECN1 in a model of cigarette smoke-induced autophagic cell death(19). Taken together, these findings indicate that NR4A1 could function as a broad inducer of autophagic cell death.…”

Clustered microRNAs: The molecular mechanism supporting the maintenance of luteal function during early pregnancy

“…NR4A1 was previously shown to regulate the transcription of cytochrome P450 C21 and C17 in rodents, consequently leading to the increased synthesis of cortisol and androgen, respectively 48,49. However, it also regulates the expression of genes involved in apoptosis or in the metabolism of progesterone (ie, AKR1C1) 36,50. Previously, NR4A1 was identified as a marker of luteolysis in the corpus luteum of cow and rat 36,37.…”

“…Although that binding was not perfectly matched, it is still possible that NR4A1 can induce the expression of AKR1C1. It was also found that NR4A1 can induce apoptosis by interacting with antiapoptotic proteins,50 suggesting that this factor can have additional roles in the porcine luteal tissue during regression. Interestingly, the expression of NR4A1 and couple other early response genes increase 30 minutes after PGF 2α injection in bovine midcycle corpus luteum, while 1.5 hours later plasma progesterone concentration decreases 52.…”