Fine spatial assembly for construction of the phenol-binding pocket to capture bisphenol A in the human nuclear receptor estrogen-related receptor γ

Various lines of evidence have shown that bisphenol A (BPA) acts as an endocrine disruptor that affects various hormones even at merely physiological levels. We demonstrated recently that BPA binds strongly to human nuclear receptor estrogen-related receptor γ (ERRγ), one of 48 nuclear receptors. Based on X-ray crystal analysis of the ERRγ ligand-binding domain (LBD)/BPA complex, we demonstrated that ERRγ receptor residues, Glu275 and Arg316, function as the intrinsic-binding site of the phenol-hydroxyl group of BPA. If these phenol-hydroxyl?Glu275 and Arg316 hydrogen bonds anchor the A-benzene ring of BPA, the benzene-phenyl group of BPA would be in a pocket constructed by specific amino acid side chain structures. In the present study, by evaluating the Ala-replaced mutant receptors, we identified such a ligand-binding pocket. Leu268, Leu271, Leu309 and Tyr326, in addition to the previously reported participants Glu275 and Arg316, were found to make a receptacle pocket for the A-ring, whereas Ile279, Ile310 and Val313 were found to assist or structurally support these residues. The results revealed that each amino acid residue is an essential structural element for the strong binding of BPA to ERRγ.     

Kaempferol is an estrogen-related receptor α and γ inverse agonist

Kaempferol is a dietary flavonoid that is thought to function as a selective estrogen receptor modulator. In this study, we established that kaempferol also functions as an inverse agonist for estrogen-related receptors alpha and gamma (ERRα and ERRγ). We demonstrated that kaempferol binds to ERRα and ERRγ and blocks their interaction with coactivator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). Kaempferol also suppressed the expressions of ERR-target genes pyruvate dehydrogenase kinase 2 and 4 (PDK2 and PDK4). This evidence suggests that kaempferol may exert some of its biological effect through both estrogen receptors and estrogen-related receptors.

Structured summary:

MINT-6824653:PGC-1 alpha (uniprotkb:Q9UBK2) and ERR gamma (uniprotkb: P62508) bind (MI:0407) by surface plasmon resonance (MI:0107)     

Estrogen‐related receptor gamma regulates dopaminergic neuronal phenotype by activating GSK3β/NFAT signaling in SH‐SY5Y cells

The orphan nuclear receptor estrogen‐related receptor gamma (ERRγ) is highly expressed in the nervous system during embryogenesis and in adult brains, but its physiological role in neuronal development remains unknown. In this study, we evaluated the relevance of ERRγ in regulating dopaminergic (DAergic) phenotype and the corresponding signaling pathway. We used retinoic acid (RA) to differentiate human neuroblastoma SH‐SY5Y cells. RA induced neurite outgrowth of SH‐SY5Y cells with an increase in DAergic neuron‐like properties, including up‐regulation of tyrosine hydroxylase, dopamine transporter, and vesicular monoamine transporter 2. ERRγ, but not ERRα, was up‐regulated by RA, and participated in RA effect on SH‐SY5Y cells. ERRγ over‐expression enhanced mature DAergic neuronal phenotype with neurite outgrowth as with RA treatment; and RA‐induced increase in DAergic phenotype was attenuated by silencing ERRγ expression. ERRγ appears to have a crucial role in morphological and functional regulation of cells that is selective for DAergic neurons. Polo‐like kinase 2 was up‐regulated in ERRγ‐over‐expressing SH‐SY5Y cells, which was involved in phosphorylation of glycogen synthase kinase 3β and resulting downstream activation of nuclear factor of activated T cells. The likely involvement of ERRγ in regulating the DAergic neuronal phenotype makes this orphan nuclear receptor a novel target for understanding DAergic neuronal differentiation.

     

N-terminal half of the cGMP phosphodiesterase gamma-subunit contributes to stabilization of the GTPase-accelerating protein complex

In the visual signal terminating transition state, the cyclic GMP phosphodiesterase (PDE6) inhibitory γ-subunit (PDEγ) stimulates GTPase activity of the α-subunit of transducin (αt) by enhancing the interaction between αt and its regulator of G protein signaling (RGS9), which is constitutively bound to the type 5 G protein β-subunit (β5). Although it is known from a crystal structure of partial molecules that the PDEγ C terminus contacts with both αt and RGS9, contributions from the intrinsically disordered PDEγ N-terminal half remain unclear. In this study, we were able to investigate this issue using a photolabel transfer strategy that allows for mapping the interface of full-length proteins. We observed label transfer from PDEγ N-terminal positions 50, 30, and 16 to RGS9·β5 in the GTPase-accelerating protein (GAP) complex composed of PDEγ·αt·RGS9·β5. In support of a direct PDEγ N-terminal interaction with RGS9·β5, the PDEγ N-terminal peptide PDEγ(1-61) abolished label transfer to RGS9·β5, and another N-terminal peptide, PDEγ(10-30), disassembled the GAP complex in label transfer and pulldown experiments. Furthermore, we determined that the PDEγ C-terminal interaction with αt was enhanced whereas the N-terminal interaction was weakened upon changing the αt conformation from the signaling state to the transition state. This "rearrangement" of PDEγ domain interactions with αt appears to facilitate the interaction of the PDEγ N-terminal half with RGS9·β5 and hence its contribution to optimal stabilization of the GAP complex.     

Estrogen-related receptor (ERR) γ protects against puromycin aminonucleoside-induced podocyte apoptosis by targeting PI3K/Akt signaling

Accumulating evidence has shown that podocyte apoptosis is of vital importance for the development of glomerulosclerosis and progressive loss of renal function. However, the molecular mechanisms leading to podocyte apoptosis are still elusive. In this study, we investigated the role of estrogen-related receptor (ERR) γ in podocyte apoptosis, as well as the underlying mechanisms. Treatment of PAN caused a dose- and time-dependent podocyte apoptosis in line with a significant downregulation of ERRγ. Interestingly, the occurrence of ERRγ downregulation appeared earlier than the onset of cell apoptosis, suggesting a potential that ERRγ reduction triggered apoptotic response in podocyte. To test this hypothesis, ERRγ siRNA was administered to the podocytes. Strikingly, ERRγ silencing resulted in a significant cell apoptosis accompanied with increased injury markers of B7-1 and cathepsin L and decreased podocyte protein nephrin. In contrast, overexpression of ERRγ remarkably attenuated PAN-induced cell apoptosis. Moreover, ERRγ overexpression stimulated PI3K/Akt signaling pathway evidenced by increased expression of PI3K subunits p85α and p110α and phosphorylated Akt. Importantly, a specific PI3K inhibitor LY294002 entirely reversed the anti-apoptotic effect of ERRγ following PAN treatment. Finally, we observed a striking downregulation of ERRγ in PAN-treated rat kidneys, suggesting that our cell model replicated the in vivo condition. Taken together, these data highly suggested that ERRγ played a novel role in modulating podocyte apoptosis by targeting PI3K/Akt signaling pathway.     

G protein-coupled receptor kinase 4gamma interacts with inactive Galpha(s) and Galpha13

G protein-coupled receptors (GPCRs) are regulated by multiple families of kinases including GPCR kinases (GRKs). GRK4 is constitutively active towards GPCRs, and polymorphisms of GRK4γ are linked to hypertension. We examined, through co-immunoprecipitation, the interactions between GRK4γ and the Gα and Gβ subunits of heterotrimeric G proteins. Because GRK4 has been shown to inhibit Gα_s-coupled GPCR signaling and lacks a PH domain, we hypothesized that GRK4γ would interact with active Gα_s, but not Gβ. Surprisingly, GRK4γ preferentially interacts with inactive Gα_s and Gβ to a greater extent than active Gα_s. GRK4γ also interacts with inactive Gα₁₃ and Gβ. Functional studies demonstrate that wild-type GRK4γ, but not kinase-dead GRK4γ, ablates isoproterenol-mediated cAMP production indicating that the kinase domain is responsible for GPCR regulation. This evidence suggests that binding to inactive Gα_s and Gβ may explain the constitutive activity of GRK4γ towards Gα_s-coupled receptors.