Regulation of p53 Stability and Apoptosis by a ROR Agonist

Activation of p53 function leading to cell-cycle arrest and/or apoptosis is a promising strategy for development of anticancer therapeutic agents. Here, we describe a novel mechanism for stabilization of p53 protein expression via activation of the orphan nuclear receptor, RORa. We demonstrate that treatment of cancer cells with a newly described synthetic ROR agonist, SR1078, leads to p53 stabilization and induction of apoptosis. These data suggest that synthetic ROR agonists may hold utility in the treatment of cancer

Ligand and Receptor Dynamics Contribute to the Mechanism of Graded PPARγ Agonism

SummaryLigand binding to proteins is not a static process, but rather involves a number of complex dynamic transitions. A flexible ligand can change conformation upon binding its target. The conformation and dynamics of a protein can change to facilitate ligand binding. The conformation of the ligand, however, is generally presumed to have one primary binding mode, shifting the protein conformational ensemble from one state to another. We report solution nuclear magnetic resonance (NMR) studies that reveal peroxisome proliferator-activated receptor γ (PPARγ) modulators can sample multiple binding modes manifesting in multiple receptor conformations in slow conformational exchange. Our NMR, hydrogen/deuterium exchange and docking studies reveal that ligand-induced receptor stabilization and binding mode occupancy correlate with the graded agonist response of the ligand. Our results suggest that ligand and receptor dynamics affect the graded transcriptional output of PPARγ modulators

Regulation of p53 Stability and Apoptosis by a ROR Agonist

Activation of p53 function leading to cell-cycle arrest and/or apoptosis is a promising strategy for development of anticancer therapeutic agents. Here, we describe a novel mechanism for stabilization of p53 protein expression via activation of the orphan nuclear receptor, RORa. We demonstrate that treatment of cancer cells with a newly described synthetic ROR agonist, SR1078, leads to p53 stabilization and induction of apoptosis. These data suggest that synthetic ROR agonists may hold utility in the treatment of cancer

Activity-Based Profiling Reveals a Regulatory Link between Oxidative Stress and Protein Arginine Phosphorylation

Protein arginine phosphorylation is a recently discovered modification that affects multiple cellular pathways in Gram-positive bacteria. In particular, the phosphorylation of arginine residues by McsB is critical for regulating the cellular stress response. Given that the highly efficient protein arginine phosphatase YwlE prevents arginine phosphorylation under non-stress conditions, we hypothesized that this enzyme negatively regulates arginine phosphorylation and acts as a sensor of cell stress. To evaluate this hypothesis, we developed the first suite of highly potent and specific SO3-amidine-based YwlE inhibitors. With these protein arginine phosphatase-specific probes, we demonstrated that YwlE activity is suppressed by oxidative stress, which consequently increases arginine phosphorylation, thereby inducing the expression of stress-response genes, which is critical for bacterial virulence. Overall, we predict that these novel chemical tools will be widely used to study the regulation of protein arginine phosphorylation in multiple organisms

RORα regulates p53 function.

<p>A.) Overexpression of RORα in HepG2 cells results in increased expression of p53 target genes including <i>p21</i>, <i>BAX</i>, and <i>PUMA</i>. In control cells where the RORα adenovirus was not included, a control LacZ adenovirus was used. *, indicates p<0.05. Experiments shown in the lower panels were performed identical to the upper panels with the exception of inclusion of siRNA treatments as indicated. B) Cell cycle analysis of control HepG2 cells infected with LacZ adenovirus (top) or RORα adenovirus (bottom). C) Analysis of the number of cells in various stages of the cell cycle in control or RORα overexpressing HepG2 cells. D) Cell cycle analysis of HepG2 cells overexpression RORα (adenovirus treatment) after treatment with either control siRNA or p53 siRNA. E) Analysis of the number of MCF-7 breast cancer cells in sub-G1 infected with control adenovirus (LacZ) or RORα adenovirus. *, indicates p<0.05.</p

Conserved sequence-specific lincRNA-steroid receptor interactions drive transcriptional repression and direct cell fate

The majority of the eukaryotic genome is transcribed, generating a significant number of long intergenic noncoding RNAs (lincRNAs). Although lincRNAs represent the most poorly understood product of transcription, recent work has shown lincRNAs fulfill important cellular functions. In addition to low sequence conservation, poor understanding of structural mechanisms driving lincRNA biology hinders systematic prediction of their function. Here we report the molecular requirements for the recognition of steroid receptors (SRs) by the lincRNA growth arrest-specific 5 (Gas5), which regulates steroid-mediated transcriptional regulation, growth arrest and apoptosis. We identify the functional Gas5-SR interface and generate point mutations that ablate the SR-Gas5 lincRNA interaction, altering Gas5-driven apoptosis in cancer cell lines. Further, we find that the Gas5 SR-recognition sequence is conserved among haplorhines, with its evolutionary origin as a splice acceptor site. This study demonstrates that lincRNAs can recognize protein targets in a conserved, sequence-specific manner in order to affect critical cell functions.Emory-NIH Graduate Training in Pharmacological Studies Grant 5T32GM008602-14; American Heart Association predoctoral fellowship (13PRE16920012); National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under award number RO1DK095750; Breast Cancer Campaign UK; Leukemia and Lymphoma Research UK; Prostate Cancer UK

Identification of a RORE in the <i>SOX4</i> promoter.

<p>A) Schematic representing the <i>SOX4</i> gene. The single exon gene is shown in the diagram the untranslated regions are also shown. The area where significant RORα occupancy was detected in the ChIP-microarray screen is indicated above the gene. The putative RORE is shown below the gene structure and alignment illustrates absolute conservation between xenopus, mouse and human sequences. The RORE is indicated by the underlined sequence. Alignment of the putative <i>SOX4</i> RORE with the prototypic RORE from the prototypic ROR target gene, <i>BMAL1</i>. Right Panel: Screen shot from genome browser indicating regions generating signal from the ChIP/chip study on both <i>SOX4</i> and the positive control <i>ARNTL</i> (<i>BMAL1</i>). B) Chromatin immunoprecipitation assessing the occupancy of RORα at the <i>SOX4</i> promoter. IgG was used as a negative control and RNA polII was used as a positive control. C) Cotransfection assay where a luciferase reporter under the direction of the <i>SOX4</i> promoter was transfected into HEK 293 cells along with a vector directing the expression of RORα. Inclusion of RORα results in stimulation of luciferase expression. The second panel demonstrates that when the RORE is mutated in the <i>SOX4</i> promoter, which inhibits the ability of RORα to bind, RORα no longer has the ability to activate transcription of this reporter. WT, indicates wild type and MT, indicates mutant. Empty expression vector was included in the control wells. D) Adenoviral overexpression of RORα in HepG2 cells results in stimulation of <i>SOX4</i> mRNA expression relative to the LacZ adenovirus control. Suppression of expression of RORα using siRNA results in a reduction of <i>SOX4</i> mRNA. *, indicates p<0.05. E) Western blot illustrating that overexpression of RORα results in increased p53 protein while suppression of RORα expression results in decreased p53 protein expression. F) Analysis of the effect of RORα overexpression on the half-life of p53. HEK293 cells overexpressing either LacZ (control) or RORα were treated with cycloheximide for various amounts of time (0, 10, 60, 90 min) and p53 protein was assessed by western analysis and normalized to tubulin expression. Densitometry was used to assess expression and was signal was normalized to tubulin and the normalized relative (to time 0) expression is indicated below the blots. p53 displayed a half-life of 22±6 min (mean±S.E.) in the absence of RORα and a half-life >90 min with RORα overexpressed.</p