Bilirubin is a new ligand for nuclear receptor Liver Receptor Homolog-1.

The nuclear receptor Liver Receptor Homolog-1 (LRH-1, NR5A2 ) binds to phospholipids that regulate important LRH-1 functions in the liver. A recent compound screen unexpectedly identified bilirubin, the product of liver heme metabolism, as a possible ligand for LRH-1. Here, we show unconjugated bilirubin directly binds LRH-1 with apparent K d =9.3uM, altering LRH-1 interaction with all transcriptional coregulator peptides tested. Bilirubin decreased LRH-1 protease sensitivity, consistent with MD simulations predicting bilirubin stably binds LRH-1 within the canonical ligand binding site. Bilirubin activated a luciferase reporter specific for LRH-1, dependent on co-expression with the bilirubin membrane transporter SLCO1B1 , but bilirubin failed to activate ligand-binding genetic mutants of LRH-1. Gene profiling in HepG2 cells shows bilirubin selectively regulated transcripts from endogenous LRH-1 ChIP-seq target genes, which was significantly attenuated by either genetic knockdown of LRH-1, or by a specific chemical competitor of LRH-1. Gene set enrichment suggests bilirubin and LRH-1 share roles in cholesterol metabolism and lipid efflux, thus we propose a new role for LRH-1 in directly sensing intracellular levels of bilirubin.

Hippo and PI5P4K signaling intersect to control the transcriptional activation of YAP.

Phosphoinositides are essential signaling molecules. The PI5P4K family of phosphoinositide kinases and their substrates and products, PI5P and PI4,5P2, respectively, are emerging as intracellular metabolic and stress sensors. We performed an unbiased screen to investigate the signals that these kinases relay and the specific upstream regulators controlling this signaling node. We found that the core Hippo pathway kinases MST1/2 phosphorylated PI5P4Ks and inhibited their signaling in vitro and in cells. We further showed that PI5P4K activity regulated several Hippo- and YAP-related phenotypes, specifically decreasing the interaction between the key Hippo proteins MOB1 and LATS and stimulating the YAP-mediated genetic program governing epithelial-to-mesenchymal transition. Mechanistically, we showed that PI5P interacted with MOB1 and enhanced its interaction with LATS, thereby providing a signaling connection between the Hippo pathway and PI5P4Ks. These findings reveal how these two important evolutionarily conserved signaling pathways are integrated to regulate metazoan development and human disease.

Steroidogenic Factor-1 form and function: From phospholipids to physiology.

Steroidogenic Factor-1 (SF-1, NR5A1) is a member of the nuclear receptor superfamily of ligand-regulated transcription factors, consisting of a DNA-binding domain (DBD) connected to a transcriptional regulatory ligand binding domain (LBD) via an unstructured hinge domain. SF-1 is a master regulator of development and adult function along the hypothalamic pituitary adrenal and gonadal axes, with strong pathophysiological association with endometriosis and adrenocortical carcinoma. SF-1 was shown to bind and be regulated by phospholipids, one of the most interesting aspects of SF-1 regulation is the manner in which SF-1 interacts with phospholipids: SF-1 buries the phospholipid acyl chains deep in the hydrophobic core of the SF-1 protein, while the lipid headgroups remain solvent-exposed on the exterior of the SF-1 protein surface. Here, we have reviewed several aspects of SF-1 structure, function and physiology, touching on other transcription factors that help regulate SF-1 target genes, non-canonical functions of SF-1, the DNA-binding properties of SF-1, the use of mass spectrometry to identify lipids that associate with SF-1, how protein phosphorylation regulates SF-1 and the structural biology of the phospholipid-ligand binding domain. Together this review summarizes the form and function of Steroidogenic Factor-1 in physiology and in human disease, with particular emphasis on adrenal cancer.

Full-length nuclear receptor allosteric regulation.

Nuclear receptors are a superfamily of transcription factors regulated by a wide range of lipids that include phospholipids, fatty acids, heme-based metabolites, and cholesterol-based steroids. Encoded as classic two-domain modular transcription factors, nuclear receptors possess a DNA-binding domain (DBD) and a lipid ligand-binding domain (LBD) containing a transcriptional activation function. Decades of structural studies on the isolated LBDs of nuclear receptors established that lipid-ligand binding allosterically regulates the conformation of the LBD, regulating transcriptional coregulator recruitment and thus nuclear receptor function. These structural studies have aided the development of several FDA-approved drugs, highlighting the importance of understanding the structure-function relationships between lipids and nuclear receptors. However, there are few published descriptions of full-length nuclear receptor structure and even fewer descriptions of how lipids might allosterically regulate full-length structure. Here, we examine multidomain interactions based on the published full-length nuclear receptor structures, evaluating the potential of interdomain interfaces within these nuclear receptors to act as inducible sites of allosteric regulation by lipids.

New High-Throughput Screen Discovers Novel Ligands of Full-Length Nuclear Receptor LRH-1.

Nuclear receptor liver receptor homolog-1 (LRH-1, NR5A2) is a lipid-regulated transcription factor and an important drug target for several liver diseases. Advances toward LRH-1 therapeutics have been driven recently by structural biology, with fewer contributions from compound screening. Standard LRH-1 screens detect compound-induced interaction between LRH-1 and a transcriptional coregulator peptide, an approach that excludes compounds that regulate LRH-1 through alternative mechanisms. Here, we developed a FRET-based LRH-1 screen that simply detects compound binding to LRH-1, applying it to discover 58 new compounds that bind the canonical ligand-binding site in LRH-1 (2.5% hit rate), also supported by computational docking. Four independent functional screens identified 15 of these 58 compounds to also regulate LRH-1 function in vitro or in living cells. Although one of these 15 compounds, abamectin, directly binds LRH-1 and regulates full-length LRH-1 in cells, abamectin failed to regulate the isolated ligand-binding domain in standard coregulator peptide recruitment assays using PGC1α, DAX-1, or SHP. Abamectin treatment of human liver HepG2 cells selectively regulated endogenous LRH-1 ChIP-seq target genes and pathways associated with known LRH-1 functions in bile acid and cholesterol metabolism. Thus, the screen reported here can discover compounds not likely to have been identified in standard LRH-1 compound screens but which bind and regulate full-length LRH-1 in cells.

Fragile X Mental Retardation Protein positively regulates PKA anchor Rugose and PKA activity to control actin assembly in learning/memory circuitry.

Recent work shows Fragile X Mental Retardation Protein (FMRP) drives the translation of very large proteins (>2000 aa) mediating neurodevelopment. Loss of function results in Fragile X syndrome (FXS), the leading heritable cause of intellectual disability (ID) and autism spectrum disorder (ASD). Using the Drosophila FXS disease model, we discover FMRP positively regulates the translation of the very large A-Kinase Anchor Protein (AKAP) Rugose (>3000 aa), homolog of ASD-associated human Neurobeachin (NBEA). In the central brain Mushroom Body (MB) circuit, where Protein Kinase A (PKA) signaling is necessary for learning/memory, FMRP loss reduces Rugose levels and targeted FMRP overexpression elevates Rugose levels. Using a new in vivo transgenic PKA activity reporter (PKA-SPARK), we find FMRP loss reduces PKA activity in MB Kenyon cells whereas FMRP overexpression elevates PKA activity. Consistently, loss of Rugose reduces PKA activity, but Rugose overexpression has no independent effect. A well-established PKA output is regulation of F-actin cytoskeleton dynamics. In the FXS disease model, F-actin is aberrantly accumulated in MB lobes and single MB Kenyon cells. Consistently, Rugose loss results in similar F-actin accumulation. Moreover, targeted FMRP, Rugose and PKA overexpression all result in increased F-actin accumulation in the MB circuit. These findings uncover a FMRP-Rugose-PKA mechanism regulating actin cytoskeleton. This study reveals a novel FMRP mechanism controlling neuronal PKA activity, and demonstrates a shared mechanistic connection between FXS and NBEA associated ASD disease states, with a common link to PKA and F-actin misregulation in brain neural circuits. SIGNIFICANCE STATEMENT: Autism spectrum disorder (ASD) arises from a wide array of genetic lesions, and it is therefore critical to identify common underlying molecular mechanisms. Here, we link two ASD states; Neurobeachin (NBEA) associated ASD and Fragile X syndrome (FXS), the most common inherited ASD. Using established Drosophila disease models, we find Fragile X Mental Retardation Protein (FMRP) positively regulates translation of NBEA homolog Rugose, consistent with a recent advance showing FMRP promotes translation of very large proteins associated with ASD. FXS exhibits reduced cAMP induction, a potent activator of PKA, and Rugose/NBEA is a PKA anchor. Consistently, we find brain PKA activity strikingly reduced in both ASD models. We discover this pathway regulation controls actin cytoskeleton dynamics in brain neural circuits.

Trend analysis of major cancer statistics according to sex and severity levels in Korea.

Existing epidemiologic reports or studies of cancer statistics in Korea lack sufficient data on cancer severity distributions and observed survival rates. This study analyzed trends in major cancer statistics according to sex and severity levels in Korea from 2006 to 2013. We included eight cancers (hepatocellular carcinoma, and thyroid, colorectal, gastric, lung, prostate, breast, and cervical cancer), using Korea Central Cancer Registry data. Severity level was classified by Surveillance, Epidemiology, and End Results (SEER) stage as follows: localized, regional, distant, or unknown. Numbers of incident cancer cases from 2006 to 2013 were described by sex and SEER stage. We estimated up to 8-year observed survival rates of major cancers by sex and SEER stage, and provided prevalence rates by sex and SEER stage in 2011, 2012, and 2013. Although increases in new cancer cases are slowing and the total number of incident cancer cases in 2013 decreased for the first time since 2006, the number of prevalent cancer cases was 663,530 in 2013, an increase of 13.3% compared to 2011. Among the five cancers affecting both sexes, sex-related differences in 5-year observed survival rates for lung cancer were greatest in the localized stage (men, 31.9%; women, 48.1%), regional stage (men, 20.0%; women, 31.3%), and unknown stage (men, 24.3%; women, 37.5%). The sum of the proportions of localized and regional stages for thyroid and breast cancer was over 90% in 2013, while the sum of the proportions of localized and regional stages for lung cancer was only 56.7% in 2013. Differences in observed survival rates between men and women were prominent in lung cancer for all SEER stages. The reported epidemiologic data from this study can be used to obtain a more valid measure of cancer burden using a summary measure of population health.