Role of NeuroD1 on the negative regulation of Pomc expression by glucocorticoid.

The mechanism of the negative regulation of proopiomelanocortin gene (Pomc) by glucocorticoids (Gcs) is still unclear in many points. Here, we demonstrated the involvement of neurogenic differentiation factor 1 (NeuroD1) in the Gc-mediated negative regulation of Pomc. Murine pituitary adrenocorticotropic hormone (ACTH) producing corticotroph tumor-derived AtT20 cells were treated with dexamethasone (DEX) (1-100 nM) and cultured for 24 hrs. Thereafter, Pomc mRNA expression was studied by quantitative real-time PCR and rat Pomc promoter (-703/+58) activity was examined by luciferase assay. Both Pomc mRNA expression and Pomc promoter activity were inhibited by DEX in a dose-dependent manner. Deletion and point mutant analyses of Pomc promoter suggested that the DEX-mediated transcriptional repression was mediated via E-box that exists at -376/-371 in the promoter. Since NeuroD1 is known to bind to and activate E-box of the Pomc promoter, we next examined the effect of DEX on NeuroD1 expression. Interestingly, DEX dose-dependently inhibited NeuroD1 mRNA expression, mouse NeuroD1 promoter (-2.2-kb) activity, and NeuroD1 protein expression in AtT20 cells. In addition, we confirmed the inhibitory effect of DEX on the interaction of NeuroD1 and E-box on Pomc promoter by chromatin immunoprecipitation (ChIP) assay. Finally, overexpression of mouse NeuroD1 could rescue the DEX-mediated inhibition of Pomc mRNA expression and Pomc promoter activity. Taken together, it is suggested that the suppression of NeuroD1 expression and the inhibition of NeuroD1/E-box interaction may play an important role in the Gc-mediated negative regulation of Pomc

Inhibitory Effects of a Novel PPAR- γ

Although therapeutic effects of the peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists rosiglitazone and pioglitazone against Cushing’s disease have been reported, their effects are still controversial and inconsistent. We therefore examined the effects of a novel PPAR-γ agonist, MEKT1, on Pomc expression/ACTH secretion using murine corticotroph-derived AtT20 cells and compared its effects with those of rosiglitazone and pioglitazone. AtT20 cells were treated with either 1 nM~10 μM MEKT1, rosiglitazone, or pioglitazone for 24 hours. Thereafter, their effects on proopiomelanocortin gene (Pomc) mRNA expression were studied by qPCR and the Pomc promoter (−703/+58) activity was demonstrated by luciferase assay. Pomc mRNA expression and promoter activity were significantly inhibited by MEKT1 at 10 μM compared to rosiglitazone and pioglitazone. SiRNA-mediated PPAR-γ knockdown significantly abrogated MEKT1-mediated Pomc mRNA suppression. ACTH secretion from AtT20 cells was also significantly inhibited by MEKT1. Deletion/point mutant analyses of Pomc promoter indicated that the MEKT1-mediated suppression was mediated via NurRE, TpitRE, and NBRE at −404/-383, −316/-309, and −69/-63, respectively. Moreover, MEKT1 significantly suppressed Nur77, Nurr1, and Tpit mRNA expression. MEKT1 also was demonstrated to inhibit the protein-DNA interaction of Nur77/Nurr1-NurRE, Tpit-TpitRE, and Nur77-NBRE by ChIP assay. Taken together, it is suggested that MEKT1 could be a novel therapeutic medication for Cushing’s disease

Endogenous Purification of NR4A2 (Nurr1) Identified Poly(ADP-Ribose) Polymerase 1 as a Prime Coregulator in Human Adrenocortical H295R Cells

Aldosterone is synthesized in zona glomerulosa of adrenal cortex in response to angiotensin II. This stimulation transcriptionally induces expression of a series of steroidogenic genes such as HSD3B and CYP11B2 via NR4A (nuclear receptor subfamily 4 group A) nuclear receptors and ATF (activating transcription factor) family transcription factors. Nurr1 belongs to the NR4A family and is regarded as an orphan nuclear receptor. The physiological significance of Nurr1 in aldosterone production in adrenal cortex has been well studied. However, coregulators supporting the Nurr1 function still remain elusive. In this study, we performed RIME (rapid immunoprecipitation mass spectrometry of endogenous proteins), a recently developed endogenous coregulator purification method, in human adrenocortical H295R cells and identified PARP1 as one of the top Nurr1-interacting proteins. Nurr1-PARP1 interaction was verified by co-immunoprecipitation. In addition, both siRNA knockdown of PARP1 and treatment of AG14361, a specific PARP1 inhibitor suppressed the angiotensin II-mediated target gene induction in H295R cells. Furthermore, PARP1 inhibitor also suppressed the aldosterone secretion in response to the angiotensin II. Together, these results suggest PARP1 is a prime coregulator for Nurr1

Identification and Functional Characterization of a Novel Androgen Receptor Coregulator, EAP1

The androgen receptor (AR) plays an essential role in the development of prostate cancer, and androgen-deprivation therapy is used as a first-line treatment for prostate cancer. However, under androgen-deprivation therapy, castration-resistant prostate cancer inevitably arises, suggesting that the interacting transcriptional coregulators of AR are promising targets for developing novel therapeutics. In this study, we used novel proteomic techniques to evaluate the AR interactome, including biochemically labile binding proteins, which might go undetected by conventional purification methods. Using rapid immunoprecipitation mass spectrometry of endogenous proteins, we identified enhanced at puberty 1 (EAP1) as a novel AR coregulator, whereas its interaction with AR could not be detected under standard biochemical conditions. EAP1 enhanced the transcriptional activity of AR via the E3 ubiquitin ligase activity, and its ubiquitination substrate proteins included AR and HDAC1. Furthermore, in prostate cancer specimens, EAP1 expression was significantly correlated with AR expression as well as a poor prognosis of prostate cancer. Together, these results suggest that EAP1 is a novel AR coregulator that promotes AR activity and potentially plays a role in prostate cancer progression

Flexible Coordination Polymers Composed of Luminescent Ruthenium(II) Metalloligands: Importance of the Position of the Coordination Site in Metalloligands

Coordination polymerization reactions between ruthenium­(II) metalloligands [Ru­(<i>n</i>,<i>n</i>′-dcbpy)]^4– (<b>[</b><i><b>n</b></i><b>Ru]</b>; <i>n</i> = 4, 5; <i>n</i>,<i>n</i>′-dcbpy = <i>n</i>,<i>n</i>′-dicarboxy-2,2′-bipyridine) and several divalent metal salts in basic aqueous solutions afforded porous luminescent complexes formulated as [Mg­(H₂O)₆]­{[Mg­(H₂O)₃]­[4Ru]·4H₂O} (<b>Mg</b>_<b>2</b><b>[4Ru]·13H</b>_<b>2</b><b>O</b>), [Mg₂(H₂O)₉]­[5Ru]·10H₂O (<b>Mg</b>_<b>2</b><b>[5Ru]·19H</b>_<b>2</b><b>O</b>), {[Sr₄(H₂O)₉]­[4Ru]₂·9H₂O} (<b>Sr</b>_<b>2</b><b>[4Ru]·9H</b>_<b>2</b><b>O</b>)₂, {[Sr₂(H₂O)₈]­[5Ru]·6H₂O} (<b>Sr</b>_<b>2</b><b>[5Ru]·14H</b>_<b>2</b><b>O</b>), and {[Cd₂(H₂O)₂]­[5Ru]·10H₂O} (<b>Cd</b>_<b>2</b><b>[5Ru]·12H</b>_<b>2</b><b>O</b>). Single-crystal X-ray structural analyses revealed that the divalent metal ions were commonly coordinated by the carboxyl groups of the <b>[</b><i><b>n</b></i><b>Ru]</b> metalloligand, forming porous frameworks with a void fraction varying from 11.4% <b>Mg</b>_<b>2</b><b>[4Ru]·13H</b>_<b>2</b><b>O</b> to 43.9% <b>Cd</b>_<b>2</b><b>[5Ru]·12H</b>_<b>2</b><b>O</b>. <b>M</b>_<b>2</b><b>[4Ru]·</b><i><b>n</b></i><b>H</b>_<b>2</b><b>O</b> showed a reversible structural transition accompanied by water and methanol vapor adsorption/desorption, while the porous structures of <b>M</b>_<b>2</b><b>[5Ru]·</b><i><b>n</b></i><b>H</b>_<b>2</b><b>O</b> were irreversibly collapsed by the removal of crystal water. The triplet metal-to-ligand charge-transfer emission energies of <b>M</b>_<b>2</b><b>[4Ru]·</b><i><b>n</b></i><b>H</b>_<b>2</b><b>O</b> were lower than those of <b>[4Ru]</b> in aqueous solution, whereas those of <b>M</b>_<b>2</b><b>[5Ru]·</b><i><b>n</b></i><b>H</b>_<b>2</b><b>O</b> were close to those of <b>[5Ru]</b> in aqueous solution. These results suggested that the position of the coordination site in the metalloligand played an important role not only on the structure of the porous framework but also on the structural flexibility involving the guest adsorption/desorption properties

Current Technologies for Complex Glycoproteomics and Their Applications to Biology/Disease-Driven Glycoproteomics

Glycoproteomics is an important recent advance in the field of glycoscience. In glycomics, glycan structures are comprehensively analyzed after glycans are released from glycoproteins. However, a major limitation of glycomics is the lack of insight into glycoprotein functions. The Biology/Disease-driven Human Proteome Project has a particular focus on biological and medical applications. Glycoproteomics technologies aimed at obtaining a comprehensive understanding of intact glycoproteins, i.e., the kind of glycan structures that are attached to particular amino acids and proteins, have been developed. This Review focuses on the recent progress of the technologies and their applications. First, the methods for large-scale identification of both N- and O-glycosylated proteins are summarized. Next, the progress of analytical methods for intact glycopeptides is outlined. MS/MS-based methods were developed for improving the sensitivity and speed of the mass spectrometer, in parallel with the software for complex spectrum assignment. In addition, a unique approach to identify intact glycopeptides using MS1-based accurate masses is introduced. Finally, as an advance of glycomics, two approaches to provide the spatial distribution of glycans in cells are described, i.e., MS imaging and lectin microarray. These methods allow rapid glycomic profiling of different types of biological samples and thus facilitate glycoproteomics

Large-Scale Identification of <i>N-</i>Glycan Glycoproteins Carrying Lewis x and Site-Specific <i>N-</i>Glycan Alterations in <i>Fut9</i> Knockout Mice

The Lewis x (Le^x) structure (Galβ1–4­(Fucα1–3)­GlcNAc-R) is a carbohydrate epitope comprising the stage-specific embryonic antigen-1 (SSEA-1) and CD15, and it is synthesized by α1,3-fucosyltransferase 9 (Fut9). <i>Fut9</i> is expressed specifically in the stomach, kidney, brain, and in leukocytes, suggesting a specific function in these tissues. In this study, the <i>N</i>-linked glycan mass spectrometry profile of wild-type mouse kidney glycoproteins revealed the presence of abundant terminal fucoses, which were lost following knockout of the <i>Fut9</i> gene; the terminal fucose was therefore concluded to be Le^x. These results suggested that Le^x presence is widespread rather than being limited to specific proteins. We endeavored to comprehensively identify the Le^x carriers in the mouse kidney. Glycopeptides carrying fucosylated glycans were collected by Aleuria aurantia lectin (AAL) affinity chromatography from kidney homogenates of wild-type and <i>Fut9</i> knockout mice. The site-specific <i>N</i>-glycomes on the glycopeptides were subsequently analyzed by adopting a new glycoproteomic technology composed of dissociation-independent assignment of glycopeptide signals and accurate mass-based prediction of the <i>N</i>-glycome on the glycopeptides. Our analyses demonstrated that 24/32 glycoproteins contained the Le^x <i>N</i>-glycan structure in wild-type kidney; of these, Le^x was lost from 21 in the knockout mice. This is the first report of large-scale identification of Le^x-carrying glycoproteins from a native sample based on the site-specific glycome analysis