Funktionelle Charakterisierung der SANT-Domänen des Korepressors N-CoR

Der Korepressor N-CoR vermittelt die Repression von nukleären Hormonrezptoren in Abwesenheit ihrer Liganden und ist darber hinaus für die embryonale Entwicklung von Säugern entscheidend. N-CoR ist in der Zelle mit Histondeacetylasen (HDACs) komplexiert. Diese Enzyme bewirken im Zusammenspiel mit ihren Gegenspielern, den Histonacetyltransferasen, durch Deacetylierung und Acetylierung von Histonen eine dynamische Modifikation des Chromatins und beeinflussen so die Transkription von Genen. Die Interaktion zwischen regulatorischen Proteinen, Histonen und histonmodifizierenden Proteinen ist ein fundamentaler und konservierter Mechanismus der Genregulation in höheren Eukaryonten. Koregulatoren (Koaktivatoren und Korepressoren) und Transkriptionsfaktoren enthalten eine Fülle noch uncharakterisierter Domänen, die in ähnlicher Weise wirken könnten. Der Korepressor N-CoR enthält beispielsweise zwei uncharakterisierte SANT-Domänen. Die SANT-Domäne ist zwischen Hefe und S„ugern konserviert und spielt vermutlich in der Chromatinmodifizierung oder Transkription eine Rolle. In der vorliegenden Arbeit wurden verschiedene Screeningmethoden zur Identifikation von Interaktionspartnern der N-CoR SANT-Domänen eingesetzt. In einem Hefe-Zwei-Hybrid Screen war es möglich, Interaktionspartner der N-CoR SANT-Domäne zu isolieren. Insgesamt 14 verschiedene Proteine wurden in dem Hefescreen identifiziert. Die Proteine PIAS1, Ubc9, TDG, Hoxa-4, TAFII250 und cDNA I interagierten in vitro mit der N-CoR SANT1-Domäne. Drei der im Hefescreen gefundenen Proteine (PIAS1, Ubc9 und Pc2) deuteten darauf hin, dass N-CoR durch die Konjugation von SUMO-Proteinen posttranslational modifiziert sein könnte. In Ko-Immunopräziptitationen konnte die Interaktion zwischen N-CoR und der SUMO-E3-Ligase PIAS1 in vivo bestätigt werden. Die N-CoR SUMO-Modifikation wurde indirekt in vivo und in einem in vitro Sumoylierungstest nachgewiesen. Endogenes N-CoR und PIAS1 kolokalisieren im Zellkern, wobei die heterologe Expression der SUMO-E3 Ligase die für N-CoR typische Aggregatbildung im Kern verhindert und eine diffusere Verteilung und eine Häufung in der Nähe der Kernmembran induziert. In Kolokalisierungsstudien konnte ferner gezeigt werden, dass eine N-CoR-DSUMO Konsensussequenzmutante eine veränderte zytoplasmatisch-nukleäre Lokalisation aufweist. Dies traf ebenfalls auf DSANT-Mutanten von N-CoR zu, die im Vergleich zum N-CoR Wildtypprotein eine unterschiedliche Lokalisation bei heterologer Expression von PIAS1 zeigten. Die Koexpression von PIAS1 reduzierte die Repression eines Luziferasereporters durch N-CoR. Andererseits war die Repression des Proteins ebenfalls durch Alanin-Substitution in potentiellen SUMO-Stellen vermindert

A Live Zebrafish-Based Screening System for Human Nuclear Receptor Ligand and Cofactor Discovery

Nuclear receptors (NRs) belong to a superfamily of transcription factors that regulate numerous homeostatic, metabolic and reproductive processes. Taken together with their modulation by small lipophilic molecules, they also represent an important and successful class of drug targets. Although many NRs have been targeted successfully, the majority have not, and one third are still orphans. Here we report the development of an in vivo GFP-based reporter system suitable for monitoring NR activities in all cells and tissues using live zebrafish (Danio rerio). The human NR fusion proteins used also contain a new affinity tag cassette allowing the purification of receptors with bound molecules from responsive tissues. We show that these constructs 1) respond as expected to endogenous zebrafish hormones and cofactors, 2) facilitate efficient receptor and cofactor purification, 3) respond robustly to NR hormones and drugs and 4) yield readily quantifiable signals. Transgenic lines representing the majority of human NRs have been established and are available for the investigation of tissue- and isoform-specific ligands and cofactors

The Structural Basis of Gas-Responsive Transcription by the Human Nuclear Hormone Receptor REV-ERBβ

Heme is a ligand for the human nuclear receptors (NR) REV-ERBα and REV-ERBβ, which are transcriptional repressors that play important roles in circadian rhythm, lipid and glucose metabolism, and diseases such as diabetes, atherosclerosis, inflammation, and cancer. Here we show that transcription repression mediated by heme-bound REV-ERBs is reversed by the addition of nitric oxide (NO), and that the heme and NO effects are mediated by the C-terminal ligand-binding domain (LBD). A 1.9 Å crystal structure of the REV-ERBβ LBD, in complex with the oxidized Fe(III) form of heme, shows that heme binds in a prototypical NR ligand-binding pocket, where the heme iron is coordinately bound by histidine 568 and cysteine 384. Under reducing conditions, spectroscopic studies of the heme-REV-ERBβ complex reveal that the Fe(II) form of the LBD transitions between penta-coordinated and hexa-coordinated structural states, neither of which possess the Cys384 bond observed in the oxidized state. In addition, the Fe(II) LBD is also able to bind either NO or CO, revealing a total of at least six structural states of the protein. The binding of known co-repressors is shown to be highly dependent upon these various liganded states. REV-ERBs are thus highly dynamic receptors that are responsive not only to heme, but also to redox and gas. Taken together, these findings suggest new mechanisms for the systemic coordination of molecular clocks and metabolism. They also raise the possibility for gas-based therapies for the many disorders associated with REV-ERB biological functions

SUMOylation of the Corepressor N-CoR Modulates Its Capacity to Repress Transcription

In the absence of ligands the corepressor N-CoR mediates transcriptional repression by some nuclear hormone receptors. Several protein–protein interactions of N-CoR are known, of which mainly complex formation with histone deacetylases (HDACs) leads to the repression of target genes. On the other hand, the role of posttranslational modifications in corepressor function is not well established. Here, we show that N-CoR is modified by Sumo-1. We found SUMO-E2–conjugating enzyme Ubc9 and SUMO-E3 ligase Pias1 as novel N-CoR interaction partners. The SANT1 domain of N-CoR was found to mediate this interaction. We show that K152, K1117, and K1330 of N-CoR can be conjugated to SUMO and that mutation of all sites is necessary to fully block SUMOylation in vitro. Because these lysine residues are located within repression domains I and III, respectively, we investigated a possible correlation between the functions of the repression domains and SUMOylation. Coexpression of Ubc9 protein resulted in enhanced N-CoR–dependent transcriptional repression. Studies using SUMOylation-deficient N-CoR RDI mutants suggest that SUMO modification contributes to repression by N-CoR. Mutation of K152 to R in RD1, for example, not only significantly reduced repression of a reporter gene, but also abolished the effect of Ubc9 on transcriptional repression

The angiotensin receptor-associated protein Atrap is a stimulator of the cardiac Ca2+-ATPase SERCA2a

The angiotensin II type 1 receptor-associated protein (Atrap) is highly expressed in the heart, but its function in the heart is unknown. We hypothesized that cardiac Atrap may interact with proteins other than the AT1 receptor. To identify potential novel interacting partners of Atrap, pull-down assays were performed. Sequencing by MALDI-MS of the isolated complexes showed that Atrap interacts with the cardiac Ca2+-ATPase SERCA2a. The interaction between Atrap and SERCA2a was confirmed by co-immunoprecipitation and by surface plasmon resonance (SPR) spectroscopy. Atrap enhanced the SERCA-dependent Ca2+ uptake in isolated SR membrane vesicles. Furthermore, sarcomere shortenings and [Ca2+](i) transients (CaTs) were determined in ventricular myocytes isolated from Atrap-/- and wild-type (WT) mice. The amplitudes of CaTs and sarcomere shortenings were similar in Atrap-/- and WT myocytes. However, the CaT decay and sarcomere re-lengthening were prolonged in Atrap-/- myocytes. To further evaluate the functional relevance of the Atrap-SERCA2a interaction in vivo, left-ventricular function was assessed in WT and Atrap-/- mice. The heart rates (564 +/- 10 b.p.m. vs. 560 +/- 11 b.p.m.; P = 0.80) and ejection fractions (71.3 +/- 1.3 vs. 72 +/- 1.8%; P = 0.79) were similar in WT and Atrap-/- mice, respectively (n = 15 for each genotype). However, the maximum filling rate (dV/dt(max)) was markedly decreased in Atrap-/- (725 +/- 48 A mu L/s) compared with WT mice (1065 +/- 122 A mu L/s; P = 0.01; n = 15). We identified Atrap as a novel regulatory protein of the cardiac Ca2+-ATPase SERCA2a. We suggest that Atrap enhances the activity of SERCA2a and, consequently, facilitates ventricular relaxation

Idebenone and coenzyme Q10 are novel PPARα/γ ligands, with potential for treatment of fatty liver diseases

Current peroxisome proliferator-activated receptor (PPAR)-targeted drugs, such as the PPARγ-directed diabetes drug rosiglitazone, are associated with undesirable side effects due to robust agonist activity in non-target tissues. To find new PPAR ligands with fewer toxic effects, we generated transgenic zebrafish that can be screened in high throughput for new tissue-selective PPAR partial agonists. A structural analog of coenzyme Q10 (idebenone) that elicits spatially restricted partial agonist activity for both PPARα and PPARγ was identified. Coenzyme Q10 was also found to bind and activate both PPARs in a similar fashion, suggesting an endogenous role in relaying the states of mitochondria, peroxisomes and cellular redox to the two receptors. Testing idebenone in a mouse model of type 2 diabetes revealed the ability to reverse fatty liver development. These findings indicate new mechanisms of action for both PPARα and PPARγ, and new potential treatment options for nonalcoholic fatty liver disease (NAFLD) and steatosis. This article has an associated First Person interview with the first author of the paper

Correction: 15-keto-prostaglandin E2 activates host peroxisome proliferator-activated receptor gamma (PPAR-γ) to promote Cryptococcus neoformans growth during infection.

[This corrects the article DOI: 10.1371/journal.ppat.1007597.]