Hepatic p53 is regulated by transcription factor FOXO1 and acutely controls glycogen homeostasis

The tumor suppressor p53 is involved in the adaptation of hepatic metabolism to nutrient availability. Acute deletion of p53 in the mouse liver affects hepatic glucose and triglyceride metabolism. However, long-term adaptations upon the loss of hepatic p53 and its transcriptional regulators are unknown. Here we show that short-term, but not chronic, liver-specific deletion of p53 in mice reduces liver glycogen levels, and we implicate the transcription factor forkhead box O1 protein (FOXO1) in the regulation of p53 and its target genes. We demonstrate that acute p53 deletion prevents glycogen accumulation upon refeeding, whereas a chronic loss of p53 associates with a compensational activation of the glycogen synthesis pathway. Moreover, we identify fasting-activated FOXO1 as a repressor of p53 transcription in hepatocytes. We show that this repression is relieved by inactivation of FOXO1 by insulin, which likely mediates the upregulation of p53 expression upon refeeding. Strikingly, we find that high-fat diet-induced insulin resistance with persistent FOXO1 activation not only blunted the regulation of p53 but also the induction of p53 target genes like p21 during fasting, indicating overlapping effects of both FOXO1 and p53 on target gene expression in a context-dependent manner. Thus, we conclude that p53 acutely controls glycogen storage in the liver and is linked to insulin signaling via FOXO1, which has important implications for our understanding of the hepatic adaptation to nutrient availability

A Three-Dimensional Model of the Yeast Transmembrane Sensor Wsc1 Obtained by SMA-Based Detergent-Free Purification and Transmission Electron Microscopy

The cell wall sensor Wsc1 belongs to a small family of transmembrane proteins, which are crucial to sustain cell integrity in yeast and other fungi. Wsc1 acts as a mechanosensor of the cell wall integrity (CWI) signal transduction pathway which responds to external stresses. Here we report on the purification of Wsc1 by its trapping in water-soluble polymer-stabilized lipid nanoparticles, obtained with an amphipathic styrene-maleic acid (SMA) copolymer. The latter was employed to transfer tagged sensors from their native yeast membranes into SMA/lipid particles (SMALPs), which allows their purification in a functional state, i.e., avoiding denaturation. The SMALPs composition was characterized by fluorescence correlation spectroscopy, followed by two-dimensional image acquisition from single particle transmission electron microscopy to build a three-dimensional model of the sensor. The latter confirms that Wsc1 consists of a large extracellular domain connected to a smaller intracellular part by a single transmembrane domain, which is embedded within the hydrophobic moiety of the lipid bilayer. The successful extraction of a sensor from the yeast plasma membrane by a detergent-free procedure into a native-like membrane environment provides new prospects for in vitro structural and functional studies of yeast plasma proteins which are likely to be applicable to other fungi, including plant and human pathogens

MitoCLox: A Novel Mitochondria-Targeted Fluorescent Probe for Tracing Lipid Peroxidation

Peroxidation of cardiolipin (CL) in the inner mitochondrial membrane plays a key role in the development of various pathologies and, probably, aging. The four fatty acid tails of CL are usually polyunsaturated, which makes CL particularly sensitive to peroxidation. Peroxidation of CL is involved in the initiation of apoptosis, as well as in some other important cellular signaling chains. However, the studies of CL peroxidation are strongly limited by the lack of methods for its tracing in living cells. We have synthesized a new mitochondria-targeted fluorescent probe sensitive to lipid peroxidation (dubbed MitoCLox), where the BODIPY fluorophore, carrying a diene-containing moiety (as in the C11-BODIPY (581/591) probe), is conjugated with a triphenylphosphonium cation (TPP+) via a long flexible linker that contains two amide bonds. The oxidation of MitoCLox could be measured either as a decrease of absorbance at 588 nm or as an increase of fluorescence in the ratiometric mode at 520/590 nm (emission). In CL-containing liposomes, MitoCLox oxidation was induced by cytochrome c and developed in parallel with cardiolipin oxidation. TPP+-based mitochondria-targeted antioxidant SkQ1, in its reduced form, inhibited oxidation of MitoCLox concurrently with the peroxidation of cardiolipin. Molecular dynamic simulations of MitoCLox in a cardiolipin-containing membrane showed affinity of positively charged MitoCLox to negatively charged CL molecules; the oxidizable diene moiety of MitoCLox resided on the same depth as the cardiolipin lipid peroxides. We suggest that MitoCLox could be used for monitoring CL oxidation in vivo and, owing to its flexible linker, also serve as a platform for producing peroxidation sensors with affinity to particular lipids

Signaling and Adaptation Modulate the Dynamics of the Photosensoric Complex of <i>Natronomonas pharaonis</i>

<div><p>Motile bacteria and archaea respond to chemical and physical stimuli seeking optimal conditions for survival. To this end transmembrane chemo- and photoreceptors organized in large arrays initiate signaling cascades and ultimately regulate the rotation of flagellar motors. To unravel the molecular mechanism of signaling in an archaeal phototaxis complex we performed coarse-grained molecular dynamics simulations of a trimer of receptor/transducer dimers, namely <i>Np</i>SRII/<i>Np</i>HtrII from <i>Natronomonas pharaonis</i>. Signaling is regulated by a reversible methylation mechanism called adaptation, which also influences the level of basal receptor activation. Mimicking two extreme methylation states in our simulations we found conformational changes for the transmembrane region of <i>Np</i>SRII/<i>Np</i>HtrII which resemble experimentally observed light-induced changes. Further downstream in the cytoplasmic domain of the transducer the signal propagates via distinct changes in the dynamics of HAMP1, HAMP2, the adaptation domain and the binding region for the kinase CheA, where conformational rearrangements were found to be subtle. Overall these observations suggest a signaling mechanism based on dynamic allostery resembling models previously proposed for <i>E</i>. <i>coli</i> chemoreceptors, indicating similar properties of signal transduction for archaeal photoreceptors and bacterial chemoreceptors.</p></div

Inter-dimeric distances for related residues of the transducer.

<p>Distances were calculated as an average over the three dimers for the methylated (black) and demethylated (red) states, shaded areas representing the standard deviation. The distance is measured between the center of mass (COM) of two related residues in one dimer and the COM of the six respective residues in the trimer-of-dimers (see inset on the lower left). The domains of the complex are depicted in colored bars; m.s and A/W indicate methylation sites and binding sites for CheA/CheW, respectively. Representative distance trajectories are depicted in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004561#pcbi.1004561.s007" target="_blank">S7 Fig</a>.</p

Dynamics of the methylated and the demethylated systems.

<p>A: Structure of the <i>Np</i>SRII/<i>Np</i>HtrII trimeric complex with colors that code for the difference between the RMSF value per residue of the demethylated and the methylated transducer. Positive values (in Å) correspond to a higher fluctuation and therefore higher mobility of the corresponding residues in the demethylated system, negative values indicate a lower mobility. B: The differences in mobility as function of residue number show distinct changes in the transmembrane region of the complex, an inversion between the two HAMP domains and in the adaptation and close to the glycine rich (293, 296) regions. This change in dynamics upon adaption includes the tip region and the binding sites for CheA (A/W). Colored bars have the same meaning as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004561#pcbi.1004561.g003" target="_blank">Fig 3</a>.</p

Conformations of the trimeric photoreceptor-transducer complexes.

<p>Cartoon with the resulting structures of the demethylated (left) and the methylated (right) trimer systems combined in the bent membrane with a schematic representation of the adaptation process. Methylation sites are shown as red and black spheres in the demethylated and methylated state, respectively.</p

Comparison of the conformational changes of the transmembrane region upon demethylation and illumination.

<p>Cytoplasmic view of a monomer of the <i>Np</i>SRII/<i>Np</i>HtrII complex with experimentally observed conformational changes upon illumination shown as arrows and corresponding numerical values from the CG MD simulations. An outward tilt of helix F at the cytoplasmic side of the membrane embedded part of the transducer by 0.6±0.3 Å (blue arrow) is accompanied by a rotation of helix TM2 of 12±8° (olive green arrow) with respect to the equilibrated methylated structure. In addition TM2 shifts with respect to the helix TM1 of the transducer by 0.7±0.5 Å (light green arrow).</p

Model for the <i>Np</i>SRII/<i>Np</i>HtrII complex activation.

<p>The regions with higher mobility are shown in diffuse representation; the arrows correspond to the domain motions (compacting/expanding within the trimer).</p