A diurnal serum lipid integrates hepatic lipogenesis and peripheral fatty acid utilization

Food intake increases the activity of hepatic de novo lipogenesis, which mediates the conversion of glucose to fats for storage or utilization. In mice, this program follows a circadian rhythm that peaks with nocturnal feeding1,2 and is repressed by Rev-erbα/β and an HDAC3-containing complex3–5 during the day. The transcriptional activators controlling rhythmic lipid synthesis in the dark cycle remain poorly defined. Disturbances in hepatic lipogenesis are also associated with systemic metabolic phenotypes6–8, suggesting that lipogenesis in the liver communicates with peripheral tissues to control energy substrate homeostasis. Here we identify a PPARδ-dependent de novo lipogenic pathway in the liver that modulates fat utilization by muscle via a circulating lipid. The nuclear receptor PPARδ controls diurnal expression of lipogenic genes in the dark/feeding cycle. Liver-specific PPARδ activation increases, while hepatocyte-Ppard deletion reduces, muscle fatty acid (FA) uptake. Unbiased metabolite profiling identifies PC(18:0/18:1), or 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), as a serum lipid regulated by diurnal hepatic PPARδ activity. PC(18:0/18:1) reduces postprandial lipid levels and increases FA utilization through muscle PPARα. High fat feeding diminishes rhythmic production of PC(18:0/18:1), whereas PC(18:0/18:1) administration in db/db mice improves metabolic homeostasis. These findings reveal an integrated regulatory circuit coupling lipid synthesis in the liver to energy utilization in muscle by coordinating the activity of two closely related nuclear receptors. These data implicate alterations in diurnal hepatic PPARδ-PC(18:0/18:1) signaling in metabolic disorders including obesity

MultiDsk:a ubiquitin-specific affinity resin

Ubiquitylation is a highly diverse and complex post-translational modification for the regulation of protein function and stability. Studies of ubiquitylation have, however, been hampered by its rapid reversal in cell extracts, for example through the action of de-ubiquitylating enzymes (DUBs). Here we describe a novel ubiquitin-binding protein reagent, MultiDsk, composed of an array of five UBA domains from the yeast ubiquitin-binding protein Dsk2, fused to GST. MultiDsk binds ubiquitylated substrates with unprecedented avidity, and can be used as both an affinity resin to study protein ubiquitylation, and to effectively protect ubiquitylated proteins from the action of DUBs and the proteasome in crude cell extracts. We use the resin to show that the Def1 protein becomes ubiquitylated in response to DNA damage, and to isolate ubiquitylated forms of RNA polymerase II

Deletion of prepl causes growth impairment and hypotonia in mice

Genetic studies of rare diseases can identify genes of unknown function that strongly impact human physiology. Prolyl endopeptidase-like (PREPL) is an uncharacterized member of the prolyl peptidase family that was discovered because of its deletion in humans with hypotonia-cystinuria syndrome (HCS). HCS is characterized by a number of physiological changes including diminished growth and neonatal hypotonia or low muscle tone. HCS patients have deletions in other genes as well, making it difficult to tease apart the specific role of PREPL. Here, we develop a PREPL null (PREPL(-/-)) mouse model to address the physiological role of this enzyme. Deletion of exon 11 from the Prepl gene, which encodes key catalytic amino acids, leads to a loss of PREPL protein as well as lower Prepl mRNA levels. PREPL(-/-) mice have a pronounced growth phenotype, being significantly shorter and lighter than their wild type (PREPL(+/+)) counterparts. A righting assay revealed that PREPL(-/-) pups took significantly longer than PREPL(+/+) pups to right themselves when placed on their backs. This deficit indicates that PREPL(-/-) mice suffer from neonatal hypotonia. According to these results, PREPL regulates growth and neonatal hypotonia in mice, which supports the idea that PREPL causes diminished growth and neonatal hypotonia in humans with HCS. These animals provide a valuable asset in deciphering the underlying biochemical, cellular and physiological pathways that link PREPL to HCS, and this may eventually lead to new insights in the treatment of this disease.status: publishe

MultiDsks can be used to characterise the kinetics of ubiquitylation of a specific protein species. A.

<p>Exponentially growing yeast cells were either treated with 10 µg/ml of 4-NQO for one hour, or left untreated, as indicated. Equal amounts of extracts were incubated with MultiDsk resin. Dilute Input extract (1%) and washes from the beads were also loaded. Proteins were analysed by Western blotting using anti-Rpb1 antibody, 4H8. <b>B</b>. Exponentially growing yeast cells were either treated with 10 µg/ml of 4-NQO for one hour, or left untreated, as indicated. Equal amounts of the extracts were incubated with agarose beads loaded with GST alone, GST-Dsk2 protein, commercial TUBE1, or MultiDsk. Proteins were eluted via boiling in sample buffer and subjected to Western blot analysis using the anti-Rpb1 antibody, 4H8 (upper panel). Ponceau S staining (lower panel) shows relative amounts of affinity proteins used. <b>C</b>. Yeast cells were harvested at the indicated time after treatment with 4-NQO (10 µg/ml), incubated with MultiDsk resin, and isolated proteins were analysed by Western blotting using either 4H8, or an anti-Def1 antibody, as indicated.</p

MultiDsk binds efficiently to ubiquitylated proteins. A

<p>. Schematic representation of the MultiDsk protein. <b>B</b>. 2 mg of yeast whole cell lysate from strain SUB592 expressing Myc-His-tagged ubiquitin was incubated with affinity beads. Differing amounts of GST protein, alone or as a mixture with GST-MultiDsk protein, were mixed with agarose beads so that equal amounts of total protein and bead bed volumes were used in each experiment. The flow-through not bound to the beads was retained and loaded at equivalent levels to the Input (Lane 1). After Western transfer, the membrane was stained with Ponceau S to reveal total protein in samples (lower panel), and Western blot was performed using anti-Myc antibodies to detect ubiquitylated species (upper panel). <b>C</b>. As in B, except performed on a human cell whole cell extract. 100 µg of protein was incubated with the indicated amounts of GST, GST-Dsk2, or MultiDsk protein and purified via agarose beads. <b>D</b>. As in B, but using purified ubiquitin chains. GST alone, full length GST-Dsk2 protein, or MultiDsk, bound to beads, were incubated with 100 µg synthetic K48- or K63-linked ubiquitin chains for 2 hours. After Western transfer, the membrane was stained with Ponceau S to reveal total protein in samples (lower panel), and Western blot was performed using anti-ubiquitin antibodies to detect ubiquitylated species (upper panel).</p

Protection of poly-ubiquitin chains in extract.

<p>Extract from strain SUB592 was incubated with equivalent amounts of GST, GST-Dsk2, commercially available TUBE-1, and MultiDsk and incubated at 30°C for the indicated time. Total protein extracts were subject to Western blot and probed using anti-myc antibody.</p

Revealing disease-associated pathways by network integration of untargeted metabolomics

Uncovering the molecular context of dysregulated metabolites is crucial to understand pathogenic pathways. However, their system-level analysis has been limited owing to challenges in global metabolite identification. Most metabolite features detected by untargeted metabolomics carried out by liquid-chromatography-mass spectrometry cannot be uniquely identified without additional, time-consuming experiments. We report a network-based approach, prize-collecting Steiner forest algorithm for integrative analysis of untargeted metabolomics (PIUMet), that infers molecular pathways and components via integrative analysis of metabolite features, without requiring their identification. We demonstrated PIUMet by analyzing changes in metabolism of sphingolipids, fatty acids and steroids in a Huntington's disease model. Additionally, PIUMet enabled us to elucidate putative identities of altered metabolite features in diseased cells, and infer experimentally undetected, disease-associated metabolites and dysregulated proteins. Finally, we established PIUMet's ability for integrative analysis of untargeted metabolomics data with proteomics data, demonstrating that this approach elicits disease-associated metabolites and proteins that cannot be inferred by individual analysis of these data.National Institutes of Health (U.S.) (grant R01-GM089903)National Institutes of Health (U.S.) (grant U54-NS091046)National Institutes of Health (U.S.) (grant U01-CA184898)National Cancer Institute (U.S.) (grant U54 CA112967)National Cancer Institute (U.S.) (grant P30 CA014051)Searle Scholars Progra

Teachers’ autonomy deconstructed: Irish and Finnish teachers’ perceptions of decision-making and control

Teacher autonomy has been a popular topic of investigation over the past decades. This article contributes to the debate by casting light on Irish and Finnish teachers’ perceptions of their professional autonomy, drawing from teacher interviews conducted in both countries. The intersection of newly introduced curriculum reforms, differing education governance models and differing control regimes make Ireland and Finland fertile points of comparison. Teacher autonomy is understood in this article as a multidimensional and context-dependent phenomenon, and the conceptualisation is presented in an analytical matrix applicable to comparative research. Findings indicate that teachers in both countries consider themselves very autonomous in their classroom practice and in their educational decisions overall. However, where much of the school-level decision-making in Finnish schools concerning educational, social and developmental issues tends to be in the hands of teachers (either collegially or as individuals), in Irish schools the senior management, and especially the principal, is reportedly more involved. Possibly the greatest difference is the ways in which teachers’ work is controlled, and in how teachers perceive it; Finnish teachers report intensified external control from the civil society, whereas on top of parental pressures Irish teachers report also increasing pressures from the state agencies

Construct design and validation.

<p>A) The genome region around PREPL's exon 11 was used to design a targeting vector for homologous recombination. This vector included a neomycin (NEO) cassette for positive selection, Frt sites flanking the NEO cassette, LoxP sites flanking exon 11, a HindIII restriction site for 5′-Southern blots, and an NdeI site for 3′-Southern blots. Homologous recombination in ES cells generated cells carrying recombinant genomic DNA, which according to standard nomenclature (<i>Gene^tm#Labcode</i>) is referred to as <i>Prepl^tm1Sagh</i>. These ES cells were then used to generate chimeric mice designated <i>Prepl^+/tm1Sagh.</i> B) The germ line transmission of the <i>Prepl^tm1Sagh</i> allele to the offspring from chimera-C57BL/6J crosses was confirmed by Southern blots of tail genomic DNA. Restriction digests of the genomic DNA with HindIII (5′) or NdeI (3′), followed by probing with a 5′ or 3′ specific probe generated a single band for <i>Prepl^+/+</i> (left lane), whereas an additional, lower molecular weight band is generated in mice carrying the <i>Prepl^tm1Sagh</i> allele (right lane). C) <i>Prepl^+/tm1Sagh</i> were then crossed with mice expressing Flp recombinase to produce <i>Prepl^+/tm1.1Sagh</i> mice (middle) lacking the NEO cassette, and finally crossed with mice ubiquitously expressing Cre recombinase, resulting in <i>Prepl^+/tm1.2Sagh</i> mice (bottom) which lack exon 11. We refer to these <i>Prepl^+/tm1.2Sagh</i> mice as PREPL^+/− mice. D) Representative PCR genotyping results for PREPL^+/+, PREPL^−/− and PREPL^+/− mice. Using this strategy, one can easily distinguish these three genotypes as well as other possible genotypes, such as the <i>Prepl^tm1.1Sagh</i> allele.</p

Grip strength and righting assay to assess hypotonia in PREPL^−/− mice.

<p>A) Six month old female mice had no difference in grip strength between genotypes when normalized to their respective body weights. B) A comparison of pups of both genders and genotypes in a righting assay. In this experiment, mice are placed on their backs and the time it takes for them to right themselves is measured. At day 5, PREPL^−/− pups are much slower at righting themselves than PREPL^+/+ mice. (Error bars show SEM and Statistical significance calculated by a Student's t-test, p-value<0.05, *, N = 4–5 for grip strength and N = 13–14 for the righting assay).</p