HP1 reshapes nucleosome core to promote phase separation of heterochromatin

Heterochromatin affects genome function at many levels. It enables heritable gene repression, maintains chromosome integrity and provides mechanical rigidity to the nucleus1,2. These diverse functions are proposed to arise in part from compaction of the underlying chromatin2. A major type of heterochromatin contains at its core the complex formed between HP1 proteins and chromatin that is methylated on histone H3, lysine 9 (H3K9me). HP1 is proposed to use oligomerization to compact chromatin into phase-separated condensates3-6. Yet, how HP1-mediated phase separation relates to chromatin compaction remains unclear. Here we show that chromatin compaction by the Schizosaccharomyces pombe HP1 protein Swi6 results in phase-separated liquid condensates. Unexpectedly, we find that Swi6 substantially increases the accessibility and dynamics of buried histone residues within a nucleosome. Restraining these dynamics impairs compaction of chromatin into liquid droplets by Swi6. Our results indicate that Swi6 couples its oligomerization to the phase separation of chromatin by a counterintuitive mechanism, namely the dynamic exposure of buried nucleosomal regions. We propose that such reshaping of the octamer core by Swi6 increases opportunities for multivalent interactions between nucleosomes, thereby promoting phase separation. This mechanism may more generally drive chromatin organization beyond heterochromatin

The nucleoporin Nup60p functions as a Gsp1p–GTP-sensitive tether for Nup2p at the nuclear pore complex

The nucleoporins Nup60p, Nup2p, and Nup1p form part of the nuclear basket structure of the Saccharomyces cerevisiae nuclear pore complex (NPC). Here, we show that these necleoporins can be isolated from yeast extracts by affinity chromatography on karyopherin Kap95p-coated beads. To characterize Nup60p further, Nup60p-coated beads were used to capture its interacting proteins from extracts. We find that Nup60p binds to Nup2p and serves as a docking site for Kap95p–Kap60p heterodimers and Kap123p. Nup60p also binds Gsp1p–GTP and its guanine nucleotide exchange factor Prp20p, and functions as a Gsp1p guanine nucleotide dissociation inhibitor by reducing the activity of Prp20p. Yeast lacking Nup60p exhibit minor defects in nuclear export of Kap60p, nuclear import of Kap95p–Kap60p-dependent cargoes, and diffusion of small proteins across the NPC. Yeast lacking Nup60p also fail to anchor Nup2p at the NPC, resulting in the mislocalization of Nup2p to the nucleoplasm and cytoplasm. Purified Nup60p and Nup2p bind each other directly, but the stability of the complex is compromised when Kap60p binds Nup2p. Gsp1p–GTP enhances by 10-fold the affinity between Nup60p and Nup2p, and restores binding of Nup2p–Kap60p complexes to Nup60p. The results suggest a dynamic interaction, controlled by the nucleoplasmic concentration of Gsp1p–GTP, between Nup60p and Nup2p at the NPC

Organic Geochemical Studies. I. Molecular Criteria for Hydrocarbon Genesis

In recent years the search for life-forms at the earliest periods of geological time has been continued not only at the morphological level but also at the molecular level. This has been possible as a result of the increase in the biochemical knowledge and with the advent of analytical techniques that are capable of describing the intimate molecular architecture of individual molecules in acute detail. The fundamental premises upon which this organic geochemical approach rest are the following: that certain molecules, possessing a characteristic structural skeleton, show a reasonable stability to degradation over long periods of geological time; that their structural specificity can be understood in terms of known biosynthetic sequences; and that their formation by any non-biological means is of negligible probability. In this manuscript it is proposed to critically re-examine these premises and to establish criteria whereby one can differentiate molecules derived from biological systems from those that have their origin in non-biological processes. The importance of establishing such criteria lies in the significance these criteria have in determining whether life exists, or has existed, on other planets. Within the very near future it may be possible to provide an initial answer to this question when the first lunar samples are returned to the earth for analysis

Ultrasound-assisted extraction of polyphenols from potato peels: profiling and kinetic modelling

peer-reviewedUltrasound‐assisted extraction (UAE) at 33 and 42 kHz has been investigated in the extraction of polyphenols from peels of two potato varieties, cream‐skinned Lady Claire (LC) and pink‐skinned Lady Rosetta (LR), commonly used in snack food production. Extraction efficacy between the UAE‐untreated (control) and the UAE‐treated extracts was assessed on the total phenolic content and antioxidant capacities (DPPH and FRAP). Application of UAE showed significantly higher recovery of phenolic compounds compared to solid–liquid extraction process alone. Lower ultrasonic frequency (33 kHz) was more effective in recovering polyphenols compared to 42 kHz ultrasonic treatment. The liquid chromatography‐tandem mass spectrometry revealed that chlorogenic acid and caffeic acid were the most prevalent phenolics in LR peels, whereas caffeic acid was dominant in LC peels. Peleg's equation showed a good correlation (R2 > 0.92) between the experimental values and the predicted values on the kinetics of UAE of phenolic compounds.The authors acknowledge financial support from the ‘NovTechIng’ project funded under the Food Institutional Research Measure (Project No. FIRM/11/F/050) by the Irish Department of Agriculture, Food and Marine

Taxation of the fat content of foods for reducing their consumption and preventing obesity or other adverse health outcomes

Background: Overweight and obesity are increasing worldwide and are considered to be a major public health issue of the 21st century. Introducing taxation of the fat content in foods is considered a potentially powerful policy tool to reduce consumption of foods high in fat or saturated fat, or both. Objectives: To assess the effects of taxation of the fat content in food on consumption of total fat and saturated fat, energy intake, overweight, obesity, and other adverse health outcomes in the general population. Search methods: We searched CENTRAL, Cochrane Database of Systematic Reviews, MEDLINE, Embase, and 15 other databases and trial registers on 12 September 2019. We handsearched the reference lists of all records of included studies, searched websites of international organizations and institutions (14 October 2019), and contacted review advisory group members to identify planned, ongoing, or unpublished studies (26 February 2020). Selection criteria: In line with Cochrane Effective Practice and Organisation of Care Group (EPOC) criteria, we included the following study types: randomized controlled trials (RCTs), cluster‐randomized controlled trials (cRCTs), non‐randomized controlled trials (nRCTs), controlled before‐after (CBA) studies, and interrupted time series studies. We included studies that evaluated the effects of taxes on the fat content in foods. Such a tax could be expressed as sales, excise, or special value added tax (VAT) on the final product or an intermediary product. Eligible interventions were taxation at any level, with no restriction on the duration or the implementation level (i.e. local, regional, national, or multinational). Eligible study populations were children (zero to 17 years) and adults (18 years or older) from any country and setting. We excluded studies that focused on specific subgroups only (e.g. people receiving pharmaceutical intervention; people undergoing a surgical intervention; ill people who are overweight or obese as a side effect, such as those with thyroiditis and depression; and people with chronic illness). Primary outcomes were total fat consumption, consumption of saturated fat, energy intake through fat, energy intake through saturated fat, total energy intake, and incidence/prevalence of overweight or obesity. We did not exclude studies based on country, setting, comparison, or population. Data collection and analysis: We used standard Cochrane methods for all phases of the review. Risk of bias of the included studies was assessed using the criteria of Cochrane’s ‘Risk of bias’ tool and the EPOC Group’s guidance. Results of the review are summarized narratively and the certainty of the evidence was assessed using the GRADE approach. These steps were done by two review authors, independently. Main results: We identified 23,281 records from searching electronic databases and 1173 records from other sources, leading to a total of 24,454 records. Two studies met the criteria for inclusion in the review. Both included studies investigated the effect the Danish tax on saturated fat contained in selected food items between 2011 and 2012. Both studies used an interrupted time series design. Neither included study had a parallel control group from another geographic area. The included studies investigated an unbalanced panel of approximately 2000 households in Denmark and the sales data from a specific Danish supermarket chain (1293 stores). Therefore, the included studies did not address individual participants, and no restriction regarding age, sex, and socioeconomic characteristics were defined. We judged the overall risk of bias of the two included studies as unclear. For the outcome total consumption of fat, a reduction of 41.8 grams per week per person in a household (P < 0.001) was estimated. For the consumption of saturated fat, one study reported a reduction of 4.2% from minced beef sales, a reduction of 5.8% from cream sales, and an increase of 0.5% to sour cream sales (no measures of statistical precision were reported for these estimates). These estimates are based on a restricted number of food types and derived from sales data; they do not measure individual intake. Moreover, these estimates do not account for other relevant sources of fat intake (e.g. packaged or processed food) or other food outlets (e.g. restaurants or cafeterias); hence, we judged the evidence on the effect of taxation on total fat consumption or saturated fat consumption to be very uncertain. We did not identify evidence on the effect of the intervention on energy intake or the incidence or prevalence of overweight or obesity. Authors' conclusions: Given the very low quality of the evidence currently available, we are unable to reliably establish whether a tax on total fat or saturated fat is effective or ineffective in reducing consumption of total fat or saturated fat. There is currently no evidence on the effect of a tax on total fat or saturated fat on total energy intake or energy intake through saturated fat or total fat, or preventing the incidence or reducing the prevalence of overweight or obesity

Cytochrome P450(cin) (CYP176A), isolation, expression, and characterization

Cytochromes P450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiologic and xenobiotic compounds in eukaryotes and prokaryotes. Studies on bacterial P450s, particularly those involved in monoterpene oxidation, have provided an integral contribution to our understanding of these proteins, away from the problems encountered with eukaryotic forms. We report here a novel cytochrome P450 (P450(cin), CYP176A1) purified from a strain of Citrobacter braakii that is capable of using cineole 1 as its sole source of carbon and energy. This enzyme has been purified to homogeneity and the amino acid sequences of three tryptic peptides determined. By using this information, a PCR-based cloning strategy was developed that allowed the isolation of a 4-kb DNA fragment containing the cytochrome P450(cin) gene (cinA). Sequencing revealed three open reading frames that were identified on the basis of sequence homology as a cytochrome P450, an NADPH-dependent flavodoxin/ferrodoxin reductase, and a flavodoxin. This arrangement suggests that P450(cin) may be the first isolated P450 to use a flavodoxin as its natural redox partner. Sequencing also identified the unprecedented substitution of a highly conserved, catalytically, important active site threonine with an asparagine residue. The P450 gene was subcloned and heterologously expressed in Escherichia coli at similar to2000 nmol/liter of original culture, and purification was achieved by standard protocols. Postulating the native E. coli flavodoxin/flavodoxin reductase system might mimic the natural redox partners of P450,in, it was expressed in E. coli in the presence of cineole 1. A product was formed in vivo that was tentatively identified by gas chromatography-mass spectrometry as 2-hydroxycineole 2. Examination of P450(cin) by UV-visible spectroscopy revealed typical spectra characteristic of P450s, a high affinity for cineole 1 (K-D = 0.7 mum), and a large spin state change of the heme iron associated with binding of cineole 1. These facts support the hypothesis that cineole 1 is the natural substrate for this enzyme and that P450(cin) catalyzes the initial monooxygenation of cineole 1 biodegradation. This constitutes the first characterization of an enzyme involved in this pathway

Receptor tyrosine kinase signaling mechanisms: Devolving TrkA responses with phosphoproteomics

Receptor tyrosine kinases (RTKs) function through protein kinase entities located in the intracellular domain of each protomer. Following activation by ligand binding, they selectively form phosphotyrosine residues by autocatalytic modification. Some of these sites are involved in maintaining the active conformation of the kinase, while others become docking sites for various adaptor/effector/scaffold proteins, which, after complexing with the receptor, then initiate further responses through cascades of post-translational modifications and the generation of lipid second messengers. Although there is substantial overlap in the pathways and activities stimulated by this superfamily, the molecular features of the endodomains of the sub-families and the moieties that they interact with to perpetrate their signals are surprisingly distinct, which may play a significant role in the regulation and responses of the individual RTK types. Some use large scaffold proteins as the basis for most, if not all, of their signal-generating interactions, while others have numerous receptor endodomain phosphotyrosine sites that are quite overlapping in specificity. The members of the Trk family of receptors each have several tyrosine residues that are phosphorylated following stimulation, including those in the kinase activation loop, but there are only two established sites (Y490 and Y785 on TrkA) that are known to be directly involved in signal propagation. Taking advantage of this limited repertoire of docking sites, we have applied phosphoproteomic methods to dissect the signaling responses of both the native protein and derivatives that have had these two sites modified. Interestingly, a clear subset that was not dependent on either docking site was identified. A comparison with a similar set of data for EGFR indicates a considerable degree of similarity in the downstream signaling profile between these two RTKs

Quantitative proteomic analysis of synaptic phosphorylation and protein expression

3nonenoneSchoepfer R; Thalhammer A; Trinidad JC & Burlingame ALSchoepfer, R; Thalhammer, A; Trinidad JC, & Burlingame A

Electron transfer dissociation (ETD): The mass spectrometric breakthrough essential for O‐GlcNAc protein site assignments—a study of the O‐GlcNAcylated protein Host Cell Factor C1

The development of electron-based, unimolecular dissociation MS, i.e. electron capture and electron transfer dissociation (ECD and ETD, respectively), has greatly increased the speed and reliability of labile PTM site assignment. The field of intracellular O-GlcNAc (O-linked N-acetylglucosamine) signaling has especially advanced with the advent of ETD MS. Only within the last five years have proteomic-scale experiments utilizing ETD allowed the assignment of hundreds of O-GlcNAc sites within cells and subcellular structures. Our ability to identify and unambiguously assign the site of O-GlcNAc modifications using ETD is rapidly increasing our understanding of this regulatory glycosylation and its potential interaction with other PTMs. Here, we discuss the advantages of using ETD, complimented with collisional-activation MS, in a study of the extensively O-GlcNAcylated protein Host Cell Factor C1 (HCF-1). HCF-1 is a transcriptional coregulator that forms a stable complex with O-GlcNAc transferase and controls cell cycle progression. ETD, along with higher energy collisional dissociation (HCD) MS, was employed to assign the PTMs of the HCF-1 protein isolated from HEK293T cells. These include 19 sites of O-GlcNAcylation, two sites of phosphorylation, and two sites bearing dimethylarginine, and showcase the residue-specific, PTM complexity of this regulator of cell proliferation