The histone chaperone Vps75 forms multiple oligomeric assemblies capable of mediating exchange between histone H3–H4 tetramers and Asf1–H3–H4 complexes

Wellcome Trust [094090, 097945, 099149]; Medical Research Council [G1100021]. Funding for open access charge: Wellcome Trust.Vps75 is a histone chaperone that has been historically characterized as homodimer by X-ray crystallography. In this study, we present a crystal structure containing two related tetrameric forms of Vps75 within the crystal lattice. We show Vps75 associates with histones in multiple oligomers. In the presence of equimolar H3-H4 and Vps75, the major species is a reconfigured Vps75 tetramer bound to a histone H3-H4 tetramer. However, in the presence of excess histones, a Vps75 dimer bound to a histone H3-H4 tetramer predominates. We show the Vps75-H3-H4 interaction is compatible with the histone chaperone Asf1 and deduce a structural model of the Vps75-Asf1-H3-H4 (VAH) co-chaperone complex using the Pulsed Electron-electron Double Resonance (PELDOR) technique and cross-linking MS/MS distance restraints. The model provides a molecular basis for the involvement of both Vps75 and Asf1 in Rtt109 catalysed histone H3 K9 acetylation. In the absence of Asf1 this model can be used to generate a complex consisting of a reconfigured Vps75 tetramer bound to a H3-H4 tetramer. This provides a structural explanation for many of the complexes detected biochemically and illustrates the ability of Vps75 to interact with dimeric or tetrameric H3-H4 using the same interaction surface.Publisher PDFPeer reviewe

The Helicase Aquarius/EMB-4 Is Required to Overcome Intronic Barriers to Allow Nuclear RNAi Pathways to Heritably Silence Transcription

Small RNAs play a crucial role in genome defense against transposable elements and guide Argonaute proteins to nascent RNA transcripts to induce co-transcriptional gene silencing. However, the molecular basis of this process remains unknown. Here, we identify the conserved RNA helicase Aquarius/EMB-4 as a direct and essential link between small RNA pathways and the transcriptional machinery in $\textit{Caenorhabditis elegans}$. Aquarius physically interacts with the germline Argonaute HRDE-1. Aquarius is required to initiate small-RNA-induced heritable gene silencing. HRDE-1 and Aquarius silence overlapping sets of genes and transposable elements. Surprisingly, removal of introns from a target gene abolishes the requirement for Aquarius, but not HRDE-1, for small RNA-dependent gene silencing. We conclude that Aquarius allows small RNA pathways to compete for access to nascent transcripts undergoing co-transcriptional splicing in order to detect and silence transposable elements. Thus, Aquarius and HRDE-1 act as gatekeepers coordinating gene expression and genome defense.A.C.B. was supported by an HFSP grant to E.A.M. (RPG0014/2015). This work was supported by Cancer Research UK (C13474/A18583, C6946/A14492), the Wellcome Trust (104640/Z/14/Z, 092096/Z/10/Z), and The European Research Council (ERC, grant 260688). The work of P.M. and X.Z. is supported by NIH grant R01GM113242 and NIH grant R01GM122080. R.M. was a Commonwealth Scholar, funded by the UK Government. J.M.C., A.N., and C.J.W. were supported by the CIHR (MOP-274660) and the Canada Research Chairs Program. A.I.L. was supported by a Wellcome Trust Programme Grant (108058/Z/15/Z) and M.L was supported by 2013/RSE/SCOTGOV/ MARIECURIE

Head-to-head comparison of BAM15, semaglutide, rosiglitazone, NEN, and calorie restriction on metabolic physiology in female <i>db/db</i> mice

Metabolic disorders such as type 2 diabetes, fatty liver disease, hyperlipidemia, and obesity commonly co-occur but clinical treatment options do not effectively target all disorders. Calorie restriction, semaglutide, rosiglitazone, and mitochondrial uncouplers have all demonstrated efficacy against one or more obesity-related metabolic disorders, but it currently remains unclear which therapeutic strategy best targets the combination of hyperglycaemia, liver fat, hypertriglyceridemia, and adiposity. Herein we performed a head-to-head comparison of 5 treatment interventions in the female db/db mouse model of severe metabolic disease. Treatments included ∼60 % calorie restriction (CR), semaglutide, rosiglitazone, BAM15, and niclosamide ethanolamine (NEN). Results showed that BAM15 and CR improved body weight and liver steatosis to levels superior to semaglutide, NEN, and rosiglitazone, while BAM15, semaglutide, and rosiglitazone improved glucose tolerance better than CR and NEN. BAM15, CR, semaglutide, and rosiglitazone all had efficacy against hypertriglyceridaemia. These data provide a comprehensive head-to-head comparison of several key treatment strategies for metabolic disease and highlight the efficacy of mitochondrial uncoupling to correct multiple facets of the metabolic disease milieu in female db/db mice.</p

Macrophage development and activation involve coordinated intron retention in key inflammatory regulators

Monocytes and macrophages are essential components of the innate immune system. Herein, we report that intron retention (IR) plays an important role in the development and function of these cells. Using Illumina mRNA sequencing, Nanopore direct cDNA sequencing and proteomics analysis, we identify IR events that affect the expression of key genes/proteins involved in macrophage development and function. We demonstrate that decreased IR in nuclear-detained mRNA is coupled with increased expression of genes encoding regulators of macrophage transcription, phagocytosis and inflammatory signalling, including ID2, IRF7, ENG and LAT. We further show that this dynamic IR program persists during the polarisation of resting macrophages into activated macrophages. In the presence of proinflammatory stimuli, intron-retaining CXCL2 and NFKBIZ transcripts are rapidly spliced, enabling timely expression of these key inflammatory regulators by macrophages. Our study provides novel insights into the molecular factors controlling vital regulators of the innate immune response

Characterization of the role of the Rab GTPase-activating protein AS160 in insulin-regulated GLUT4 trafficking

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. In the present study we have conducted a comprehensive proteomic analysis of affinity-purified GLUT4 vesicles from 3T3-L1 adipocytes to discover potential regulators of GLUT4 trafficking. In addition to previously identified components of GLUT4 storage vesicles including the insulin-regulated aminopeptidase insulin-regulated aminopeptidase and the vesicle soluble N-ethylmaleimide factor attachment protein (v-SNARE) VAMP2, we have identified three new Rab proteins, Rab10, Rab11, and Rab14, on GLUT4 vesicles. We have also found that the putative Rab GTPase-activating protein AS160 (Akt substrate of 160 kDa) is associated with GLUT4 vesicles in the basal state and dissociates in response to insulin. This association is likely to be mediated by the cytosolic tail of insulin-regulated aminopeptidase, which interacted both in vitro and in vivo with AS160. Consistent with an inhibitory role of AS160 in the basal state, reduced expression of AS160 in adipocytes using short hairpin RNA increased plasma membrane levels of GLUT4 in an insulin-independent manner. These findings support an important role for AS160 in the insulin regulated trafficking of GLUT4

Rab4b Is a Small GTPase Involved in the Control of the Glucose Transporter GLUT4 Localization in Adipocyte

Endosomal small GTPases of the Rab family, among them Rab4a, play an essential role in the control of the glucose transporter GLUT4 trafficking, which is essential for insulin-mediated glucose uptake. We found that adipocytes also expressed Rab4b and we observed a consistent decrease in the expression of Rab4b mRNA in human and mice adipose tissue in obese diabetic states. These results led us to study this poorly characterized Rab member and its potential role in glucose transport.We used 3T3-L1 adipocytes to study by imaging approaches the localization of Rab4b and to determine the consequence of its down regulation on glucose uptake and endogenous GLUT4 location. We found that Rab4b was localized in endosomal structures in preadipocytes whereas in adipocytes it was localized in GLUT4 and in VAMP2-positive compartments, and also in endosomal compartments containing the transferrin receptor (TfR). When Rab4b expression was decreased with specific siRNAs by two fold, an extent similar to its decrease in obese diabetic subjects, we observed a small increase (25%) in basal deoxyglucose uptake and a more sustained increase (40%) in presence of submaximal and maximal insulin concentrations. This increase occurred without any change in GLUT4 and GLUT1 expression levels and in the insulin signaling pathways. Concomitantly, GLUT4 but not TfR amounts were increased at the plasma membrane of basal and insulin-stimulated adipocytes. GLUT4 seemed to be targeted towards its non-endosomal sequestration compartment.Taken our results together, we conclude that Rab4b is a new important player in the control of GLUT4 trafficking in adipocytes and speculate that difference in its expression in obese diabetic states could act as a compensatory effect to minimize the glucose transport defect in their adipocytes

Phenotypic screen for oxygen consumption rate identifies an anti-cancer naphthoquinone that induces mitochondrial oxidative stress.

A hallmark of cancer cells is their ability to reprogram nutrient metabolism. Thus, disruption to this phenotype is a potential avenue for anti-cancer therapy. Herein we used a phenotypic chemical library screening approach to identify molecules that disrupted nutrient metabolism (by increasing cellular oxygen consumption rate) and were toxic to cancer cells. From this screen we discovered a 1,4-Naphthoquinone (referred to as BH10) that is toxic to a broad range of cancer cell types. BH10 has improved cancer-selective toxicity compared to doxorubicin, 17-AAG, vitamin K3, and other known anti-cancer quinones. BH10 increases glucose oxidation via both mitochondrial and pentose phosphate pathways, decreases glycolysis, lowers GSH:GSSG and NAPDH/NAPD+ ratios exclusively in cancer cells, and induces necrosis. BH10 targets mitochondrial redox defence as evidenced by increased mitochondrial peroxiredoxin 3 oxidation and decreased mitochondrial aconitase activity, without changes in markers of cytosolic or nuclear damage. Over-expression of mitochondria-targeted catalase protects cells from BH10-mediated toxicity, while the thioredoxin reductase inhibitor auranofin synergistically enhances BH10-induced peroxiredoxin 3 oxidation and cytotoxicity. Overall, BH10 represents a 1,4-Naphthoquinone with an improved cancer-selective cytotoxicity profile via its mitochondrial specificity