Nuclear translocation of vitellogenin in the honey bee (Apis mellifera)

Vitellogenin (Vg) is a conserved protein used by nearly all oviparous animals to produce eggs. It is also pleiotropic and performs functions in oxidative stress resistance, immunity, and, in honey bees, behavioral development of the worker caste. It has remained enigmatic how Vg affects multiple traits. Here, we asked whether Vg enters the nucleus and acts via DNA-binding. We used cell fractionation, immunohistology, and cell culture to show that a structural subunit of honey bee Vg translocates into cell nuclei. We then demonstrated Vg-DNA binding theoretically and empirically with prediction software and chromatin immunoprecipitation with sequencing (ChIP-seq), finding binding sites at genes influencing immunity and behavior. Finally, we investigated the immunological and enzymatic conditions affecting Vg cleavage and nuclear translocation and constructed a 3D structural model. Our data are the first to show Vg in the nucleus and suggest a new fundamental regulatory role for this ubiquitous protein.Peer reviewe

Titin domains with reduced core hydrophobicity cause dilated cardiomyopathy.

The underlying genetic defect in most cases of dilated cardiomyopathy (DCM), a common inherited heart disease, remains unknown. Intriguingly, many patients carry single missense variants of uncertain pathogenicity targeting the giant protein titin, a fundamental sarcomere component. To explore the deleterious potential of these variants, we first solved the wild-type and mutant crystal structures of I21, the titin domain targeted by pathogenic variant p.C3575S. Although both structures are remarkably similar, the reduced hydrophobicity of deeply buried position 3575 strongly destabilizes the mutant domain, a scenario supported by molecular dynamics simulations and by biochemical assays that show no disulfide involving C3575. Prompted by these observations, we have found that thousands of similar hydrophobicity-reducing variants associate specifically with DCM. Hence, our results imply that titin domain destabilization causes DCM, a conceptual framework that not only informs pathogenicity assessment of gene variants but also points to therapeutic strategies counterbalancing protein destabilization.S

Mechanical unfolding of long human telomeric RNA (TERRA)

[EN] We report the first single molecule investigation of TERRA molecules. By using optical-tweezers and other biophysical techniques, we have found that long RNA constructions of up to 25 GGGUUA repeats form higher order structures comprised of single parallel G-quadruplex blocks, which unfold at lower forces than their DNA counterparts.This work was supported by grants from the Spanish Ministry of Science and Innovation (grants RYC2007-01765 to JRA-G, BFU2011-30295-C02-01 to AV, and CTQ2010-21567-C02-02 to CG). MG was supported by the FPI fellowship BES-2009-027909. RB and EH-G were supported by Comunidad de Madrid, grant CAM-S2009MAT-1507. AV acknowledges an institutional grant from the Fundacion Ramon Areces to the CBMSO. JRA-G wants to thank Prof. J. L. Carrascosa and Prof. J. M. Valpuesta (CNB-CSIC) for their continuous support and encouragement in this research. We also acknowledge the excellent technical assistance of Beatriz de Pablos (CBMSO).Garavís, M.; Bocanegra, R.; Herrero-Galán, E.; González, C.; Villasante, A.; Arias-Gonzalez, JR. (2013). Mechanical unfolding of long human telomeric RNA (TERRA). Chemical Communications. 49(57):6397-6399. https://doi.org/10.1039/c3cc42981dS639763994957De Lange, T. (2005). Shelterin: the protein complex that shapes and safeguards human telomeres. Genes & Development, 19(18), 2100-2110. doi:10.1101/gad.1346005Blackburn, E. H. (1991). Structure and function of telomeres. Nature, 350(6319), 569-573. doi:10.1038/350569a0Biffi, G., Tannahill, D., McCafferty, J., & Balasubramanian, S. (2013). Quantitative visualization of DNA G-quadruplex structures in human cells. Nature Chemistry, 5(3), 182-186. doi:10.1038/nchem.1548Paeschke, K., Simonsson, T., Postberg, J., Rhodes, D., & Lipps, H. J. (2005). Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo. Nature Structural & Molecular Biology, 12(10), 847-854. doi:10.1038/nsmb982Hwang, H., Buncher, N., Opresko, P. L., & Myong, S. (2012). POT1-TPP1 Regulates Telomeric Overhang Structural Dynamics. Structure, 20(11), 1872-1880. doi:10.1016/j.str.2012.08.018Azzalin, C. M., Reichenbach, P., Khoriauli, L., Giulotto, E., & Lingner, J. (2007). Telomeric Repeat Containing RNA and RNA Surveillance Factors at Mammalian Chromosome Ends. Science, 318(5851), 798-801. doi:10.1126/science.1147182Schoeftner, S., & Blasco, M. A. (2007). Developmentally regulated transcription of mammalian telomeres by DNA-dependent RNA polymerase II. Nature Cell Biology, 10(2), 228-236. doi:10.1038/ncb1685Porro, A., Feuerhahn, S., Reichenbach, P., & Lingner, J. (2010). Molecular Dissection of Telomeric Repeat-Containing RNA Biogenesis Unveils the Presence of Distinct and Multiple Regulatory Pathways. Molecular and Cellular Biology, 30(20), 4808-4817. doi:10.1128/mcb.00460-10Deng, Z., Norseen, J., Wiedmer, A., Riethman, H., & Lieberman, P. M. (2009). TERRA RNA Binding to TRF2 Facilitates Heterochromatin Formation and ORC Recruitment at Telomeres. Molecular Cell, 35(4), 403-413. doi:10.1016/j.molcel.2009.06.025De Silanes, I. L., d’ Alcontres, M. S., & Blasco, M. A. (2010). TERRA transcripts are bound by a complex array of RNA-binding proteins. Nature Communications, 1(1). doi:10.1038/ncomms1032Xu, Y., Suzuki, Y., Ito, K., & Komiyama, M. (2010). Telomeric repeat-containing RNA structure in living cells. Proceedings of the National Academy of Sciences, 107(33), 14579-14584. doi:10.1073/pnas.1001177107Martadinata, H., & Phan, A. T. (2009). Structure of Propeller-Type Parallel-Stranded RNA G-Quadruplexes, Formed by Human Telomeric RNA Sequences in K+Solution. Journal of the American Chemical Society, 131(7), 2570-2578. doi:10.1021/ja806592zXu, Y., Kaminaga, K., & Komiyama, M. (2008). G-Quadruplex Formation by Human Telomeric Repeats-Containing RNA in Na+Solution. Journal of the American Chemical Society, 130(33), 11179-11184. doi:10.1021/ja8031532Collie, G. W., Haider, S. M., Neidle, S., & Parkinson, G. N. (2010). A crystallographic and modelling study of a human telomeric RNA (TERRA) quadruplex. Nucleic Acids Research, 38(16), 5569-5580. doi:10.1093/nar/gkq259Collie, G. W., Parkinson, G. N., Neidle, S., Rosu, F., De Pauw, E., & Gabelica, V. (2010). Electrospray Mass Spectrometry of Telomeric RNA (TERRA) Reveals the Formation of Stable Multimeric G-Quadruplex Structures. Journal of the American Chemical Society, 132(27), 9328-9334. doi:10.1021/ja100345zMartadinata, H., Heddi, B., Lim, K. W., & Phan, A. T. (2011). Structure of Long Human Telomeric RNA (TERRA): G-Quadruplexes Formed by Four and Eight UUAGGG Repeats Are Stable Building Blocks. Biochemistry, 50(29), 6455-6461. doi:10.1021/bi200569fArora, A., & Maiti, S. (2009). Differential Biophysical Behavior of Human Telomeric RNA and DNA Quadruplex. The Journal of Physical Chemistry B, 113(30), 10515-10520. doi:10.1021/jp810638nJoachimi, A., Benz, A., & Hartig, J. S. (2009). A comparison of DNA and RNA quadruplex structures and stabilities. Bioorganic & Medicinal Chemistry, 17(19), 6811-6815. doi:10.1016/j.bmc.2009.08.043Qi, J., & Shafer, R. H. (2007). Human Telomere Quadruplex:  Refolding and Selection of Individual Conformers via RNA/DNA Chimeric Editing†. Biochemistry, 46(25), 7599-7606. doi:10.1021/bi602392uRandall, A., & Griffith, J. D. (2009). Structure of Long Telomeric RNA Transcripts. Journal of Biological Chemistry, 284(21), 13980-13986. doi:10.1074/jbc.m900631200Kumari, S., Bugaut, A., & Balasubramanian, S. (2008). Position and Stability Are Determining Factors for Translation Repression by an RNA G-Quadruplex-Forming Sequence within the 5′ UTR of theNRASProto-oncogene†. Biochemistry, 47(48), 12664-12669. doi:10.1021/bi8010797McKenna, S. A., Kim, I., Puglisi, E. V., Lindhout, D. A., Aitken, C. E., Marshall, R. A., & Puglisi, J. D. (2007). Purification and characterization of transcribed RNAs using gel filtration chromatography. Nature Protocols, 2(12), 3270-3277. doi:10.1038/nprot.2007.480Parkinson, G. N., Lee, M. P. H., & Neidle, S. (2002). Crystal structure of parallel quadruplexes from human telomeric DNA. Nature, 417(6891), 876-880. doi:10.1038/nature755Herrero-Galán, E., Fuentes-Perez, M. E., Carrasco, C., Valpuesta, J. M., Carrascosa, J. L., Moreno-Herrero, F., & Arias-Gonzalez, J. R. (2012). Mechanical Identities of RNA and DNA Double Helices Unveiled at the Single-Molecule Level. Journal of the American Chemical Society, 135(1), 122-131. doi:10.1021/ja3054755Bustamante, C., Bryant, Z., & Smith, S. B. (2003). Ten years of tension: single-molecule DNA mechanics. Nature, 421(6921), 423-427. doi:10.1038/nature01405Yu, Z., Schonhoft, J. D., Dhakal, S., Bajracharya, R., Hegde, R., Basu, S., & Mao, H. (2009). ILPR G-Quadruplexes Formed in Seconds Demonstrate High Mechanical Stabilities. Journal of the American Chemical Society, 131(5), 1876-1882. doi:10.1021/ja806782sKoirala, D., Dhakal, S., Ashbridge, B., Sannohe, Y., Rodriguez, R., Sugiyama, H., … Mao, H. (2011). A single-molecule platform for investigation of interactions between G-quadruplexes and small-molecule ligands. Nature Chemistry, 3(10), 782-787. doi:10.1038/nchem.1126Dhakal, S., Cui, Y., Koirala, D., Ghimire, C., Kushwaha, S., Yu, Z., … Mao, H. (2013). Structural and mechanical properties of individual human telomeric G-quadruplexes in molecularly crowded solutions. Nucleic Acids Research, 41(6), 3915-3923. doi:10.1093/nar/gkt038De Messieres, M., Chang, J.-C., Brawn-Cinani, B., & La Porta, A. (2012). Single-Molecule Study ofG-Quadruplex Disruption Using Dynamic Force Spectroscopy. Physical Review Letters, 109(5). doi:10.1103/physrevlett.109.058101Schonhoft, J. D., Bajracharya, R., Dhakal, S., Yu, Z., Mao, H., & Basu, S. (2009). Direct experimental evidence for quadruplex–quadruplex interaction within the human ILPR. Nucleic Acids Research, 37(10), 3310-3320. doi:10.1093/nar/gkp181Carrion-Vazquez, M., Oberhauser, A. F., Fowler, S. B., Marszalek, P. E., Broedel, S. E., Clarke, J., & Fernandez, J. M. (1999). Mechanical and chemical unfolding of a single protein: A comparison. Proceedings of the National Academy of Sciences, 96(7), 3694-3699. doi:10.1073/pnas.96.7.369

Functional Characterization of MODY2 Mutations Highlights the Importance of the Fine-Tuning of Glucokinase and Its Role in Glucose Sensing

Glucokinase (GK) acts as a glucose sensor in the pancreatic beta-cell and regulates insulin secretion. Heterozygous mutations in the human GK-encoding GCK gene that reduce the activity index increase the glucose-stimulated insulin secretion threshold and cause familial, mild fasting hyperglycaemia, also known as Maturity Onset Diabetes of the Young type 2 (MODY2). Here we describe the biochemical characterization of five missense GK mutations: p.Ile130Thr, p.Asp205His, p.Gly223Ser, p.His416Arg and p.Ala449Thr. The enzymatic analysis of the corresponding bacterially expressed GST-GK mutant proteins show that all of them impair the kinetic characteristics of the enzyme. In keeping with their position within the protein, mutations p.Ile130Thr, p.Asp205His, p.Gly223Ser, and p.His416Arg strongly decrease the activity index of GK, affecting to one or more kinetic parameters. In contrast, the p.Ala449Thr mutation, which is located in the allosteric activator site, does not affect significantly the activity index of GK, but dramatically modifies the main kinetic parameters responsible for the function of this enzyme as a glucose sensor. The reduced Kcat of the mutant (3.21±0.28 s−1 vs 47.86±2.78 s−1) is balanced by an increased glucose affinity (S0.5 = 1.33±0.08 mM vs 7.86±0.09 mM) and loss of cooperativity for this substrate. We further studied the mechanism by which this mutation impaired GK kinetics by measuring the differential effects of several competitive inhibitors and one allosteric activator on the mutant protein. Our results suggest that this mutation alters the equilibrium between the conformational states of glucokinase and highlights the importance of the fine-tuning of GK and its role in glucose sensing

Using Prior Information from the Medical Literature in GWAS of Oral Cancer Identifies Novel Susceptibility Variant on Chromosome 4 - the AdAPT Method

Background: Genome-wide association studies (GWAS) require large sample sizes to obtain adequate statistical power, but it may be possible to increase the power by incorporating complementary data. In this study we investigated the feasibility of automatically retrieving information from the medical literature and leveraging this information in GWAS. Methods: We developed a method that searches through PubMed abstracts for pre-assigned keywords and key concepts, and uses this information to assign prior probabilities of association for each single nucleotide polymorphism (SNP) with the phenotype of interest - the Adjusting Association Priors with Text (AdAPT) method. Association results from a GWAS can subsequently be ranked in the context of these priors using the Bayes False Discovery Probability (BFDP) framework. We initially tested AdAPT by comparing rankings of known susceptibility alleles in a previous lung cancer GWAS, and subsequently applied it in a two-phase GWAS of oral cancer. Results: Known lung cancer susceptibility SNPs were consistently ranked higher by AdAPT BFDPs than by p-values. In the oral cancer GWAS, we sought to replicate the top five SNPs as ranked by AdAPT BFDPs, of which rs991316, located in the ADH gene region of 4q23, displayed a statistically significant association with oral cancer risk in the replication phase (per-rare-allele log additive p-value [p(trend)] = 2.5 x 10(-3)). The combined OR for having one additional rare allele was 0.83 (95% CI: 0.76-0.90), and this association was independent of previously identified susceptibility SNPs that are associated with overall UADT cancer in this gene region. We also investigated if rs991316 was associated with other cancers of the upper aerodigestive tract (UADT), but no additional association signal was found. Conclusion: This study highlights the potential utility of systematically incorporating prior knowledge from the medical literature in genome-wide analyses using the AdAPT methodology. AdAPT is available online (url: http://services.gate.ac.uk/lld/gwas/service/config)

Participation of Candida albicans transcription factor Rlm1 in cell wall biogenesis and virulence

Candida albicans cell wall is important for growth and interaction with the environment. RLM1 is one of the putative transcription factors involved in the cell wall integrity pathway, which plays an important role in the maintenance of the cell wall integrity. In this work we investigated the involvement of RLM1 in the cell wall biogenesis and in virulence. Newly constructed C. albicans Δ/Δrlm1 mutants showed typical cell wall weakening phenotypes, such as hypersensitivity to Congo Red, Calcofluor White, and caspofungin (phenotype reverted in the presence of sorbitol), confirming the involvement of RLM1 in the cell wall integrity. Additionally, the cell wall of C. albicans Δ/Δrlm1 showed a significant increase in chitin (213%) and reduction in mannans (60%), in comparison with the wild-type, results that are consistent with cell wall remodelling. Microarray analysis in the absence of any stress showed that deletion of RLM1 in C. albicans significantly down-regulated genes involved in carbohydrate catabolism such as DAK2, GLK4, NHT1 and TPS1, up-regulated genes involved in the utilization of alternative carbon sources, like AGP2, SOU1, SAP6, CIT1 or GAL4, and genes involved in cell adhesion like ECE1, ALS1, ALS3, HWP1 or RBT1. In agreement with the microarray results adhesion assays showed an increased amount of adhering cells and total biomass in the mutant strain, in comparison with the wild-type. C. albicans mutant Δ/Δrlm1 strain was also found to be less virulent than the wild-type and complemented strains in the murine model of disseminated candidiasis. Overall, we showed that in the absence of RLM1 the modifications in the cell wall composition alter yeast interaction with the environment, with consequences in adhesion ability and virulence. The gene expression findings suggest that this gene participates in the cell wall biogenesis, with the mutant rearranging its metabolic pathways to allow the use of alternative carbon sources.This work was supported by CBMA (Centre of Molecular and Environmental Biology) through the FCT (Fundacao para a Ciencia e Tecnologia) project PEst-C/BIA/UI4050/2011. Yolanda Delgado-Silva was supported by an ALbAN scholarship (No E07D400922PE), and Alexandra Correia by SFRH/BD/31354/2006 fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie