Role of a ribosomal RNA phosphate oxygen during the EF-G–triggered GTP hydrolysis
Miriam Koch, Sara Flür, Christoph Kreutz, Eric Ennifar, Ronald Micura, and Norbert Polacek
Translational GTPases are key players in ribosomal protein synthesis. Their intrinsic GTPase activity is low and is stimulated by ribosome association. Although it has been shown by structural and biochemical studies that the sarcin-ricin loop of ribosomal RNA (rRNA) closely approaches the supposedly catalytic His of elongation factor (EF)-G and EF-Tu and thereby may contribute to GTP hydrolysis, the exact mechanism of activation remains unclear and is a matter of controversial debate. Using the atomic mutagenesis approach that allows incorporation of nonnatural 23S rRNA nucleotides into 70S particles, we show that a nonbridging phosphate oxygen of rRNA is key for GTP hydrolysis of translational GTPases (pp. E2561–E2568). To date, this is only the second rRNA backbone residue shown to be critical in ribosome function.
Genetically encoded sensors of protein hydrodynamics and molecular proximity
Alexander C. Hoepker, Ariel Wang, Alix Le Marois, Klaus Suhling, Yuling Yan, and Gerard Marriott
The lumazine binding protein (LUMP) emits a cyan-colored fluorescence and has the longest average fluorescence lifetime of any genetically encoded fluorescent protein complex. Coupled with a small mass of 20 kDa, LUMP and its fusion with capture sequences are exploited as unique sensors of protein hydrodynamics and are shown (pp. E2569–E2574) to enable quantitative fluorescence anisotropy imaging of specific target proteins in vitro and in vivo. Moreover, the surface location of the lumazine probe is shown to improve the efficiency of Förster resonance energy transfer (FRET) with the Venus acceptor protein compared with CFP, which is used in the development of a new class of FRET-based sensor.
Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair
Dylan A. Reid, Sarah Keegan, Alejandra Leo-Macias, Go Watanabe, Natasha T. Strande, Howard H. Chang, Betul Akgol Oksuz, David Fenyo, Michael R. Lieber, Dale A. Ramsden, and Eli Rothenberg
Nonhomologous end-joining (NHEJ) is the main pathway for repair of DNA double-strand breaks (DSBs), the most cytotoxic form of DNA damage resulting from ionizing radiation, chemotherapeutics, and normal cellular processes. The mechanisms that control NHEJ play key roles in development, in immunity, and in response to cancer therapy; however, the current state of knowledge regarding the physical nature of the NHEJ repair process is limited. Here (pp. E2575–E2584) we used super-resolution microscopy to define the organization of NHEJ complexes in cells, showing that long filaments form at either side of the break. Single-molecule FRET revealed dynamic behavior in which breaks can pair in an adjacent, non–end-to-end configuration.
Ligand deconstruction: Why some fragment binding positions are conserved and others are not
Dima Kozakov, David R. Hall, Sefan Jehle, Lingqi Luo, Stefan O. Ochiana, Elizabeth V. Jones, Michael Pollastri, Karen N. Allen, Adrian Whitty, and Sandor Vajda
Fragment-based drug discovery (FBDD), in which initial screening is done with low-molecular-weight compounds called fragments, relies on the premise that the fragment binding mode will be conserved on subsequent expansion to a larger ligand. We describe (pp. E2585–E2594) a remarkably simple condition for fragment binding conservation that can be tested computationally. The condition can be used for detecting whether a protein is suitable for FBDD, for predicting the size of fragments required for screening, and for determining if a fragment hit can be extended into a higher affinity ligand. The findings also reveal general properties of binding sites, highlighting the role that critical interactions between anchor sites and anchor fragments play in protein–ligand interactions in general.
Mechanical stimulation induces formin-dependent assembly of a perinuclear actin rim
Xiaowei Shao, Qingsen Li, Alex Mogilner, Alexander D. Bershadsky, and G. V. Shivashankar
Cells can sense and adapt to their physical microenvironment through specific mechanosensing mechanisms. These properties are often mediated by the actin cytoskeleton, which can be affected by a wide range of forces, including fluid shear stress, cyclic stretch, and optical or magnetic force. However, the immediate effects of force on the assembly of actin structures distal from the sites of force application were not assessed. Our work (pp. E2595–E2601) reveals a previously unidentified actin structure, a perinuclear actin rim, which is induced by mechanical stimulation of cells. We show that, on local force application to the cell periphery, a distal effect emerges at the perinuclear region. Such distal effects have potential implications in modulating nuclear functions by local mechanical signals from the cell periphery.
Therapeutic effects of cell-permeant peptides that activate G proteins downstream of growth factors
Gary S. Ma, Nicolas Aznar, Nicholas Kalogriopoulos, Krishna K. Midde, Inmaculada Lopez-Sanchez, Emi Sato, Ying Dunkel, Richard L. Gallo, and Pradipta Ghosh
Most common diseases (e.g., cancer, inflammatory disorders, diabetes) are driven by not one, but multiple cell surface receptors that trigger and sustain a pathologic signaling network. The largest fraction of therapeutic agents that target individual receptors/pathways often eventually fail due to the emergence of compensatory mechanisms. Recently, we identified GIV protein as a central platform for receptor cross-talk which integrates signals downstream of a myriad of upstream receptors, and modulates several key pathways within downstream signaling network, all via activation of trimeric G proteins. Here (pp. E2602–E2610) we provide the proof-of-concept that nongenetic exogenous modulation of the GIV-Gi signaling interface using cell-penetrable GIV-derived peptides is an effective strategy to reset pathologic signaling networks downstream of multiple receptors in a diverse array of pathophysiologic conditions.
Apolipoprotein CIII links islet insulin resistance to β-cell failure in diabetes
Karin Åvall, Yusuf Ali, Ingo B. Leibiger, Barbara Leibiger, Tilo Moede, Meike Paschen, Andrea Dicker, Elisabetta Daré, Martin Köhler, Erwin Ilegems, Midhat H. Abdulreda, Mark Graham, Rosanne M. Crooke, Vanessa S. Y. Tay, Essam Refai, Stefan K. Nilsson, Stefan Jacob, Lars Selander, Per-Olof Berggren, and Lisa Juntti-Berggren
Insulin resistance and β-cell failure are the major defects in type 2 diabetes. We now demonstrate that local insulin resistance-induced increase in apolipoprotein CIII (apoCIII) within pancreatic islets causes promotion of an intraislet inflammatory milieu, increased mitochondrial metabolism, deranged regulation of β-cell cytoplasmic free Ca2+ concentration ([Ca2+]i), and apoptosis (pp. E2611–E2619). Decreasing apoCIII in vivo in animals with insulin resistance improves glucose tolerance, and apoCIII knockout islets transplanted into diabetic mice, with high systemic levels of apoCIII, demonstrate a normal [Ca2+]i response pattern and no hallmarks of inflammation. Hence, under conditions of islet insulin resistance, locally produced apoCIII is an important diabetogenic factor involved in impairment of β-cell function and may thus constitute a novel target for the treatment of type 2 diabetes.
Dictyostelium discoideum has a highly Q/N-rich proteome and shows an unusual resilience to protein aggregation
Liliana Malinovska, Sandra Palm, Kimberley Gibson, Jean-Marc Verbavatz, and Simon Alberti
Proteins carrying aggregation-prone prion-like domains cause many neurodegenerative diseases. The presence of prions in yeast has provided important insights into these disease processes and mechanisms of cellular proteostasis. However, it is not known whether these findings extend to other organisms. In this paper, we show that Dictyostelium discoideum has the highest content of prion-like proteins of all organisms investigated to date. Remarkably, overexpressed prion-like proteins remain soluble and are innocuous to D. discoideum, in contrast to yeast and other organisms, where they form cytotoxic cytosolic aggregates. However, when exposed to conditions that compromise proteostasis, these proteins aggregate and become cytotoxic. This indicates that D. discoideum has undergone specific adaptations in its proteostasis machinery to control its highly aggregation-prone prion-like proteome (pp. E2620–E2629).
Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells
Andrea Milenkovic, Caroline Brandl, Vladimir M. Milenkovic, Thomas Jendryke, Lalida Sirianant, Potchanart Wanitchakool, Stephanie Zimmermann, Charlotte M. Reiff, Franziska Horling, Heinrich Schrewe, Rainer Schreiber, Karl Kunzelmann, Christian H. Wetzel, and Bernhard H. F. Weber
First insight into the molecular identity of volume-regulated anion channel (VRAC) emerged only recently by demonstrating a role for leucine-rich repeats containing 8A (LRRC8A) in channel activity. Our results (pp. E2630–E2639) now expand on VRAC biology, suggesting a model where VRAC subunit composition is cell type- or tissue-specific rather than a single ubiquitous channel formed solely by LRRC8A. Here, we show that bestrophin 1 (BEST1), but not LRCC8A, is crucial in cell volume regulation in retinal pigment epithelium (RPE) cells differentiated from human-induced pluripotent stem cells (hiPSCs). VRAC-mediated currents were strongly reduced in hiPSC-RPE from macular dystrophy patients with pathologic BEST1 mutations. Our model is further supported by in vivo effects of Best1 deficiency in the mouse that manifest as severe subfertility phenotype due to enhanced abnormal sperm morphology related to impaired volume regulation.
Metabolic theory and taxonomic identity predict nutrient recycling in a diverse food web
Jacob Edward Allgeier, Seth J. Wenger, Amy D. Rosemond, Daniel E. Schindler, and Craig A. Layman
A fundamental dilemma in ecology is to reconcile the degree to which ecological processes are generalizable among taxa and ecosystems or determined primarily by taxonomic identity. We apply a unique dataset of organisms from a diverse marine community to test the applicability of two theories, metabolic theory of ecology (MTE) and ecological stoichiometry (EST), and the role of taxonomic identity for predicting nutrient excretion rates by fishes and macroinvertebrates. Excretion rates were principally explained by body mass and taxonomic identity, providing strong support for MTE, but also highlighting the intrinsic importance of taxonomic identity. Little support for basic predictions of EST was found. This research (pp. E2640–E2647) reveals animal-mediated nutrient cycling is largely generalizable by metabolic processes, but refined predictions require taxa-specific understanding.
Supplementary feeding restructures urban bird communities
Josie A. Galbraith, Jacqueline R. Beggs, Darryl N. Jones, and Margaret C. Stanley
Bird feeding is essentially a massive global supplementary feeding experiment, yet few studies have attempted to explore its ecological effects. In this study (pp. E2648–E2657) we use an in situ experimental approach to investigate the impacts of bird feeding on the structure of local bird assemblages. We present vital evidence that bird feeding contributes to the bird community patterns we observe in urban areas. In particular, the study demonstrates that common feeding practices can encourage higher densities of introduced birds, with potential negative consequences for native birds.
Obstruction of adaptation in diploids by recessive, strongly deleterious alleles
Zoe June Assaf, Dmitri A. Petrov, and Jamie R. Blundell
A deleterious mutation that is recessive is hidden in individuals containing only one copy (i.e., heterozygotes); however, individuals containing two copies (i.e., homozygotes) suffer negative effects. This class of mutation is responsible for a number of human genetic disorders, including cystic fibrosis and Tay-Sachs, in addition to causing the widespread phenomenon of inbreeding depression. Evidence suggests that recessive deleterious mutations may be abundant in nature, likely due to their ability to persist for long timescales at moderate frequencies (pp. E2658–E2666). It is thus possible that when an adaptive mutation occurs, it lands on a chromosome containing a recessive deleterious mutation. We propose a model for this and find that recessive deleterious mutations can significantly slow adaptation and alter signatures of adaptation.
Lack of pairing during meiosis triggers multigenerational transgene silencing in Caenorhabditis elegans
Luciana E. Leopold, Bree N. Heestand, Soobin Seong, Ludmila Shtessel, and Shawn Ahmed
Transgenes can be permanently silenced in a single generation via a previously described small RNA-induced epigenetic silencing (RNAe) mechanism, which is promoted by the presence of a perfect Piwi-interacting RNA (piRNA) target site. In this study (pp. E2667–E2676), we identify a previously unidentified mechanism capable of silencing single-copy transgenes that lack perfect piRNA target sites and that is triggered by a lack of chromosomal pairing during meiosis for multiple generations. Multigenerational RNAe can lead to reversible or permanent transgene silencing and may provide insight into variability in the expression of single-copy transgenes or single-copy genomic insertions, which are commonly used in experimental biology. Our analysis of “multigenerational RNAe” also offers new insights into potentially common epigenetic silencing events relevant to genome expression in the germline and embryo.
Interleukin-6/interleukin-21 signaling axis is critical in the pathogenesis of pulmonary arterial hypertension
Takahiro Hashimoto-Kataoka, Naoki Hosen, Takashi Sonobe, Yoh Arita, Taku Yasui, Takeshi Masaki, Masato Minami, Tadakatsu Inagaki, Shigeru Miyagawa, Yoshiki Sawa, Masaaki Murakami, Atsushi Kumanogoh, Keiko Yamauchi-Takihara, Meinoshin Okumura, Tadamitsu Kishimoto, Issei Komuro, Mikiyasu Shirai, Yasushi Sakata, and Yoshikazu Nakaoka
Pulmonary arterial hypertension (PAH) is a serious disease characterized by vascular remodeling in pulmonary arteries. Although an elevated IL-6 serum level correlates with poor prognosis of PAH patients, it is unclear how IL-6 promotes PAH. Here (pp. E2677–E2686) we identified IL-21 as a downstream target of IL-6 signaling in PAH. In mice with hypoxia-induced pulmonary hypertension (HPH), Th17 cells and M2 macrophages accumulate in the lungs after hypoxia exposure. IL-21 primarily derived from Th17 cells promotes M2 macrophage polarization. Consistently, IL-21 receptor-deficient mice show resistance to HPH with no accumulation of M2 macrophages in the lungs. IL-21 and M2 macrophage markers were upregulated in the lungs of patients with end-stage idiopathic PAH. These findings suggest promising therapeutic strategies for PAH targeting IL-6/IL-21–signaling axis.
CD4 mimetics sensitize HIV-1-infected cells to ADCC
Jonathan Richard, Maxime Veillette, Nathalie Brassard, Shilpa S. Iyer, Michel Roger, Loïc Martin, Marzena Pazgier, Arne Schön, Ernesto Freire, Jean-Pierre Routy, Amos B. Smith III, Jongwoo Park, David M. Jones, Joel R. Courter, Bruno N. Melillo, Daniel E. Kaufmann, Beatrice H. Hahn, Sallie R. Permar, Barton F. Haynes, Navid Madani, Joseph G. Sodroski, and Andrés Finzi
The prevention of HIV-1 transmission and progression likely requires approaches that can specifically eliminate HIV-1-infected cells. Rationally designed CD4-mimetic compounds (CD4mc) have been shown to efficiently inhibit viral entry and sensitize HIV-1 particles to neutralization by otherwise nonneutralizing CD4-induced antibodies. Here (pp. E2687–E2694) we found that CD4mc can also sensitize HIV-1-infected cells to antibody-dependent cell-mediated cytotoxicity (ADCC). Indeed, CD4mc induced the CD4-bound conformation of HIV-1 envelope glycoproteins, exposing CD4-induced epitopes recognized by easy-to-elicit antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we provide evidence that CD4mc can efficiently sensitize primary CD4 T cells from HIV-1-infected individuals to ADCC mediated by autologous sera and effector cells. Therefore, CD4mc might represent an attractive approach to prevent and control HIV-1 infection.
Connectivity reveals relationship of brain areas for reward-guided learning and decision making in human and monkey frontal cortex
Franz-Xaver Neubert, Rogier B. Mars, Jérôme Sallet, and Matthew F. S. Rushworth
Because of the interest in reward-guided learning and decision making, these neural mechanisms have been studied in both humans and monkeys. But whether and how key brain areas correspond between the two species has been uncertain. Areas in the two species can be compared as a function of the brain circuits in which they participate, which can be estimated from patterns of correlation in brain activity measured with functional MRI. Taking such measurements in 38 humans and 25 macaques, we identified fundamental similarities between the species and one human frontal area with no monkey counterpart. Altogether these findings (pp. E2695–E2704) suggest that everyday human decision-making capitalizes on a neural apparatus similar to the one that supports monkeys when foraging in the wild.
Modulation of extrasynaptic NMDA receptors by synaptic and tonic zinc
Charles T. Anderson, Robert J. Radford, Melissa L. Zastrow, Daniel Y. Zhang, Ulf-Peter Apfel, Stephen J. Lippard, and Thanos Tzounopoulos
As an essential element for living organisms, zinc is a cofactor in many enzymes and regulatory proteins. After the surprising discovery of mobile zinc in synaptic vesicles throughout many areas of the brain, numerous investigators have studied its possible roles during neurotransmission. Nonetheless, knowledge of the physiology of zinc at the synapse is still in its infancy. Here, we show that synaptic and tonic zinc inhibit extrasynaptic NMDA receptors (NMDARs), which are widely distributed in the CNS and are important for normal and pathological excitatory signaling. Our work (pp. E2705–E2714) indicates that this newly discovered interaction between zinc and extrasynaptic NMDARs can provide a general mechanism for controlling neuronal excitability in the CNS.
The phototransduction machinery in the rod outer segment has a strong efficacy gradient
Monica Mazzolini, Giuseppe Facchetti, Laura Andolfi, Remo Proietti Zaccaria, Salvatore Tuccio, Johannes Treu, Claudio Altafini, Enzo M. Di Fabrizio, Marco Lazzarino, Gert Rapp, and Vincent Torre
Phototransduction is now considered to be a quite thoroughly understood phenomenon. It is well known that new discs are continuously generated at the base of the outer segments (OSs) and old discs are shed at their tip, but the rod OSs are considered a well-stirred compartment with minor inhomogeneities. To verify this assumption and to better understand the machinery within the OS, we developed a new methodology to deliver highly localized lights. We found that (pp. E2715–E2724), as the light stimulus is moved from the OS base toward its tip, the amplitude of saturating and single photon responses decreased by 5–10 times. This gradient of efficacy is attributed to a progressive loss of phosphodiesterase. Therefore, OSs are highly inhomogeneous compartments.
Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro
Cedric Bardy, Mark van den Hurk, Tameji Eames, Cynthia Marchand, Ruben V. Hernandez, Mariko Kellogg, Mark Gorris, Ben Galet, Vanessa Palomares, Joshua Brown, Anne G. Bang, Jerome Mertens, Lena Böhnke, Leah Boyer, Suzanne Simon, and Fred H. Gage
Neuronal cultures are very valuable to learn about basic principles of the nervous system. In vivo, neural electrical activity is the essence of nervous system function, controlling emotion, memory, sensory modalities, and behavior. In this study (pp. E2725–E2734), we discovered that many crucial neurophysiological properties were strongly altered in classic culture media that are widely used by the research community. To overcome this problem, we designed and tested a new tissue culture neuromedium that adequately supports in vitro neuronal activity. The improvements made in this medium reduce the gap between in vivo brain physiological conditions and neuronal models in vitro. Improving physiological conditions in vitro may lead to more successful translation from bench to clinics.