RESUMEN
Phosphofructokinase-1 (PFK1) catalyzes the rate-limiting step of glycolysis, committing glucose to conversion into cellular energy. PFK1 is highly regulated to respond to the changing energy needs of the cell. In bacteria, the structural basis of PFK1 regulation is a textbook example of allostery; molecular signals of low and high cellular energy promote transition between an active R-state and inactive T-state conformation, respectively. Little is known, however, about the structural basis for regulation of eukaryotic PFK1. Here, we determine structures of the human liver isoform of PFK1 (PFKL) in the R- and T-state by cryoEM, providing insight into eukaryotic PFK1 allosteric regulatory mechanisms. The T-state structure reveals conformational differences between the bacterial and eukaryotic enzyme, the mechanisms of allosteric inhibition by ATP binding at multiple sites, and an autoinhibitory role of the C-terminus in stabilizing the T-state. We also determine structures of PFKL filaments that define the mechanism of higher-order assembly and demonstrate that these structures are necessary for higher-order assembly of PFKL in cells.
Asunto(s)
Adenosina Trifosfato , Fosfofructoquinasa-1 , Humanos , Regulación Alostérica , Fosfofructoquinasa-1/metabolismo , Fosfofructoquinasa-1/química , Fosfofructoquinasa-1/genética , Adenosina Trifosfato/metabolismo , Microscopía por Crioelectrón , Modelos Moleculares , Conformación Proteica , Glucólisis , Hígado/enzimología , Hígado/metabolismoRESUMEN
The final and rate-limiting enzyme in pyrimidine biosynthesis, CTP synthase (CTPS) , is essential for the viability of Mycobacterium tuberculosis and other mycobacteria. Its product, CTP, is critical for RNA, DNA, lipid and cell wall synthesis, and is involved in chromosome segregation. In various organisms across the tree of life, CTPS assembles into higher-order filaments, leading us to hypothesize that M. tuberculosis CTPS (mtCTPS) also forms higher-order structures. Here, we show that mtCTPS does assemble into filaments but with an unusual architecture not seen in other organisms. Through a combination of structural, biochemical, and cellular techniques, we show that polymerization stabilizes the active conformation of the enzyme and resists product inhibition, potentially allowing for the highly localized production of CTP within the cell. Indeed, CTPS filaments localize near the CTP-dependent complex needed for chromosome segregation, and cells expressing mutant enzymes unable to polymerize are altered in their ability to robustly form this complex. Intriguingly, mutants that alter filament formation are under positive selection in clinical isolates of M. tuberculosis, pointing to a critical role needed to withstand pressures imposed by the host and/or antibiotics. Taken together, our data reveal an unexpected mechanism for the spatially organized production of a critical nucleotide in M. tuberculosis, which may represent a vulnerability of the pathogen that can be exploited with chemotherapy.
RESUMEN
Phosphofructokinase-1 (PFK1) catalyzes the rate-limiting step of glycolysis, committing glucose to conversion into cellular energy. PFK1 is highly regulated to respond to the changing energy needs of the cell. In bacteria, the structural basis of PFK1 regulation is a textbook example of allostery; molecular signals of low and high cellular energy promote transition between an active R-state and inactive T-state conformation, respectively Little is known, however, about the structural basis for regulation of eukaryotic PFK1. Here, we determine structures of the human liver isoform of PFK1 (PFKL) in the R- and T-state by cryoEM, providing insight into eukaryotic PFK1 allosteric regulatory mechanisms. The T-state structure reveals conformational differences between the bacterial and eukaryotic enzyme, the mechanisms of allosteric inhibition by ATP binding at multiple sites, and an autoinhibitory role of the C-terminus in stabilizing the T-state. We also determine structures of PFKL filaments that define the mechanism of higher-order assembly and demonstrate that these structures are necessary for higher-order assembly of PFKL in cells.
RESUMEN
Biological evolution has led to precise and dynamic nanostructures that reconfigure in response to pH and other environmental conditions. However, designing micrometre-scale protein nanostructures that are environmentally responsive remains a challenge. Here we describe the de novo design of pH-responsive protein filaments built from subunits containing six or nine buried histidine residues that assemble into micrometre-scale, well-ordered fibres at neutral pH. The cryogenic electron microscopy structure of an optimized design is nearly identical to the computational design model for both the subunit internal geometry and the subunit packing into the fibre. Electron, fluorescent and atomic force microscopy characterization reveal a sharp and reversible transition from assembled to disassembled fibres over 0.3 pH units, and rapid fibre disassembly in less than 1 s following a drop in pH. The midpoint of the transition can be tuned by modulating buried histidine-containing hydrogen bond networks. Computational protein design thus provides a route to creating unbound nanomaterials that rapidly respond to small pH changes.
Asunto(s)
Histidina , Concentración de Iones de Hidrógeno , Histidina/química , Proteínas/química , Nanoestructuras/química , Modelos Moleculares , Enlace de Hidrógeno , Microscopía por CrioelectrónRESUMEN
Many metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the molecular level and what their role is in the yeast starvation response. CTP Synthase (CTPS) assembles into metabolic filaments across many species. Here, we characterize in vitro polymerization and investigate in vivo consequences of CTPS assembly in yeast. Cryo-EM structures reveal a pH-sensitive assembly mechanism and highly ordered filament bundles that stabilize an inactive state of the enzyme, features unique to yeast CTPS. Disruption of filaments in cells with non-assembly or pH-insensitive mutations decreases growth rate, reflecting the importance of regulated CTPS filament assembly in homeotstasis.
Asunto(s)
Ligasas de Carbono-Nitrógeno/química , Saccharomyces cerevisiae/enzimología , Microscopía por Crioelectrón , Concentración de Iones de Hidrógeno , Conformación Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Cytidine triphosphate synthase 1 (CTPS1) is necessary for an effective immune response, as revealed by severe immunodeficiency in CTPS1-deficient individuals [E. Martin et al], [Nature] [510], [288-292] ([2014]). CTPS1 expression is up-regulated in activated lymphocytes to expand CTP pools [E. Martin et al], [Nature] [510], [288-292] ([2014]), satisfying increased demand for nucleic acid and lipid synthesis [L. D. Fairbanks, M. Bofill, K. Ruckemann, H. A. Simmonds], [J. Biol. Chem. ] [270], [29682-29689] ([1995]). Demand for CTP in other tissues is met by the CTPS2 isoform and nucleoside salvage pathways [E. Martin et al], [Nature] [510], [288-292] ([2014]). Selective inhibition of the proliferative CTPS1 isoform is therefore desirable in the treatment of immune disorders and lymphocyte cancers, but little is known about differences in regulation of the isoforms or mechanisms of known inhibitors. We show that CTP regulates both isoforms by binding in two sites that clash with substrates. CTPS1 is less sensitive to CTP feedback inhibition, consistent with its role in increasing CTP levels in proliferation. We also characterize recently reported small-molecule inhibitors, both CTPS1 selective and nonselective. Cryo-electron microscopy (cryo-EM) structures reveal these inhibitors mimic CTP binding in one inhibitory site, where a single amino acid substitution explains selectivity for CTPS1. The inhibitors bind to CTPS assembled into large-scale filaments, which for CTPS1 normally represents a hyperactive form of the enzyme [E. M. Lynch et al], [Nat. Struct. Mol. Biol.] [24], [507-514] ([2017]). This highlights the utility of cryo-EM in drug discovery, particularly for cases in which targets form large multimeric assemblies not amenable to structure determination by other techniques. Both inhibitors also inhibit the proliferation of human primary T cells. The mechanisms of selective inhibition of CTPS1 lay the foundation for the design of immunosuppressive therapies.
Asunto(s)
Ligasas de Carbono-Nitrógeno/metabolismo , Isoformas de Proteínas/metabolismo , Proliferación Celular/fisiología , Humanos , Síndromes de Inmunodeficiencia/metabolismo , Linfocitos T/metabolismoRESUMEN
In neutrophils, nicotinamide adenine dinucleotide phosphate (NADPH) generated via the pentose phosphate pathway fuels NADPH oxidase NOX2 to produce reactive oxygen species for killing invading pathogens. However, excessive NOX2 activity can exacerbate inflammation, as in acute respiratory distress syndrome (ARDS). Here, we use two unbiased chemical proteomic strategies to show that small-molecule LDC7559, or a more potent designed analog NA-11, inhibits the NOX2-dependent oxidative burst in neutrophils by activating the glycolytic enzyme phosphofructokinase-1 liver type (PFKL) and dampening flux through the pentose phosphate pathway. Accordingly, neutrophils treated with NA-11 had reduced NOX2-dependent outputs, including neutrophil cell death (NETosis) and tissue damage. A high-resolution structure of PFKL confirmed binding of NA-11 to the AMP/ADP allosteric activation site and explained why NA-11 failed to agonize phosphofructokinase-1 platelet type (PFKP) or muscle type (PFKM). Thus, NA-11 represents a tool for selective activation of PFKL, the main phosphofructokinase-1 isoform expressed in immune cells.
Asunto(s)
Fagocitosis , Fosfofructoquinasa-1 Tipo Hepático/metabolismo , Estallido Respiratorio , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Regulación Alostérica/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Glucólisis/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cinética , Viabilidad Microbiana/efectos de los fármacos , Modelos Moleculares , NADPH Oxidasas/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Fagocitosis/efectos de los fármacos , Proteínas de Unión a Fosfato/metabolismo , Fosfofructoquinasa-1 Tipo Hepático/antagonistas & inhibidores , Fosfofructoquinasa-1 Tipo Hepático/ultraestructura , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Recombinantes/aislamiento & purificación , Estallido Respiratorio/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Multimeric immunoglobulin-like molecules arose early in vertebrate evolution, yet the unique contributions of multimeric IgM antibodies to infection control are not well understood. This is partially due to the difficulty of distinguishing low-affinity IgM, secreted rapidly by plasmablasts, from high-affinity antibodies derived from later-arising memory cells. We developed a pipeline to express B cell receptors (BCRs) from Plasmodium falciparum-specific IgM+ and IgG+ human memory B cells (MBCs) as both IgM and IgG molecules. BCRs from both subsets were somatically hypermutated and exhibited comparable monomeric affinity. Crystallization of one IgM+ MBC-derived antibody complexed with antigen defined a linear epitope within a conserved Plasmodium protein. In its physiological multimeric state, this antibody displayed exponentially higher antigen binding than a clonally identical IgG monomer, and more effectively inhibited P. falciparum invasion. Forced multimerization of this IgG significantly improved both antigen binding and parasite restriction, underscoring how avidity can alter antibody function. This work demonstrates the potential of high-avidity IgM in both therapeutics and vaccines.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Linfocitos B/inmunología , Inmunoglobulina M/química , Inmunoglobulina M/inmunología , Memoria Inmunológica , Malaria Falciparum/inmunología , Plasmodium falciparum/inmunología , Multimerización de Proteína/inmunología , Adolescente , Afinidad de Anticuerpos , Células Cultivadas , Niño , Estudios de Cohortes , Epítopos de Linfocito B/inmunología , Femenino , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Malaria Falciparum/sangre , Malaria Falciparum/parasitología , Masculino , Malí , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunologíaRESUMEN
Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2-4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7,8 and in redesigning naturally occurring channel-containing proteins9,10. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge11,12. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.
Asunto(s)
Simulación por Computador , Genes Sintéticos/genética , Canales Iónicos/química , Canales Iónicos/genética , Modelos Moleculares , Biología Sintética , Línea Celular , Microscopía por Crioelectrón , Cristalografía por Rayos X , Conductividad Eléctrica , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrazinas , Canales Iónicos/metabolismo , Transporte Iónico , Liposomas/metabolismo , Técnicas de Placa-Clamp , Porinas/química , Porinas/genética , Porinas/metabolismo , Ingeniería de Proteínas , Estructura Secundaria de Proteína , Solubilidad , Agua/químicaRESUMEN
Polymerization of metabolic enzymes into micron-scale assemblies is an emerging mechanism for regulating their activity. CTP synthase (CTPS) is an essential enzyme in the biosynthesis of the nucleotide CTP and undergoes regulated and reversible assembly into large filamentous structures in organisms from bacteria to humans. The purpose of these assemblies is unclear. A major challenge to addressing this question has been the inability to abolish assembly without eliminating CTPS protein. Here we demonstrate that a recently reported point mutant in CTPS, Histidine 355A (H355A), prevents CTPS filament assembly in vivo and dominantly inhibits the assembly of endogenous wild-type CTPS in the Drosophila ovary. Expressing this mutant in ovarian germline cells, we show that disruption of CTPS assembly in early stage egg chambers reduces egg production. This effect is exacerbated in flies fed the glutamine antagonist 6-diazo-5-oxo-L-norleucine, which inhibits de novo CTP synthesis. These findings introduce a general approach to blocking the assembly of polymerizing enzymes without eliminating their catalytic activity and demonstrate a role for CTPS assembly in supporting egg production, particularly under conditions of limited glutamine metabolism.This article has an associated First Person interview with the first author of the paper.
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Ligasas de Carbono-Nitrógeno/metabolismo , Drosophila/fisiología , Células Germinativas/metabolismo , Multimerización de Proteína , Reproducción , Animales , Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/genética , Metabolismo Energético , Técnica del Anticuerpo Fluorescente , Expresión Génica , Glutamina/metabolismo , MutaciónRESUMEN
Compartmentalization of metabolic enzymes through protein-protein interactions is an emerging mechanism for localizing and regulating metabolic activity. Self-assembly into linear filaments is a common strategy for cellular compartmentalization of enzymes. Polymerization is often driven by changes in the metabolic state of the cell, suggesting that it is a strategy for shifting metabolic flux in response to cellular demand. Although polymerization of metabolic enzymes is widespread, observed from bacteria to humans, we are just beginning to appreciate their role in regulating cellular metabolism. In most cases, one functional role of metabolic enzyme filaments is allosteric control of enzyme activity. Here, we highlight recent findings, providing insight into the structural and functional significance of filamentation of metabolic enzymes in cells.
Asunto(s)
Citoesqueleto/metabolismo , Enzimas/metabolismo , Metabolismo , Enzimas/química , Humanos , PolimerizacionRESUMEN
The actin fold is found in cytoskeletal polymers, chaperones, and various metabolic enzymes. Many actin-fold proteins, such as the carbohydrate kinases, do not polymerize. We found that Glk1, a Saccharomyces cerevisiae glucokinase, forms two-stranded filaments with ultrastructure that is distinct from that of cytoskeletal polymers. In cells, Glk1 polymerized upon sugar addition and depolymerized upon sugar withdrawal. Polymerization inhibits enzymatic activity; the Glk1 monomer-polymer equilibrium sets a maximum rate of glucose phosphorylation regardless of Glk1 concentration. A mutation that eliminated Glk1 polymerization alleviated concentration-dependent enzyme inhibition. Yeast containing nonpolymerizing Glk1 were less fit when growing on sugars and more likely to die when refed glucose. Glk1 polymerization arose independently from other actin-related filaments and may allow yeast to rapidly modulate glucokinase activity as nutrient availability changes.
Asunto(s)
Actinas/química , Adenosina Trifosfatasas/química , Glucoquinasa/química , Hexoquinasa/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Adenosina Trifosfatasas/genética , Glucoquinasa/genética , Hexoquinasa/genética , Polimerizacion , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Many enzymes assemble into defined oligomers, providing a mechanism for cooperatively regulating activity. Recent studies have described a mode of regulation in which enzyme activity is modulated by polymerization into large-scale filaments. Here we describe an ultrasensitive form of polymerization-based regulation employed by human CTP synthase 2 (CTPS2). Cryo-EM structures reveal that CTPS2 filaments dynamically switch between active and inactive forms in response to changes in substrate and product levels. Linking the conformational state of many CTPS2 subunits in a filament results in highly cooperative regulation, greatly exceeding the limits of cooperativity for the CTPS2 tetramer alone. The structures reveal a link between conformation and control of ammonia channeling between the enzyme's active sites, and explain differences in regulation of human CTPS isoforms. This filament-based mechanism of enhanced cooperativity demonstrates how the widespread phenomenon of enzyme polymerization can be adapted to achieve different regulatory outcomes.
Asunto(s)
Ligasas de Carbono-Nitrógeno/química , Multimerización de Proteína , Ligasas de Carbono-Nitrógeno/metabolismo , Ligasas de Carbono-Nitrógeno/ultraestructura , Dominio Catalítico , Microscopía por Crioelectrón , Activación Enzimática , Humanos , Modelos Moleculares , Conformación Proteica , Especificidad por SustratoRESUMEN
Microtubule (MT) nucleation depends on the γ-tubulin complex (γ-TuC), in which multiple copies of the heterotetrameric γ-tubulin small complex (γ-TuSC) associate to form a ring-like structure (in metazoans, γ-tubulin ring complex; γ-TuRC) [1-7]. Additional conserved regulators of the γ-TuC include the small protein Mzt1 (MOZART1 in human; GIP1/1B and GIP2/1A in plants) [8-13] and proteins containing a Centrosomin Motif 1 (CM1) domain [10, 14-19]. Many insights into γ-TuC regulators have come from in vivo analysis in fission yeast Schizosaccharomyces pombe. The S. pombe CM1 protein Mto1 recruits the γ-TuC to microtubule-organizing centers (MTOCs) [14, 20-22], and analysis of Mto1[bonsai], a truncated version of Mto1 that cannot localize to MTOCs, has shown that Mto1 also has a role in γ-TuC activation [23]. S. pombe Mzt1 interacts with γ-TuSC and is essential for γ-TuC function and localization to MTOCs [11, 12]. However, the mechanisms by which Mzt1 functions remain unclear. Here we describe reconstitution of MT nucleation using purified recombinant Mto1[bonsai], the Mto1 partner protein Mto2, γ-TuSC, and Mzt1. Multiple copies of the six proteins involved coassemble to form a 34-40S ring-like "MGM" holocomplex that is a potent MT nucleator in vitro. Using purified MGM and subcomplexes, we investigate the role of Mzt1 in MT nucleation. Our results suggest that Mzt1 is critical to stabilize Alp6, the S. pombe homolog of human γ-TuSC protein GCP3, in an "interaction-competent" form within the γ-TuSC. This is essential for MGM to become a functional nucleator.
Asunto(s)
Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
We describe a general computational approach to designing self-assembling helical filaments from monomeric proteins and use this approach to design proteins that assemble into micrometer-scale filaments with a wide range of geometries in vivo and in vitro. Cryo-electron microscopy structures of six designs are close to the computational design models. The filament building blocks are idealized repeat proteins, and thus the diameter of the filaments can be systematically tuned by varying the number of repeat units. The assembly and disassembly of the filaments can be controlled by engineered anchor and capping units built from monomers lacking one of the interaction surfaces. The ability to generate dynamic, highly ordered structures that span micrometers from protein monomers opens up possibilities for the fabrication of new multiscale metamaterials.
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Biología Computacional/métodos , Ingeniería de Proteínas/métodos , Proteínas/química , Microscopía por Crioelectrón , Escherichia coli , Conformación Proteica en Hélice alfa , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas/genéticaRESUMEN
Non-centrosomal microtubule organizing centers (MTOCs) are important for microtubule organization in many cell types. In fission yeast Schizosaccharomyces pombe, the protein Mto1, together with partner protein Mto2 (Mto1/2 complex), recruits the γ-tubulin complex to multiple non-centrosomal MTOCs, including the nuclear envelope (NE). Here, we develop a comparative-interactome mass spectrometry approach to determine how Mto1 localizes to the NE. Surprisingly, we find that Mto1, a constitutively cytoplasmic protein, docks at nuclear pore complexes (NPCs), via interaction with exportin Crm1 and cytoplasmic FG-nucleoporin Nup146. Although Mto1 is not a nuclear export cargo, it binds Crm1 via a nuclear export signal-like sequence, and docking requires both Ran in the GTP-bound state and Nup146 FG repeats. In addition to determining the mechanism of MTOC formation at the NE, our results reveal a novel role for Crm1 and the nuclear export machinery in the stable docking of a cytoplasmic protein complex at NPCs.
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Carioferinas/metabolismo , Centro Organizador de los Microtúbulos/fisiología , Microtúbulos/metabolismo , Poro Nuclear/fisiología , Receptores Citoplasmáticos y Nucleares/metabolismo , Schizosaccharomyces/fisiología , Transporte Activo de Núcleo Celular , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Citoplasma/metabolismo , Carioferinas/química , Carioferinas/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Simulación del Acoplamiento Molecular , Membrana Nuclear , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Huso Acromático , Proteína Exportina 1RESUMEN
BACKGROUND: Noise-induced hearing loss is a leading cause of occupational and recreational injury and disease, and a major determinant of age-related hearing loss. No therapeutic agent has been approved for the prevention or treatment of this disorder. In animal models, glutathione peroxidase 1 (GPx1) activity is reduced after acute noise exposure. Ebselen, a novel GPx1 mimic, has been shown to reduce both temporary and permanent noise-induced hearing loss in preclinical studies. We assessed the safety and efficacy of ebselen for the prevention of noise-induced hearing loss in young adults in a phase 2 clinical trial. METHODS: In this single-centre, randomised, double-blind, placebo-controlled phase 2 trial, healthy adults aged 18-31 years were randomly assigned (1:1:1:1) at the University of Florida (Gainsville, FL, USA) to receive ebselen 200 mg, 400 mg, or 600 mg, or placebo orally twice daily for 4 days, beginning 2 days before a calibrated sound challenge (4 h of pre-recorded music delivered by insert earphones). Randomisation was done with an allocation sequence generated by an independent third party. The primary outcome was mean temporary threshold shift (TTS) at 4 kHz measured 15 min after the calibrated sound challenge by pure tone audiometry; a reduction of 50% in an ebselen dose group compared with the placebo group was judged to be clinically relevant. All participants who received the calibrated sound challenge and at least one dose of study drug were included in the efficacy analysis. All randomly assigned patients were included in the safety analysis. This trial is registered with ClinicalTrials.gov, number NCT01444846. FINDINGS: Between Jan 11, 2013, and March 24, 2014, 83 participants were enrolled and randomly assigned to receive ebselen 200 mg (n=22), 400 mg (n=20), or 600 mg (n=21), or placebo (n=20). Two participants in the 200 mg ebselen group were discontinued from the study before the calibrated sound challenge because they no longer met the inclusion criteria; these participants were excluded from the efficacy analysis. Mean TTS at 4 kHz was 1·32 dB (SE 0·91) in the 400 mg ebselen group compared with 4·07 dB (0·90) in the placebo group, representing a significant reduction of 68% (difference -2·75 dB, 95% CI -4·54 to -0·97; p=0·0025). Compared with placebo, TTS at 4 kHz was non-significantly reduced by 21% in the 200 mg ebselen group (3·23 dB [SE 0·91] vs 4·07 dB [0·90] in the placebo group; difference -0·84 dB, 95% CI -2·63 to 0·94; p=0·3542) and by 7% in the 600 mg ebselen group (3·81 dB [0·90] vs 4·07 dB [0·90] in the placebo group; difference -0·27, 95% CI -2·03 to 1·50; p=0·7659). Ebselen treatment was well tolerated across all doses and no significant differences were seen in any haematological, serum chemistry, or radiological assessments between the ebselen groups and the placebo group. INTERPRETATION: Treatment with ebselen was safe and effective at a dose of 400 mg twice daily in preventing a noise-induced TTS. These data lend support to a role of GPx1 activity in acute noise-induced hearing loss. FUNDING: Sound Pharmaceuticals.
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Antiinflamatorios no Esteroideos/administración & dosificación , Azoles/administración & dosificación , Pérdida Auditiva Provocada por Ruido/prevención & control , Compuestos de Organoselenio/administración & dosificación , Adolescente , Adulto , Método Doble Ciego , Femenino , Humanos , Isoindoles , Masculino , Música , Resultado del Tratamiento , Adulto JovenRESUMEN
The universally conserved enzyme CTP synthase (CTPS) forms filaments in bacteria and eukaryotes. In bacteria, polymerization inhibits CTPS activity and is required for nucleotide homeostasis. Here we show that for human CTPS, polymerization increases catalytic activity. The cryo-EM structures of bacterial and human CTPS filaments differ considerably in overall architecture and in the conformation of the CTPS protomer, explaining the divergent consequences of polymerization on activity. The structure of human CTPS filament, the first structure of the full-length human enzyme, reveals a novel active conformation. The filament structures elucidate allosteric mechanisms of assembly and regulation that rely on a conserved conformational equilibrium. The findings may provide a mechanism for increasing human CTPS activity in response to metabolic state and challenge the assumption that metabolic filaments are generally storage forms of inactive enzymes. Allosteric regulation of CTPS polymerization by ligands likely represents a fundamental mechanism underlying assembly of other metabolic filaments.