RESUMEN
The glycine transporter 1 (GlyT1) plays a crucial role in the regulation of both inhibitory and excitatory neurotransmission by removing glycine from the synaptic cleft. Given its close association with glutamate/glycine co-activated NMDA receptors (NMDARs), GlyT1 has emerged as a central target for the treatment of schizophrenia, which is often linked to hypofunctional NMDARs. Here, we report the cryo-EM structures of GlyT1 bound with substrate glycine and drugs ALX-5407, SSR504734, and PF-03463275. These structures, captured at three fundamental states of the transport cycle-outward-facing, occluded, and inward-facing-enable us to illustrate a comprehensive blueprint of the conformational change associated with glycine reuptake. Additionally, we identified three specific pockets accommodating drugs, providing clear insights into the structural basis of their inhibitory mechanism and selectivity. Collectively, these structures offer significant insights into the transport mechanism and recognition of substrate and anti-schizophrenia drugs, thus providing a platform to design small molecules to treat schizophrenia.
Asunto(s)
Proteínas de Transporte de Glicina en la Membrana Plasmática , Humanos , Transporte Biológico , Glicina/metabolismo , Proteínas de Transporte de Glicina en la Membrana Plasmática/química , Proteínas de Transporte de Glicina en la Membrana Plasmática/metabolismo , Proteínas de Transporte de Glicina en la Membrana Plasmática/ultraestructura , Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/metabolismo , Transmisión Sináptica , Imidazoles/química , Sarcosina/análogos & derivados , Piperidinas/químicaRESUMEN
Sulfonates include diverse natural products and anthropogenic chemicals and are widespread in the environment. Many bacteria can degrade sulfonates and obtain sulfur, carbon, and energy for growth, playing important roles in the biogeochemical sulfur cycle. Cleavage of the inert sulfonate C-S bond involves a variety of enzymes, cofactors, and oxygen-dependent and oxygen-independent catalytic mechanisms. Sulfonate degradation by strictly anaerobic bacteria was recently found to involve C-S bond cleavage through O2-sensitive free radical chemistry, catalyzed by glycyl radical enzymes (GREs). The associated discoveries of new enzymes and metabolic pathways for sulfonate metabolism in diverse anaerobic bacteria have enriched our understanding of sulfonate chemistry in the anaerobic biosphere. An anaerobic environment of particular interest is the human gut microbiome, where sulfonate degradation by sulfate- and sulfite-reducing bacteria (SSRB) produces H2S, a process linked to certain chronic diseases and conditions.
Asunto(s)
Liasas de Carbono-Carbono/metabolismo , Microbioma Gastrointestinal/fisiología , Ácidos Sulfónicos/metabolismo , Acetiltransferasas/química , Acetiltransferasas/metabolismo , Alcanosulfonatos/metabolismo , Anaerobiosis , Bacterias/metabolismo , Liasas de Carbono-Carbono/química , Glicina/metabolismo , Humanos , Sulfuro de Hidrógeno/metabolismo , Ácido Isetiónico/metabolismo , Microbiota/fisiología , Taurina/metabolismoRESUMEN
Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant.
Asunto(s)
Sustitución de Aminoácidos , COVID-19/transmisión , COVID-19/virología , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/genética , Ácido Aspártico/análisis , Ácido Aspártico/genética , COVID-19/epidemiología , Genoma Viral , Glicina/análisis , Glicina/genética , Humanos , Mutación , SARS-CoV-2/crecimiento & desarrollo , Reino Unido/epidemiología , Virulencia , Secuenciación Completa del GenomaRESUMEN
Ligand-gated ion channels mediate signal transduction at chemical synapses and transition between resting, open, and desensitized states in response to neurotransmitter binding. Neurotransmitters that produce maximum open channel probabilities (Po) are full agonists, whereas those that yield lower than maximum Po are partial agonists. Cys-loop receptors are an important class of neurotransmitter receptors, yet a structure-based understanding of the mechanism of partial agonist action has proven elusive. Here, we study the glycine receptor with the full agonist glycine and the partial agonists taurine and γ-amino butyric acid (GABA). We use electrophysiology to show how partial agonists populate agonist-bound, closed channel states and cryo-EM reconstructions to illuminate the structures of intermediate, pre-open states, providing insights into previously unseen conformational states along the receptor reaction pathway. We further correlate agonist-induced conformational changes to Po across members of the receptor family, providing a hypothetical mechanism for partial and full agonist action at Cys-loop receptors.
Asunto(s)
Activación del Canal Iónico , Receptores de Glicina/agonistas , Receptores de Glicina/metabolismo , Animales , Sitios de Unión , Línea Celular , Microscopía por Crioelectrón , Glicina , Células HEK293 , Humanos , Imagenología Tridimensional , Maleatos/química , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Neurotransmisores/metabolismo , Dominios Proteicos , Receptores de Glicina/genética , Receptores de Glicina/ultraestructura , Estireno/química , Pez Cebra , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Excitatory neurotransmission meditated by glutamate receptors including N-methyl-D-aspartate receptors (NMDARs) is pivotal to brain development and function. NMDARs are heterotetramers composed of GluN1 and GluN2 subunits, which bind glycine and glutamate, respectively, to activate their ion channels. Despite importance in brain physiology, the precise mechanisms by which activation and inhibition occur via subunit-specific binding of agonists and antagonists remain largely unknown. Here, we show the detailed patterns of conformational changes and inter-subunit and -domain reorientation leading to agonist-gating and subunit-dependent competitive inhibition by providing multiple structures in distinct ligand states at 4 Å or better. The structures reveal that activation and competitive inhibition by both GluN1 and GluN2 antagonists occur by controlling the tension of the linker between the ligand-binding domain and the transmembrane ion channel of the GluN2 subunit. Our results provide detailed mechanistic insights into NMDAR pharmacology, activation, and inhibition, which are fundamental to the brain physiology.
Asunto(s)
Receptores de N-Metil-D-Aspartato/metabolismo , Sitios de Unión , Unión Competitiva , Microscopía por Crioelectrón , Cristalografía por Rayos X , Dimerización , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Glicina/química , Glicina/metabolismo , Humanos , Ligandos , Simulación de Dinámica Molecular , Estructura Cuaternaria de Proteína , Subunidades de Proteína/agonistas , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Erythropoietin (EPO) production in the kidney is regulated by the oxygen-sensing transcription factor HIF-1α, which is degraded under normoxic conditions by HIF-prolyl hydroxylase (HIF-PHD). Inhibition of HIF-PHD by roxadustat leads to increased EPO production, better iron absorption, and amelioration of anemia in chronic kidney disease (CKD).
Asunto(s)
Anemia/terapia , Glicina/análogos & derivados , Factor 1 Inducible por Hipoxia/metabolismo , Isoquinolinas/uso terapéutico , Anemia/metabolismo , Glicina/uso terapéutico , Humanos , Factor 1 Inducible por Hipoxia/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Prolil Hidroxilasas/efectos de los fármacos , Prolil Hidroxilasas/metabolismo , Insuficiencia Renal Crónica/tratamiento farmacológico , Insuficiencia Renal Crónica/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) tumors have a nutrient-poor, desmoplastic, and highly innervated tumor microenvironment. Although neurons can release stimulatory factors to accelerate PDAC tumorigenesis, the metabolic contribution of peripheral axons has not been explored. We found that peripheral axons release serine (Ser) to support the growth of exogenous Ser (exSer)-dependent PDAC cells during Ser/Gly (glycine) deprivation. Ser deprivation resulted in ribosomal stalling on two of the six Ser codons, TCC and TCT, and allowed the selective translation and secretion of nerve growth factor (NGF) by PDAC cells to promote tumor innervation. Consistent with this, exSer-dependent PDAC tumors grew slower and displayed enhanced innervation in mice on a Ser/Gly-free diet. Blockade of compensatory neuronal innervation using LOXO-101, a Trk-NGF inhibitor, further decreased PDAC tumor growth. Our data indicate that axonal-cancer metabolic crosstalk is a critical adaptation to support PDAC growth in nutrient poor environments.
Asunto(s)
Neuronas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Biosíntesis de Proteínas , Serina/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Anciano , Animales , Axones/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proliferación Celular , Codón/genética , Femenino , Glicina/metabolismo , Humanos , Masculino , Ratones , Persona de Mediana Edad , Mitocondrias/metabolismo , Tejido Nervioso/patología , Consumo de Oxígeno , Neoplasias Pancreáticas/patología , Pirazoles , Pirimidinas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia/genética , RatasRESUMEN
RNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of which have been characterized on structural grounds. Here, we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We have determined the cryoelectron microscopy (cryo-EM) structure of human SHMT1 in its free- and RNA-bound states, and we show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzyme's reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in evolution of eukaryotic SHMT1 and in compartmentalization of one-carbon metabolism. Our findings provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway.
Asunto(s)
Microscopía por Crioelectrón , Glicina Hidroximetiltransferasa , Glicina Hidroximetiltransferasa/metabolismo , Glicina Hidroximetiltransferasa/genética , Glicina Hidroximetiltransferasa/química , Humanos , ARN/metabolismo , ARN/genética , Serina/metabolismo , Regulación Alostérica , Unión Proteica , Filogenia , Modelos Moleculares , Conformación Proteica , Relación Estructura-Actividad , Glicina/metabolismo , Glicina/química , Sitios de UniónRESUMEN
Oncogenic activation of RAS genes via point mutations occurs in 20%-30% of human cancers. The development of effective RAS inhibitors has been challenging, necessitating new approaches to inhibit this oncogenic protein. Functional studies have shown that the switch region of RAS interacts with a large number of effector proteins containing a common RAS-binding domain (RBD). Because RBD-mediated interactions are essential for RAS signaling, blocking RBD association with small molecules constitutes an attractive therapeutic approach. Here, we present evidence that rigosertib, a styryl-benzyl sulfone, acts as a RAS-mimetic and interacts with the RBDs of RAF kinases, resulting in their inability to bind to RAS, disruption of RAF activation, and inhibition of the RAS-RAF-MEK pathway. We also find that ribosertib binds to the RBDs of Ral-GDS and PI3Ks. These results suggest that targeting of RBDs across multiple signaling pathways by rigosertib may represent an effective strategy for inactivation of RAS signaling.
Asunto(s)
Glicina/análogos & derivados , Proteínas de Unión al ARN/química , Transducción de Señal/efectos de los fármacos , Sulfonas/farmacología , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Transformación Celular Neoplásica/efectos de los fármacos , Cristalografía por Rayos X , Dimerización , Glicina/administración & dosificación , Glicina/química , Glicina/farmacología , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Desnudos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Neoplasias Pancreáticas/tratamiento farmacológico , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia , Sulfonas/administración & dosificación , Sulfonas/química , Proteínas ras/metabolismo , Quinasa Tipo Polo 1RESUMEN
The emergence of biopolymer building blocks is a crucial step during the origins of life1-6. However, all known formation pathways rely on rare pure feedstocks and demand successive purification and mixing steps to suppress unwanted side reactions and enable high product yields. Here we show that heat flows through thin, crack-like geo-compartments could have provided a widely available yet selective mechanism that separates more than 50 prebiotically relevant building blocks from complex mixtures of amino acids, nucleobases, nucleotides, polyphosphates and 2-aminoazoles. Using measured thermophoretic properties7,8, we numerically model and experimentally prove the advantageous effect of geological networks of interconnected cracks9,10 that purify the previously mixed compounds, boosting their concentration ratios by up to three orders of magnitude. The importance for prebiotic chemistry is shown by the dimerization of glycine11,12, in which the selective purification of trimetaphosphate (TMP)13,14 increased reaction yields by five orders of magnitude. The observed effect is robust under various crack sizes, pH values, solvents and temperatures. Our results demonstrate how geologically driven non-equilibria could have explored highly parallelized reaction conditions to foster prebiotic chemistry.
Asunto(s)
Biopolímeros , Evolución Química , Calor , Origen de la Vida , Biopolímeros/química , Dimerización , Glicina/química , Concentración de Iones de Hidrógeno , Nucleótidos/química , Polifosfatos/química , Solventes/químicaRESUMEN
Photobiocatalysis-where light is used to expand the reactivity of an enzyme-has recently emerged as a powerful strategy to develop chemistries that are new to nature. These systems have shown potential in asymmetric radical reactions that have long eluded small-molecule catalysts1. So far, unnatural photobiocatalytic reactions are limited to overall reductive and redox-neutral processes2-9. Here we report photobiocatalytic asymmetric sp3-sp3 oxidative cross-coupling between organoboron reagents and amino acids. This reaction requires the cooperative use of engineered pyridoxal biocatalysts, photoredox catalysts and an oxidizing agent. We repurpose a family of pyridoxal-5'-phosphate-dependent enzymes, threonine aldolases10-12, for the α-C-H functionalization of glycine and α-branched amino acid substrates by a radical mechanism, giving rise to a range of α-tri- and tetrasubstituted non-canonical amino acids 13-15 possessing up to two contiguous stereocentres. Directed evolution of pyridoxal radical enzymes allowed primary and secondary radical precursors, including benzyl, allyl and alkylboron reagents, to be coupled in an enantio- and diastereocontrolled fashion. Cooperative photoredox-pyridoxal biocatalysis provides a platform for sp3-sp3 oxidative coupling16, permitting the stereoselective, intermolecular free-radical transformations that are unknown to chemistry or biology.
Asunto(s)
Aminoácidos , Biocatálisis , Acoplamiento Oxidativo , Procesos Fotoquímicos , Aminoácidos/biosíntesis , Aminoácidos/química , Aminoácidos/metabolismo , Biocatálisis/efectos de la radiación , Evolución Molecular Dirigida , Radicales Libres/química , Radicales Libres/metabolismo , Glicina/química , Glicina/metabolismo , Glicina Hidroximetiltransferasa/metabolismo , Glicina Hidroximetiltransferasa/química , Indicadores y Reactivos , Luz , Acoplamiento Oxidativo/efectos de la radiación , Fosfato de Piridoxal/metabolismo , Estereoisomerismo , Aminoácidos de Cadena Ramificada/química , Aminoácidos de Cadena Ramificada/metabolismoRESUMEN
Glutamate transmission and activation of ionotropic glutamate receptors are the fundamental means by which neurons control their excitability and neuroplasticity1. The N-methyl-D-aspartate receptor (NMDAR) is unique among all ligand-gated channels, requiring two ligands-glutamate and glycine-for activation. These receptors function as heterotetrameric ion channels, with the channel opening dependent on the simultaneous binding of glycine and glutamate to the extracellular ligand-binding domains (LBDs) of the GluN1 and GluN2 subunits, respectively2,3. The exact molecular mechanism for channel gating by the two ligands has been unclear, particularly without structures representing the open channel and apo states. Here we show that the channel gate opening requires tension in the linker connecting the LBD and transmembrane domain (TMD) and rotation of the extracellular domain relative to the TMD. Using electron cryomicroscopy, we captured the structure of the GluN1-GluN2B (GluN1-2B) NMDAR in its open state bound to a positive allosteric modulator. This process rotates and bends the pore-forming helices in GluN1 and GluN2B, altering the symmetry of the TMD channel from pseudofourfold to twofold. Structures of GluN1-2B NMDAR in apo and single-liganded states showed that binding of either glycine or glutamate alone leads to distinct GluN1-2B dimer arrangements but insufficient tension in the LBD-TMD linker for channel opening. This mechanistic framework identifies a key determinant for channel gating and a potential pharmacological strategy for modulating NMDAR activity.
Asunto(s)
Ácido Glutámico , Glicina , Activación del Canal Iónico , Receptores de N-Metil-D-Aspartato , Animales , Ratas , Regulación Alostérica , Microscopía por Crioelectrón , Ácido Glutámico/metabolismo , Glicina/metabolismo , Ligandos , Modelos Moleculares , Oocitos/metabolismo , Dominios Proteicos , Multimerización de Proteína , Subunidades de Proteína/metabolismo , Subunidades de Proteína/química , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/ultraestructura , Rotación , Xenopus laevisRESUMEN
HIV-1 infection requires nuclear entry of the viral genome. Previous evidence suggests that this entry proceeds through nuclear pore complexes (NPCs), with the 120 × 60 nm capsid squeezing through an approximately 60-nm-wide central channel1 and crossing the permeability barrier of the NPC. This barrier can be described as an FG phase2 that is assembled from cohesively interacting phenylalanine-glycine (FG) repeats3 and is selectively permeable to cargo captured by nuclear transport receptors (NTRs). Here we show that HIV-1 capsid assemblies can target NPCs efficiently in an NTR-independent manner and bind directly to several types of FG repeats, including barrier-forming cohesive repeats. Like NTRs, the capsid readily partitions into an in vitro assembled cohesive FG phase that can serve as an NPC mimic and excludes much smaller inert probes such as mCherry. Indeed, entry of the capsid protein into such an FG phase is greatly enhanced by capsid assembly, which also allows the encapsulated clients to enter. Thus, our data indicate that the HIV-1 capsid behaves like an NTR, with its interior serving as a cargo container. Because capsid-coating with trans-acting NTRs would increase the diameter by 10 nm or more, we suggest that such a 'self-translocating' capsid undermines the size restrictions imposed by the NPC scaffold, thereby bypassing an otherwise effective barrier to viral infection.
Asunto(s)
Proteínas de la Cápside , Cápside , Glicina , VIH-1 , Proteínas de Complejo Poro Nuclear , Poro Nuclear , Fenilalanina , Humanos , Transporte Activo de Núcleo Celular , Cápside/química , Cápside/metabolismo , Glicina/metabolismo , VIH-1/química , VIH-1/genética , VIH-1/metabolismo , Poro Nuclear/química , Poro Nuclear/metabolismo , Poro Nuclear/virología , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Permeabilidad , Fenilalanina/metabolismo , Solubilidad , Internalización del Virus , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismoRESUMEN
HIV can infect non-dividing cells because the viral capsid can overcome the selective barrier of the nuclear pore complex and deliver the genome directly into the nucleus1,2. Remarkably, the intact HIV capsid is more than 1,000 times larger than the size limit prescribed by the diffusion barrier of the nuclear pore3. This barrier in the central channel of the nuclear pore is composed of intrinsically disordered nucleoporin domains enriched in phenylalanine-glycine (FG) dipeptides. Through multivalent FG interactions, cellular karyopherins and their bound cargoes solubilize in this phase to drive nucleocytoplasmic transport4. By performing an in vitro dissection of the nuclear pore complex, we show that a pocket on the surface of the HIV capsid similarly interacts with FG motifs from multiple nucleoporins and that this interaction licences capsids to penetrate FG-nucleoporin condensates. This karyopherin mimicry model addresses a key conceptual challenge for the role of the HIV capsid in nuclear entry and offers an explanation as to how an exogenous entity much larger than any known cellular cargo may be able to non-destructively breach the nuclear envelope.
Asunto(s)
Proteínas de la Cápside , Glicina , VIH , Carioferinas , Imitación Molecular , Proteínas de Complejo Poro Nuclear , Poro Nuclear , Fenilalanina , Humanos , Transporte Activo de Núcleo Celular , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Difusión , Dipéptidos/química , Dipéptidos/metabolismo , Glicina/metabolismo , VIH/química , VIH/metabolismo , Técnicas In Vitro , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Carioferinas/metabolismo , Poro Nuclear/química , Poro Nuclear/metabolismo , Poro Nuclear/virología , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Permeabilidad , Fenilalanina/metabolismo , Solubilidad , Internalización del Virus , Cápside/química , Cápside/metabolismoRESUMEN
The outer membrane in Gram-negative bacteria consists of an asymmetric phospholipid-lipopolysaccharide bilayer that is densely packed with outer-membrane ß-barrel proteins (OMPs) and lipoproteins1. The architecture and composition of this bilayer is closely monitored and is essential to cell integrity and survival2-4. Here we find that SlyB, a lipoprotein in the PhoPQ stress regulon, forms stable stress-induced complexes with the outer-membrane proteome. SlyB comprises a 10 kDa periplasmic ß-sandwich domain and a glycine zipper domain that forms a transmembrane α-helical hairpin with discrete phospholipid- and lipopolysaccharide-binding sites. After loss in lipid asymmetry, SlyB oligomerizes into ring-shaped transmembrane complexes that encapsulate ß-barrel proteins into lipid nanodomains of variable size. We find that the formation of SlyB nanodomains is essential during lipopolysaccharide destabilization by antimicrobial peptides or acute cation shortage, conditions that result in a loss of OMPs and compromised outer-membrane barrier function in the absence of a functional SlyB. Our data reveal that SlyB is a compartmentalizing transmembrane guard protein that is involved in cell-envelope proteostasis and integrity, and suggest that SlyB represents a larger family of broadly conserved lipoproteins with 2TM glycine zipper domains with the ability to form lipid nanodomains.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Membrana Celular , Bacterias Gramnegativas , Membrana Dobles de Lípidos , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Glicina/metabolismo , Lipopolisacáridos/metabolismo , Lipoproteínas/química , Lipoproteínas/metabolismo , Fosfolípidos/metabolismo , Sitios de Unión , Proteostasis , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Proteoma/química , Proteoma/metabolismo , Regulón , Dominios Proteicos , Péptidos Antimicrobianos/metabolismo , Bacterias Gramnegativas/química , Bacterias Gramnegativas/citología , Bacterias Gramnegativas/metabolismoRESUMEN
Neurotransmission mediated by diverse subtypes of N-methyl-D-aspartate receptors (NMDARs) is fundamental for basic brain functions and development as well as neuropsychiatric diseases and disorders. NMDARs are glycine- and glutamate-gated ion channels that exist as heterotetramers composed of obligatory GluN1 and GluN2(A-D) and/or GluN3(A-B). The GluN2C and GluN2D subunits form ion channels with distinct properties and spatio-temporal expression patterns. Here, we provide the structures of the agonist-bound human GluN1-2C NMDAR in the presence and absence of the GluN2C-selective positive allosteric potentiator (PAM), PYD-106, the agonist-bound GluN1-2A-2C tri-heteromeric NMDAR, and agonist-bound GluN1-2D NMDARs by single-particle electron cryomicroscopy. Our analysis shows unique inter-subunit and domain arrangements of the GluN2C NMDARs, which contribute to functional regulation and formation of the PAM binding pocket and is distinct from GluN2D NMDARs. Our findings here provide the fundamental blueprint to study GluN2C- and GluN2D-containing NMDARs, which are uniquely involved in neuropsychiatric disorders.
Asunto(s)
Ácido Glutámico , Receptores de N-Metil-D-Aspartato , Humanos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Ácido Glutámico/metabolismo , Glicina/metabolismo , Transmisión Sináptica , Subunidades de Proteína/metabolismoRESUMEN
Diabetes represents a spectrum of disease in which metabolic dysfunction damages multiple organ systems including liver, kidneys and peripheral nerves1,2. Although the onset and progression of these co-morbidities are linked with insulin resistance, hyperglycaemia and dyslipidaemia3-7, aberrant non-essential amino acid (NEAA) metabolism also contributes to the pathogenesis of diabetes8-10. Serine and glycine are closely related NEAAs whose levels are consistently reduced in patients with metabolic syndrome10-14, but the mechanistic drivers and downstream consequences of this metabotype remain unclear. Low systemic serine and glycine are also emerging as a hallmark of macular and peripheral nerve disorders, correlating with impaired visual acuity and peripheral neuropathy15,16. Here we demonstrate that aberrant serine homeostasis drives serine and glycine deficiencies in diabetic mice, which can be diagnosed with a serine tolerance test that quantifies serine uptake and disposal. Mimicking these metabolic alterations in young mice by dietary serine or glycine restriction together with high fat intake markedly accelerates the onset of small fibre neuropathy while reducing adiposity. Normalization of serine by dietary supplementation and mitigation of dyslipidaemia with myriocin both alleviate neuropathy in diabetic mice, linking serine-associated peripheral neuropathy to sphingolipid metabolism. These findings identify systemic serine deficiency and dyslipidaemia as novel risk factors for peripheral neuropathy that may be exploited therapeutically.
Asunto(s)
Diabetes Mellitus Experimental , Insulina , Metabolismo de los Lípidos , Enfermedades del Sistema Nervioso Periférico , Serina , Animales , Ratones , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Glicina/metabolismo , Insulina/metabolismo , Enfermedades del Sistema Nervioso Periférico/metabolismo , Serina/metabolismo , Dieta Alta en Grasa , Adiposidad , Esfingolípidos/metabolismo , Neuropatía de Fibras Pequeñas , DislipidemiasRESUMEN
The nuclear pore complex (NPC) is the bidirectional gate that mediates the exchange of macromolecules or their assemblies between nucleus and cytoplasm1-3. The assembly intermediates of the ribosomal subunits, pre-60S and pre-40S particles, are among the largest cargoes of the NPC and the export of these gigantic ribonucleoproteins requires numerous export factors4,5. Here we report the cryo-electron microscopy structure of native pre-60S particles trapped in the channel of yeast NPCs. In addition to known assembly factors, multiple factors with export functions are also included in the structure. These factors in general bind to either the flexible regions or subunit interface of the pre-60S particle, and virtually form many anchor sites for NPC binding. Through interactions with phenylalanine-glycine (FG) repeats from various nucleoporins of NPC, these factors collectively facilitate the passage of the pre-60S particle through the central FG repeat network of the NPC. Moreover, in silico analysis of the axial and radial distribution of pre-60S particles within the NPC shows that a single NPC can take up to four pre-60S particles simultaneously, and pre-60S particles are enriched in the inner ring regions close to the wall of the NPC with the solvent-exposed surface facing the centre of the nuclear pore. Our data suggest a translocation model for the export of pre-60S particles through the NPC.
Asunto(s)
Transporte Activo de Núcleo Celular , Poro Nuclear , Saccharomyces cerevisiae , Microscopía por Crioelectrón , Poro Nuclear/química , Poro Nuclear/metabolismo , Poro Nuclear/ultraestructura , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/ultraestructura , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructura , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Fenilalanina , Glicina , Simulación por Computador , SolventesRESUMEN
N-degron pathways are a set of proteolytic systems that target the N-terminal destabilizing residues of substrates for proteasomal degradation. Recently, the Gly/N-degron pathway has been identified as a new branch of the N-degron pathway. The N-terminal glycine degron (Gly/N-degron) is recognized by ZYG11B and ZER1, the substrate receptors of the Cullin 2-RING E3 ubiquitin ligase (CRL2). Here we present the crystal structures of ZYG11B and ZER1 bound to various Gly/N-degrons. The structures reveal that ZYG11B and ZER1 utilize their armadillo (ARM) repeats forming a deep and narrow cavity to engage mainly the first four residues of Gly/N-degrons. The α-amino group of the Gly/N-degron is accommodated in an acidic pocket by five conserved hydrogen bonds. These structures, together with biochemical studies, decipher the molecular basis for the specific recognition of the Gly/N-degron by ZYG11B and ZER1, providing key information for future structure-based chemical probe design.
Asunto(s)
Proteínas de Ciclo Celular/ultraestructura , Glicina/química , Conformación Proteica , Receptores de Citocinas/ultraestructura , Secuencia de Aminoácidos/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cristalografía por Rayos X , Glicina/genética , Células HEK293 , Humanos , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/ultraestructura , Unión Proteica/genética , Dominios Proteicos/genética , Proteolisis , Receptores de Citocinas/química , Receptores de Citocinas/genética , Especificidad por Sustrato , Ubiquitina/genéticaRESUMEN
Cellular senescence is a state of stable proliferative arrest triggered by damaging signals. Senescent cells persist during aging and promote age-related pathologies via the pro-inflammatory senescence-associated secretory phenotype (SASP), whose regulation depends on environmental factors. In vivo, a major environmental variable is oxygenation, which varies among and within tissues. Here, we demonstrate that senescent cells express lower levels of detrimental pro-inflammatory SASP factors in physiologically hypoxic environments, as measured in culture and in tissues. Mechanistically, exposure of senescent cells to low-oxygen conditions leads to AMPK activation and AMPK-mediated suppression of the mTOR-NF-κB signaling loop. Finally, we demonstrate that treatment with hypoxia-mimetic compounds reduces SASP in cells and tissues and improves strength in chemotherapy-treated and aged mice. Our findings highlight the importance of oxygen as a determinant for pro-inflammatory SASP expression and offer a potential new strategy to reduce detrimental paracrine effects of senescent cells.