RESUMEN
Multiple proteins act co-operatively in mammalian clathrin-mediated endocytosis (CME) to generate endocytic vesicles from the plasma membrane. The principles controlling the activation and organization of the actin cytoskeleton during mammalian CME are, however, not fully understood. Here, we show that the protein FCHSD2 is a major activator of actin polymerization during CME. FCHSD2 deletion leads to decreased ligand uptake caused by slowed pit maturation. FCHSD2 is recruited to endocytic pits by the scaffold protein intersectin via an unusual SH3-SH3 interaction. Here, its flat F-BAR domain binds to the planar region of the plasma membrane surrounding the developing pit forming an annulus. When bound to the membrane, FCHSD2 activates actin polymerization by a mechanism that combines oligomerization and recruitment of N-WASP to PI(4,5)P2, thus promoting pit maturation. Our data therefore describe a molecular mechanism for linking spatiotemporally the plasma membrane to a force-generating actin platform guiding endocytic vesicle maturation.
Asunto(s)
Citoesqueleto de Actina/fisiología , Proteínas Portadoras/metabolismo , Clatrina/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Membrana Celular/química , Membrana Celular/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Endocitosis , Células HeLa , Humanos , Liposomas/química , Liposomas/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Microscopía Fluorescente , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína Neuronal del Síndrome de Wiskott-Aldrich/química , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismo , Dominios Homologos srcRESUMEN
A defining pathological feature of most neurodegenerative diseases is the assembly of proteins into amyloid that form disease-specific structures1. In Alzheimer's disease, this is characterized by the deposition of ß-amyloid and tau with disease-specific conformations. The in situ structure of amyloid in the human brain is unknown. Here, using cryo-fluorescence microscopy-targeted cryo-sectioning, cryo-focused ion beam-scanning electron microscopy lift-out and cryo-electron tomography, we determined in-tissue architectures of ß-amyloid and tau pathology in a postmortem Alzheimer's disease donor brain. ß-amyloid plaques contained a mixture of fibrils, some of which were branched, and protofilaments, arranged in parallel arrays and lattice-like structures. Extracellular vesicles and cuboidal particles defined the non-amyloid constituents of ß-amyloid plaques. By contrast, tau inclusions formed parallel clusters of unbranched filaments. Subtomogram averaging a cluster of 136 tau filaments in a single tomogram revealed the polypeptide backbone conformation and filament polarity orientation of paired helical filaments within tissue. Filaments within most clusters were similar to each other, but were different between clusters, showing amyloid heterogeneity that is spatially organized by subcellular location. The in situ structural approaches outlined here for human donor tissues have applications to a broad range of neurodegenerative diseases.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Encéfalo , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Placa Amiloide , Proteínas tau , Humanos , Masculino , Ratones , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/ultraestructura , Autopsia , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/ultraestructura , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/química , Vesículas Extracelulares/ultraestructura , Placa Amiloide/metabolismo , Placa Amiloide/patología , Placa Amiloide/química , Placa Amiloide/ultraestructura , Proteínas tau/química , Proteínas tau/metabolismo , Proteínas tau/ultraestructuraRESUMEN
The amount of any given protein in the brain is determined by the rates of its synthesis and destruction, which are regulated by different cellular mechanisms. Here, we combine metabolic labeling in live mice with global proteomic profiling to simultaneously quantify both the flux and amount of proteins in mouse models of neurodegeneration. In multiple models, protein turnover increases were associated with increasing pathology. This method distinguishes changes in protein expression mediated by synthesis from those mediated by degradation. In the AppNL-F knockin mouse model of Alzheimer's disease, increased turnover resulted from imbalances in both synthesis and degradation, converging on proteins associated with synaptic vesicle recycling (Dnm1, Cltc, Rims1) and mitochondria (Fis1, Ndufv1). In contrast to disease models, aging in wild-type mice caused a widespread decrease in protein recycling associated with a decrease in autophagic flux. Overall, this simple multidimensional approach enables a comprehensive mapping of proteome dynamics and identifies affected proteins in mouse models of disease and other live animal test settings.
Asunto(s)
Enfermedad de Alzheimer , Proteoma , Envejecimiento , Enfermedad de Alzheimer/metabolismo , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Mamíferos/metabolismo , Ratones , Ratones Transgénicos , Proteoma/metabolismo , Proteómica/métodosRESUMEN
Resolving atomic structures of isolated proteins has uncovered mechanisms and fundamental processes in biology. However, many functions can only be tested in the context of intact cells and tissues that are many orders of magnitude larger than the macromolecules on which they depend. Therefore, methods that interrogate macromolecular structure in situ provide a means of directly relating structure to function across length scales. Here, we developed several workflows using cryogenic correlated light and electron microscopy (cryoCLEM) and electron tomography (cryoET) that can bridge this gap to reveal the molecular infrastructure that underlies higher order functions within cells and tissues. We also describe experimental design considerations, including cryoCLEM labelling, sample preparation, and quality control, for determining the in situ molecular architectures within native, hydrated cells and tissues.
Asunto(s)
Tomografía con Microscopio Electrónico , Electrones , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Microscopía Electrónica , Sustancias Macromoleculares/químicaRESUMEN
PSD95 is an abundant postsynaptic scaffold protein in glutamatergic synapses that assembles into supercomplexes composed of over 80 proteins including neurotransmitter receptors, ion channels and adhesion proteins. How these diverse constituents are organized into PSD95 supercomplexes in vivo is poorly understood. Here, we dissected the supercomplexes in mice combining endogenous gene-tagging, targeted mutations and quantitative biochemical assays. Generating compound heterozygous mice with two different gene-tags, one on each Psd95 allele, showed that each ~1.5 MDa PSD95-containing supercomplex contains on average two PSD95 molecules. Gene-tagging the endogenous GluN1 and PSD95 with identical Flag tags revealed N-methyl D-aspartic acid receptors (NMDARs) containing supercomplexes that represent only 3% of the total population of PSD95 supercomplexes, suggesting there are many other subtypes. To determine whether this extended population of different PSD95 supercomplexes use genetically defined mechanisms to specify their assembly, we tested the effect of five targeted mouse mutations on the assembly of known PSD95 interactors, Kir2.3, Arc, IQsec2/BRAG1 and Adam22. Unexpectedly, some mutations were highly selective, whereas others caused widespread disruption, indicating that PSD95 interacting proteins are organized hierarchically into distinct subfamilies of ~1.5 MDa supercomplexes, including a subpopulation of Kir2.3-NMDAR ion channel-channel supercomplexes. Kir2.3-NMDAR ion channel-channel supercomplexes were found to be anatomically restricted to particular brain regions. These data provide new insight into the mechanisms that govern the constituents of postsynaptic supercomplexes and the diversity of synapse types. Read the Editorial Highlight for this article on page 500. Cover Image for this issue: doi. 10.1111/jnc.13811.
Asunto(s)
Guanilato-Quinasas/genética , Guanilato-Quinasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Sinapsis/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Homólogo 4 de la Proteína Discs Large , Masculino , Ratones , Ratones Transgénicos , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/genéticaRESUMEN
Amyloid plaques composed of Aß fibrils are a hallmark of Alzheimer's disease (AD). However, the molecular architecture of amyloid plaques in the context of fresh mammalian brain tissue is unknown. Here, using cryogenic correlated light and electron tomography we report the in situ molecular architecture of Aß fibrils in the AppNL-G-F familial AD mouse model containing the Arctic mutation and an atomic model of ex vivo purified Arctic Aß fibrils. We show that in-tissue Aß fibrils are arranged in a lattice or parallel bundles, and are interdigitated by subcellular compartments, extracellular vesicles, extracellular droplets and extracellular multilamellar bodies. The Arctic Aß fibril differs significantly from an earlier AppNL-F fibril structure, indicating a striking effect of the Arctic mutation. These structural data also revealed an ensemble of additional fibrillar species, including thin protofilament-like rods and branched fibrils. Together, these results provide a structural model for the dense network architecture that characterises ß-amyloid plaque pathology.
Asunto(s)
Enfermedad de Alzheimer , Péptidos beta-Amiloides , Ratones , Animales , Péptidos beta-Amiloides/metabolismo , Placa Amiloide/patología , Enfermedad de Alzheimer/genética , Encéfalo/metabolismo , Mutación , Mamíferos/metabolismoRESUMEN
Ionotropic receptors, including the NMDAR (N-methyl-D-aspartate receptor) mediate fast neurotransmission, neurodevelopment, neuronal excitability and learning. In the present article, the structure and function of the NMDAR is reviewed with the aim to condense our current understanding and highlight frontiers where important questions regarding the biology of this receptor remain unanswered. In the second part of the present review, new biochemical and genetic approaches for the investigation of ion channel receptor complexes will be discussed.
Asunto(s)
Canales Iónicos/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Animales , Agonistas de Aminoácidos Excitadores/metabolismo , Canales Iónicos/química , Modelos Moleculares , N-Metilaspartato/metabolismo , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/fisiología , Receptores de N-Metil-D-Aspartato/química , Transmisión Sináptica/fisiologíaRESUMEN
The pyruvate dehydrogenase multienzyme assembly (PDH) generates acetyl coenzyme A and reducing equivalents from pyruvate in a multiple-step process that is a nexus of central metabolism. We report crystal structures of the Geobacillus stearothermophilus PDH E1p subunit with ligands that mimic the prereaction complex and the postdecarboxylation product. The structures implicate residues that help to orient substrates, nurture intermediates, and organize surface loops so that they can engage a mobile lipoyl domain that receives the acetyl group and shuttles it to the next active site. The structural and enzymatic data suggest that H128beta performs a dual role: first, as electrostatic catalyst of the reaction of pyruvate with the thiamine cofactor; and second, as a proton donor in the second reaction of acetyl group with the lipoate. We also identify I206alpha as a key residue in mediating the conformation of active-site loops. We propose that a simple conformational flip of the H271alpha side chain assists transfer of the acetyl group from thiamine cofactor to lipoyl domain in synchrony with reduction of the dithiolane ring.
Asunto(s)
Geobacillus stearothermophilus/enzimología , Complejo Piruvato Deshidrogenasa/metabolismo , Acetilcoenzima A/biosíntesis , Acetilcoenzima A/genética , Secuencia de Aminoácidos , Sitios de Unión/genética , Catálisis , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Modelos Biológicos , Modelos Moleculares , Mutación , Unión Proteica/genética , Estructura Cuaternaria de Proteína/genética , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Complejo Piruvato Deshidrogenasa/química , Complejo Piruvato Deshidrogenasa/genética , Relación Estructura-Actividad , Especificidad por Sustrato/genéticaRESUMEN
The catalytic cofactor thiamine diphosphate is found in many enzymes of central metabolism and is essential in all extant forms of life. We demonstrate the presence of an oxygen-dependent free radical in the thiamine diphosphate-dependent Escherichia coli 2-oxoglutarate dehydrogenase, which is a key component of the tricarboxylic acid (Krebs) cycle. The radical was sufficiently long-lived to be trapped by freezing in liquid nitrogen, and its electronic structure was investigated by electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR). Taken together, the spectroscopic results revealed a delocalized pi radical on the enamine-thiazolium intermediate within the enzyme active site. The radical is generated as an intermediate during substrate turnover by a side reaction with molecular oxygen, resulting in the continuous production of reactive oxygen species under aerobic conditions. This off-pathway reaction may account for metabolic dysfunction associated with several neurodegenerative diseases. The possibility that the on-pathway reaction may proceed via a radical mechanism is discussed.
Asunto(s)
Ciclo del Ácido Cítrico , Oxígeno/química , Oxígeno/metabolismo , Tiamina/química , Tiamina/metabolismo , Catálisis , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/química , Radicales Libres/metabolismo , Geobacillus stearothermophilus/enzimología , Modelos Moleculares , Estructura Molecular , Peróxidos/metabolismoRESUMEN
The thiamine-dependent E1o component (EC 1.2.4.2) of the 2-oxoglutarate dehydrogenase complex catalyses a rate-limiting step of the tricarboxylic acid cycle (TCA) of aerobically respiring organisms. We describe the crystal structure of Escherichia coli E1o in its apo and holo forms at 2.6 A and 3.5 A resolution, respectively. The structures reveal the characteristic fold that binds thiamine diphosphate and resemble closely the alpha(2)beta(2) hetero-tetrameric E1 components of other 2-oxo acid dehydrogenase complexes, except that in E1o, the alpha and beta subunits are fused as a single polypeptide. The extended segment that links the alpha-like and beta-like domains forms a pocket occupied by AMP, which is recognised specifically. Also distinctive to E1o are N-terminal extensions to the core fold, and which may mediate interactions with other components of the 2-oxoglutarate dehydrogenase multienzyme complex. The active site pocket contains a group of three histidine residues and one serine that appear to confer substrate specificity and the capacity to accommodate the TCA metabolite oxaloacetate. Oxaloacetate inhibits E1o activity at physiological concentrations, and we suggest that the inhibition may allow coordinated activity within the TCA cycle. We discuss the implications for metabolic control in facultative anaerobes, and for energy homeostasis of the mammalian brain.
Asunto(s)
Proteínas de Escherichia coli/química , Complejo Cetoglutarato Deshidrogenasa/química , Modelos Moleculares , Adenosina Monofosfato/química , Sitios de Unión , Ciclo del Ácido Cítrico , Proteínas de Escherichia coli/genética , Complejo Cetoglutarato Deshidrogenasa/genética , Mutación , Ácido Oxaloacético/química , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Tiamina Pirofosfato/químicaRESUMEN
The pyruvate dehydrogenase (PDH) multienzyme complex is central to oxidative metabolism. We present the first crystal structure of a complex between pyruvate decarboxylase (E1) and the peripheral subunit binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2). The interface is dominated by a "charge zipper" of networked salt bridges. Remarkably, the PSBD uses essentially the same zipper to alternately recognize the dihydrolipoyl dehydrogenase (E3) component of the PDH assembly. The PSBD achieves this dual recognition largely through the addition of a network of interfacial water molecules unique to the E1-PSBD complex. These structural comparisons illuminate our observations that the formation of this water-rich E1-E2 interface is largely enthalpy driven, whereas that of the E3-PSBD complex (from which water is excluded) is entropy driven. Interfacial water molecules thus diversify surface complementarity and contribute to avidity, enthalpically. Additionally, the E1-PSBD structure provides insight into the organization and active site coupling within the approximately 9 MDa PDH complex.
Asunto(s)
Complejo Piruvato Deshidrogenasa/química , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Geobacillus stearothermophilus/enzimología , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Pseudomonas putida/enzimología , Complejo Piruvato Deshidrogenasa/metabolismoRESUMEN
Arc is an activity-regulated neuronal protein, but little is known about its interactions, assembly into multiprotein complexes, and role in human disease and cognition. We applied an integrated proteomic and genetic strategy by targeting a tandem affinity purification (TAP) tag and Venus fluorescent protein into the endogenous Arc gene in mice. This allowed biochemical and proteomic characterization of native complexes in wild-type and knockout mice. We identified many Arc-interacting proteins, of which PSD95 was the most abundant. PSD95 was essential for Arc assembly into 1.5-MDa complexes and activity-dependent recruitment to excitatory synapses. Integrating human genetic data with proteomic data showed that Arc-PSD95 complexes are enriched in schizophrenia, intellectual disability, autism, and epilepsy mutations and normal variants in intelligence. We propose that Arc-PSD95 postsynaptic complexes potentially affect human cognitive function.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Homólogo 4 de la Proteína Discs Large/metabolismo , Inteligencia , Proteínas del Tejido Nervioso/metabolismo , Sistema Nervioso/metabolismo , Sistema Nervioso/fisiopatología , Sinapsis/metabolismo , Animales , Técnicas de Sustitución del Gen , Humanos , Ratones Noqueados , ProteómicaRESUMEN
How neuronal proteomes self-organize is poorly understood because of their inherent molecular and cellular complexity. Here, focusing on mammalian synapses we use blue-native PAGE and 'gene-tagging' of GluN1 to report the first biochemical purification of endogenous NMDA receptors (NMDARs) directly from adult mouse brain. We show that NMDARs partition between two discrete populations of receptor complexes and â¼1.5 MDa supercomplexes. We tested the assembly mechanism with six mouse mutants, which indicates a tripartite requirement of GluN2B, PSD93 and PSD95 gate the incorporation of receptors into â¼1.5 MDa supercomplexes, independent of either canonical PDZ-ligands or GluN2A. Supporting the essential role of GluN2B, quantitative gene-tagging revealed a fourfold molar excess of GluN2B over GluN2A in adult forebrain. NMDAR supercomplexes are assembled late in postnatal development and triggered by synapse maturation involving epigenetic and activity-dependent mechanisms. Finally, screening the quaternary organization of 60 native proteins identified numerous discrete supercomplexes that populate the mammalian synapse.
Asunto(s)
Complejos Multiproteicos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , Animales , Células Cultivadas , Células HEK293 , Humanos , Ratones Transgénicos , Complejos Multiproteicos/genética , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Prosencéfalo/citología , Prosencéfalo/embriología , Prosencéfalo/metabolismo , Unión Proteica , Proteoma/metabolismo , Proteómica , Receptores de N-Metil-D-Aspartato/genética , Sinapsis/genéticaRESUMEN
Current models of schizophrenia and bipolar disorder implicate multiple genes, however their biological relationships remain elusive. To test the genetic role of glutamate receptors and their interacting scaffold proteins, the exons of ten glutamatergic 'hub' genes in 1304 individuals were re-sequenced in case and control samples. No significant difference in the overall number of non-synonymous single nucleotide polymorphisms (nsSNPs) was observed between cases and controls. However, cluster analysis of nsSNPs identified two exons encoding the cysteine-rich domain and first transmembrane helix of GRM1 as a risk locus with five mutations highly enriched within these domains. A new splice variant lacking the transmembrane GPCR domain of GRM1 was discovered in the human brain and the GRM1 mutation cluster could perturb the regulation of this variant. The predicted effect on individuals harbouring multiple mutations distributed in their ten hub genes was also examined. Diseased individuals possessed an increased load of deleteriousness from multiple concurrent rare and common coding variants. Together, these data suggest a disease model in which the interplay of compound genetic coding variants, distributed among glutamate receptors and their interacting proteins, contribute to the pathogenesis of schizophrenia and bipolar disorders.
Asunto(s)
Trastorno Bipolar/genética , Receptores de Glutamato/genética , Esquizofrenia/genética , Estudios de Casos y Controles , Análisis por Conglomerados , Exones , Predisposición Genética a la Enfermedad , Variación Genética , Genotipo , Humanos , Mutación , Polimorfismo de Nucleótido Simple , Estructura Terciaria de Proteína , Análisis de Secuencia de ADNRESUMEN
Thiamine diphosphate (ThDP) is used as a cofactor in many key metabolic enzymes. We present evidence that the ThDPs in the two active sites of the E1 (EC 1.2.4.1) component of the pyruvate dehydrogenase complex communicate over a distance of 20 angstroms by reversibly shuttling a proton through an acidic tunnel in the protein. This "proton wire" permits the co-factors to serve reciprocally as general acid/base in catalysis and to switch the conformation of crucial active-site peptide loops. This synchronizes the progression of chemical events and can account for the oligomeric organization, conformational asymmetry, and "ping-pong" kinetic properties of E1 and other thiamine-dependent enzymes.