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1.
Mol Biol Evol ; 40(3)2023 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-36857092

RESUMO

Amino acids evolve at different speeds within protein sequences, because their functional and structural roles are different. Notably, amino acids located at the surface of proteins are known to evolve more rapidly than those in the core. In particular, amino acids at the N- and C-termini of protein sequences are likely to be more exposed than those at the core of the folded protein due to their location in the peptidic chain, and they are known to be less structured. Because of these reasons, we would expect that amino acids located at protein termini would evolve faster than residues located inside the chain. Here we test this hypothesis and found that amino acids evolve almost twice as fast at protein termini compared with those in the center, hinting at a strong topological bias along the sequence length. We further show that the distribution of solvent-accessible residues and functional domains in proteins readily explain how structural and functional constraints are weaker at their termini, leading to the observed excess of amino acid substitutions. Finally, we show that the specific evolutionary rates at protein termini may have direct consequences, notably misleading in silico methods used to infer sites under positive selection within genes. These results suggest that accounting for positional information should improve evolutionary models.


Assuntos
Aminoácidos , Proteínas , Proteínas/genética , Proteínas/química , Sequência de Aminoácidos , Aminoácidos/genética , Aminoácidos/química , Éxons , Substituição de Aminoácidos , Evolução Molecular
2.
EMBO J ; 38(2)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30396997

RESUMO

NMDA receptors (NMDARs) are glutamate-gated ion channels that are key mediators of excitatory neurotransmission and synaptic plasticity throughout the central nervous system. They form massive heterotetrameric complexes endowed with unique allosteric capacity provided by eight extracellular clamshell-like domains arranged as two superimposed layers. Despite an increasing number of full-length NMDAR structures, how these domains cooperate in an intact receptor to control its activity remains poorly understood. Here, combining single-molecule and macroscopic electrophysiological recordings, cysteine biochemistry, and in silico analysis, we identify a rolling motion at a yet unexplored interface between the two constitute dimers in the agonist-binding domain (ABD) layer as a key structural determinant in NMDAR activation and allosteric modulation. This rotation acts as a gating switch that tunes channel opening depending on the conformation of the membrane-distal N-terminal domain (NTD) layer. Remarkably, receptors locked in a rolled state display "super-activity" and resistance to NTD-mediated allosteric modulators. Our work unveils how NMDAR domains move in a concerted manner to transduce long-range conformational changes between layers and command receptor channel activity.


Assuntos
Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Regulação Alostérica , Animais , Simulação por Computador , Cisteína/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Receptores de N-Metil-D-Aspartato/genética , Transdução de Sinais , Imagem Individual de Molécula , Xenopus laevis
3.
Proc Natl Acad Sci U S A ; 117(34): 20898-20907, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32788354

RESUMO

The voltage-gated proton channel Hv1 is a member of the voltage-gated ion channel superfamily, which stands out in design: It is a dimer of two voltage-sensing domains (VSDs), each containing a pore pathway, a voltage sensor (S4), and a gate (S1) and forming its own ion channel. Opening of the two channels in the dimer is cooperative. Part of the cooperativity is due to association between coiled-coil domains that extend intracellularly from the S4s. Interactions between the transmembrane portions of the subunits may also contribute, but the nature of transmembrane packing is unclear. Using functional analysis of a mutagenesis scan, biochemistry, and modeling, we find that the subunits form a dimer interface along the entire length of S1, and also have intersubunit contacts between S1 and S4. These interactions exert a strong effect on gating, in particular on the stability of the open state. Our results suggest that gating in Hv1 is tuned by extensive VSD-VSD interactions between the gates and voltage sensors of the dimeric channel.


Assuntos
Canais Iônicos/metabolismo , Sequência de Aminoácidos , Humanos , Ativação do Canal Iônico , Canais Iônicos/química , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Prótons
4.
J Physiol ; 599(10): 2615-2638, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-32786006

RESUMO

Ionotropic glutamate receptors (iGluRs) are a major class of ligand-gated ion channels that are widespread in the living kingdom. Their critical role in excitatory neurotransmission and brain function of arthropods and vertebrates has made them a compelling subject of interest for neurophysiologists and pharmacologists. This is particularly true for NMDA receptor (NMDARs), a subclass of iGluRs that act as central drivers of synaptic plasticity in the CNS. How and when the unique properties of NMDARs arose during evolution, and how they relate to the evolution of the nervous system, remain open questions. Recent years have witnessed a boom in both genomic and structural data, such that it is now possible to analyse the evolution of iGluR genes on an unprecedented scale and within a solid molecular framework. In this review, combining insights from phylogeny, atomic structure and physiological and mechanistic data, we discuss how evolution of NMDAR motifs and sequences shaped their architecture and functionalities. We trace differences and commonalities between NMDARs and other iGluRs, emphasizing a few distinctive properties of the former regarding ligand binding and gating, permeation, allosteric modulation and intracellular signalling. Finally, we speculate on how specific molecular properties of iGuRs arose to supply new functions to the evolving structure of the nervous system, from early metazoan to present mammals.


Assuntos
Canais Iônicos de Abertura Ativada por Ligante , Receptores de N-Metil-D-Aspartato , Animais , Fenômenos Biofísicos , Receptores Ionotrópicos de Glutamato/genética , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica
5.
Eur Biophys J ; 50(3-4): 313-330, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33792745

RESUMO

Biophysical quantification of protein interactions is central to unveil the molecular mechanisms of cellular processes. Researchers can choose from a wide panel of biophysical methods that quantify molecular interactions in different ways, including both classical and more novel techniques. We report the outcome of an ARBRE-MOBIEU training school held in June 2019 in Gif-sur-Yvette, France ( https://mosbio.sciencesconf.org/ ). Twenty European students benefited from a week's training with theoretical and practical sessions in six complementary approaches: (1) analytical ultracentrifugation with or without a fluorescence detector system (AUC-FDS), (2) isothermal titration calorimetry (ITC), (3) size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), (4) bio-layer interferometry (BLI), (5) microscale thermophoresis (MST) and, (6) switchSENSE. They implemented all these methods on two examples of macromolecular interactions with nanomolar affinity: first, a protein-protein interaction between an artificial alphaRep binder, and its target protein, also an alphaRep; second, a protein-DNA interaction between a DNA repair complex, Ku70/Ku80 (hereafter called Ku), and its cognate DNA ligand. We report the approaches used to analyze the two systems under study and thereby showcase application of each of the six techniques. The workshop provided students with improved understanding of the advantages and limitations of different methods, enabling future choices concerning approaches that are most relevant or informative for specific kinds of sample and interaction.


Assuntos
Substâncias Macromoleculares/análise , Calorimetria , DNA , Humanos , Ligantes , Proteínas
6.
Mol Pharmacol ; 89(5): 541-51, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26912815

RESUMO

N-methyl-d-aspartate receptors (NMDARs) are glutamate-gated ion channels that play key roles in brain physiology and pathology. Because numerous pathologic conditions involve NMDAR overactivation, subunit-selective antagonists hold strong therapeutic potential, although clinical successes remain limited. Among the most promising NMDAR-targeting drugs are allosteric inhibitors of GluN2B-containing receptors. Since the discovery of ifenprodil, a range of GluN2B-selective compounds with strikingly different structural motifs have been identified. This molecular diversity raises the possibility of distinct binding sites, although supporting data are lacking. Using X-ray crystallography, we show that EVT-101, a GluN2B antagonist structurally unrelated to the classic phenylethanolamine pharmacophore, binds at the same GluN1/GluN2B dimer interface as ifenprodil but adopts a remarkably different binding mode involving a distinct subcavity and receptor interactions. Mutagenesis experiments demonstrate that this novel binding site is physiologically relevant. Moreover, in silico docking unveils that GluN2B-selective antagonists broadly divide into two distinct classes according to binding pose. These data widen the allosteric and pharmacological landscape of NMDARs and offer a renewed structural framework for designing next-generation GluN2B antagonists with therapeutic value for brain disorders.


Assuntos
Antagonistas Adrenérgicos alfa/metabolismo , Drogas em Investigação/metabolismo , Moduladores de Transporte de Membrana/metabolismo , Modelos Moleculares , Receptores de N-Metil-D-Aspartato/metabolismo , Antagonistas Adrenérgicos alfa/química , Antagonistas Adrenérgicos alfa/farmacologia , Regulação Alostérica/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Biologia Computacional , Drogas em Investigação/química , Drogas em Investigação/farmacologia , Sistemas Inteligentes , Humanos , Imidazóis/química , Imidazóis/metabolismo , Imidazóis/farmacologia , Ligantes , Moduladores de Transporte de Membrana/química , Moduladores de Transporte de Membrana/farmacologia , Conformação Molecular , Simulação de Acoplamento Molecular , Mutação , Oxidiazóis/química , Oxidiazóis/metabolismo , Oxidiazóis/farmacologia , Piperidinas/química , Piperidinas/metabolismo , Piperidinas/farmacologia , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Piridazinas/química , Piridazinas/metabolismo , Piridazinas/farmacologia , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo
8.
J Neurosci ; 34(50): 16630-6, 2014 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-25505316

RESUMO

Ligand-gated ion channels (LGICs) mediate fast synaptic transmission in the CNS. Typically, these membrane proteins are multimeric complexes associating several homologous subunits around a central pore. Because of the large repertoire of subunits within each family, LGICs exist in vivo as multiple subtypes that differ in subunit composition and functional properties. Establishing the specific properties of individual receptor subtypes remains a major goal in the field of neuroscience and molecular pharmacology. However, isolating specific receptor subtype in recombinant systems can be problematic because of the mixture of receptor populations. This is the case for NMDA receptors (NMDARs), a large family of tetrameric glutamate-gated ion channels that play key roles in brain physiology and pathology. A significant fraction of native NMDARs are triheteromers composed of two GluN1 subunits and two different GluN2 subunits (GluN2A-D). We developed a method based on dual retention signals adapted from G-protein-coupled GABA-B receptors allowing exclusive cell surface expression of triheteromeric rat NMDARs while coexpressed diheteromeric receptors (which contain a single type of GluN2 subunit) are retained intracellularly. Using this approach, we determined the functional properties of GluN1/GluN2A/GluN2B triheteromers, one of the most abundant NMDAR subtypes in the adult forebrain, revealing their unique gating and pharmacological attributes. We envision applicability of the retention signal approach for the study of a variety of heteromeric glutamate-gated ion channel receptors with defined subunit composition.


Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Feminino , Ácido Glutâmico/farmacologia , Glicina/farmacologia , Dados de Sequência Molecular , Subunidades Proteicas/agonistas , Subunidades Proteicas/fisiologia , Ratos , Receptores de N-Metil-D-Aspartato/agonistas , Transdução de Sinais/efeitos dos fármacos , Xenopus laevis
10.
J Biol Chem ; 288(2): 778-84, 2013 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-23223336

RESUMO

NMDA receptors are widely expressed in the central nervous system and play a major role in excitatory synaptic transmission and plasticity. Here, we used atomic force microscopy (AFM) imaging to visualize activation-induced structural changes in the GluN1/GluN2A NMDA receptor reconstituted into a lipid bilayer. In the absence of agonist, AFM imaging revealed two populations of particles with heights above the bilayer surface of 8.6 and 3.4 nm. The taller, but not the shorter, particles could be specifically decorated by an anti-GluN1 antibody, which recognizes the S2 segment of the agonist-binding domain, indicating that the two populations represent the extracellular and intracellular regions of the receptor, respectively. In the presence of glycine and glutamate, there was a reduction in the height of the extracellular region to 7.3 nm. In contrast, the height of the intracellular domain was unaffected. Fast-scan AFM imaging combined with UV photolysis of caged glutamate permitted the detection of a rapid reduction in the height of individual NMDA receptors. The reduction in height did not occur in the absence of the co-agonist glycine or in the presence of the selective NMDA receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid, indicating that the observed structural change was caused by receptor activation. These results represent the first demonstration of an activation-induced effect on the structure of the NMDA receptor at the single-molecule level. A change in receptor size following activation could have important functional implications, in particular by affecting interactions between the NMDA receptor and its extracellular synaptic partners.


Assuntos
Microscopia de Força Atômica/métodos , Receptores de N-Metil-D-Aspartato/metabolismo , Células HEK293 , Humanos , Conformação Proteica , Receptores de N-Metil-D-Aspartato/química
11.
Science ; 382(6677): 1389-1394, 2023 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-38060673

RESUMO

Fast synaptic neurotransmission in the vertebrate central nervous system relies primarily on ionotropic glutamate receptors (iGluRs), which drive neuronal excitation, and type A γ-aminobutyric acid receptors (GABAARs), which are responsible for neuronal inhibition. However, the GluD1 receptor, an iGluR family member, is present at both excitatory and inhibitory synapses. Whether and how GluD1 activation may affect inhibitory neurotransmission is unknown. In this work, by using a combination of biochemical, structural, and functional analyses, we demonstrate that GluD1 binds GABA, a previously unknown feature of iGluRs. GluD1 activation produces long-lasting enhancement of GABAergic synaptic currents in the adult mouse hippocampus through a non-ionotropic mechanism that is dependent on trans-synaptic anchoring. The identification of GluD1 as a GABA receptor that controls inhibitory synaptic plasticity challenges the classical dichotomy between glutamatergic and GABAergic receptors.


Assuntos
Inibição Neural , Plasticidade Neuronal , Receptores de GABA , Transmissão Sináptica , Ácido gama-Aminobutírico , Animais , Camundongos , Ácido gama-Aminobutírico/metabolismo , Glutamato Desidrogenase/metabolismo , Hipocampo/metabolismo , Receptores de GABA/metabolismo , Sinapses/fisiologia , Camundongos Knockout , Racemases e Epimerases/genética
12.
Neuron ; 57(1): 80-93, 2008 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-18184566

RESUMO

Ionotropic glutamate receptor (iGluR) subunits contain a large N-terminal domain (NTD) that precedes the agonist-binding domain (ABD) and participates in subunit oligomerization. In NMDA receptors (NMDARs), the NTDs of NR2A and NR2B subunits also form binding sites for the endogenous inhibitor Zn(2+) ion. Although these allosteric sites have been characterized in detail, the molecular mechanisms by which the NTDs communicate with the rest of the receptor to promote its inhibition remain unknown. Here, we identify the ABD dimer interface as a major structural determinant that permits coupling between the NTDs and the channel gate. The strength of this interface also controls proton inhibition, another form of allosteric modulation of NMDARs. Conformational rearrangements at the ABD dimer interface thus appear to be a key mechanism conserved in all iGluR subfamilies, but have evolved to fulfill different functions: fast desensitization at AMPA and kainate receptors, allosteric inhibition at NMDARs.


Assuntos
Estrutura Terciária de Proteína , Receptores de N-Metil-D-Aspartato/química , Regulação Alostérica , Animais , Relação Dose-Resposta a Droga , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Larva , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Microinjeções , Modelos Moleculares , Biologia Molecular/métodos , Mutação , Oócitos , Oxirredução , Técnicas de Patch-Clamp/métodos , Ligação Proteica/efeitos dos fármacos , Prótons , Receptores de N-Metil-D-Aspartato/fisiologia , Xenopus , Zinco/farmacologia
13.
Neuropharmacology ; 193: 108631, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34058193

RESUMO

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate the majority of excitatory neurotransmission in the vertebrate CNS. Classified as AMPA, kainate, delta and NMDA receptors, iGluRs are central drivers of synaptic plasticity widely considered as a major cellular substrate of learning and memory. Surprisingly however, five out of the eighteen vertebrate iGluR subunits do not bind glutamate but glycine, a neurotransmitter known to mediate inhibitory neurotransmission through its action on pentameric glycine receptors (GlyRs). This is the case of GluN1, GluN3A, GluN3B, GluD1 and GluD2 subunits, all also binding the D amino acid d-serine endogenously present in many brain regions. Glycine and d-serine action and affinities broadly differ between glycinergic iGluR subtypes. On 'conventional' GluN1/GluN2 NMDA receptors, glycine (or d-serine) acts in concert with glutamate as a mandatory co-agonist to set the level of receptor activity. It also regulates the receptor's trafficking and expression independently of glutamate. On 'unconventional' GluN1/GluN3 NMDARs, glycine acts as the sole agonist directly triggering opening of excitatory glycinergic channels recently shown to be physiologically relevant. On GluD receptors, d-serine on its own mediates non-ionotropic signaling involved in excitatory and inhibitory synaptogenesis, further reinforcing the concept of glutamate-insensitive iGluRs. Here we present an overview of our current knowledge on glycine and d-serine agonism in iGluRs emphasizing aspects related to molecular mechanisms, cellular function and pharmacological profile. The growing appreciation of the critical influence of glycine and d-serine on iGluR biology reshapes our understanding of iGluR signaling diversity and complexity, with important implications in neuropharmacology.


Assuntos
Glicina/agonistas , Receptores Ionotrópicos de Glutamato/fisiologia , Animais , Sítios de Ligação , Ácido Glutâmico/metabolismo , Humanos , Ligantes , Camundongos , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/metabolismo , Transmissão Sináptica/fisiologia , Xenopus laevis/metabolismo
14.
Nat Commun ; 12(1): 4709, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354080

RESUMO

Allostery represents a fundamental mechanism of biological regulation that involves long-range communication between distant protein sites. It also provides a powerful framework for novel therapeutics. NMDA receptors (NMDARs), glutamate-gated ionotropic receptors that play central roles in synapse maturation and plasticity, are prototypical allosteric machines harboring large extracellular N-terminal domains (NTDs) that provide allosteric control of key receptor properties with impact on cognition and behavior. It is commonly thought that GluN2A and GluN2B receptors, the two predominant NMDAR subtypes in the adult brain, share similar allosteric transitions. Here, combining functional and structural interrogation, we reveal that GluN2A and GluN2B receptors utilize different long-distance allosteric mechanisms involving distinct subunit-subunit interfaces and molecular rearrangements. NMDARs have thus evolved multiple levels of subunit-specific allosteric control over their transmembrane ion channel pore. Our results uncover an unsuspected diversity in NMDAR molecular mechanisms with important implications for receptor physiology and precision drug development.


Assuntos
Receptores de N-Metil-D-Aspartato/metabolismo , Regulação Alostérica , Animais , Feminino , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Fotoquímica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas , Ratos , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevis
15.
Neuron ; 109(15): 2443-2456.e5, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34186027

RESUMO

N-methyl-D-aspartate (NMDA) receptors are glutamate-gated calcium-permeable ion channels that are widely implicated in synaptic transmission and plasticity. Here, we report a gallery of cryo-electron microscopy (cryo-EM) structures of the human GluN1-GluN2A NMDA receptor at an overall resolution of 4 Å in complex with distinct ligands or modulators. In the full-length context of GluN1-GluN2A receptors, we visualize the competitive antagonists bound to the ligand-binding domains (LBDs) of GluN1 and GluN2A subunits, respectively. We reveal that the binding of positive allosteric modulator shortens the distance between LBDs and the transmembrane domain (TMD), which further stretches the opening of the gate. In addition, we unexpectedly visualize the binding cavity of the "foot-in-the-door" blocker 9-aminoacridine within the LBD-TMD linker region, differing from the conventional "trapping" blocker binding site at the vestibule within the TMD. Our study provides molecular insights into the crosstalk between LBDs and TMD during channel activation, inhibition, and allosteric transition.


Assuntos
Modelos Moleculares , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/ultraestrutura , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/ultraestrutura , Regulação Alostérica , Microscopia Crioeletrônica , Humanos , Domínios Proteicos/fisiologia
16.
Nature ; 426(6965): 413-8, 2003 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-14647374

RESUMO

Photosystems I and II (PSI and II) are reaction centres that capture light energy in order to drive oxygenic photosynthesis; however, they can only do so by interacting with the multisubunit cytochrome b(6)f complex. This complex receives electrons from PSII and passes them to PSI, pumping protons across the membrane and powering the Q-cycle. Unlike the mitochondrial and bacterial homologue cytochrome bc(1), cytochrome b(6)f can switch to a cyclic mode of electron transfer around PSI using an unknown pathway. Here we present the X-ray structure at 3.1 A of cytochrome b(6)f from the alga Chlamydomonas reinhardtii. The structure bears similarities to cytochrome bc(1) but also exhibits some unique features, such as binding chlorophyll, beta-carotene and an unexpected haem sharing a quinone site. This haem is atypical as it is covalently bound by one thioether linkage and has no axial amino acid ligand. This haem may be the missing link in oxygenic photosynthesis.


Assuntos
Chlamydomonas reinhardtii/enzimologia , Complexo Citocromos b6f/química , Complexo Citocromos b6f/metabolismo , Heme/metabolismo , Animais , Benzoquinonas/metabolismo , Sítios de Ligação , Clorofila/metabolismo , Cristalografia por Raios X , Dimerização , Ligantes , Metabolismo dos Lipídeos , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , beta Caroteno/metabolismo
17.
Genome Biol ; 20(1): 100, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31113491

RESUMO

BACKGROUND: The functional determinants of H3K4me3, their potential dependency on histone H2B monoubiquitination, and their contribution to defining transcriptional regimes are poorly defined in plant systems. Unlike in Saccharomyces cerevisiae, where a single SET1 protein catalyzes H3K4me3 as part of COMPlex of proteins ASsociated with Set1 (COMPASS), in Arabidopsis thaliana, this activity involves multiple histone methyltransferases. Among these, the plant-specific SET DOMAIN GROUP 2 (SDG2) has a prominent role. RESULTS: We report that SDG2 co-regulates hundreds of genes with SWD2-like b (S2Lb), a plant ortholog of the Swd2 axillary subunit of yeast COMPASS. We show that S2Lb co-purifies with the AtCOMPASS core subunit WDR5, and both S2Lb and SDG2 directly influence H3K4me3 enrichment over highly transcribed genes. S2Lb knockout triggers pleiotropic developmental phenotypes at the vegetative and reproductive stages, including reduced fertility and seed dormancy. However, s2lb seedlings display little transcriptomic defects as compared to the large repertoire of genes targeted by S2Lb, SDG2, or H3K4me3, suggesting that H3K4me3 enrichment is important for optimal gene induction during cellular transitions rather than for determining on/off transcriptional status. Moreover, unlike in budding yeast, most of the S2Lb and H3K4me3 genomic distribution does not rely on a trans-histone crosstalk with histone H2B monoubiquitination. CONCLUSIONS: Collectively, this study unveils that the evolutionarily conserved COMPASS-like complex has been co-opted by the plant-specific SDG2 histone methyltransferase and mediates H3K4me3 deposition through an H2B monoubiquitination-independent pathway in Arabidopsis.


Assuntos
Arabidopsis/metabolismo , Histona Metiltransferases/metabolismo , Histonas/metabolismo , Ubiquitinação
18.
Biochim Biophys Acta ; 1768(6): 1567-73, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17467658

RESUMO

We have used analytical ultracentrifugation to explore the oligomeric states of AcrB and CusA in micellar solution of detergent. These two proteins belong to the resistance, nodulation and cell division (RND) family of efflux proteins that are involved in multiple drug and heavy metal resistance. Only the structure of AcrB has been determined so far. Although functional RND proteins should assemble as trimers as AcrB does, both AcrB and CusA form a mixture of quaternary structures (from monomer to heavy oligomer) in detergent solution. The distribution of the oligomeric states was studied as a function of different parameters: nature and concentration of the detergent, ionic strength, pH, protein concentration. This pseudo-heterogeneity does not hamper the crystallization of AcrB as a homotrimer.


Assuntos
Detergentes/química , Proteínas de Escherichia coli/química , Proteínas de Membrana Transportadoras/química , Micelas , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Cristalização , Concentração de Íons de Hidrogênio , Ligação Proteica , Conformação Proteica , Ultracentrifugação
19.
Curr Opin Physiol ; 2: 1-12, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29682629

RESUMO

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that play crucial roles in brain development and synaptic plasticity. They are also therapeutic targets of interest since their dysfunction is associated with multiple neurological and psychiatric disorders. In vivo, NMDARs exist as multiple subtypes that differ in their subunit composition, anatomical distribution, functional properties, as well as signaling capacities. While much is known about diheteromeric NMDARs composed of two GluN1 subunits and two identical GluN2 (or GluN3) subunits, the majority of native NMDARs are triheteromers containing two GluN1 and two different GluN2 (or a combination of GluN2 and GluN3). Knowledge about triheteromeric NMDARs has recently boomed, with the first decoding of their atomic structure and the development of a new methodology allowing selective expression of recombinant triheteromers at the cell-surface without confounding co-expression of diheteromers. Here we review these progresses and highlight the unique attributes of triheteromers. Particular emphasis is put on GluN1/GluN2A/GluN2B triheteromers, presumably the most abundant NMDARs in the adult forebrain and critical actors of synaptic plasticity. Better understanding triheteromeric NMDAR structure and function is of major interest for brain physiology and drug discovery.

20.
Elife ; 72018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30192741

RESUMO

DE-ETIOLATED 1 (DET1) is an evolutionarily conserved component of the ubiquitination machinery that mediates the destabilization of key regulators of cell differentiation and proliferation in multicellular organisms. In this study, we provide evidence from Arabidopsis that DET1 is essential for the regulation of histone H2B monoubiquitination (H2Bub) over most genes by controlling the stability of a deubiquitination module (DUBm). In contrast with yeast and metazoan DUB modules that are associated with the large SAGA complex, the Arabidopsis DUBm only comprises three proteins (hereafter named SGF11, ENY2 and UBP22) and appears to act independently as a major H2Bub deubiquitinase activity. Our study further unveils that DET1-DDB1-Associated-1 (DDA1) protein interacts with SGF11 in vivo, linking the DET1 complex to light-dependent ubiquitin-mediated proteolytic degradation of the DUBm. Collectively, these findings uncover a signaling path controlling DUBm availability, potentially adjusting H2Bub turnover capacity to the cell transcriptional status.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Histonas/metabolismo , Homeostase , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Proteólise , Ubiquitinação , Sequência de Aminoácidos , Arabidopsis/genética , Genes de Plantas , Peptídeos e Proteínas de Sinalização Intracelular , Luz , Mutação/genética , Fases de Leitura Aberta/genética , Peptídeos/química , Ligação Proteica , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/metabolismo
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