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1.
PLoS One ; 15(9): e0239870, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32991626

RESUMO

The activity-regulated cytoskeleton-associate protein Arc (or Arg3.1) is specifically linked to memory formation and a number of cognitive disorders, including Alzheimer's disease and schizophrenia. Since the discovery of Arc in 1995, extensive research has been conducted on the protein to identify its function and mechanisms of action, with solving the structure of Arc as a major goal. However, the Arc protein tends to self-oligomerize in vitro, and is difficult to crystallize. These properties have hindered efforts to obtain the structure of the full-length, whole protein Arc. As an alternative approach, we and others, have sought to solve the structures of various subdomain proteins of Arc, including the N-lobe, C-lobe, and capsid domain (N-lobe + C-lobe). In this study, we characterized the C-terminal tail of Arc using integrated bioinformatic and structural biology techniques. We compared the sequences of Arc proteins in different mammal species and found that the amino-acid composition in the C-terminal tail region has a significantly higher degree of variation rate than the rest of the protein. Structural prediction programs suggested that the C-terminal tail is structurally disordered. Chemical shift analysis based on solution NMR spectra confirmed that the C-terminal tail has a random coil (disordered) structure, and the tail starts from the residue D357. Furthermore, the NMR spectra showed that the C-terminal tail has minimum (if any) interaction with its neighboring capsid domain in Arc. This study fills gaps in our specific understanding of the structural nature and functional contributions of the Arc C-terminus.


Assuntos
Proteínas do Citoesqueleto/química , Proteínas do Tecido Nervoso/química , Animais , Bovinos , Proteínas do Citoesqueleto/genética , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas do Tecido Nervoso/genética , Primatas , Domínios Proteicos , Roedores , Homologia de Sequência
2.
Science ; 369(6507)2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32855309

RESUMO

Neuronal synapses undergo structural and functional changes throughout life, which are essential for nervous system physiology. However, these changes may also perturb the excitatory-inhibitory neurotransmission balance and trigger neuropsychiatric and neurological disorders. Molecular tools to restore this balance are highly desirable. Here, we designed and characterized CPTX, a synthetic synaptic organizer combining structural elements from cerebellin-1 and neuronal pentraxin-1. CPTX can interact with presynaptic neurexins and postsynaptic AMPA-type ionotropic glutamate receptors and induced the formation of excitatory synapses both in vitro and in vivo. CPTX restored synaptic functions, motor coordination, spatial and contextual memories, and locomotion in mouse models for cerebellar ataxia, Alzheimer's disease, and spinal cord injury, respectively. Thus, CPTX represents a prototype for structure-guided biologics that can efficiently repair or remodel neuronal circuits.


Assuntos
Proteína C-Reativa/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Vias Neurais/efeitos dos fármacos , Precursores de Proteínas/farmacologia , Receptores de AMPA/metabolismo , Proteínas Recombinantes/farmacologia , Sinapses/efeitos dos fármacos , Doença de Alzheimer/terapia , Animais , Proteína C-Reativa/química , Proteína C-Reativa/uso terapêutico , Ataxia Cerebelar/terapia , Modelos Animais de Doenças , Células HEK293 , Hipocampo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/uso terapêutico , Domínios Proteicos , Precursores de Proteínas/química , Precursores de Proteínas/uso terapêutico , Receptores de Glutamato/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapêutico , Coluna Vertebral/efeitos dos fármacos , Coluna Vertebral/fisiologia
3.
Nucleic Acids Res ; 48(18): 10329-10341, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32663306

RESUMO

The recently characterized mammalian writer (methyltransferase) and eraser (demethylase) of the DNA N6-methyladenine (N6mA) methyl mark act on single-stranded (ss) and transiently-unpaired DNA. As YTH domain-containing proteins bind N6mA-containing RNA in mammalian cells, we investigated whether mammalian YTH domains are also methyl mark readers of N6mA DNA. Here, we show that the YTH domain of YTHDC1 (known to localize in the nucleus) binds ssDNA containing N6mA, with a 10 nM dissociation constant. This binding is stronger by a factor of 5 than in an RNA context, tested under the same conditions. However, the YTH domains of YTHDF2 and YTHDF1 (predominantly cytoplasmic) exhibited the opposite effect with ∼1.5-2נstronger binding to ssRNA containing N6mA than to the corresponding DNA. We determined two structures of the YTH domain of YTHDC1 in complex with N6mA-containing ssDNA, which illustrated that YTHDC1 binds the methylated adenine in a single-stranded region flanked by duplexed DNA. We discuss the hypothesis that the writer-reader-eraser of N6mA-containining ssDNA is associated with maintaining genome stability. Structural comparison of YTH and SRA domains (the latter a DNA 5-methylcytosine reader) revealed them to be diverse members of a larger family of DNA/RNA modification readers, apparently having originated from bacterial modification-dependent restriction enzymes.


Assuntos
Adenina/química , Complexos Multiproteicos/química , Proteínas do Tecido Nervoso/química , Conformação Proteica , Fatores de Processamento de RNA/química , DNA/química , DNA/genética , DNA/ultraestrutura , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Histona Desmetilases/genética , Humanos , Metilação , Metiltransferases/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/ultraestrutura , Domínios Proteicos/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/ultraestrutura , Proteínas de Ligação a RNA/genética
4.
Nature ; 584(7822): 646-651, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32494015

RESUMO

Pannexin 1 (PANX1) is an ATP-permeable channel with critical roles in a variety of physiological functions such as blood pressure regulation1, apoptotic cell clearance2 and human oocyte development3. Here we present several structures of human PANX1 in a heptameric assembly at resolutions of up to 2.8 angström, including an apo state, a caspase-7-cleaved state and a carbenoxolone-bound state. We reveal a gating mechanism that involves two ion-conducting pathways. Under normal cellular conditions, the intracellular entry of the wide main pore is physically plugged by the C-terminal tail. Small anions are conducted through narrow tunnels in the intracellular domain. These tunnels connect to the main pore and are gated by a long linker between the N-terminal helix and the first transmembrane helix. During apoptosis, the C-terminal tail is cleaved by caspase, allowing the release of ATP through the main pore. We identified a carbenoxolone-binding site embraced by W74 in the extracellular entrance and a role for carbenoxolone as a channel blocker. We identified a gap-junction-like structure using a glycosylation-deficient mutant, N255A. Our studies provide a solid foundation for understanding the molecular mechanisms underlying the channel gating and inhibition of PANX1 and related large-pore channels.


Assuntos
Conexinas/química , Conexinas/metabolismo , Microscopia Crioeletrônica , Ativação do Canal Iônico , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Patch-Clamp , Trifosfato de Adenosina/metabolismo , Animais , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Apoptose , Sítios de Ligação/efeitos dos fármacos , Carbenoxolona/química , Carbenoxolona/metabolismo , Carbenoxolona/farmacologia , Caspase 7/metabolismo , Linhagem Celular , Conexinas/ultraestrutura , Junções Comunicantes , Glicosilação , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Modelos Moleculares , Mutação , Proteínas do Tecido Nervoso/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Células Sf9
5.
Hum Genet ; 139(12): 1513-1529, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32529326

RESUMO

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder caused by the loss of function of a set of imprinted genes on chromosome 15q11-15q13. One of these genes, NDN, encodes necdin, a protein that is important for neuronal differentiation and survival. Loss of Ndn in mice causes defects in the formation and function of the nervous system. Necdin is a member of the melanoma-associated antigen gene (MAGE) protein family. The functions of MAGE proteins depend highly on their interactions with other proteins, and in particular MAGE proteins interact with E3 ubiquitin ligases and deubiquitinases to form MAGE-RING E3 ligase-deubiquitinase complexes. Here, we used proximity-dependent biotin identification (BioID) and mass spectrometry (MS) to determine the network of protein-protein interactions (interactome) of the necdin protein. This process yielded novel as well as known necdin-proximate proteins that cluster into a protein network. Next, we used BioID-MS to define the interactomes of necdin proteins carrying coding variants. Variant necdin proteins had interactomes that were distinct from wildtype necdin. BioID-MS is not only a useful tool to identify protein-protein interactions, but also to analyze the effects of variants of unknown significance on the interactomes of proteins involved in genetic disease.


Assuntos
Substituição de Aminoácidos/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Mapas de Interação de Proteínas/genética , Ubiquitina-Proteína Ligases/genética , Animais , Biotinilação/genética , Diferenciação Celular/genética , Enzimas Desubiquitinantes/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Espectrometria de Massas/métodos , Camundongos , Mutação/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/ultraestrutura , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/patologia , Neurônios/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/ultraestrutura , Proteínas de Ligação a Poli(A)/química , Proteínas de Ligação a Poli(A)/genética , Síndrome de Prader-Willi/genética , Conformação Proteica , Relação Estrutura-Atividade , Ubiquitina-Proteína Ligases/química
6.
Proc Natl Acad Sci U S A ; 117(25): 14178-14186, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513706

RESUMO

The interaction of the neuronal protein α-synuclein with lipid membranes appears crucial in the context of Parkinson's disease, but the underlying mechanistic details, including the roles of different lipids in pathogenic protein aggregation and membrane disruption, remain elusive. Here, we used single-vesicle resolution fluorescence and label-free scattering microscopy to investigate the interaction kinetics of monomeric α-synuclein with surface-tethered vesicles composed of different negatively charged lipids. Supported by a theoretical model to account for structural changes in scattering properties of surface-tethered lipid vesicles, the data demonstrate stepwise vesicle disruption and asymmetric membrane deformation upon α-synuclein binding to phosphatidylglycerol vesicles at protein concentrations down to 10 nM (∼100 proteins per vesicle). In contrast, phosphatidylserine vesicles were only marginally affected. These insights into structural consequences of α-synuclein interaction with lipid vesicles highlight the contrasting roles of different anionic lipids, which may be of mechanistic relevance for both normal protein function (e.g., synaptic vesicle binding) and dysfunction (e.g., mitochondrial membrane interaction).


Assuntos
Lipídeos de Membrana/metabolismo , Membranas/metabolismo , alfa-Sinucleína/metabolismo , Fluoresceínas , Humanos , Cinética , Bicamadas Lipídicas/química , Proteínas do Tecido Nervoso/química , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Fosfatidilgliceróis/química , Ligação Proteica , alfa-Sinucleína/genética
7.
Nature ; 582(7812): 443-447, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32499642

RESUMO

TWIK-related acid-sensitive potassium (TASK) channels-members of the two pore domain potassium (K2P) channel family-are found in neurons1, cardiomyocytes2-4 and vascular smooth muscle cells5, where they are involved in the regulation of heart rate6, pulmonary artery tone5,7, sleep/wake cycles8 and responses to volatile anaesthetics8-11. K2P channels regulate the resting membrane potential, providing background K+ currents controlled by numerous physiological stimuli12-15. Unlike other K2P channels, TASK channels are able to bind inhibitors with high affinity, exceptional selectivity and very slow compound washout rates. As such, these channels are attractive drug targets, and TASK-1 inhibitors are currently in clinical trials for obstructive sleep apnoea and atrial fibrillation16. In general, potassium channels have an intramembrane vestibule with a selectivity filter situated above and a gate with four parallel helices located below; however, the K2P channels studied so far all lack a lower gate. Here we present the X-ray crystal structure of TASK-1, and show that it contains a lower gate-which we designate as an 'X-gate'-created by interaction of the two crossed C-terminal M4 transmembrane helices at the vestibule entrance. This structure is formed by six residues (243VLRFMT248) that are essential for responses to volatile anaesthetics10, neurotransmitters13 and G-protein-coupled receptors13. Mutations within the X-gate and the surrounding regions markedly affect both the channel-open probability and the activation of the channel by anaesthetics. Structures of TASK-1 bound to two high-affinity inhibitors show that both compounds bind below the selectivity filter and are trapped in the vestibule by the X-gate, which explains their exceptionally low washout rates. The presence of the X-gate in TASK channels explains many aspects of their physiological and pharmacological behaviour, which will be beneficial for the future development and optimization of TASK modulators for the treatment of heart, lung and sleep disorders.


Assuntos
Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/química , Anestésicos/farmacologia , Animais , Cristalografia por Raios X , Condutividade Elétrica , Feminino , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Modelos Moleculares , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Xenopus laevis
8.
Nat Commun ; 11(1): 2140, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358586

RESUMO

The trans-synaptic interaction of the cell-adhesion molecules teneurins (TENs) with latrophilins (LPHNs/ADGRLs) promotes excitatory synapse formation when LPHNs simultaneously interact with FLRTs. Insertion of a short alternatively-spliced region within TENs abolishes the TEN-LPHN interaction and switches TEN function to specify inhibitory synapses. How alternative-splicing regulates TEN-LPHN interaction remains unclear. Here, we report the 2.9 Å resolution cryo-EM structure of the TEN2-LPHN3 complex, and describe the trimeric TEN2-LPHN3-FLRT3 complex. The structure reveals that the N-terminal lectin domain of LPHN3 binds to the TEN2 barrel at a site far away from the alternatively spliced region. Alternative-splicing regulates the TEN2-LPHN3 interaction by hindering access to the LPHN-binding surface rather than altering it. Strikingly, mutagenesis of the LPHN-binding surface of TEN2 abolishes the LPHN3 interaction and impairs excitatory but not inhibitory synapse formation. These results suggest that a multi-level coincident binding mechanism mediated by a cryptic adhesion complex between TENs and LPHNs regulates synapse specificity.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Sinapses/metabolismo , Processamento Alternativo/genética , Processamento Alternativo/fisiologia , Sítios de Ligação/genética , Células HEK293 , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutação/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Ligação Proteica/genética , Estrutura Secundária de Proteína , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/genética , Receptores de Peptídeos/química , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Sinapses/fisiologia
9.
Proc Natl Acad Sci U S A ; 117(23): 12826-12835, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32461371

RESUMO

Complete cancer regression occurs in a subset of patients following adoptive T cell therapy (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TILs). However, the low success rate presents a great challenge to broader clinical application. To provide insight into TIL-based immunotherapy, we studied a successful case of ACT where regression was observed against tumors carrying the hotspot mutation G12D in the KRAS oncogene. Four T cell receptors (TCRs) made up the TIL infusion and recognized two KRAS-G12D neoantigens, a nonamer and a decamer, all restricted by human leukocyte antigen (HLA) C*08:02. Three of them (TCR9a, 9b, and 9c) were nonamer-specific, while one was decamer-specific (TCR10). We show that only mutant G12D but not the wild-type peptides stabilized HLA-C*08:02 due to the formation of a critical anchor salt bridge to HLA-C. Therapeutic TCRs exhibited high affinities, ranging from nanomolar to low micromolar. Intriguingly, TCR binding affinities to HLA-C inversely correlated with their persistence in vivo, suggesting the importance of antigenic affinity in the function of therapeutic T cells. Crystal structures of TCR-HLA-C complexes revealed that TCR9a to 9c recognized G12D nonamer with multiple conserved contacts through shared CDR2ß and CDR3α. This allowed CDR3ß variation to confer different affinities via a variable HLA-C contact, generating an oligoclonal response. TCR10 recognized an induced and distinct G12D decamer conformation. Thus, this successful case of ACT included oligoclonal TCRs of high affinity recognizing distinct conformations of neoantigens. Our study revealed the potential of a structural approach to inform clinical efforts in targeting KRAS-G12D tumors by immunotherapy and has general implications for T cell-based immunotherapies.


Assuntos
Antígenos de Neoplasias/imunologia , Imunoterapia Adotiva/métodos , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Apresentação do Antígeno , Antígenos de Neoplasias/química , Sítios de Ligação , Antígenos HLA-C/química , Antígenos HLA-C/imunologia , Humanos , Células Jurkat , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/imunologia , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Receptores de Antígenos de Linfócitos T/química
10.
Am J Hum Genet ; 106(6): 859-871, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32470375

RESUMO

Congenital cone-rod synaptic disorder (CRSD), also known as incomplete congenital stationary night blindness (iCSNB), is a non-progressive inherited retinal disease (IRD) characterized by night blindness, photophobia, and nystagmus, and distinctive electroretinographic features. Here, we report bi-allelic RIMS2 variants in seven CRSD-affected individuals from four unrelated families. Apart from CRSD, neurodevelopmental disease was observed in all affected individuals, and abnormal glucose homeostasis was observed in the eldest affected individual. RIMS2 regulates synaptic membrane exocytosis. Data mining of human adult bulk and single-cell retinal transcriptional datasets revealed predominant expression in rod photoreceptors, and immunostaining demonstrated RIMS2 localization in the human retinal outer plexiform layer, Purkinje cells, and pancreatic islets. Additionally, nonsense variants were shown to result in truncated RIMS2 and decreased insulin secretion in mammalian cells. The identification of a syndromic stationary congenital IRD has a major impact on the differential diagnosis of syndromic congenital IRD, which has previously been exclusively linked with degenerative IRD.


Assuntos
Oftalmopatias Hereditárias/genética , Proteínas de Ligação ao GTP/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Mutação com Perda de Função , Miopia/genética , Proteínas do Tecido Nervoso/genética , Cegueira Noturna/genética , Adulto , Alelos , Processamento Alternativo , Encéfalo/metabolismo , Linhagem Celular , Criança , Pré-Escolar , Diagnóstico Diferencial , Saúde da Família , Feminino , França , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Glucose/metabolismo , Humanos , Secreção de Insulina , Masculino , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Pâncreas/metabolismo , Linhagem , Retina/metabolismo , Arábia Saudita , Senegal
11.
Nat Struct Mol Biol ; 27(4): 373-381, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32231289

RESUMO

The plasma membrane adenosine triphosphate (ATP) release channel pannexin 1 (PANX1) has been implicated in many physiological and pathophysiological processes associated with purinergic signaling, including cancer progression, apoptotic cell clearance, inflammation, blood pressure regulation, oocyte development, epilepsy and neuropathic pain. Here we present near-atomic-resolution structures of human and frog PANX1 determined by cryo-electron microscopy that revealed a heptameric channel architecture. Compatible with ATP permeation, the transmembrane pore and cytoplasmic vestibule were exceptionally wide. An extracellular tryptophan ring located at the outer pore created a constriction site, potentially functioning as a molecular sieve that restricts the size of permeable substrates. The amino and carboxyl termini, not resolved in the density map, appeared to be structurally dynamic and might contribute to narrowing of the pore during channel gating. In combination with functional characterization, this work elucidates the previously unknown architecture of pannexin channels and establishes a foundation for understanding their unique channel properties.


Assuntos
Trifosfato de Adenosina/química , Membrana Celular/ultraestrutura , Conexinas/ultraestrutura , Microscopia Crioeletrônica , Proteínas do Tecido Nervoso/ultraestrutura , Trifosfato de Adenosina/genética , Animais , Anuros/genética , Membrana Celular/química , Membrana Celular/genética , Conexinas/química , Conexinas/genética , Humanos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Conformação Proteica , Transdução de Sinais/genética
12.
Nat Commun ; 11(1): 1506, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198400

RESUMO

Sorting nexins (SNX) are a family of PX domain-containing proteins with pivotal roles in trafficking and signaling. SNX-BARs, which also have a curvature-generating Bin/Amphiphysin/Rvs (BAR) domain, have membrane-remodeling functions, particularly at the endosome. The minimal PX-BAR module is a dimer mediated by BAR-BAR interactions. Many SNX-BAR proteins, however, additionally have low-complexity N-terminal regions of unknown function. Here, we present the cryo-EM structure of the full-length SNX-BAR Mvp1, which is an autoinhibited tetramer. The tetramer is a dimer of dimers, wherein the membrane-interacting BAR surfaces are sequestered and the PX lipid-binding sites are occluded. The N-terminal low-complexity region of Mvp1 is essential for tetramerization. Mvp1 lacking its N-terminus is dimeric and exhibits enhanced membrane association. Membrane binding and remodeling by Mvp1 therefore requires unmasking of the PX and BAR domain lipid-interacting surfaces. This work reveals a tetrameric configuration of a SNX-BAR protein that provides critical insight into SNX-BAR function and regulation.


Assuntos
Microscopia Crioeletrônica/métodos , Prolapso da Valva Mitral/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nexinas de Classificação/metabolismo , Sítios de Ligação , Biofísica , Membrana Celular/metabolismo , Endossomos/metabolismo , Humanos , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Domínios Proteicos/genética , Transporte Proteico , Saccharomyces cerevisiae/genética
13.
Nat Commun ; 11(1): 1489, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198364

RESUMO

Axon pathfinding is critical for nervous system development, and it is orchestrated by molecular cues that activate receptors on the axonal growth cone. Robo family receptors bind Slit guidance cues to mediate axon repulsion. In mammals, the divergent family member Robo3 does not bind Slits, but instead signals axon repulsion from its own ligand, NELL2. Conversely, canonical Robos do not mediate NELL2 signaling. Here, we present the structures of NELL-Robo3 complexes, identifying a mode of ligand engagement for Robos that is orthogonal to Slit binding. We elucidate the structural basis for differential binding between NELL and Robo family members and show that NELL2 repulsive activity is a function of its Robo3 affinity and is enhanced by ligand trimerization. Our results reveal a mechanism of oligomerization-induced Robo activation for axon guidance and shed light on Robo family member ligand binding specificity, conformational variability, divergent modes of signaling, and evolution.


Assuntos
Orientação de Axônios/fisiologia , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Animais , Axônios/metabolismo , Células COS , Chlorocebus aethiops , Cristalografia por Raios X , Drosophila , Proteínas de Drosophila/metabolismo , Mamíferos , Camundongos , Modelos Moleculares , Proteínas do Tecido Nervoso/genética , Receptores de Superfície Celular/genética , Espalhamento de Radiação , Transdução de Sinais
14.
ScientificWorldJournal ; 2020: 7910702, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32148468

RESUMO

Dye-Sensitized Solar Cell (DSSC) constitutes a solar cell using natural dyes from plants that are adsorbed in semiconductors to convert solar energy into electrical energy. DSSC has relatively inexpensive fabrication costs, is easy to produce, works in visible light, and is environmentally friendly. The disadvantage of DSSC is that its efficiency is still low compared to silicon solar cells. This low efficiency is due to obstacles in the flow of electric current on DSSC. In this study, DSSC has been successfully fabricated with the deposition of clathrin protein from cow brain. The zwitterions effect of protein on cow brain is able to reduce resistance and increase electric current on DSSC. The zwitterions effect of cow brain protein that fills gaps or empty spaces between TiO2 particles generates acidic reactions (capturing electrons) and bases (releasing electrons); hence, proteins in the cow brain are able to function as electron bridges between TiO2 molecules and generate an increase in electric current in DSSC. The method used in this research was to deposit clathrin protein from cow brain in a porous TiO2 semiconductor with a concentration of 0%, 25%, 50%, and 75%. Tests carried out on DSSC that have been performed were X-Ray Diffractometer (XRD) testing to determine the crystal structure formed, Fourier Transform Infrared Spectroscopy (FTIR) testing to determine the functional groups formed on DSSC, Scanning Electron Microscopy (SEM) testing to determine the surface morphological characteristics of the DSSC layer, and testing the efficiency using AM 1.5 G solar simulator (1000 W/m2) to determine the efficiency changes that occur in DSSC. From the XRD test results by increasing the concentration of cow brain protein in DSSC, the structure of amino acid crystals also increased and the crystal size increased with the largest crystal size of 42.25 nm at the addition of 75% of cow brain protein. FTIR test results show that the addition of cow brain protein will form functional protein-forming amino groups on DSSC. FTIR analysis shows the sharp absorption of energy by protein functional groups in the FTIR spectrum with increasing concentration of cow brain protein in DSSC. The SEM test results show that the concentration of additional molecules of protein deposited into TiO2 increases and the cavity or pore between the TiO2 molecules decreases. The reduction of cavities in the layers indicates that protein molecules fill cavities that exist between TiO2 molecules. From the results of testing using AM 1.5 G solar simulator (1000 W/m2), the highest efficiency value is 1.465% with the addition of 75% brain protein concentration.


Assuntos
Encéfalo , Corantes , Proteínas do Tecido Nervoso/farmacologia , Compostos de Amônio Quaternário/farmacologia , Energia Solar , Algoritmos , Animais , Encéfalo/metabolismo , Bovinos , Corantes/metabolismo , Fontes de Energia Elétrica , Modelos Teóricos , Proteínas do Tecido Nervoso/química , Compostos de Amônio Quaternário/química , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X
15.
Biochemistry ; 59(13): 1367-1377, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32207963

RESUMO

More than 80 loss-of-function (LOF) mutations in the SLC6A8 creatine transporter (hCRT1) are responsible for cerebral creatine deficiency syndrome (CCDS), which gives rise to a spectrum of neurological defects, including intellectual disability, epilepsy, and autism spectrum disorder. To gain insight into the nature of the molecular defects caused by these mutations, we quantitatively profiled the cellular processing, trafficking, expression, and function of eight pathogenic CCDS variants in relation to the wild type (WT) and one neutral isoform. All eight CCDS variants exhibit measurable proteostatic deficiencies that likely contribute to the observed LOF. However, the magnitudes of their specific effects on the expression and trafficking of hCRT1 vary considerably, and we find that the LOF associated with two of these variants primarily arises from the disruption of the substrate-binding pocket. In conjunction with an analysis of structural models of the transporter, we use these data to suggest mechanistic classifications for these variants. To evaluate potential avenues for therapeutic intervention, we assessed the sensitivity of these variants to temperature and measured their response to the proteostasis regulator 4-phenylbutyrate (4-PBA). Only one of the tested variants (G132V) is sensitive to temperature, though its response to 4-PBA is negligible. Nevertheless, 4-PBA significantly enhances the activity of WT hCRT1 in HEK293T cells, which suggests it may be worth evaluating as a therapeutic for female intellectual disability patients carrying a single CCDS mutation. Together, these findings reveal that pathogenic SLC6A8 mutations cause a spectrum of molecular defects that should be taken into consideration in future efforts to develop CCDS therapeutics.


Assuntos
Encefalopatias Metabólicas Congênitas/metabolismo , Creatina/deficiência , Retardo Mental Ligado ao Cromossomo X/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/deficiência , Encefalopatias Metabólicas Congênitas/genética , Creatina/genética , Creatina/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Retardo Mental Ligado ao Cromossomo X/genética , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/química , Fenilbutiratos/metabolismo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/química , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo
16.
Phytomedicine ; 68: 153149, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32032836

RESUMO

BACKGROUND: Mast cells (MCs) are crucial effectors in allergic disorders by secreting inflammatory mediators. The Mas-related G-protein-coupled receptor X2 (Mrgprx2) was shown to have a key role in IgE-independent allergic reactions. Therefore, potential drug candidates that directly target Mrgprx2 could be used to treat pseudo-allergic diseases. Shikonin, an active ingredient derived from Lithospermum erythrorhizon Sieb. et Zucc has been used for its anti-inflammatory properties since ancient China. PURPOSE: To investigate the inhibitory effects of Shikonin on IgE-independent allergy both in vitro and in vivo, as well as the mechanism underlying its effects. METHODS/STUDY DESIGNS: The anti-anaphylactoid activity of Shikonin was evaluated in PCA and systemic anaphylaxis models, Calcium imaging was used to assess intracellular Ca2+ mobilization. The release of cytokines and chemokines was measured using enzyme immunoassay kits. Western blot analysis was conducted to investigate the molecules of PLCγ-PKC-IP3 signaling pathway. The analytical method of surface plasmon resonance was employed to study the interaction between Shikonin and potential target protein Mrgprx2. RESULTS: Shikonin can suppress compound 48/80 (C48/80)-induced PCA, active systemic anaphylaxis, and MCs degranulation in mice in a dose-dependent manner. In addition, Shikonin reduced C48/80-induced calcium flux and suppressed LAD2 cell degranulation via PLCγ-PKC-IP3 signaling pathway. Moreover, Shikonin was found to inhibit C48/80-induced Mrgprx2 expression in HEK cells, displaying specific interactions with the Mrgprx2 protein. CONCLUSION: Shikonin could be a potential antagonist of Mrgprx2, thereby inhibiting pseudo-allergic reactions through Ca2+ mobilization.


Assuntos
Anafilaxia/tratamento farmacológico , Hipersensibilidade/tratamento farmacológico , Naftoquinonas/farmacologia , Proteínas do Tecido Nervoso/imunologia , Receptores Acoplados a Proteínas-G/imunologia , Receptores de Neuropeptídeos/imunologia , Anafilaxia/induzido quimicamente , Animais , Cálcio/metabolismo , Degranulação Celular/efeitos dos fármacos , Linhagem Celular , Quimiocinas/metabolismo , Citocinas/metabolismo , Humanos , Hipersensibilidade/imunologia , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos Endogâmicos C57BL , Naftoquinonas/química , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Fosfolipase C gama/metabolismo , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Neuropeptídeos/antagonistas & inibidores , Receptores de Neuropeptídeos/química , Receptores de Neuropeptídeos/metabolismo , Secretagogos/toxicidade , p-Metoxi-N-metilfenetilamina/toxicidade
17.
Proc Natl Acad Sci U S A ; 117(8): 4199-4210, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32029586

RESUMO

The transcription factor Sox10 is a key regulator in the fate determination of a subpopulation of multipotent trunk neural crest (NC) progenitors toward glial cells instead of sensory neurons in the dorsal root ganglia (DRG). However, the mechanism by which Sox10 regulates glial cell fate commitment during lineage segregation remains poorly understood. In our study, we showed that the neurogenic determinant Neurogenin 2 (Neurog2) exhibited transient overlapping expression with Sox10 in avian trunk NC progenitors, which progressively underwent lineage segregation during migration toward the forming DRG. Gain- and loss-of-function studies revealed that the temporary expression of Neurog2 was due to Sox10 regulation of its protein stability. Transcriptional profiling identified Sox10-regulated F-box only protein (Fbxo9), which is an SCF (Skp1-Cul-F-box)-type ubiquitin ligase for Neurog2. Consistently, overexpression of Fbxo9 in NC progenitors down-regulated Neurog2 protein expression through ubiquitination and promoted the glial lineage at the expense of neuronal differentiation, whereas Fbxo9 knockdown resulted in the opposite phenomenon. Mechanistically, we found that Fbxo9 interacted with Neurog2 to promote its destabilization through the F-box motif. Finally, epistasis analysis further demonstrated that Fbxo9 and probably other F-box members mediated the role of Sox10 in destabilizing Neurog2 protein and directing the lineage of NC progenitors toward glial cells rather than sensory neurons. Altogether, these findings unravel a Sox10-Fbxo9 regulatory axis in promoting the glial fate of NC progenitors through Neurog2 destabilization.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas F-Box/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fatores de Transcrição SOXE/metabolismo , Raízes Nervosas Espinhais/metabolismo , Motivos de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Embrião de Galinha , Proteínas F-Box/química , Proteínas F-Box/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Crista Neural/citologia , Crista Neural/metabolismo , Neurogênese , Ligação Proteica , Estabilidade Proteica , Raízes Nervosas Espinhais/citologia
18.
Proc Natl Acad Sci U S A ; 117(2): 1036-1041, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31888993

RESUMO

Munc13-1 is a large multifunctional protein essential for synaptic vesicle fusion and neurotransmitter release. Its dysfunction has been linked to many neurological disorders. Evidence suggests that the MUN domain of Munc13-1 collaborates with Munc18-1 to initiate SNARE assembly, thereby priming vesicles for fast calcium-triggered vesicle fusion. The underlying molecular mechanism, however, is poorly understood. Recently, it was found that Munc18-1 catalyzes neuronal SNARE assembly through an obligate template complex intermediate containing Munc18-1 and 2 SNARE proteins-syntaxin 1 and VAMP2. Here, using single-molecule force spectroscopy, we discovered that the MUN domain of Munc13-1 stabilizes the template complex by ∼2.1 kBT. The MUN-bound template complex enhances SNAP-25 binding to the templated SNAREs and subsequent full SNARE assembly. Mutational studies suggest that the MUN-bound template complex is functionally important for SNARE assembly and neurotransmitter release. Taken together, our observations provide a potential molecular mechanism by which Munc13-1 and Munc18-1 cooperatively chaperone SNARE folding and assembly, thereby regulating synaptic vesicle fusion.


Assuntos
Chaperonas Moleculares/metabolismo , Proteínas Munc18/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas SNARE/metabolismo , Exocitose/fisiologia , Cinética , Fusão de Membrana/fisiologia , Chaperonas Moleculares/química , Proteínas Munc18/química , Proteínas do Tecido Nervoso/química , Neurônios/metabolismo , Pinças Ópticas , Ligação Proteica , Domínios Proteicos , Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/química , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Proteína 25 Associada a Sinaptossoma/química , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo
19.
Proc Natl Acad Sci U S A ; 117(6): 3214-3219, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31974314

RESUMO

Which neural circuits undergo synaptic changes when an animal learns? Although it is widely accepted that changes in synaptic strength underlie many forms of learning and memory, it remains challenging to connect changes in synaptic strength at specific neural pathways to specific behaviors and memories. Here we introduce SYNPLA (synaptic proximity ligation assay), a synapse-specific, high-throughput, and potentially brain-wide method capable of detecting circuit-specific learning-induced synaptic plasticity.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Aprendizagem/fisiologia , Plasticidade Neuronal/fisiologia , Mapeamento de Interação de Proteínas/métodos , Sinapses , Animais , Córtex Auditivo/química , Córtex Auditivo/citologia , Córtex Auditivo/metabolismo , Células Cultivadas , Condicionamento Psicológico/fisiologia , Corpos Geniculados/química , Corpos Geniculados/citologia , Corpos Geniculados/metabolismo , Hipocampo/química , Hipocampo/citologia , Hipocampo/metabolismo , Camundongos , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Ratos , Sinapses/química , Sinapses/metabolismo
20.
Nat Commun ; 11(1): 277, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937754

RESUMO

Migration of meiosis-I (MI) spindle from the cell center to a sub-cortical location is a critical step for mouse oocytes to undergo asymmetric meiotic cell division. In this study, we investigate the mechanism by which formin-2 (FMN2) orchestrates the initial movement of MI spindle. By defining protein domains responsible for targeting FMN2, we show that spindle-periphery localized FMN2 is required for spindle migration. The spindle-peripheral FMN2 nucleates short actin bundles from vesicles derived likely from the endoplasmic reticulum (ER) and concentrated in a layer outside the spindle. This layer is in turn surrounded by mitochondria. A model based on polymerizing actin filaments pushing against mitochondria, thus generating a counter force on the spindle, demonstrated an inherent ability of this system to break symmetry and evolve directional spindle motion. The model is further supported through experiments involving spatially biasing actin nucleation via optogenetics and disruption of mitochondrial distribution and dynamics.


Assuntos
Actinas/metabolismo , Meiose , Oócitos/citologia , Organelas/metabolismo , Fuso Acromático/metabolismo , Animais , Divisão Celular Assimétrica , Vesículas Citoplasmáticas/metabolismo , Retículo Endoplasmático/metabolismo , Feminino , Forminas/química , Forminas/genética , Forminas/metabolismo , Camundongos , Mitocôndrias/metabolismo , Modelos Biológicos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Oócitos/metabolismo , Domínios Proteicos , Deleção de Sequência
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