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1.
Cells ; 10(12)2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34943900

RESUMO

Glycine is an important neurotransmitter in vertebrates, performing both excitatory and inhibitory actions. Synaptic levels of glycine are tightly controlled by the action of two glycine transporters, GlyT1 and GlyT2, located on the surface of glial cells and neurons, respectively. Only limited information is available on glycinergic neurotransmission in invertebrates, and the evolution of glycinergic neurotransmission is poorly understood. Here, by combining phylogenetic and gene expression analyses, we characterized the glycine transporter complement of amphioxus, an important invertebrate model for studying the evolution of chordates. We show that amphioxus possess three glycine transporter genes. Two of these (GlyT2.1 and GlyT2.2) are closely related to GlyT2 of vertebrates, whereas the third (GlyT) is a member of an ancestral clade of deuterostome glycine transporters. GlyT2.2 expression is predominantly non-neural, whereas GlyT and GlyT2.1 are widely expressed in the amphioxus nervous system and are differentially expressed, respectively, in neurons and glia. Vertebrate glycinergic neurons express GlyT2 and glia GlyT1, suggesting that the evolution of the chordate glycinergic system was accompanied by a paralog-specific inversion of gene expression. Despite this genetic divergence between amphioxus and vertebrates, we found strong evidence for conservation in the role glycinergic neurotransmission plays during larval swimming, the implication being that the neural networks controlling the rhythmic movement of chordate bodies may be homologous.


Assuntos
Evolução Molecular , Proteínas da Membrana Plasmática de Transporte de Glicina/genética , Glicina/genética , Transmissão Sináptica/genética , Animais , Cordados/genética , Cordados/crescimento & desenvolvimento , Regulação da Expressão Gênica/genética , Variação Genética/genética , Glicina/metabolismo , Anfioxos/genética , Larva/genética , Larva/crescimento & desenvolvimento , Neuroglia/metabolismo , Neurônios/metabolismo , Filogenia
2.
Nat Commun ; 12(1): 6176, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34702840

RESUMO

Serine is a non-essential amino acid that is critical for tumour proliferation and depletion of circulating serine results in reduced tumour growth and increased survival in various cancer models. While many cancer cells cultured in a standard tissue culture medium depend on exogenous serine for optimal growth, here we report that these cells are less sensitive to serine/glycine depletion in medium containing physiological levels of metabolites. The lower requirement for exogenous serine under these culture conditions reflects both increased de novo serine synthesis and the use of hypoxanthine (not present in the standard medium) to support purine synthesis. Limiting serine availability leads to increased uptake of extracellular hypoxanthine, sparing available serine for other pathways such as glutathione synthesis. Taken together these results improve our understanding of serine metabolism in physiologically relevant nutrient conditions and allow us to predict interventions that may enhance the therapeutic response to dietary serine/glycine limitation.


Assuntos
Neoplasias/metabolismo , Serina/metabolismo , Vias Biossintéticas , Linhagem Celular Tumoral , Proliferação de Células , Meios de Cultura/química , Meios de Cultura/metabolismo , Glicina/análise , Glicina/metabolismo , Humanos , Hipoxantina/análise , Hipoxantina/metabolismo , Neoplasias/dietoterapia , Neoplasias/patologia , Purinas/biossíntese , Serina/análise , Regulação para Cima
3.
Appl Environ Microbiol ; 87(20): e0133521, 2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34347519

RESUMO

ß-Hydroxy-α-amino acids are useful compounds for pharmaceutical development. Enzymatic synthesis of ß-hydroxy-α-amino acids has attracted considerable interest as a selective, sustainable, and environmentally benign process. In this study, we identified a novel amino acid hydroxylase, AEP14369, from Sulfobacillus thermotolerans Y0017, which is included in a previously constructed CAS-like superfamily protein library, to widen the variety of amino acid hydroxylases. The detailed structures determined by nuclear magnetic resonance and X-ray crystallography analysis of the enzymatically produced compounds revealed that AEP14369 catalyzed threo-ß-selective hydroxylation of l-His and l-Gln in a 2-oxoglutarate-dependent manner. Furthermore, the production of l-threo-ß-hydroxy-His and l-threo-ß-hydroxy-Gln was achieved using Escherichia coli expressing the gene encoding AEP14369 as a whole-cell biocatalyst. Under optimized reaction conditions, 137 mM (23.4 g liter-1) l-threo-ß-hydroxy-His and 150 mM l-threo-ß-hydroxy-Gln (24.3 g liter-1) were obtained, indicating that the enzyme is applicable for preparative-scale production. AEP14369, an l-His/l-Gln threo-ß-hydroxylase, increases the availability of 2-oxoglutarate-dependent hydroxylase and opens the way for the practical production of ß-hydroxy-α-amino acids in the future. The amino acids produced in this study would also contribute to the structural diversification of pharmaceuticals that affect important bioactivities. IMPORTANCE Owing to an increasing concern for sustainability, enzymatic approaches for producing industrially useful compounds have attracted considerable attention as a powerful complement to chemical synthesis for environment-friendly synthesis. In this study, we developed a bioproduction method for ß-hydroxy-α-amino acid synthesis using a newly discovered enzyme. AEP14369 from the moderate thermophilic bacterium Sulfobacillus thermotolerans Y0017 catalyzed the hydroxylation of l-His and l-Gln in a regioselective and stereoselective fashion. Furthermore, we biotechnologically synthesized both l-threo-ß-hydroxy-His and l-threo-ß-hydroxy-Gln with a titer of over 20 g liter-1 through whole-cell bioconversion using recombinant Escherichia coli cells. As ß-hydroxy-α-amino acids are important compounds for pharmaceutical development, this achievement would facilitate future sustainable and economical industrial applications.


Assuntos
Proteínas de Bactérias/metabolismo , Clostridiales/enzimologia , Glicina/metabolismo , Histidina/metabolismo , Ácidos Cetoglutáricos/metabolismo , Oxigenases de Função Mista/metabolismo , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Glicina/análogos & derivados , Histidina/análogos & derivados , Hidroxilação , Oxigenases de Função Mista/genética
4.
Food Microbiol ; 100: 103854, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34416957

RESUMO

This study investigated the effects of combinations of acetic or malic acid and various solutes (salt, glucose, glycine, or sucrose) on the survival of Escherichia coli O157:H7 in laboratory broth. Additionally, the effectiveness of combining organic acids and various concentrations of salt (0-18%) or sucrose (0-100%) with different water activity values against E. coli O157:H7 were evaluated. For treatment of 1% malic acid, the addition of 3% salt showed synergistic effect. Whereas, when 3% salt, glucose, glycine, or sucrose was added to 1% acetic acid, the solutes antagonized the action of the acid against E. coli O157:H7. Acetic, lactic, or propionic acid combined with salt at either 7 or 9% or sucrose at 60, 80, or 100% resulted in the highest resistance of E. coli O157:H7. From a result of evaluating the membrane fatty acid (MFA) composition of cells, salt or sucrose significantly increased levels of saturated fatty acids (SFAs) or SFAs and cyclopropane fatty acids, respectively. From the results of this study, the addition of solutes and organic compounds may increase the tolerance of E. coli O157:H7 to acetic, lactic, and propionic acid treatments and that the salt or sucrose significantly affects cell MFA composition.


Assuntos
Ácido Acético/farmacologia , Escherichia coli O157/efeitos dos fármacos , Escherichia coli O157/crescimento & desenvolvimento , Glucose/metabolismo , Malatos/farmacologia , Propionatos/farmacologia , Cloreto de Sódio/metabolismo , Sacarose/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Meios de Cultura/química , Meios de Cultura/metabolismo , Escherichia coli O157/metabolismo , Ácidos Graxos/metabolismo , Glicina/metabolismo
5.
Biochem Pharmacol ; 192: 114668, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34216603

RESUMO

GABAA receptors (GABAARs) play a crucial role in mediating inhibition in adult mammalian brains. In the recent years, an impressive progress in revealing the static structure of GABAARs was achieved but the molecular mechanisms underlying their conformational transitions remain elusive. Phenylalanine 64 (α1F64) is located at the loop D of the orthosteric binding site of GABAAR and was found to directly interact with GABA molecule. Mutations of α1F64 were demonstrated to affect not only binding but also some gating properties. Loop D is a rigid ß strand which seems to be particularly suitable to convey activatory signaling from the ligand binding site (LBS) to the gate at the channel pore. To test this scenario, we have investigated the substitution of α1F64 with glycine, the smallest amino acid, widely recognized as a rigidity "reducer" of protein structures. To this end, we assessed the impact of the α1F64G mutation in the α1ß2γ2L type of GABAARs on gating properties by analyzing both macroscopic responses to rapid agonist applications and single-channel currents. We found that this substitution dramatically altered all gating features of the receptor (opening/closing, preactivation and desensitization) which contrasts with markedly weaker effects of previously considered substitutions (α1F64L and α1F64A). In particular, α1F64G mutation practically abolished the desensitization process. At the same time, the α1F64G mutant maintained gating integrity manifested as single-channel activity in the form of clusters. We conclude that rigidity of the loop D plays a crucial role in conveying the activation signal from the LBS to the channel gate.


Assuntos
Glicina/genética , Glicina/metabolismo , Ativação do Canal Iônico/fisiologia , Mutação/fisiologia , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Relação Dose-Resposta a Droga , Agonistas GABAérgicos/metabolismo , Agonistas GABAérgicos/farmacologia , Glicina/química , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Mutação/efeitos dos fármacos , Estrutura Secundária de Proteína , Ratos , Receptores de GABA-A/química , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/farmacologia
6.
Environ Toxicol Pharmacol ; 87: 103703, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34265456

RESUMO

Potential endpoint biomarkers were evaluated in the assessment of exposure to triazoles, in the southern region of Minas Gerais, Brazil. Volunteers were divided into three groups: occupationally exposed and rural residents (n = 21), non-occupationally exposed and rural residents (n = 35) and non-occupationally exposed and urban residents (n = 30). Of all endpoints evaluated, plasma concentration of androstenedione (p < 0.001) and glycine-conjugated bile acids presented statistical differences in the three studied groups (p < 0.05). However, our findings concerning oxidative stress and testosterone levels, plus that related to unconjugated and taurine conjugated bile acids, suggested that more studies are necessary to evaluate their potential as biomarkers for triazole exposure, as statistical significance was not attained between the groups. Our human population data contributes to the development of triazole exposure risk assessment with respect to these potential effect biomarkers, in potentially vulnerable groups and individuals.


Assuntos
Disruptores Endócrinos , Fungicidas Industriais , Exposição Ocupacional , Triazóis , Adulto , Androstenodiona/sangue , Ácidos e Sais Biliares/sangue , Ácidos e Sais Biliares/metabolismo , Monitoramento Biológico , Biomarcadores/sangue , Glicina/metabolismo , Humanos , Pessoa de Meia-Idade , Estresse Oxidativo , População Rural , Testosterona/sangue , População Urbana
7.
Commun Biol ; 4(1): 904, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294851

RESUMO

Though a definitive link between small colony variants (SCVs) and implant-related staphylococcal infections has been well-established, the specific underlying mechanism remains an ill-explored field. The present study analyzes the role SCVs play in catheter infection by performing genomic and metabolic analyses, as well as analyzing biofilm formation and impacts of glycine on growth and peptidoglycan-linking rate, on a clinically typical Staphylococcus epidermidis case harboring stable SCV, normal counterpart (NC) and nonstable SCV. Our findings reveal that S. epidermidis stable SCV carries mutations involved in various metabolic processes. Metabolome analyses demonstrate that two biosynthetic pathways are apparently disturbed in SCV. One is glycine biosynthesis, which contributes to remarkable glycine shortage, and supplementation of glycine restores growth and peptidoglycan-linking rate of SCV. The other is overflow of pyruvic acid and acetyl-CoA, leading to excessive acetate. SCV demonstrates higher biofilm-forming ability due to rapid autolysis and subsequent eDNA release. Despite a remarkable decline in cell viability, SCV can facilitate in vitro biofilm formation and in vivo survival of NC when co-infected with its normal counterparts. This work illustrates an intriguing strategy utilized by a glycine-auxotrophic clinical S. epidermidis SCV isolate to facilitate biofilm-related infections, and casts a new light on the role of SCV in persistent infections.


Assuntos
Infecções Relacionadas a Cateter/epidemiologia , DNA Bacteriano/metabolismo , Glicina/metabolismo , Infecções Estafilocócicas/epidemiologia , Staphylococcus epidermidis/fisiologia , Adolescente , Adulto , Idoso , Animais , Infecções Relacionadas a Cateter/microbiologia , Criança , China/epidemiologia , Cidades , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Prevalência , Infecções Estafilocócicas/microbiologia , Adulto Jovem
8.
Nature ; 596(7871): 301-305, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34321660

RESUMO

Ketamine is a non-competitive channel blocker of N-methyl-D-aspartate (NMDA) receptors1. A single sub-anaesthetic dose of ketamine produces rapid (within hours) and long-lasting antidepressant effects in patients who are resistant to other antidepressants2,3. Ketamine is a racemic mixture of S- and R-ketamine enantiomers, with S-ketamine isomer being the more active antidepressant4. Here we describe the cryo-electron microscope structures of human GluN1-GluN2A and GluN1-GluN2B NMDA receptors in complex with S-ketamine, glycine and glutamate. Both electron density maps uncovered the binding pocket for S-ketamine in the central vestibule between the channel gate and selectivity filter. Molecular dynamics simulation showed that S-ketamine moves between two distinct locations within the binding pocket. Two amino acids-leucine 642 on GluN2A (homologous to leucine 643 on GluN2B) and asparagine 616 on GluN1-were identified as key residues that form hydrophobic and hydrogen-bond interactions with ketamine, and mutations at these residues reduced the potency of ketamine in blocking NMDA receptor channel activity. These findings show structurally how ketamine binds to and acts on human NMDA receptors, and pave the way for the future development of ketamine-based antidepressants.


Assuntos
Microscopia Crioeletrônica , Ketamina/química , Ketamina/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/ultraestrutura , Antidepressivos/química , Antidepressivos/metabolismo , Antidepressivos/farmacologia , Asparagina/química , Asparagina/metabolismo , Sítios de Ligação , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Glicina/química , Glicina/metabolismo , Glicina/farmacologia , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ketamina/metabolismo , Leucina/química , Leucina/metabolismo , Simulação de Dinâmica Molecular , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/ultraestrutura , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo
9.
Biomolecules ; 11(6)2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200954

RESUMO

Among the myriad of cellular and molecular processes identified as contributing to pathological pain, disinhibition of spinal cord nociceptive signaling to higher cortical centers plays a critical role. Importantly, evidence suggests that impaired glycinergic neurotransmission develops in the dorsal horn of the spinal cord in inflammatory and neuropathic pain models and is a key maladaptive mechanism causing mechanical hyperalgesia and allodynia. Thus, it has been hypothesized that pharmacological agents capable of augmenting glycinergic tone within the dorsal horn may be able to blunt or block aberrant nociceptor signaling to the brain and serve as a novel class of analgesics for various pathological pain states. Indeed, drugs that enhance dysfunctional glycinergic transmission, and in particular inhibitors of the glycine transporters (GlyT1 and GlyT2), are generating widespread interest as a potential class of novel analgesics. The GlyTs are Na+/Cl--dependent transporters of the solute carrier 6 (SLC6) family and it has been proposed that the inhibition of them presents a possible mechanism by which to increase spinal extracellular glycine concentrations and enhance GlyR-mediated inhibitory neurotransmission in the dorsal horn. Various inhibitors of both GlyT1 and GlyT2 have demonstrated broad analgesic efficacy in several preclinical models of acute and chronic pain, providing promise for the approach to deliver a first-in-class non-opioid analgesic with a mechanism of action differentiated from current standard of care. This review will highlight the therapeutic potential of GlyT inhibitors as a novel class of analgesics, present recent advances reported for the field, and discuss the key challenges associated with the development of a GlyT inhibitor into a safe and effective agent to treat pain.


Assuntos
Dor Crônica , Glicina/metabolismo , Neuralgia , Nociceptividade , Transdução de Sinais , Medula Espinal/metabolismo , Transmissão Sináptica , Animais , Dor Crônica/metabolismo , Dor Crônica/patologia , Dor Crônica/terapia , Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Humanos , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Neuralgia/patologia , Medula Espinal/patologia
10.
Nat Commun ; 12(1): 4227, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244482

RESUMO

Glycine decarboxylase (GLDC) is a key enzyme of glycine cleavage system that converts glycine into one-carbon units. GLDC is commonly up-regulated and plays important roles in many human cancers. Whether and how GLDC is regulated by post-translational modifications is unknown. Here we report that mechanistic target of rapamycin complex 1 (mTORC1) signal inhibits GLDC acetylation at lysine (K) 514 by inducing transcription of the deacetylase sirtuin 3 (SIRT3). Upon inhibition of mTORC1, the acetyltransferase acetyl-CoA acetyltransferase 1 (ACAT1) catalyzes GLDC K514 acetylation. This acetylation of GLDC impairs its enzymatic activity. In addition, this acetylation of GLDC primes for its K33-linked polyubiquitination at K544 by the ubiquitin ligase NF-X1, leading to its degradation by the proteasomal pathway. Finally, we find that GLDC K514 acetylation inhibits glycine catabolism, pyrimidines synthesis and glioma tumorigenesis. Our finding reveals critical roles of post-translational modifications of GLDC in regulation of its enzymatic activity, glycine metabolism and tumorigenesis, and provides potential targets for therapeutics of cancers such as glioma.


Assuntos
Carcinogênese/genética , Glioma/genética , Glicina Desidrogenase (Descarboxilante)/metabolismo , Glicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Acetil-CoA C-Acetiltransferase/metabolismo , Acetilação , Animais , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Glioma/patologia , Células HEK293 , Humanos , Masculino , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Pirimidinas/biossíntese , Proteínas Repressoras/metabolismo , Sirtuína 3/genética , Sirtuína 3/metabolismo , Ativação Transcricional , Ubiquitinação/genética , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Am J Chin Med ; 49(6): 1437-1448, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34247560

RESUMO

Linalool, a major odorous constituent in essential oils extracted from lavender, is known to have a wide range of physiological effects on humans including pain management. The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) is involved in transmission of orofacial nociceptive responses through thin myelinated A[Formula: see text] and unmyelinated C primary afferent fibers. Up to date, the orofacial antinociceptive mechanism of linalool concerning SG neurons of the Vc has not been completely clarified yet. To fill this knowledge gap, whole-cell patch-clamp technique was used in this study to examine how linalool acted on SG neurons of the Vc in mice. Under a high chloride pipette solution, non-desensitizing and repeatable linalool-induced inward currents were preserved in the presence of tetrodotoxin (a voltage-gated Na[Formula: see text]channel blocker), CNQX (a non-NMDA glutamate receptor antagonist), and DL-AP5 (an NMDA receptor antagonist). However, linalool-induced inward currents were partially suppressed by picrotoxin (a GABA[Formula: see text] receptor antagonist) or strychnine (a glycine receptor antagonist). These responses were almost blocked in the presence of picrotoxin and strychnine. It was also found that linalool exhibited potentiation with GABA- and glycine-induced responses. Taken together, these data show that linalool has GABA- and glycine-mimetic effects, suggesting that it can be a promising target molecule for orofacial pain management by activating inhibitory neurotransmission in the SG area of the Vc.


Assuntos
Monoterpenos Acíclicos/farmacologia , Glicina/metabolismo , Manejo da Dor/métodos , Substância Gelatinosa/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Núcleo Inferior Caudal do Nervo Trigêmeo/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos
12.
Biophys J ; 120(16): 3382-3391, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34237287

RESUMO

FG nucleoporins (FG Nups) are intrinsically disordered proteins and are the putative regulators of nucleocytoplasmic transport. They allow fast, yet selective, transport of molecules through the nuclear pore complex, but the underlying mechanism of nucleocytoplasmic transport is not yet fully discovered. As a result, FG Nups have been the subject of extensive research in the past two decades. Although most studies have been focused on analyzing the conformation and function of FG Nups from a biophysical standpoint, some recent studies have investigated the sequence-function relationship of FG Nups, with a few investigating amino acid sequences of a large number of FG Nups to understand common characteristics that might enable their function. Previously, we identified an evolutionarily conserved feature in FG Nup sequences, which are extended subsequences with low charge density, containing only positive charges, and located toward the N-terminus of FG Nups. We named these patterns longest positive like charge regions (lpLCRs). These patterns are specific to positively charged residues, and negatively charged residues do not demonstrate such a pattern. In this study, we compare FG Nups with other disordered proteins obtained from the DisProt and UniProt database in terms of presence of lpLCRs. Our results show that the lpLCRs are virtually exclusive to FG Nups and are not observed in other disordered proteins. Also, lpLCRs are what differentiate FG Nups from DisProt proteins in terms of charge distribution, meaning that excluding lpLCRs from the sequences of FG Nups make them similar to DisProt proteins in terms of charge distribution. We also previously showed the biophysical effect of lpLCRs in conformation of FG Nups. The results of this study are in line with our previous findings and imply that lpLCRs are virtually exclusive and functionally significant characteristics of FG Nups and nucleocytoplasmic transport.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Fenilalanina , Transporte Ativo do Núcleo Celular , Glicina/metabolismo , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fenilalanina/metabolismo
13.
Elife ; 102021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-34121662

RESUMO

Locomotion generates adventitious sounds which enable detection and localization of predators and prey. Such sounds contain brisk changes or transients in amplitude. We investigated the hypothesis that ill-understood temporal specializations in binaural circuits subserve lateralization of such sound transients, based on different time of arrival at the ears (interaural time differences, ITDs). We find that Lateral Superior Olive (LSO) neurons show exquisite ITD-sensitivity, reflecting extreme precision and reliability of excitatory and inhibitory postsynaptic potentials, in contrast to Medial Superior Olive neurons, traditionally viewed as the ultimate ITD-detectors. In vivo, inhibition blocks LSO excitation over an extremely short window, which, in vitro, required synaptically evoked inhibition. Light and electron microscopy revealed inhibitory synapses on the axon initial segment as the structural basis of this observation. These results reveal a neural vetoing mechanism with extreme temporal and spatial precision and establish the LSO as the primary nucleus for binaural processing of sound transients.


Assuntos
Neurônios/fisiologia , Núcleo Olivar , Localização de Som/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Gerbillinae , Glicina/metabolismo , Potenciais Pós-Sinápticos Inibidores/fisiologia , Masculino , Núcleo Olivar/citologia , Núcleo Olivar/fisiologia
15.
RNA ; 27(9): 1046-1067, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34162742

RESUMO

RNA exosomopathies, a growing family of diseases, are linked to missense mutations in genes encoding structural subunits of the evolutionarily conserved, 10-subunit exoribonuclease complex, the RNA exosome. This complex consists of a three-subunit cap, a six-subunit, barrel-shaped core, and a catalytic base subunit. While a number of mutations in RNA exosome genes cause pontocerebellar hypoplasia, mutations in the cap subunit gene EXOSC2 cause an apparently distinct clinical presentation that has been defined as a novel syndrome SHRF (short stature, hearing loss, retinitis pigmentosa, and distinctive facies). We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by modeling pathogenic EXOSC2 missense mutations (p.Gly30Val and p.Gly198Asp) in the orthologous S. cerevisiae gene RRP4 The resulting rrp4 mutant cells show defects in cell growth and RNA exosome function. Consistent with altered RNA exosome function, we detect significant transcriptomic changes in both coding and noncoding RNAs in rrp4-G226D cells that model EXOSC2 p.Gly198Asp, suggesting defects in nuclear surveillance. Biochemical and genetic analyses suggest that the Rrp4 G226D variant subunit shows impaired interactions with key RNA exosome cofactors that modulate the function of the complex. These results provide the first in vivo evidence that pathogenic missense mutations present in EXOSC2 impair the function of the RNA exosome. This study also sets the stage to compare exosomopathy models to understand how defects in RNA exosome function underlie distinct pathologies.


Assuntos
Exorribonucleases/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Mutação de Sentido Incorreto , RNA Fúngico/genética , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Nanismo/enzimologia , Nanismo/genética , Nanismo/patologia , Exorribonucleases/química , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/química , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Facies , Expressão Gênica , Glicina/química , Glicina/metabolismo , Perda Auditiva/enzimologia , Perda Auditiva/genética , Perda Auditiva/patologia , Humanos , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , RNA Fúngico/química , RNA Fúngico/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Retinite Pigmentosa/enzimologia , Retinite Pigmentosa/genética , Retinite Pigmentosa/patologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Síndrome
16.
Bioconjug Chem ; 32(6): 1167-1174, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34060308

RESUMO

Multiple, site-specific protein conjugation is increasingly attractive for the generation of antibody-drug conjugates (ADCs). As it is important to control the number and position of cargoes in an ADC, position-selective generation of reactive sites in the protein of interest is required. Formylglycine (FGly) residues are generated by enzymatic conversion of cysteine residues embedded in a certain amino acid sequence motif with a formylglycine-generating enzyme (FGE). The addition of copper ions increases FGE activity leading to the conversion of cysteines within less readily accepted sequences. With this tuned enzyme activity, it is possible to address two different recognition sequences using two aerobic formylglycine-generating enzymes. We demonstrate an improved and facile strategy for the functionalization of a DARPin (designed ankyrin repeat protein) and the single-chain antibody scFv425-Fc, both directed against the epidermal growth factor receptor (EGFR). The single-chain antibody was conjugated with monomethyl auristatin E (MMAE) and carboxyfluorescein (CF) and successfully tested for receptor binding, internalization, and cytotoxicity in cell culture, respectively.


Assuntos
Enzimas/metabolismo , Glicina/análogos & derivados , Imunoconjugados/química , Imunoconjugados/metabolismo , Aerobiose , Repetição de Anquirina , Cobre/química , Fluoresceínas/química , Glicina/metabolismo , Oligopeptídeos/química
17.
PLoS Biol ; 19(5): e3001182, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33979323

RESUMO

Melanin, a black-brown pigment found throughout all kingdoms of life, has diverse biological functions including UV protection, thermoregulation, oxidant scavenging, arthropod immunity, and microbial virulence. Given melanin's broad roles in the biosphere, particularly in insect immune defenses, it is important to understand how exposure to ubiquitous environmental contaminants affects melanization. Glyphosate-the most widely used herbicide globally-inhibits melanin production, which could have wide-ranging implications in the health of many organisms, including insects. Here, we demonstrate that glyphosate has deleterious effects on insect health in 2 evolutionary distant species, Galleria mellonella (Lepidoptera: Pyralidae) and Anopheles gambiae (Diptera: Culicidae), suggesting a broad effect in insects. Glyphosate reduced survival of G. mellonella caterpillars following infection with the fungus Cryptococcus neoformans and decreased the size of melanized nodules formed in hemolymph, which normally help eliminate infection. Glyphosate also increased the burden of the malaria-causing parasite Plasmodium falciparum in A. gambiae mosquitoes, altered uninfected mosquito survival, and perturbed the microbial composition of adult mosquito midguts. Our results show that glyphosate's mechanism of melanin inhibition involves antioxidant synergy and disruption of the reaction oxidation-reduction balance. Overall, these findings suggest that glyphosate's environmental accumulation could render insects more susceptible to microbial pathogens due to melanin inhibition, immune impairment, and perturbations in microbiota composition, potentially contributing to declines in insect populations.


Assuntos
Anopheles/efeitos dos fármacos , Glicina/análogos & derivados , Melaninas/metabolismo , Mariposas/efeitos dos fármacos , Animais , Anopheles/imunologia , Cryptococcus neoformans/patogenicidade , Dípteros/efeitos dos fármacos , Dípteros/imunologia , Glicina/metabolismo , Glicina/farmacologia , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Infecções/imunologia , Infecções/metabolismo , Infecções/fisiopatologia , Insetos/efeitos dos fármacos , Insetos/imunologia , Lepidópteros/efeitos dos fármacos , Lepidópteros/imunologia , Mariposas/imunologia , Plasmodium falciparum/patogenicidade , Virulência
18.
Nat Commun ; 12(1): 2694, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976221

RESUMO

N-Methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors essential for synaptic plasticity and memory. Receptor activation involves glycine- and glutamate-stabilized closure of the GluN1 and GluN2 subunit ligand binding domains that is allosterically regulated by the amino-terminal domain (ATD). Using single molecule fluorescence resonance energy transfer (smFRET) to monitor subunit rearrangements in real-time, we observe a stable ATD inter-dimer distance in the Apo state and test the effects of agonists and antagonists. We find that GluN1 and GluN2 have distinct gating functions. Glutamate binding to GluN2 subunits elicits two identical, sequential steps of ATD dimer separation. Glycine binding to GluN1 has no detectable effect, but unlocks the receptor for activation so that glycine and glutamate together drive an altered activation trajectory that is consistent with ATD dimer separation and rotation. We find that protons exert allosteric inhibition by suppressing the glutamate-driven ATD separation steps, and that greater ATD separation translates into greater rotation and higher open probability.


Assuntos
Conformação Proteica , Multimerização Proteica , Receptores de N-Metil-D-Aspartato/química , Regulação Alostérica , Transferência Ressonante de Energia de Fluorescência/métodos , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Glicina/química , Glicina/metabolismo , Células HEK293 , Humanos , Cinética , Microscopia Confocal , Modelos Moleculares , Ligação Proteica , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo
19.
Commun Biol ; 4(1): 554, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976379

RESUMO

Glyphosate is widely used as a herbicide, but recent studies begin to reveal its detrimental side effects on animals by targeting the shikimate pathway of associated gut microorganisms. However, its impact on nutritional endosymbionts in insects remains poorly understood. Here, we sequenced the tiny, shikimate pathway encoding symbiont genome of the sawtoothed grain beetle Oryzaephilus surinamensis. Decreased titers of the aromatic amino acid tyrosine in symbiont-depleted beetles underscore the symbionts' ability to synthesize prephenate as the precursor for host tyrosine synthesis and its importance for cuticle sclerotization and melanization. Glyphosate exposure inhibited symbiont establishment during host development and abolished the mutualistic benefit on cuticle synthesis in adults, which could be partially rescued by dietary tyrosine supplementation. Furthermore, phylogenetic analyses indicate that the shikimate pathways of many nutritional endosymbionts likewise contain a glyphosate sensitive 5-enolpyruvylshikimate-3-phosphate synthase. These findings highlight the importance of symbiont-mediated tyrosine supplementation for cuticle biosynthesis in insects, but also paint an alarming scenario regarding the use of glyphosate in light of recent declines in insect populations.


Assuntos
Besouros/metabolismo , Glicina/análogos & derivados , Simbiose/fisiologia , Escamas de Animais/metabolismo , Animais , Besouros/fisiologia , Glicina/metabolismo , Glicina/farmacologia , Herbicidas , Filogenia , Ácido Chiquímico/metabolismo , Simbiose/efeitos dos fármacos
20.
Int J Mol Sci ; 22(6)2021 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-33803916

RESUMO

The glycine conjugation pathway in humans is involved in the metabolism of natural substrates and the detoxification of xenobiotics. The interactions between the various substrates in this pathway and their competition for the pathway enzymes are currently unknown. The pathway consists of a mitochondrial xenobiotic/medium-chain fatty acid: coenzyme A (CoA) ligase (ACSM2B) and glycine N-acyltransferase (GLYAT). The catalytic mechanism and substrate specificity of both of these enzymes have not been thoroughly characterised. In this study, the level of evolutionary conservation of GLYAT missense variants and haplotypes were analysed. From these data, haplotype variants were selected (156Asn > Ser, [17Ser > Thr,156Asn > Ser] and [156Asn > Ser,199Arg > Cys]) in order to characterise the kinetic mechanism of the enzyme over a wide range of substrate concentrations. The 156Asn > Ser haplotype has the highest frequency and the highest relative enzyme activity in all populations studied, and hence was used as the reference in this study. Cooperative substrate binding was observed, and the kinetic data were fitted to a two-substrate Hill equation. The coding region of the GLYAT gene was found to be highly conserved and the rare 156Asn > Ser,199Arg > Cys variant negatively affected the relative enzyme activity. Even though the 156Asn > Ser,199Arg > Cys variant had a higher affinity for benzoyl-CoA (s0.5,benz = 61.2 µM), kcat was reduced to 9.8% of the most abundant haplotype 156Asn > Ser (s0.5,benz = 96.6 µM), while the activity of 17Ser > Thr,156Asn > Ser (s0.5,benz = 118 µM) was 73% of 156Asn > Ser. The in vitro kinetic analyses of the effect of the 156Asn > Ser,199Arg > Cys variant on human GLYAT enzyme activity indicated that individuals with this haplotype might have a decreased ability to metabolise benzoate when compared to individuals with the 156Asn > Ser variant. Furthermore, the accumulation of acyl-CoA intermediates can inhibit ACSM2B leading to a reduction in mitochondrial energy production.


Assuntos
Acil Coenzima A/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Glicina/metabolismo , Mutação/genética , Animais , Sequência Conservada/genética , Frequência do Gene/genética , Humanos , Cinética , Filogenia
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