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1.
PLoS Biol ; 18(7): e3000811, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32735558

RESUMO

One of the earliest and most prevalent barriers to successful reproduction is polyspermy, or fertilization of an egg by multiple sperm. To prevent these supernumerary fertilizations, eggs have evolved multiple mechanisms. It has recently been proposed that zinc released by mammalian eggs at fertilization may block additional sperm from entering. Here, we demonstrate that eggs from amphibia and teleost fish also release zinc. Using Xenopus laevis as a model, we document that zinc reversibly blocks fertilization. Finally, we demonstrate that extracellular zinc similarly disrupts early embryonic development in eggs from diverse phyla, including Cnidaria, Echinodermata, and Chordata. Our study reveals that a fundamental strategy protecting human eggs from fertilization by multiple sperm may have evolved more than 650 million years ago.


Assuntos
Fertilização , Oócitos/metabolismo , Zinco/metabolismo , Ambystoma mexicanum , Animais , Feminino , Hidrozoários , Masculino , Strongylocentrotus purpuratus , Xenopus laevis , Peixe-Zebra
2.
Proc Natl Acad Sci U S A ; 117(33): 20298-20304, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747539

RESUMO

In mammals, temperature-sensitive TRP channels make membrane conductance of cells extremely temperature dependent, allowing the detection of temperature ranging from noxious cold to noxious heat. We progressively deleted the distal carboxyl terminus domain (CTD) of the cold-activated melastatin receptor channel, TRPM8. We found that the enthalpy change associated with channel gating is proportional to the length of the CTD. Deletion of the last 36 amino acids of the CTD transforms TRPM8 into a reduced temperature-sensitivity channel (Q10 ∼4). Exposing the intracellular domain to a denaturing agent increases the energy required to open the channel indicating that cold drives channel gating by stabilizing the folded state of the CTD. Experiments in the presence of an osmoticant agent suggest that channel gating involves a change in solute-inaccessible volume in the CTD of ∼1,900 Å3 This volume matches the void space inside the coiled coil according to the cryogenic electron microscopy structure of TRPM8. The results indicate that a folding-unfolding reaction of a specialized temperature-sensitive structure is coupled to TRPM8 gating.


Assuntos
Domínios Proteicos , Dobramento de Proteína , Canais de Cátion TRPM/química , Animais , Temperatura Baixa , Microscopia Crioeletrônica , Humanos , Ativação do Canal Iônico , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Oócitos , Conformação Proteica , Canais de Cátion TRPM/metabolismo , Termodinâmica , Xenopus laevis
3.
Nat Commun ; 11(1): 3922, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764664

RESUMO

The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is a key contributor to multidrug resistance and is also essential for the survival of the malaria parasite, yet its natural function remains unresolved. We identify host-derived peptides of 4-11 residues, varying in both charge and composition, as the substrates of PfCRT in vitro and in situ, and show that PfCRT does not mediate the non-specific transport of other metabolites and/or ions. We find that drug-resistance-conferring mutations reduce both the peptide transport capacity and substrate range of PfCRT, explaining the impaired fitness of drug-resistant parasites. Our results indicate that PfCRT transports peptides from the lumen of the parasite's digestive vacuole to the cytosol, thereby providing a source of amino acids for parasite metabolism and preventing osmotic stress of this organelle. The resolution of PfCRT's native substrates will aid the development of drugs that target PfCRT and/or restore the efficacy of existing antimalarials.


Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Transporte Biológico Ativo , Resistência a Medicamentos/genética , Feminino , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/fisiologia , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Modelos Biológicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oligopeptídeos/metabolismo , Oócitos/metabolismo , Plasmodium falciparum/genética , Transporte Proteico , Proteínas de Protozoários/genética , Xenopus laevis
4.
PLoS One ; 15(8): e0236515, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764757

RESUMO

With the increasing availability of high quality genomic data, there is opportunity to deeply explore the genealogical relationships of different gene loci between closely related species. In this study, we utilized genomes of Xenopus laevis (XLA, a tetraploid species with (L) and (S) sub-genomes) and X. tropicalis (XTR, a diploid species) to investigate whether synonymous substitution rates among orthologous or homoeologous genes displayed any heterogeneity. From over 1500 orthologous/homoeologous genes collected, we calculated proportion of synonymous substitutions between genomes/sub-genomes (k) and found variation within and between chromosomes. Within most chromosomes, we identified higher k with distance from the centromere, likely attributed to higher substitution rates and recombination in these regions. Using maximum likelihood methods, we identified further evidence supporting rate heterogeneity, and estimated species divergence times and ancestral population sizes. Estimated species divergence times (XLA.L-XLA.S: ~25.5 mya; XLA-XTR: ~33.0 mya) were slightly younger compared to a past study, attributed to consideration of population size in our study. Meanwhile, we found very large estimated population size in the ancestral populations of the two species (NA = 2.55 x 106). Local hybridization and population structure, which have not yet been well elucidated in frogs, may be a contributing factor to these possible large population sizes.


Assuntos
Evolução Molecular , Genoma/genética , Mutação Silenciosa/genética , Xenopus laevis/genética , Animais , Cromossomos , Heterogeneidade Genética , Hibridização Genética , Hibridização in Situ Fluorescente , Filogenia
5.
Science ; 369(6499): 59-64, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32631887

RESUMO

Eukaryotic histone H3-H4 tetramers contain a putative copper (Cu2+) binding site at the H3-H3' dimerization interface with unknown function. The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis. We report that the recombinant Xenopus laevis H3-H4 tetramer is an oxidoreductase enzyme that binds Cu2+ and catalyzes its reduction to Cu1+ in vitro. Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu1+ abundance and copper-dependent mitochondrial respiration and Sod1 function in the yeast Saccharomyces cerevisiae The histone H3-H4 tetramer, therefore, has a role other than chromatin compaction or epigenetic regulation and generates biousable Cu1+ ions in eukaryotes.


Assuntos
Cobre/metabolismo , Histonas/química , Oxirredutases/química , Multimerização Proteica , Animais , Biocatálise , Domínio Catalítico/genética , Mutação com Ganho de Função , Histonas/genética , Histonas/metabolismo , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Superóxido Dismutase-1/química , Fatores de Transcrição/metabolismo , Xenopus laevis
6.
PLoS One ; 15(7): e0235433, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32726316

RESUMO

ADP-ribosylhydrolase-like 1 (Adprhl1) is a pseudoenzyme expressed in the developing heart myocardium of all vertebrates. In the amphibian Xenopus laevis, knockdown of the two cardiac Adprhl1 protein species (40 and 23 kDa) causes failure of chamber outgrowth but this has only been demonstrated using antisense morpholinos that interfere with RNA-splicing. Transgenic production of 40 kDa Adprhl1 provides only part rescue of these defects. CRISPR/Cas9 technology now enables targeted mutation of the adprhl1 gene in G0-generation embryos with routine cleavage of all alleles. Testing multiple gRNAs distributed across the locus reveals exonic locations that encode critical amino acids for Adprhl1 function. The gRNA recording the highest frequency of a specific ventricle outgrowth phenotype directs Cas9 cleavage of an exon 6 sequence, where microhomology mediated end-joining biases subsequent DNA repairs towards three small in-frame deletions. Mutant alleles encode discrete loss of 1, 3 or 4 amino acids from a di-arginine (Arg271-Arg272) containing peptide loop at the centre of the ancestral ADP-ribosylhydrolase site. Thus despite lacking catalytic activity, it is the modified (adenosine-ribose) substrate binding cleft of Adprhl1 that fulfils an essential role during heart formation. Mutation results in striking loss of myofibril assembly in ventricle cardiomyocytes. The defects suggest Adprhl1 participation from the earliest stage of cardiac myofibrillogenesis and are consistent with previous MO results and Adprhl1 protein localization to actin filament Z-disc boundaries. A single nucleotide change to the gRNA sequence renders it inactive. Mice lacking Adprhl1 exons 3-4 are normal but production of the smaller ADPRHL1 species is unaffected, providing further evidence that cardiac activity is concentrated at the C-terminal protein portion.


Assuntos
Ventrículos do Coração/crescimento & desenvolvimento , Coração/crescimento & desenvolvimento , Desenvolvimento Muscular/genética , N-Glicosil Hidrolases/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Catálise , Domínio Catalítico/genética , Coração/fisiopatologia , Ventrículos do Coração/patologia , Humanos , Camundongos , Camundongos Knockout , Morfolinos/genética , Oligodesoxirribonucleotídeos Antissenso/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Organogênese/genética , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimento
7.
Aquat Toxicol ; 226: 105557, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32645606

RESUMO

Extensive studies have shown that estrogenic endocrine-disrupting chemicals (EDCs) can disrupt testis differentiation and even cause feminization in vertebrates. However, little is known about the mechanisms by which estrogenic EDCs disrupt testis differentiation. Here, we employed Xenopus laevis, a model amphibian species sensitive to estrogenic EDCs, to explore the molecular and cellular events by which 17ß-estradiol (E2) disrupts testis differentiation and causes feminization. Following waterborne exposure to E2 from stage 45/46, genetically male X. laevis were confirmed to undergo testis differentiation inhibition and ovary differentiation activation at stages 52 and 53, ultimately displaying gonadal feminization at stage 66. Using a time-course RNA sequencing approach, we then identified thousands of differentially expressed transcripts (DETs) in genetically male gonad-mesonephros complexes at stages 48, 50 and 52 (the window for testis differentiation) between E2 treatment and the control. Enrichment analysis suggests alterations in cell proliferation, extracellular matrix, and cell motility following E2 exposure. Further verification by multiple methods demonstrated that E2 inhibited cell proliferation, disrupted extracellular matrix, and altered cell motility in the genetically male gonads compared with controls, implying that these events together contributed to testis differentiation disruptions and feminization in X. laevis. This study for the first time uncovered some of the early molecular and cellular events by which estrogen disrupts testicular differentiation and causes feminization in X. laevis. These new findings improve our understanding of the mechanisms by which estrogenic EDCs disrupt testicular differentiation in vertebrates.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , Estradiol/toxicidade , Feminização , Testículo/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Feminino , Feminização/induzido quimicamente , Feminização/genética , Perfilação da Expressão Gênica , Humanos , Larva/efeitos dos fármacos , Larva/genética , Masculino , Ovário/efeitos dos fármacos , Xenopus laevis
8.
Chemosphere ; 259: 127415, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32603964

RESUMO

Amphibians are the most endangered class of vertebrates. In this study, Xenopus laevis frogs were exposed to 0, 1 and 10 mg/L of triadimefon or triadimenol. After 14 or 28 days of exposure, high levels of triadimefon or triadimenol obstructed the growth of frogs. However, low levels of triadimefon induced the growth of females after the longer period of exposure. We also found that the antioxidant enzyme activity and LDH levels in males were higher than those in females after 28-days exposure. In histopathology tests, triadimenol exerted more effect on the ovary while triadimefon exerted more effect on the testes. Additionally, the levels of Estradiol in all 14-day treatments, except 1 mg/L triadimenol, were significantly decreased, however, there was no difference in testosterone levels. Furthermore, triadimefon and triadimenol disrupted the expression of genes controlling hormone homeostasis and reproduction, and this effect depended on the exposure time and the gender of the organism. Our experiments explored the effect of triadimefon and its metabolite on the gonads of frogs and highlighted the role that pesticides are likely to play in the global decline of amphibians.


Assuntos
Fungicidas Industriais/toxicidade , Gônadas/crescimento & desenvolvimento , Triazóis/toxicidade , Xenopus laevis/fisiologia , Animais , Feminino , Fungicidas Industriais/metabolismo , Gônadas/anatomia & histologia , Gônadas/efeitos dos fármacos , Gônadas/metabolismo , Masculino , Xenopus laevis/metabolismo
9.
Mol Cell ; 79(2): 221-233.e5, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32603710

RESUMO

Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Unbiased detection through proximity labeling of transient protein interactions in cell-free Xenopus laevis egg extract identified the dimeric histone chaperone facilitates chromatin transcription (FACT) as an interactor of substrate-bound Cas9. FACT is both necessary and sufficient to displace dCas9, and FACT immunodepletion converts Cas9's activity from multi-turnover to single turnover. In human cells, FACT depletion extends dCas9 residence times, delays genome editing, and alters the balance between indel formation and homology-directed repair. FACT knockdown also increases epigenetic marking by dCas9-based transcriptional effectors with a concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genoma Humano , Proteínas de Grupo de Alta Mobilidade/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Animais , Proteínas Associadas a CRISPR/metabolismo , Linhagem Celular , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Epigênese Genética , Edição de Genes , Técnicas de Silenciamento de Genes , Humanos , Nucleossomos/metabolismo , Xenopus laevis
10.
Proc Natl Acad Sci U S A ; 117(28): 16154-16159, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601228

RESUMO

The metaphase spindle is a dynamic structure orchestrating chromosome segregation during cell division. Recently, soft matter approaches have shown that the spindle behaves as an active liquid crystal. Still, it remains unclear how active force generation contributes to its characteristic spindle-like shape. Here we combine theory and experiments to show that molecular motor-driven forces shape the structure through a barreling-type instability. We test our physical model by titrating dynein activity in Xenopus egg extract spindles and quantifying the shape and microtubule orientation. We conclude that spindles are shaped by the interplay between surface tension, nematic elasticity, and motor-driven active forces. Our study reveals how motor proteins can mold liquid crystalline droplets and has implications for the design of active soft materials.


Assuntos
Metáfase/fisiologia , Fuso Acromático/fisiologia , Animais , Fenômenos Biomecânicos , Dineínas/antagonistas & inibidores , Dineínas/metabolismo , Elasticidade , Cristais Líquidos , Metáfase/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Microtúbulos/fisiologia , Mitose , Fuso Acromático/química , Fuso Acromático/efeitos dos fármacos , Tensão Superficial , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/metabolismo , Xenopus laevis
11.
Proc Natl Acad Sci U S A ; 117(28): 16283-16291, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32611810

RESUMO

The difficulty of achieving robust functional expression of insect nicotinic acetylcholine receptors (nAChRs) has hampered our understanding of these important molecular targets of globally deployed neonicotinoid insecticides at a time when concerns have grown regarding the toxicity of this chemotype to insect pollinators. We show that thioredoxin-related transmembrane protein 3 (TMX3) is essential to enable robust expression in Xenopus laevis oocytes of honeybee (Apis mellifera) and bumblebee (Bombus terrestris) as well as fruit fly (Drosophila melanogaster) nAChR heteromers targeted by neonicotinoids and not hitherto robustly expressed. This has enabled the characterization of picomolar target site actions of neonicotinoids, findings important in understanding their toxicity.


Assuntos
Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Neonicotinoides/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacologia , Animais , Abelhas/metabolismo , Relação Dose-Resposta a Droga , Drosophila melanogaster/metabolismo , Proteínas de Insetos/agonistas , Proteínas de Insetos/genética , Oócitos/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/genética , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Xenopus laevis
12.
PLoS Biol ; 18(7): e3000750, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32667916

RESUMO

Photoreceptors are specialized cells devoted to the transduction of the incoming visual signals. Rods are able also to shed from their tip old disks and to synthesize at the base of the outer segment (OS) new disks. By combining electrophysiology, optical tweezers (OTs), and biochemistry, we investigate mechanosensitivity in the rods of Xenopus laevis, and we show that 1) mechanosensitive channels (MSCs), transient receptor potential canonical 1 (TRPC1), and Piezo1 are present in rod inner segments (ISs); 2) mechanical stimulation-of the order of 10 pN-applied briefly to either the OS or IS evokes calcium transients; 3) inhibition of MSCs decreases the duration of photoresponses to bright flashes; 4) bright flashes of light induce a rapid shortening of the OS; and 5) the genes encoding the TRPC family have an ancient association with the genes encoding families of protein involved in phototransduction. These results suggest that MSCs play an integral role in rods' phototransduction.


Assuntos
Transdução de Sinal Luminoso , Mecanotransdução Celular , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Xenopus laevis/metabolismo , Animais , Cálcio/metabolismo , Fluorescência , Luz , Transdução de Sinal Luminoso/efeitos da radiação , Mecanotransdução Celular/efeitos da radiação , Família Multigênica , Estimulação Luminosa , Células Fotorreceptoras Retinianas Bastonetes/efeitos da radiação , Canais de Cátion TRPC/genética , Proteínas de Xenopus/genética
13.
PLoS Pathog ; 16(7): e1008715, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32716968

RESUMO

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels mostly located in the post-synaptic membrane of cholinergic synapses. The natural neurotransmitter is acetylcholine, but they are also the direct targets for neonicotinoids, chemicals widely used against ectoparasites, arthropod vectors and agricultural pests. There are significant concerns regarding adverse effects of neonicotinoids on beneficial insects. In arthropods, functional nAChRs made of α subunits have been expressed from Drosophila genes, and hybrid receptors (sometimes also referred to as chimeric receptors) using species-specific α subunits and vertebrate ß subunits have been expressed ex-vivo. Arthropod-specific nAChRs made of both α and ß subunits from the target species have not been expressed ex-vivo. The aim of the current study was to express such receptors in Xenopus oocytes using only genes from Lepeophtheirus salmonis, to characterize them and study their modulation. Genes encoding α and ß subunits of the nAChRs and three ancillary proteins, RIC-3, UNC-50 and UNC-74 were identified in the L. salmonis genome, subjected to RACE-PCR, cloned into an expression vector and the cRNA produced was then injected into Xenopus laevis oocytes. Co-expression of the ancillary proteins was essential for the successful expression of the L. salmonis nAChRs with both α and ß subunits. Two functional nAChRs were identified: Lsa-nAChR1 consisting of α1, α2, ß1 and ß2 subunits, reconstituted to one distinct receptor, while Lsa-nAChR2, consisting of α3, ß1 and ß2 subunits reconstitutes receptors with two distinct characteristics. Out of seven neonicotinoids tested, six worked as partial agonist of Lsa-nAChR1 while only three did so for Lsa-nAChR2. Four non-neonicotinoid compounds tested had no effect on either of the nAChRs. The study demonstrated that fully functional, non-hybrid nAChRs containing both α and ß subunits from an arthropod can be reconstituted ex-vivo by co-expression of essential ancillary proteins. Such models would be valuable for in-depth studies of effects by neonicotinoids and other compounds on target pests, as well as for studies of adverse effects on non-target arthropods.


Assuntos
Copépodes/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Copépodes/efeitos dos fármacos , Inseticidas/farmacologia , Neonicotinoides/farmacologia , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/efeitos dos fármacos , Xenopus laevis
14.
Proc Natl Acad Sci U S A ; 117(24): 13437-13446, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32482881

RESUMO

Pentameric ligand-gated ion channels (pLGICs) are allosteric receptors that mediate rapid electrochemical signal transduction in the animal nervous system through the opening of an ion pore upon binding of neurotransmitters. Orthologs have been found and characterized in prokaryotes and they display highly similar structure-function relationships to eukaryotic pLGICs; however, they often encode greater architectural diversity involving additional amino-terminal domains (NTDs). Here we report structural, functional, and normal-mode analysis of two conformational states of a multidomain pLGIC, called DeCLIC, from a Desulfofustis deltaproteobacterium, including a periplasmic NTD fused to the conventional ligand-binding domain (LBD). X-ray structure determination revealed an NTD consisting of two jelly-roll domains interacting across each subunit interface. Binding of Ca2+ at the LBD subunit interface was associated with a closed transmembrane pore, with resolved monovalent cations intracellular to the hydrophobic gate. Accordingly, DeCLIC-injected oocytes conducted currents only upon depletion of extracellular Ca2+; these were insensitive to quaternary ammonium block. Furthermore, DeCLIC crystallized in the absence of Ca2+ with a wide-open pore and remodeled periplasmic domains, including increased contacts between the NTD and classic LBD agonist-binding sites. Functional, structural, and dynamical properties of DeCLIC paralleled those of sTeLIC, a pLGIC from another symbiotic prokaryote. Based on these DeCLIC structures, we would reclassify the previous structure of bacterial ELIC (the first high-resolution structure of a pLGIC) as a "locally closed" conformation. Taken together, structures of DeCLIC in multiple conformations illustrate dramatic conformational state transitions and diverse regulatory mechanisms available to ion channels in pLGICs, particularly involving Ca2+ modulation and periplasmic NTDs.


Assuntos
Proteínas de Bactérias/química , Canais Iônicos de Abertura Ativada por Ligante/química , Regulação Alostérica , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Cristalografia por Raios X , Deltaproteobacteria/química , Deltaproteobacteria/metabolismo , Canais Iônicos de Abertura Ativada por Ligante/genética , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Ligantes , Modelos Moleculares , Oócitos/metabolismo , Periplasma/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Quaternária de Proteína , Relação Estrutura-Atividade , Xenopus laevis
15.
Mol Pharmacol ; 98(2): 168-180, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32474444

RESUMO

The two major nicotinic acetylcholine receptors (nAChRs) in the brain are the α4ß2 and α7 subtypes. A "methyl scan" of the pyrrolidinium ring was used to detect differences in nicotine's interactions with these two receptors. Each methylnicotine was investigated using voltage-clamp and radioligand binding techniques. Methylation at each ring carbon elicited unique changes in nicotine's receptor interactions. Replacing the 1'-N-methyl with an ethyl group or adding a second 1'-N-methyl group significantly reduced interaction with α4ß2 but not α7 receptors. The 2'-methylation uniquely enhanced binding and agonist potency at α7 receptors. Although 3'- and 5'-trans-methylations were much better tolerated by α7 receptors than α4ß2 receptors, 4'-methylation decreased potency and efficacy at α7 receptors much more than at α4ß2 receptors. Whereas cis-5'-methylnicotine lacked agonist activity and displayed a low affinity at both receptors, trans-5'-methylnicotine retained considerable α7 receptor activity. Differences between the two 5'-methylated analogs of the potent pyridyl oxymethylene-bridged nicotine analog A84543 were consistent with what was found for the 5'-methylnicotines. Computer docking of the methylnicotines to the Lymnaea acetylcholine binding protein crystal structure containing two persistent waters predicted most of the changes in receptor affinity that were observed with methylation, particularly the lower affinities of the cis-methylnicotines. The much smaller effects of 1'-, 3'-, and 5'-methylations and the greater effects of 2'- and 4'-methylations on nicotine α7 nAChR interaction might be exploited for the design of new drugs based on the nicotine scaffold. SIGNIFICANCE STATEMENT: Using a comprehensive "methyl scan" approach, we show that the orthosteric binding sites for acetylcholine and nicotine in the two major brain nicotinic acetylcholine receptors interact differently with the pyrrolidinium ring of nicotine, and we suggest reasons for the higher affinity of nicotine for the heteromeric receptor. Potential sites for nicotine structure modification were identified that may be useful in the design of new drugs targeting these receptors.


Assuntos
Nicotina/análogos & derivados , Piridinas/síntese química , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Sítios de Ligação , Masculino , Metilação , Simulação de Acoplamento Molecular , Estrutura Molecular , Nicotina/química , Piridinas/química , Piridinas/farmacologia , Ratos , Relação Estrutura-Atividade , Xenopus laevis
16.
Am J Physiol Cell Physiol ; 319(2): C371-C380, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32579473

RESUMO

Cation-coupled chloride cotransporters (CCC) play a role in modulating intracellular chloride concentration ([Cl-]i) and cell volume. Cell shrinkage and cell swelling are accompanied by an increase or decrease in [Cl-]i, respectively. Cell shrinkage and a decrease in [Cl-]i increase the activity of NKCCs (Na-K-Cl cotransporters: NKCC1, NKCC2, and Na-Cl) and inhibit the activity of KCCs (K-Cl cotransporters: KCC1 to KCC4), wheras cell swelling and an increase in [Cl-]i activate KCCs and inhibit NKCCs; thus, it is unlikely that the same kinase is responsible for both effects. WNK1 and WNK4 are chloride-sensitive kinases that modulate the activity of CCC in response to changes in [Cl-]i. Here, we showed that WNK3, another member of the serine-threonine kinase WNK family with known effects on CCC, is not sensitive to [Cl-]i but can be regulated by changes in extracellular tonicity. In contrast, WNK4 is highly sensitive to [Cl-]i but is not regulated by changes in cell volume. The activity of WNK3 toward NaCl cotransporter is not affected by eliminating the chloride-binding site of WNK3, further confirming that the kinase is not sensitive to chloride. Chimeric WNK3/WNK4 proteins were produced, and analysis of the chimeras suggests that sequences within the WNK's carboxy-terminal end may modulate the chloride affinity. We propose that WNK3 is a cell volume-sensitive kinase that translates changes in cell volume into phosphorylation of CCC.


Assuntos
Tamanho Celular , Proteínas Serina-Treonina Quinases/genética , Simportadores de Cloreto de Sódio/metabolismo , Proteínas de Xenopus/genética , Animais , Cloretos/química , Cloretos/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Humanos , Oócitos/química , Oócitos/metabolismo , Fosforilação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores de Cloreto de Sódio/química , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo
17.
Am J Physiol Heart Circ Physiol ; 319(2): H251-H261, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32559136

RESUMO

Human ether-à-go-go related gene (hERG) K+ channels are important in cardiac repolarization, and their dysfunction causes prolongation of the ventricular action potential, long QT syndrome, and arrhythmia. As such, approaches to augment hERG channel function, such as activator compounds, have been of significant interest due to their marked therapeutic potential. Activator compounds that hinder channel inactivation abbreviate action potential duration (APD) but carry risk of overcorrection leading to short QT syndrome. Enhanced risk by overcorrection of the APD may be tempered by activator-induced increased refractoriness; however, investigation of the cumulative effect of hERG activator compounds on the balance of these effects in whole organ systems is lacking. Here, we have investigated the antiarrhythmic capability of a hERG activator, RPR260243, which primarily augments channel function by slowing deactivation kinetics in ex vivo zebrafish whole hearts. We show that RPR260243 abbreviates the ventricular APD, reduces triangulation, and steepens the slope of the electrical restitution curve. In addition, RPR260243 increases the post-repolarization refractory period. We provide evidence that this latter effect arises from RPR260243-induced enhancement of hERG channel-protective currents flowing early in the refractory period. Finally, the cumulative effect of RPR260243 on arrhythmogenicity in whole organ zebrafish hearts is demonstrated by the restoration of normal rhythm in hearts presenting dofetilide-induced arrhythmia. These findings in a whole organ model demonstrate the antiarrhythmic benefit of hERG activator compounds that modify both APD and refractoriness. Furthermore, our results demonstrate that targeted slowing of hERG channel deactivation and enhancement of protective currents may provide an effective antiarrhythmic approach.NEW & NOTEWORTHY hERG channel dysfunction causes long QT syndrome and arrhythmia. Activator compounds have been of significant interest due to their therapeutic potential. We used the whole organ zebrafish heart model to demonstrate the antiarrhythmic benefit of the hERG activator, RPR260243. The activator abbreviated APD and increased refractoriness, the combined effect of which rescued induced ventricular arrhythmia. Our findings show that the targeted slowing of hERG channel deactivation and enhancement of protective currents caused by the RPR260243 activator may provide an effective antiarrhythmic approach.


Assuntos
Antiarrítmicos/farmacologia , Arritmias Cardíacas/prevenção & controle , Canal de Potássio ERG1/agonistas , Canais de Potássio Éter-A-Go-Go/agonistas , Frequência Cardíaca/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Piperidinas/farmacologia , Quinolinas/farmacologia , Proteínas de Peixe-Zebra/agonistas , Potenciais de Ação , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Modelos Animais de Doenças , Canal de Potássio ERG1/genética , Canal de Potássio ERG1/metabolismo , Canais de Potássio Éter-A-Go-Go/metabolismo , Cinética , Miócitos Cardíacos/metabolismo , Oócitos , Período Refratário Eletrofisiológico , Transdução de Sinais , Xenopus laevis , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
18.
Nat Cell Biol ; 22(7): 803-814, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32572169

RESUMO

Cell shape is controlled by the submembranous cortex, an actomyosin network mainly generated by two actin nucleators: the Arp2/3 complex and the formin mDia1. Changes in relative nucleator activity may alter cortical organization, mechanics and cell shape. Here we investigate how nucleation-promoting factors mediate interactions between nucleators. In vitro, the nucleation-promoting factor SPIN90 promotes formation of unbranched filaments by Arp2/3, a process thought to provide the initial filament for generation of dendritic networks. Paradoxically, in cells, SPIN90 appears to favour a formin-dominated cortex. Our in vitro experiments reveal that this feature stems mainly from two mechanisms: efficient recruitment of mDia1 to SPIN90-Arp2/3 nucleated filaments and formation of a ternary SPIN90-Arp2/3-mDia1 complex that greatly enhances filament nucleation. Both mechanisms yield rapidly elongating filaments with mDia1 at their barbed ends and SPIN90-Arp2/3 at their pointed ends. Thus, in networks, SPIN90 lowers branching densities and increases the proportion of long filaments elongated by mDia1.


Assuntos
Citoesqueleto de Actina/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Forminas/metabolismo , Melanoma/patologia , Proteínas Musculares/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Blástula/citologia , Blástula/metabolismo , Forma Celular , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Forminas/genética , Humanos , Melanoma/genética , Melanoma/metabolismo , Proteínas Musculares/genética , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismo
19.
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
20.
Proc Natl Acad Sci U S A ; 117(26): 15075-15084, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32532919

RESUMO

Some lineage-determining transcription factors are overwhelmingly important in directing embryonic cells to a particular differentiation pathway, such as Ascl1 for nerve. They also have an exceptionally strong ability to force cells to change from an unrelated pathway to one preferred by their action. Transcription factors are believed to have a very short residence time of only a few seconds on their specific DNA or chromatin-binding sites. We have developed a procedure in which DNA containing one copy of the binding site for the neural-inducing factor Ascl1 is injected directly into a Xenopus oocyte nucleus which has been preloaded with a limiting amount of the Ascl1 transcription factor protein. This is followed by a further injection of DNA as a competitor, either in a plasmid or in chromosomal DNA, containing the same binding site but with a different reporter. Importantly, expression of the reporter provides a measure of the function of the transcription factor in addition to its residence time. The same long residence time and resistance to competition are seen with the estrogen receptor and its DNA response elements. We find that in this nondividing oocyte, the nerve-inducing factor Ascl1 can remain bound to a specific chromatin site for hours or days and thereby help to stabilize gene expression. This stability of transcription factor binding to chromatin is a necessary part of its action because removal of this factor causes discontinuation of its effect on gene expression. Stable transcription factor binding may be a characteristic of nondividing cells.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cromatina/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Cromatina/genética , DNA/genética , DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Ligação Proteica , Xenopus laevis/embriologia , Xenopus laevis/genética , Xenopus laevis/metabolismo
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