RESUMO
Reversible phase separation underpins the role of FUS in ribonucleoprotein granules and other membrane-free organelles and is, in part, driven by the intrinsically disordered low-complexity (LC) domain of FUS. Here, we report that cooperative cation-π interactions between tyrosines in the LC domain and arginines in structured C-terminal domains also contribute to phase separation. These interactions are modulated by post-translational arginine methylation, wherein arginine hypomethylation strongly promotes phase separation and gelation. Indeed, significant hypomethylation, which occurs in FUS-associated frontotemporal lobar degeneration (FTLD), induces FUS condensation into stable intermolecular ß-sheet-rich hydrogels that disrupt RNP granule function and impair new protein synthesis in neuron terminals. We show that transportin acts as a physiological molecular chaperone of FUS in neuron terminals, reducing phase separation and gelation of methylated and hypomethylated FUS and rescuing protein synthesis. These results demonstrate how FUS condensation is physiologically regulated and how perturbations in these mechanisms can lead to disease.
Assuntos
Arginina/química , Chaperonas Moleculares/química , Proteína FUS de Ligação a RNA/química , Esclerose Lateral Amiotrófica/metabolismo , Animais , Cátions , Metilação de DNA , Demência Frontotemporal/metabolismo , Degeneração Lobar Frontotemporal/metabolismo , Humanos , Microscopia de Força Atômica , Microscopia de Fluorescência , Ligação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Proteína FUS de Ligação a RNA/metabolismo , Tirosina/química , Xenopus laevisRESUMO
Resting CD4+ T cells are highly resistant to the production of human immunodeficiency virus type 1 (HIV-1). However, the mechanism by which resting CD4+ T cells restrict such production in the late viral replication phase of infection has remained unclear. In this study, we found that the cell membrane metalloprotease TRAB domain-containing protein 2A (TRABD2A) inhibited this production in resting CD4+ T cells by degrading the virion structural precursor polyprotein Gag at the plasma membrane. Depletion or inhibition of metalloprotease activity by TRABD2A profoundly enhanced HIV-1 production in resting CD4+ T cells. TRABD2A expression was much higher in resting CD4+ T cells than in activated CD4+ T cells and was considerably reduced by T cell activation. Moreover, reexpressing TRABD2A reinforced the resistance of activated CD4+ T cells to the production of HIV-1 progeny. Collectively, these results elucidate the molecular mechanism employed by resting CD4+ T cells to potently restrict the assembly and production of HIV-1 progeny.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/fisiologia , Metaloproteases/genética , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Cátions , Linhagem Celular , Ativação Enzimática , Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Proteínas de Membrana/metabolismo , Metaloproteases/antagonistas & inibidores , Metaloproteases/metabolismo , Família Multigênica , Proteólise , Proteína 1 com Domínio SAM e Domínio HD/genética , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Carga ViralRESUMO
Methods for selective covalent modification of amino acids on proteins can enable a diverse array of applications, spanning probes and modulators of protein function to proteomics1-3. Owing to their high nucleophilicity, cysteine and lysine residues are the most common points of attachment for protein bioconjugation chemistry through acid-base reactivity3,4. Here we report a redox-based strategy for bioconjugation of tryptophan, the rarest amino acid, using oxaziridine reagents that mimic oxidative cyclization reactions in indole-based alkaloid biosynthetic pathways to achieve highly efficient and specific tryptophan labelling. We establish the broad use of this method, termed tryptophan chemical ligation by cyclization (Trp-CLiC), for selectively appending payloads to tryptophan residues on peptides and proteins with reaction rates that rival traditional click reactions and enabling global profiling of hyper-reactive tryptophan sites across whole proteomes. Notably, these reagents reveal a systematic map of tryptophan residues that participate in cation-π interactions, including functional sites that can regulate protein-mediated phase-separation processes.
Assuntos
Cátions , Ciclização , Indicadores e Reagentes , Proteínas , Triptofano , Cátions/química , Indicadores e Reagentes/química , Oxirredução , Proteoma/química , Triptofano/química , Peptídeos/química , Química Click , Proteínas/químicaRESUMO
Nitrosopumilus maritimus is an ammonia-oxidizing archaeon that is crucial to the global nitrogen cycle1,2. A critical step for nitrogen oxidation is the entrapment of ammonium ions from a dilute marine environment at the cell surface and their subsequent channelling to the cell membrane of N. maritimus. Here we elucidate the structure of the molecular machinery responsible for this process, comprising the surface layer (S-layer), using electron cryotomography and subtomogram averaging from cells. We supplemented our in situ structure of the ammonium-binding S-layer array with a single-particle electron cryomicroscopy structure, revealing detailed features of this immunoglobulin-rich and glycan-decorated S-layer. Biochemical analyses showed strong ammonium binding by the cell surface, which was lost after S-layer disassembly. Sensitive bioinformatic analyses identified similar S-layers in many ammonia-oxidizing archaea, with conserved sequence and structural characteristics. Moreover, molecular simulations and structure determination of ammonium-enriched specimens enabled us to examine the cation-binding properties of the S-layer, revealing how it concentrates ammonium ions on its cell-facing side, effectively acting as a multichannel sieve on the cell membrane. This in situ structural study illuminates the biogeochemically essential process of ammonium binding and channelling, common to many marine microorganisms that are fundamental to the nitrogen cycle.
Assuntos
Amônia , Organismos Aquáticos , Archaea , Membrana Celular , Amônia/química , Amônia/metabolismo , Organismos Aquáticos/química , Organismos Aquáticos/metabolismo , Organismos Aquáticos/ultraestrutura , Archaea/química , Archaea/metabolismo , Archaea/ultraestrutura , Cátions/química , Cátions/metabolismo , Microscopia Crioeletrônica , Modelos Moleculares , Oxirredução , Polissacarídeos/metabolismo , Polissacarídeos/químicaRESUMO
Recent years have seen revived interest in computer-assisted organic synthesis1,2. The use of reaction- and neural-network algorithms that can plan multistep synthetic pathways have revolutionized this field1,3-7, including examples leading to advanced natural products6,7. Such methods typically operate on full, literature-derived 'substrate(s)-to-product' reaction rules and cannot be easily extended to the analysis of reaction mechanisms. Here we show that computers equipped with a comprehensive knowledge-base of mechanistic steps augmented by physical-organic chemistry rules, as well as quantum mechanical and kinetic calculations, can use a reaction-network approach to analyse the mechanisms of some of the most complex organic transformations: namely, cationic rearrangements. Such rearrangements are a cornerstone of organic chemistry textbooks and entail notable changes in the molecule's carbon skeleton8-12. The algorithm we describe and deploy at https://HopCat.allchemy.net/ generates, within minutes, networks of possible mechanistic steps, traces plausible step sequences and calculates expected product distributions. We validate this algorithm by three sets of experiments whose analysis would probably prove challenging even to highly trained chemists: (1) predicting the outcomes of tail-to-head terpene (THT) cyclizations in which substantially different outcomes are encoded in modular precursors differing in minute structural details; (2) comparing the outcome of THT cyclizations in solution or in a supramolecular capsule; and (3) analysing complex reaction mixtures. Our results support a vision in which computers no longer just manipulate known reaction types1-7 but will help rationalize and discover new, mechanistically complex transformations.
Assuntos
Algoritmos , Técnicas de Química Sintética , Ciclização , Redes Neurais de Computação , Terpenos , Cátions/química , Bases de Conhecimento , Terpenos/química , Técnicas de Química Sintética/métodos , Produtos Biológicos/síntese química , Produtos Biológicos/química , Reprodutibilidade dos Testes , SoluçõesRESUMO
Human feline leukaemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and FLVCR2) are members of the major facilitator superfamily1. Their dysfunction is linked to several clinical disorders, including PCARP, HSAN and Fowler syndrome2-7. Earlier studies concluded that FLVCR1 may function as a haem exporter8-12, whereas FLVCR2 was suggested to act as a haem importer13, yet conclusive biochemical and detailed molecular evidence remained elusive for the function of both transporters14-16. Here, we show that FLVCR1 and FLVCR2 facilitate the transport of choline and ethanolamine across the plasma membrane, using a concentration-driven substrate translocation process. Through structural and computational analyses, we have identified distinct conformational states of FLVCRs and unravelled the coordination chemistry underlying their substrate interactions. Fully conserved tryptophan and tyrosine residues form the binding pocket of both transporters and confer selectivity for choline and ethanolamine through cation-π interactions. Our findings clarify the mechanisms of choline and ethanolamine transport by FLVCR1 and FLVCR2, enhance our comprehension of disease-associated mutations that interfere with these vital processes and shed light on the conformational dynamics of these major facilitator superfamily proteins during the transport cycle.
Assuntos
Colina , Etanolamina , Proteínas de Membrana Transportadoras , Humanos , Sítios de Ligação , Transporte Biológico , Cátions/química , Cátions/metabolismo , Membrana Celular/metabolismo , Membrana Celular/química , Colina/metabolismo , Colina/química , Etanolamina/metabolismo , Etanolamina/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Conformação Proteica , Receptores Virais/metabolismo , Receptores Virais/química , Especificidade por Substrato , Triptofano/metabolismo , Triptofano/química , Tirosina/metabolismo , Tirosina/química , MutaçãoRESUMO
The success of colloidal semiconductor nanocrystals (NCs) in science and optoelectronics is inextricable from their surfaces. The functionalization of lead halide perovskite NCs1-5 poses a formidable challenge because of their structural lability, unlike the well-established covalent ligand capping of conventional semiconductor NCs6,7. We posited that the vast and facile molecular engineering of phospholipids as zwitterionic surfactants can deliver highly customized surface chemistries for metal halide NCs. Molecular dynamics simulations implied that ligand-NC surface affinity is primarily governed by the structure of the zwitterionic head group, particularly by the geometric fitness of the anionic and cationic moieties into the surface lattice sites, as corroborated by the nuclear magnetic resonance and Fourier-transform infrared spectroscopy data. Lattice-matched primary-ammonium phospholipids enhance the structural and colloidal integrity of hybrid organic-inorganic lead halide perovskites (FAPbBr3 and MAPbBr3 (FA, formamidinium; MA, methylammonium)) and lead-free metal halide NCs. The molecular structure of the organic ligand tail governs the long-term colloidal stability and compatibility with solvents of diverse polarity, from hydrocarbons to acetone and alcohols. These NCs exhibit photoluminescence quantum yield of more than 96% in solution and solids and minimal photoluminescence intermittency at the single particle level with an average ON fraction as high as 94%, as well as bright and high-purity (about 95%) single-photon emission.
Assuntos
Desenho de Fármacos , Ligantes , Nanopartículas Metálicas , Pontos Quânticos , Acetona/química , Álcoois/química , Ânions , Compostos de Cálcio/química , Cátions , Coloides/química , Chumbo , Medições Luminescentes , Espectroscopia de Ressonância Magnética , Nanopartículas Metálicas/química , Simulação de Dinâmica Molecular , Óxidos/química , Fosfolipídeos/química , Pontos Quânticos/química , Solventes/química , Espectroscopia de Infravermelho com Transformada de Fourier , Titânio/químicaRESUMO
Ions surround nucleic acids in what is referred to as an ion atmosphere. As a result, the folding and dynamics of RNA and DNA and their complexes with proteins and with each other cannot be understood without a reasonably sophisticated appreciation of these ions' electrostatic interactions. However, the underlying behavior of the ion atmosphere follows physical rules that are distinct from the rules of site binding that biochemists are most familiar and comfortable with. The main goal of this review is to familiarize nucleic acid experimentalists with the physical concepts that underlie nucleic acid-ion interactions. Throughout, we provide practical strategies for interpreting and analyzing nucleic acid experiments that avoid pitfalls from oversimplified or incorrect models. We briefly review the status of theories that predict or simulate nucleic acid-ion interactions and experiments that test these theories. Finally, we describe opportunities for going beyond phenomenological fits to a next-generation, truly predictive understanding of nucleic acid-ion interactions.
Assuntos
Íons/química , Ácidos Nucleicos/química , Algoritmos , Sítios de Ligação , Cátions , Cristalografia por Raios X , DNA/química , Magnésio/química , Metais/química , Modelos Teóricos , Conformação de Ácido Nucleico , Distribuição de Poisson , RNA/química , Software , Eletricidade Estática , TermodinâmicaRESUMO
Ion channels establish the voltage gradient across cellular membranes by providing aqueous pathways for ions to selectively diffuse down their concentration gradients. The selectivity of any given channel for its favored ions has conventionally been viewed as a stable property, and in many cation channels, it is determined by an ion-selectivity filter within the external end of the ion-permeation pathway. In several instances, including voltage-activated K+ (Kv) channels, ATP-activated P2X receptor channels, and transient receptor potential (TRP) channels, the ion-permeation pathways have been proposed to dilate in response to persistent activation, dynamically altering ion permeation. Here, we discuss evidence for dynamic ion selectivity, examples where ion selectivity filters exhibit structural plasticity, and opportunities to fill gaps in our current understanding.
Assuntos
Canais Iônicos , Humanos , Canais Iônicos/metabolismo , Canais Iônicos/química , Cátions/metabolismo , Cátions/química , Animais , Ativação do Canal IônicoRESUMO
The membrane translocation of hydrophilic substances constitutes a challenge for their application as therapeutic compounds and labelling probes1-4. To remedy this, charged amphiphilic molecules have been classically used as carriers3,5. However, such amphiphilic carriers may cause aggregation and non-specific membrane lysis6,7. Here we show that globular dodecaborate clusters, and prominently B12Br122-, can function as anionic inorganic membrane carriers for a broad range of hydrophilic cargo molecules (with molecular mass of 146-4,500 Da). We show that cationic and neutral peptides, amino acids, neurotransmitters, vitamins, antibiotics and drugs can be carried across liposomal membranes. Mechanistic transport studies reveal that the carrier activity is related to the superchaotropic nature of these cluster anions8-12. We demonstrate that B12Br122- affects cytosolic uptake of different small bioactive molecules, including the antineoplastic monomethyl auristatin F, the proteolysis targeting chimera dBET1 and the phalloidin toxin, which has been successfully delivered in living cells for cytoskeleton labelling. We anticipate the broad and distinct delivery spectrum of our superchaotropic carriers to be the starting point of conceptually distinct cell-biological, neurobiological, physiological and pharmaceutical studies.
Assuntos
Boro , Peptídeos , Ânions/química , Transporte Biológico , Cátions , Portadores de Fármacos/química , Interações Hidrofóbicas e Hidrofílicas , Peptídeos/química , Preparações FarmacêuticasRESUMO
The productivity of rainforests growing on highly weathered tropical soils is expected to be limited by phosphorus availability1. Yet, controlled fertilization experiments have been unable to demonstrate a dominant role for phosphorus in controlling tropical forest net primary productivity. Recent syntheses have demonstrated that responses to nitrogen addition are as large as to phosphorus2, and adaptations to low phosphorus availability appear to enable net primary productivity to be maintained across major soil phosphorus gradients3. Thus, the extent to which phosphorus availability limits tropical forest productivity is highly uncertain. The majority of the Amazonia, however, is characterized by soils that are more depleted in phosphorus than those in which most tropical fertilization experiments have taken place2. Thus, we established a phosphorus, nitrogen and base cation addition experiment in an old growth Amazon rainforest, with a low soil phosphorus content that is representative of approximately 60% of the Amazon basin. Here we show that net primary productivity increased exclusively with phosphorus addition. After 2 years, strong responses were observed in fine root (+29%) and canopy productivity (+19%), but not stem growth. The direct evidence of phosphorus limitation of net primary productivity suggests that phosphorus availability may restrict Amazon forest responses to CO2 fertilization4, with major implications for future carbon sequestration and forest resilience to climate change.
Assuntos
Mudança Climática , Fósforo , Floresta Úmida , Solo , Árvores , Clima Tropical , Aclimatação , Dióxido de Carbono/metabolismo , Dióxido de Carbono/farmacologia , Sequestro de Carbono , Cátions/metabolismo , Cátions/farmacologia , Mudança Climática/estatística & dados numéricos , Modelos Biológicos , Nitrogênio/metabolismo , Nitrogênio/farmacologia , Fósforo/metabolismo , Fósforo/farmacologia , Solo/química , Árvores/efeitos dos fármacos , Árvores/metabolismo , IncertezaRESUMO
Plant intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) detect pathogen effectors to trigger immune responses1. Indirect recognition of a pathogen effector by the dicotyledonous Arabidopsis thaliana coiled-coil domain containing NLR (CNL) ZAR1 induces the formation of a large hetero-oligomeric protein complex, termed the ZAR1 resistosome, which functions as a calcium channel required for ZAR1-mediated immunity2-4. Whether the resistosome and channel activities are conserved among plant CNLs remains unknown. Here we report the cryo-electron microscopy structure of the wheat CNL Sr355 in complex with the effector AvrSr356 of the wheat stem rust pathogen. Direct effector binding to the leucine-rich repeats of Sr35 results in the formation of a pentameric Sr35-AvrSr35 complex, which we term the Sr35 resistosome. Wheat Sr35 and Arabidopsis ZAR1 resistosomes bear striking structural similarities, including an arginine cluster in the leucine-rich repeats domain not previously recognized as conserved, which co-occurs and forms intramolecular interactions with the 'EDVID' motif in the coiled-coil domain. Electrophysiological measurements show that the Sr35 resistosome exhibits non-selective cation channel activity. These structural insights allowed us to generate new variants of closely related wheat and barley orphan NLRs that recognize AvrSr35. Our data support the evolutionary conservation of CNL resistosomes in plants and demonstrate proof of principle for structure-based engineering of NLRs for crop improvement.
Assuntos
Canais de Cálcio , Microscopia Crioeletrônica , Proteínas NLR , Proteínas de Plantas , Receptores Imunológicos , Triticum , Arabidopsis/imunologia , Arabidopsis/metabolismo , Arginina , Canais de Cálcio/química , Canais de Cálcio/imunologia , Canais de Cálcio/metabolismo , Cátions/metabolismo , Leucina , Proteínas NLR/química , Proteínas NLR/imunologia , Proteínas NLR/metabolismo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Triticum/imunologia , Triticum/metabolismo , Motivos de Aminoácidos , Sequência Conservada , EletrofisiologiaRESUMO
Localized ion fluxes at the plasma membrane provide electrochemical gradients at the cell surface that contribute to cell polarization, migration, and division. Ion transporters, local pH gradients, membrane potential, and organization are emerging as important factors in cell polarization mechanisms. The power of electrochemical effects is illustrated by the ability of exogenous electric fields to redirect polarization in cells ranging from bacteria, fungi, and amoebas to keratocytes and neurons. Electric fields normally surround cells and tissues and thus have been proposed to guide cell polarity in development, cancer, and wound healing. Recent studies on electric field responses in model systems and development of new biosensors provide new avenues to dissect molecular mechanisms. Here, we review recent advances that bring molecular understanding of how electrochemistry contributes to cell polarity in various contexts.
Assuntos
Polaridade Celular/fisiologia , Animais , Ânions/metabolismo , Cátions/metabolismo , Divisão Celular , Movimento Celular , Forma Celular , Dictyostelium/citologia , Eletroquímica , Campos Eletromagnéticos , Peixes , Fungos/citologia , Concentração de Íons de Hidrogênio , Líquido Intracelular/química , Transporte de Íons/fisiologia , Potenciais da Membrana/fisiologia , Regeneração , Eletricidade Estática , CicatrizaçãoRESUMO
Control of molecular chirality is a fundamental challenge in organic synthesis. Whereas methods to construct carbon stereocentres enantioselectively are well established, routes to synthesize enriched heteroatomic stereocentres have garnered less attention1-5. Of those atoms commonly present in organic molecules, nitrogen is the most difficult to control stereochemically. Although a limited number of resolution processes have been demonstrated6-8, no general methodology exists to enantioselectively prepare a nitrogen stereocentre. Here we show that control of the chirality of ammonium cations is easily achieved through a supramolecular recognition process. By combining enantioselective ammonium recognition mediated by 1,1'-bi-2-naphthol scaffolds with conditions that allow the nitrogen stereocentre to racemize, chiral ammonium cations can be produced in excellent yields and selectivities. Mechanistic investigations demonstrate that, through a combination of solution and solid-phase recognition, a thermodynamically driven adductive crystallization process is responsible for the observed selectivity. Distinct from processes based on dynamic and kinetic resolution, which are under kinetic control, this allows for increased selectivity over time by a self-corrective process. The importance of nitrogen stereocentres can be revealed through a stereoselective supramolecular recognition, which is not possible with naturally occurring pseudoenantiomeric Cinchona alkaloids. With practical access to the enantiomeric forms of ammonium cations, this previously ignored stereocentre is now available to be explored.
Assuntos
Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/síntese química , Cátions/síntese química , Cátions/química , Alcaloides de Cinchona/química , Nitrogênio/química , Estereoisomerismo , TermodinâmicaRESUMO
Adding a cationic helper lipid to a lipid nanoparticle (LNP) can increase lung delivery and decrease liver delivery. However, it remains unclear whether charge-dependent tropism is universal or, alternatively, whether it depends on the component that is charged. Here, we report evidence that cationic cholesterol-dependent tropism can differ from cationic helper lipid-dependent tropism. By testing how 196 LNPs delivered mRNA to 22 cell types, we found that charged cholesterols led to a different lung:liver delivery ratio than charged helper lipids. We also found that combining cationic cholesterol with a cationic helper lipid led to mRNA delivery in the heart as well as several lung cell types, including stem cell-like populations. These data highlight the utility of exploring charge-dependent LNP tropism.
Assuntos
Fígado , Células-Tronco , Coração , Cátions , Colesterol , RNA MensageiroRESUMO
CodB is a cytosine transporter from the Nucleobase-Cation-Symport-1 (NCS1) transporter family, a member of the widespread LeuT superfamily. Previous experiments with the nosocomial pathogen Pseudomonas aeruginosa have shown CodB as also important for the uptake of 5-fluorocytosine, which has been suggested as a novel drug to combat antimicrobial resistance by suppressing virulence. Here we solve the crystal structure of CodB from Proteus vulgaris, at 2.4 Å resolution in complex with cytosine. We show that CodB carries out the sodium-dependent uptake of cytosine and can bind 5-fluorocytosine. Comparison of the substrate-bound structures of CodB and the hydantoin transporter Mhp1, the only other NCS1 family member for which the structure is known, highlight the importance of the hydrogen bonds that the substrates make with the main chain at the breakpoint in the discontinuous helix, TM6. In contrast to other LeuT superfamily members, neither CodB nor Mhp1 makes specific interactions with residues on TM1. Comparison of the structures provides insight into the intricate mechanisms of how these proteins transport substrates across the plasma membrane.
Assuntos
Simportadores , Transporte Biológico , Cátions , Citosina , Flucitosina , Proteínas de Membrana Transportadoras , Simportadores/genéticaRESUMO
The cyclic GMP-AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway is instrumental to antitumor immunity, yet the underlying molecular and cellular mechanisms are complex and still unfolding. A new paradigm suggests that cancer cells' cGAS-synthesized cGAMP can be transferred to tumor-infiltrating immune cells, eliciting STING-dependent IFN-ß response for antitumor immunity. Nevertheless, how the tumor microenvironment may shape this process remains unclear. In this study, we found that extracellular ATP, an immune regulatory molecule widely present in the tumor microenvironment, can potentiate cGAMP transfer, thereby boosting the STING signaling and IFN-ß response in murine macrophages and fibroblasts. Notably, genetic ablation or chemical inhibition of murine volume-regulation anion channel LRRC8/volume-regulated anion channel (VRAC), a recently identified cGAMP transporter, abolished ATP-potentiated cGAMP transfer and STING-dependent IFN-ß response, revealing a crucial role of LRRC8/VRAC in the cross-talk of extracellular ATP and cGAMP. Mechanistically, ATP activation of the P2X family receptors triggered Ca2+ influx and K+ efflux, promoting reactive oxygen species production. Moreover, ATP-evoked K+ efflux alleviated the phosphorylation of VRAC's obligate subunit LRRC8A/SWELL1 on S174. Mutagenesis studies indicated that the phosphorylation of S174 on LRRC8A could act as a checkpoint for VRAC in the steady state and a rheostat of ATP responsiveness. In an MC38-transplanted tumor model, systemically blocking CD39 and ENPP1, hydroxylases of extracellular ATP and cGAMP, respectively, elevated antitumor NK, NKT, and CD8+ T cell responses and restrained tumor growth in mice. Altogether, this study establishes a crucial role of ATP in facilitating LRRC8/VRAC transport cGAMP in the tumor microenvironment and provides new insight into harnessing cGAMP transfer for antitumor immunity.
Assuntos
Trifosfato de Adenosina , Proteínas de Membrana , Nucleotídeos Cíclicos , Microambiente Tumoral , Animais , Nucleotídeos Cíclicos/metabolismo , Camundongos , Trifosfato de Adenosina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/imunologia , Microambiente Tumoral/imunologia , Interferon beta/metabolismo , Interferon beta/imunologia , Camundongos Endogâmicos C57BL , Humanos , Transdução de Sinais/imunologia , Camundongos Knockout , Linhagem Celular Tumoral , Cátions/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Nucleotidiltransferases/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismoRESUMO
Tertiary stereogenic centres containing one fluorine atom are valuable for medicinal chemistry because they mimic common tertiary stereogenic centres containing one hydrogen atom, but they possess distinct charge distribution, lipophilicity, conformation and metabolic stability1-3. Although tertiary stereogenic centres containing one hydrogen atom are often set by enantioselective desymmetrization reactions at one of the two carbon-hydrogen (C-H) bonds of a methylene group, tertiary stereocentres containing fluorine have not yet been constructed by the analogous desymmetrization reaction at one of the two carbon-fluorine (C-F) bonds of a difluoromethylene group3. Fluorine atoms are similar in size to hydrogen atoms but have distinct electronic properties, causing C-F bonds to be exceptionally strong and geminal C-F bonds to strengthen one another4. Thus, exhaustive defluorination typically dominates over the selective replacement of a single C-F bond, hindering the development of the enantioselective substitution of one fluorine atom to form a stereogenic centre5,6. Here we report the catalytic, enantioselective activation of a single C-F bond in an allylic difluoromethylene group to provide a broad range of products containing a monofluorinated tertiary stereogenic centre. By combining a tailored chiral iridium phosphoramidite catalyst, which controls regioselectivity, chemoselectivity and enantioselectivity, with a fluorophilic activator, which assists the oxidative addition of the C-F bond, these reactions occur in high yield and selectivity. The design principles proposed in this work extend to palladium-catalysed benzylic substitution, demonstrating the generality of the approach.
Assuntos
Carbono/química , Flúor/química , Alcenos/química , Catálise , Cátions , Halogenação , Hidrogênio/química , Irídio/química , Compostos Organofosforados/química , Oxirredução , Paládio/químicaRESUMO
Transient receptor potential vanilloid member 1 (TRPV1) is a heat and capsaicin receptor that allows cations to permeate and cause pain. As the molecular basis for temperature sensing, the heat capacity (ΔCp) model [D. E. Clapham, C. Miller, Proc. Natl. Acad. Sci. U.S.A. 108, 19492-19497 (2011).] has been proposed and experimentally supported. Theoretically, heat capacity is proportional to a variance in enthalpy, presumably related to structural fluctuation; however, the fluctuation of TRPV1 has not been directly visualized. In this study, we directly visualized single-molecule structural fluctuations of the TRPV1 channels in a lipid bilayer with the ligands resiniferatoxin (agonist, 1,000 times hotter than capsaicin) and capsazepine (antagonist) by high-speed atomic force microscopy. We observed the structural fluctuations of TRPV1 in an apo state and found that RTX binding enhances structural fluctuations, while CPZ binding suppresses fluctuations. These ligand-dependent differences in structural fluctuation would play a key role in the gating of TRPV1.
Assuntos
Diterpenos , Canais de Potencial de Receptor Transitório , Capsaicina/farmacologia , Capsaicina/metabolismo , Canais de Cátion TRPV/metabolismo , Temperatura Alta , Cátions/metabolismo , Diterpenos/metabolismoRESUMO
Although the tremendous progress has been made for mRNA delivery based on classical cationic carriers, the excess cationic charge density of lipids was necessary to compress mRNA through electrostatic interaction, and with it comes inevitably adverse events including the highly inflammatory and cytotoxic effects. How to develop the disruptive technologies to overcome cationic nature of lipids remains a major challenge for safe and efficient mRNA delivery. Here, we prepared noncationic thiourea lipids nanoparticles (NC-TNP) to compress mRNA by strong hydrogen bonds interaction between thiourea groups of NC-TNP and the phosphate groups of mRNA, abandoning the hidebound and traditional electrostatic force to construct mRNA-cationic lipids formulation. NC-TNP was a delivery system for mRNA with simple, convenient, and repeatable preparation technology and showed negligible inflammatory and cytotoxicity side effects. Furthermore, we found that NC-TNP could escape the recycling pathway to inhibit the egress of internalized nanoparticles from the intracellular compartment to the extracellular milieu which was a common fact in mRNA-LNP (lipid nanoparticles) formulation. Therefore, NC-TNP-encapsulated mRNA showed higher gene transfection efficiency in vitro and in vivo than mRNA-LNP formulation. Unexpectedly, NC-TNP showed spleen targeting delivery ability with higher accumulation ratio (spleen/liver), compared with traditional LNP. Spleen-targeting NC-TNP with mRNA exhibited high mRNA-encoded antigen expression in spleen and elicited robust immune responses. Overall, our work establishes a proof of concept for the construction of a noncationic system for mRNA delivery with good inflammatory safety profiles, high gene transfection efficiency, and spleen-targeting delivery to induce permanent and robust humoral and cell-mediated immunity for disease treatments.