RESUMO
Hearing involves two fundamental processes: mechano-electrical transduction and signal amplification. Despite decades of studies, the molecular bases for both remain elusive. Here, we show how prestin, the electromotive molecule of outer hair cells (OHCs) that senses both voltage and membrane tension, mediates signal amplification by coupling conformational changes to alterations in membrane surface area. Cryoelectron microscopy (cryo-EM) structures of human prestin bound with chloride or salicylate at a common "anion site" adopt contracted or expanded states, respectively. Prestin is ensconced within a perimeter of well-ordered lipids, through which it induces dramatic deformation in the membrane and couples protein conformational changes to the bulk membrane. Together with computational studies, we illustrate how the anion site is allosterically coupled to changes in the transmembrane domain cross-sectional area and the surrounding membrane. These studies provide insight into OHC electromotility by providing a structure-based mechanism of the membrane motor prestin.
Assuntos
Fenômenos Eletrofisiológicos , Transportadores de Sulfato/metabolismo , Ânions , Sítios de Ligação , Cloretos/metabolismo , Microscopia Crioeletrônica , Células HEK293 , Humanos , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Domínios Proteicos , Multimerização Proteica , Estabilidade Proteica , Ácido Salicílico/metabolismo , Homologia Estrutural de Proteína , Transportadores de Sulfato/química , Transportadores de Sulfato/ultraestruturaRESUMO
Plant immunity is activated upon pathogen perception and often affects growth and yield when it is constitutively active. How plants fine-tune immune homeostasis in their natural habitats remains elusive. Here, we discover a conserved immune suppression network in cereals that orchestrates immune homeostasis, centering on a Ca2+-sensor, RESISTANCE OF RICE TO DISEASES1 (ROD1). ROD1 promotes reactive oxygen species (ROS) scavenging by stimulating catalase activity, and its protein stability is regulated by ubiquitination. ROD1 disruption confers resistance to multiple pathogens, whereas a natural ROD1 allele prevalent in indica rice with agroecology-specific distribution enhances resistance without yield penalty. The fungal effector AvrPiz-t structurally mimics ROD1 and activates the same ROS-scavenging cascade to suppress host immunity and promote virulence. We thus reveal a molecular framework adopted by both host and pathogen that integrates Ca2+ sensing and ROS homeostasis to suppress plant immunity, suggesting a principle for breeding disease-resistant, high-yield crops.
Assuntos
Cálcio/metabolismo , Sequestradores de Radicais Livres/metabolismo , Proteínas Fúngicas/metabolismo , Oryza/imunologia , Imunidade Vegetal , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sistemas CRISPR-Cas/genética , Membrana Celular/metabolismo , Resistência à Doença/genética , Modelos Biológicos , Oryza/genética , Doenças das Plantas/imunologia , Proteínas de Plantas/genética , Ligação Proteica , Estabilidade Proteica , Reprodução , Especificidade da Espécie , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Zea mays/imunologiaRESUMO
Defects in translation lead to changes in the expression of proteins that can serve as drivers of cancer formation. Here, we show that cytosolic NAD+ synthesis plays an essential role in ovarian cancer by regulating translation and maintaining protein homeostasis. Expression of NMNAT-2, a cytosolic NAD+ synthase, is highly upregulated in ovarian cancers. NMNAT-2 supports the catalytic activity of the mono(ADP-ribosyl) transferase (MART) PARP-16, which mono(ADP-ribosyl)ates (MARylates) ribosomal proteins. Depletion of NMNAT-2 or PARP-16 leads to inhibition of MARylation, increased polysome association and enhanced translation of specific mRNAs, aggregation of their translated protein products, and reduced growth of ovarian cancer cells. Furthermore, MARylation of the ribosomal proteins, such as RPL24 and RPS6, inhibits polysome assembly by stabilizing eIF6 binding to ribosomes. Collectively, our results demonstrate that ribosome MARylation promotes protein homeostasis in cancers by fine-tuning the levels of protein synthesis and preventing toxic protein aggregation.
Assuntos
ADP-Ribosilação , Neoplasias Ovarianas/metabolismo , Biossíntese de Proteínas , Proteostase , Ribossomos/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Estresse do Retículo Endoplasmático , Tubas Uterinas/metabolismo , Feminino , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , NAD/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase , Conformação de Ácido Nucleico , Neoplasias Ovarianas/patologia , Poli(ADP-Ribose) Polimerases/metabolismo , Polirribossomos/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Ribossômicas/metabolismoRESUMO
Serotonin plays a central role in cognition and is the target of most pharmaceuticals for psychiatric disorders. Existing drugs have limited efficacy; creation of improved versions will require better understanding of serotonergic circuitry, which has been hampered by our inability to monitor serotonin release and transport with high spatial and temporal resolution. We developed and applied a binding-pocket redesign strategy, guided by machine learning, to create a high-performance, soluble, fluorescent serotonin sensor (iSeroSnFR), enabling optical detection of millisecond-scale serotonin transients. We demonstrate that iSeroSnFR can be used to detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep/wake transitions. We also developed a robust assay of serotonin transporter function and modulation by drugs. We expect that both machine-learning-guided binding-pocket redesign and iSeroSnFR will have broad utility for the development of other sensors and in vitro and in vivo serotonin detection, respectively.
Assuntos
Evolução Molecular Direcionada , Aprendizado de Máquina , Serotonina/metabolismo , Algoritmos , Sequência de Aminoácidos , Tonsila do Cerebelo/fisiologia , Animais , Comportamento Animal , Sítios de Ligação , Encéfalo/metabolismo , Células HEK293 , Humanos , Cinética , Modelos Lineares , Camundongos , Camundongos Endogâmicos C57BL , Fótons , Ligação Proteica , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Sono/fisiologia , Vigília/fisiologiaRESUMO
Reactive oxygen species (ROS) encompass a collection of intricately linked chemical entities characterized by individually distinct physicochemical properties and biological reactivities. Although excessive ROS generation is well known to underpin disease development, it has become increasingly evident that ROS also play central roles in redox regulation and normal physiology. A major challenge in uncovering the relevant biological mechanisms and deconvoluting the apparently paradoxical roles of distinct ROS in human health and disease lies in the selective and sensitive detection of these transient species in the complex biological milieu. Small-molecule-based fluorescent sensors enable molecular imaging of ROS with great spatial and temporal resolution and have thus been appreciated as excellent tools for aiding discoveries in modern redox biology. We review a selection of state-of-the-art sensors with demonstrated utility in biological systems. By providing a systematic overview based on underlying chemical sensing mechanisms, we wish to highlight the strengths and weaknesses in prior sensor works and propose some guiding principles for the development of future probes.
Assuntos
Técnicas Biossensoriais/métodos , Espécies Reativas de Oxigênio/análise , Corantes Fluorescentes , Imagem Óptica , Oxirredução , Estresse OxidativoRESUMO
Varying pH of luminal fluid along the female reproductive tract is a physiological cue that modulates sperm motility. CatSper is a sperm-specific, pH-sensitive calcium channel essential for hyperactivated motility and male fertility. Multi-subunit CatSper channel complexes organize linear Ca2+ signaling nanodomains along the sperm tail. Here, we identify EF-hand calcium-binding domain-containing protein 9 (EFCAB9) as a bifunctional, cytoplasmic machine modulating the channel activity and the domain organization of CatSper. Knockout mice studies demonstrate that EFCAB9, in complex with the CatSper subunit, CATSPERζ, is essential for pH-dependent and Ca2+-sensitive activation of the CatSper channel. In the absence of EFCAB9, sperm motility and fertility is compromised, and the linear arrangement of the Ca2+ signaling domains is disrupted. EFCAB9 interacts directly with CATSPERζ in a Ca2+-dependent manner and dissociates at elevated pH. These observations suggest that EFCAB9 is a long-sought, intracellular, pH-dependent Ca2+ sensor that triggers changes in sperm motility.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Motilidade dos Espermatozoides/fisiologia , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Proteínas de Ligação ao Cálcio/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Fertilidade , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espermatozoides/metabolismoRESUMO
Voltage-gated sodium (NaV) channels initiate action potentials in nerve, muscle, and other electrically excitable cells. The structural basis of voltage gating is uncertain because the resting state exists only at deeply negative membrane potentials. To stabilize the resting conformation, we inserted voltage-shifting mutations and introduced a disulfide crosslink in the VS of the ancestral bacterial sodium channel NaVAb. Here, we present a cryo-EM structure of the resting state and a complete voltage-dependent gating mechanism. The S4 segment of the VS is drawn intracellularly, with three gating charges passing through the transmembrane electric field. This movement forms an elbow connecting S4 to the S4-S5 linker, tightens the collar around the S6 activation gate, and prevents its opening. Our structure supports the classical "sliding helix" mechanism of voltage sensing and provides a complete gating mechanism for voltage sensor function, pore opening, and activation-gate closure based on high-resolution structures of a single sodium channel protein.
Assuntos
Potenciais de Ação/fisiologia , Membrana Externa Bacteriana/metabolismo , Escherichia coli/metabolismo , Ativação do Canal Iônico/fisiologia , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Linhagem Celular , Microscopia Crioeletrônica , Cristalografia por Raios X , Mutação , Conformação Proteica em alfa-Hélice , Sódio/metabolismo , Spodoptera/citologia , Canais de Sódio Disparados por Voltagem/químicaRESUMO
Dopamine (DA) is a central monoamine neurotransmitter involved in many physiological and pathological processes. A longstanding yet largely unmet goal is to measure DA changes reliably and specifically with high spatiotemporal precision, particularly in animals executing complex behaviors. Here, we report the development of genetically encoded GPCR-activation-based-DA (GRABDA) sensors that enable these measurements. In response to extracellular DA, GRABDA sensors exhibit large fluorescence increases (ΔF/F0 â¼90%) with subcellular resolution, subsecond kinetics, nanomolar to submicromolar affinities, and excellent molecular specificity. GRABDA sensors can resolve a single-electrical-stimulus-evoked DA release in mouse brain slices and detect endogenous DA release in living flies, fish, and mice. In freely behaving mice, GRABDA sensors readily report optogenetically elicited nigrostriatal DA release and depict dynamic mesoaccumbens DA signaling during Pavlovian conditioning or during sexual behaviors. Thus, GRABDA sensors enable spatiotemporally precise measurements of DA dynamics in a variety of model organisms while exhibiting complex behaviors.
Assuntos
Dopamina/análise , Drosophila/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Comportamento Animal , Dopamina/metabolismo , Feminino , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Neurônios/citologia , Neurônios/metabolismo , Optogenética/métodos , Receptores Acoplados a Proteínas G/genética , Canais de Cátion TRPV/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.
Assuntos
Encéfalo/metabolismo , Técnicas de Inativação de Genes/métodos , Genes Reporter , Animais , Encéfalo/citologia , Cálcio/metabolismo , Linhagem Celular , Hibridização in Situ Fluorescente , Luz , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Neurônios/metabolismo , Optogenética , RNA não Traduzido/genética , Transgenes/genéticaRESUMO
Viruses employ elaborate strategies to coopt the cellular processes they require to replicate while simultaneously thwarting host antiviral responses. In many instances, how this is accomplished remains poorly understood. Here, we identify a protein, F17 encoded by cytoplasmically replicating poxviruses, that binds and sequesters Raptor and Rictor, regulators of mammalian target of rapamycin complexes mTORC1 and mTORC2, respectively. This disrupts mTORC1-mTORC2 crosstalk that coordinates host responses to poxvirus infection. During infection with poxvirus lacking F17, cGAS accumulates together with endoplasmic reticulum vesicles around the Golgi, where activated STING puncta form, leading to interferon-stimulated gene expression. By contrast, poxvirus expressing F17 dysregulates mTOR, which localizes to the Golgi and blocks these antiviral responses in part through mTOR-dependent cGAS degradation. Ancestral conservation of Raptor/Rictor across eukaryotes, along with expression of F17 across poxviruses, suggests that mTOR dysregulation forms a conserved poxvirus strategy to counter cytosolic sensing while maintaining the metabolic benefits of mTOR activity.
Assuntos
Citosol/química , Poxviridae/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Proteína Regulatória Associada a mTOR/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Células HEK293 , Homeostase , Humanos , Imunidade Inata , Interferons/metabolismo , Cinética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
Hedgehog protein signals mediate tissue patterning and maintenance by binding to and inactivating their common receptor Patched, a 12-transmembrane protein that otherwise would suppress the activity of the 7-transmembrane protein Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. A central hydrophobic conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Cholesterol activity in the inner leaflet of the plasma membrane is reduced by PTCH1 expression but rapidly restored by Hedgehog stimulation, suggesting that PTCH1 regulates Smoothened by controlling cholesterol availability.
Assuntos
Colesterol/metabolismo , Proteínas Hedgehog/metabolismo , Receptor Patched-1/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Microscopia Crioeletrônica , Dimerização , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Evolução Molecular , Células HEK293 , Proteínas Hedgehog/química , Proteínas Hedgehog/genética , Humanos , Camundongos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Receptor Patched-1/química , Receptor Patched-1/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Transdução de SinaisRESUMO
Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) critically contribute to the efficacy of anti-tumor therapeutic antibodies. We report here an unexpected finding that macrophages after ADCP inhibit NK cell-mediated ADCC and T cell-mediated cytotoxicity in breast cancers and lymphomas. Mechanistically, AIM2 is recruited to the phagosomes by FcγR signaling following ADCP and activated by sensing the phagocytosed tumor DNAs through the disrupted phagosomal membrane, which subsequently upregulates PD-L1 and IDO and causes immunosuppression. Combined treatment with anti-HER2 antibody and inhibitors of PD-L1 and IDO enhances anti-tumor immunity and anti-HER2 therapeutic efficacy in mouse models. Furthermore, neoadjuvant trastuzumab therapy significantly upregulates PD-L1 and IDO in the tumor-associated macrophages (TAMs) of HER2+ breast cancer patients, correlating with poor trastuzumab response. Collectively, our findings unveil a deleterious role of ADCP macrophages in cancer immunosuppression and suggest that therapeutic antibody plus immune checkpoint blockade may provide synergistic effects in cancer treatment.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/imunologia , Citofagocitose/imunologia , Macrófagos/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos/fisiologia , Antígeno B7-H1/genética , Antígeno B7-H1/fisiologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Citofagocitose/fisiologia , Proteínas de Ligação a DNA/fisiologia , Modelos Animais de Doenças , Feminino , Humanos , Imunoterapia , Células Matadoras Naturais/fisiologia , Linfoma/imunologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fagocitose/imunologia , Fagocitose/fisiologia , Fagossomos/fisiologia , Receptores de IgG/imunologiaRESUMO
Mitochondrial dynamics regulated by mitochondrial fusion and fission maintain mitochondrial functions, whose alterations underline various human diseases. Here, we show that inositol is a critical metabolite directly restricting AMPK-dependent mitochondrial fission independently of its classical mode as a precursor for phosphoinositide generation. Inositol decline by IMPA1/2 deficiency elicits AMPK activation and mitochondrial fission without affecting ATP level, whereas inositol accumulation prevents AMPK-dependent mitochondrial fission. Metabolic stress or mitochondrial damage causes inositol decline in cells and mice to elicit AMPK-dependent mitochondrial fission. Inositol directly binds to AMPKγ and competes with AMP for AMPKγ binding, leading to restriction of AMPK activation and mitochondrial fission. Our study suggests that the AMP/inositol ratio is a critical determinant for AMPK activation and establishes a model in which AMPK activation requires inositol decline to release AMPKγ for AMP binding. Hence, AMPK is an inositol sensor, whose inactivation by inositol serves as a mechanism to restrict mitochondrial fission.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Inositol/metabolismo , Dinâmica Mitocondrial/fisiologia , Proteínas Quinases Ativadas por AMP/fisiologia , Animais , Linhagem Celular , Humanos , Inositol/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Células PC-3 , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Estresse Fisiológico/fisiologiaRESUMO
Sex determination in animals is not only determined by karyotype but can also be modulated by environmental cues like temperature via unclear transduction mechanisms. Moreover, in contrast to earlier views that sex may exclusively be determined by either karyotype or temperature, recent observations suggest that these factors rather co-regulate sex, posing another mechanistic mystery. Here, we discovered that certain wild-isolated and mutant C. elegans strains displayed genotypic germline sex determination (GGSD), but with a temperature-override mechanism. Further, we found that BiP, an ER chaperone, transduces temperature information into a germline sex-governing signal, thereby enabling the coexistence of GGSD and temperature-dependent germline sex determination (TGSD). At the molecular level, increased ER protein-folding requirements upon increased temperatures lead to BiP sequestration, resulting in ERAD-dependent degradation of the oocyte fate-driving factor, TRA-2, thus promoting male germline fate. Remarkably, experimentally manipulating BiP or TRA-2 expression allows to switch between GGSD and TGSD. Physiologically, TGSD allows C. elegans hermaphrodites to maintain brood size at warmer temperatures. Moreover, BiP can also influence germline sex determination in a different, non-hermaphroditic nematode species. Collectively, our findings identify thermosensitive BiP as a conserved temperature sensor in TGSD, and provide mechanistic insights into the transition between GGSD and TGSD.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Células Germinativas , Processos de Determinação Sexual , Temperatura , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Masculino , Células Germinativas/metabolismo , Feminino , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genéticaRESUMO
Detection of cytosolic DNA by the enzyme cGAS triggers the production of cGAMP, a second messenger that binds and activates the adaptor protein STING, which leads to interferon (IFN) production. Here, we found that in vivo natural killer (NK) cell killing of tumor cells, but not of normal cells, depends on STING expression in non-tumor cells. Experiments using transplantable tumor models in STING- and cGAS-deficient mice revealed that cGAS expression by tumor cells was critical for tumor rejection by NK cells. In contrast, cGAS expression by host cells was dispensable, suggesting that tumor-derived cGAMP is transferred to non-tumor cells, where it activates STING. cGAMP administration triggered STING activation and IFN-ß production in myeloid cells and B cells but not NK cells. Our results reveal that the anti-tumor response of NK cells critically depends on the cytosolic DNA sensing pathway, similar to its role in defense against pathogens, and identify tumor-derived cGAMP as a major determinant of tumor immunogenicity with implications for cancer immunotherapy.
Assuntos
Interferons/imunologia , Células Matadoras Naturais/imunologia , Proteínas de Membrana/imunologia , Neoplasias/imunologia , Nucleotídeos Cíclicos/imunologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Regulação da Expressão Gênica/imunologia , Humanos , Interferons/metabolismo , Células Matadoras Naturais/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/genética , Neoplasias/metabolismo , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Nucleotidiltransferases/metabolismo , Transdução de Sinais/imunologiaRESUMO
We developed a significantly improved genetically encoded quantitative adenosine triphosphate (ATP) sensor to provide real-time dynamics of ATP levels in subcellular compartments. iATPSnFR2 is a variant of iATPSnFR1, a previously developed sensor that has circularly permuted superfolder green fluorescent protein (GFP) inserted between the ATP-binding helices of the ε-subunit of a bacterial F0-F1 ATPase. Optimizing the linkers joining the two domains resulted in a ~fivefold to sixfold improvement in the dynamic range compared to the previous-generation sensor, with excellent discrimination against other analytes, and affinity variants varying from 4 µM to 500 µM. A chimeric version of this sensor fused to either the HaloTag protein or a suitable spectrally separated fluorescent protein provides an optional ratiometric readout allowing comparisons of ATP across cellular regions. Subcellular targeting the sensor to nerve terminals reveals previously uncharacterized single-synapse metabolic signatures, while targeting to the mitochondrial matrix allowed direct quantitative probing of oxidative phosphorylation dynamics.
Assuntos
Trifosfato de Adenosina , Proteínas de Fluorescência Verde , Animais , Humanos , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Técnicas Biossensoriais/métodos , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Fosforilação Oxidativa , ATPases Translocadoras de Prótons/metabolismo , ATPases Translocadoras de Prótons/genéticaRESUMO
G protein-coupled receptors (GPCRs) transduce the effects of many neuromodulators including dopamine, serotonin, epinephrine, acetylcholine, and opioids. The localization of synthetic or endogenous GPCR agonists impacts their action on specific neuronal pathways. In this paper, we show a series of single-protein chain integrator sensors that are highly modular and could potentially be used to determine GPCR agonist localization across the brain. We previously engineered integrator sensors for the mu- and kappa-opioid receptor agonists called M- and K-Single-chain Protein-based Opioid Transmission Indicator Tool (SPOTIT), respectively. Here, we engineered red versions of the SPOTIT sensors for multiplexed imaging of GPCR agonists. We also modified SPOTIT to create an integrator sensor design platform called SPOTIT for all GPCRs (SPOTall). We used the SPOTall platform to engineer sensors for the beta 2-adrenergic receptor (B2AR), the dopamine receptor D1, and the cholinergic receptor muscarinic 2 agonists. Finally, we demonstrated the application of M-SPOTIT and B2AR-SPOTall in detecting exogenously administered morphine, isoproterenol, and epinephrine in the mouse brain via locally injected viruses. The SPOTIT and SPOTall sensor design platform has the potential for unbiased agonist detection of many synthetic and endogenous neuromodulators across the brain.
Assuntos
Receptores Acoplados a Proteínas G , Animais , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Humanos , Camundongos , Células HEK293 , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/metabolismo , Isoproterenol/farmacologia , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Morfina/farmacologia , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/diagnóstico por imagem , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/metabolismo , Técnicas Biossensoriais/métodosRESUMO
Light enhances or disrupts circadian rhythms, depending on the timing of exposure. Circadian disruption contributes to poor health outcomes that increase mortality risk. Whether personal light exposure predicts mortality risk has not been established. We therefore investigated whether personal day and night light, and light patterns that disrupt circadian rhythms, predicted mortality risk. UK Biobank participants (N = 88,905, 62.4 ± 7.8 y, 57% female) wore light sensors for 1 wk. Day and night light exposures were defined by factor analysis of 24-h light profiles. A computational model of the human circadian pacemaker was applied to model circadian amplitude and phase from light data. Cause-specific mortality was recorded in 3,750 participants across a mean (±SD) follow-up period of 8.0 ± 1.0 y. Individuals with brighter day light had incrementally lower all-cause mortality risk (adjusted-HR ranges: 0.84 to 0.90 [50 to 70th light exposure percentiles], 0.74 to 0.84 [70 to 90th], and 0.66 to 0.83 [90 to 100th]), and those with brighter night light had incrementally higher all-cause mortality risk (aHR ranges: 1.15 to 1.18 [70 to 90th], and 1.21 to 1.34 [90 to 100th]), compared to individuals in darker environments (0 to 50th percentiles). Individuals with lower circadian amplitude (aHR range: 0.90 to 0.96 per SD), earlier circadian phase (aHR range: 1.16 to 1.30), or later circadian phase (aHR range: 1.13 to 1.20) had higher all-cause mortality risks. Day light, night light, and circadian amplitude predicted cardiometabolic mortality, with larger hazard ratios than for mortality by other causes. Findings were robust to adjustment for age, sex, ethnicity, photoperiod, and sociodemographic and lifestyle factors. Minimizing night light, maximizing day light, and keeping regular light-dark patterns that enhance circadian rhythms may promote cardiometabolic health and longevity.
Assuntos
Ritmo Circadiano , Luz , Humanos , Feminino , Masculino , Pessoa de Meia-Idade , Ritmo Circadiano/fisiologia , Idoso , Estudos Prospectivos , Mortalidade , Fatores de Risco , Reino Unido/epidemiologiaRESUMO
Autism spectrum disorder (ASD) is a complex neurodevelopmental condition characterized by social and communication deficits and repetitive behaviors. The genetic heterogeneity of ASD presents a challenge to the development of an effective treatment targeting the underlying molecular defects. ASD gating charge mutations in the KCNQ/KV7 potassium channel cause gating pore currents (Igp) and impair action potential (AP) firing of dopaminergic neurons in brain slices. Here, we investigated ASD gating charge mutations of the voltage-gated SCN2A/NaV1.2 brain sodium channel, which ranked high among the ion channel genes with mutations in individuals with ASD. Our results show that ASD mutations in the gating charges R2 in Domain-II (R853Q), and R1 (R1626Q) and R2 (R1629H) in Domain-IV of NaV1.2 caused Igp in the resting state of ~0.1% of the amplitude of central pore current. The R1626Q mutant also caused significant changes in the voltage dependence of fast inactivation, and the R1629H mutant conducted proton-selective Igp. These potentially pathogenic Igp were exacerbated by the absence of the extracellular Mg2+ and Ca2+. In silico simulation of the effects of these mutations in a conductance-based single-compartment cortical neuron model suggests that the inward Igp reduces the time to peak for the first AP in a train, increases AP rates during a train of stimuli, and reduces the interstimulus interval between consecutive APs, consistent with increased neural excitability and altered input/output relationships. Understanding this common pathophysiological mechanism among different voltage-gated ion channels at the circuit level will give insights into the underlying mechanisms of ASD.
Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Canais de Sódio Disparados por Voltagem , Humanos , Transtorno do Espectro Autista/genética , Transtorno Autístico/genética , Encéfalo , MutaçãoRESUMO
Artificial skins or flexible pressure sensors that mimic human cutaneous mechanoreceptors transduce tactile stimuli to quantitative electrical signals. Conventional trial-and-error designs for such devices follow a forward structure-to-property routine, which is usually time-consuming and determines one possible solution in one run. Data-driven inverse design can precisely target desired functions while showing far higher productivity, however, it is still absent for flexible pressure sensors because of the difficulties in acquiring a large amount of data. Here, we report a property-to-structure inverse design of flexible pressure sensors, exhibiting a significantly greater efficiency than the conventional routine. We use a reduced-order model that analytically constrains the design scope and an iterative "jumping-selection" method together with a surrogate model that enhances data screening. As an exemplary scenario, hundreds of solutions that overcome the intrinsic signal saturation have been predicted by the inverse method, validating for a variety of material systems. The success in property design on multiple indicators demonstrates that the proposed inverse design is an efficient and powerful tool to target multifarious applications of flexible pressure sensors, which can potentially advance the fields of intelligent robots, advanced healthcare, and human-machine interfaces.