RESUMO
G protein-coupled receptors (GPCRs) relay extracellular stimuli into specific cellular functions. Cells express many different GPCRs, but all these GPCRs signal to only a few second messengers such as cAMP. It is largely unknown how cells distinguish between signals triggered by different GPCRs to orchestrate their complex functions. Here, we demonstrate that individual GPCRs signal via receptor-associated independent cAMP nanodomains (RAINs) that constitute self-sufficient, independent cell signaling units. Low concentrations of glucagon-like peptide 1 (GLP-1) and isoproterenol exclusively generate highly localized cAMP pools around GLP-1- and ß2-adrenergic receptors, respectively, which are protected from cAMP originating from other receptors and cell compartments. Mapping local cAMP concentrations with engineered GPCR nanorulers reveals gradients over only tens of nanometers that define the size of individual RAINs. The coexistence of many such RAINs allows a single cell to operate thousands of independent cellular signals simultaneously, rather than function as a simple "on/off" switch.
Assuntos
Receptores Acoplados a Proteínas G , Transdução de Sinais , Fenômenos Fisiológicos Celulares , AMP Cíclico , Peptídeo 1 Semelhante ao Glucagon , Receptores Adrenérgicos beta 2 , Receptores Acoplados a Proteínas G/química , Sistemas do Segundo MensageiroRESUMO
Cells relay a plethora of extracellular signals to specific cellular responses by using only a few second messengers, such as cAMP. To explain signaling specificity, cAMP-degrading phosphodiesterases (PDEs) have been suggested to confine cAMP to distinct cellular compartments. However, measured rates of fast cAMP diffusion and slow PDE activity render cAMP compartmentalization essentially impossible. Using fluorescence spectroscopy, we show that, contrary to earlier data, cAMP at physiological concentrations is predominantly bound to cAMP binding sites and, thus, immobile. Binding and unbinding results in largely reduced cAMP dynamics, which we term "buffered diffusion." With a large fraction of cAMP being buffered, PDEs can create nanometer-size domains of low cAMP concentrations. Using FRET-cAMP nanorulers, we directly map cAMP gradients at the nanoscale around PDE molecules and the areas of resulting downstream activation of cAMP-dependent protein kinase (PKA). Our study reveals that spatiotemporal cAMP signaling is under precise control of nanometer-size domains shaped by PDEs that gate activation of downstream effectors.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Transdução de Sinais , Análise de Célula Única/métodos , Simulação por Computador , AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/química , Citoplasma/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Modelos Moleculares , Diester Fosfórico Hidrolases/química , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes , Análise Espaço-Temporal , Espectrometria de FluorescênciaRESUMO
Development of microbiota-directed foods (MDFs) that selectively increase the abundance of beneficial human gut microbes, and their expressed functions, requires knowledge of both the bioactive components of MDFs and the mechanisms underlying microbe-microbe interactions. Here, gnotobiotic mice were colonized with a defined consortium of human-gut-derived bacterial strains and fed different combinations of 34 food-grade fibers added to a representative low-fiber diet consumed in the United States. Bioactive carbohydrates in fiber preparations targeting particular Bacteroides species were identified using community-wide quantitative proteomic analyses of bacterial gene expression coupled with forward genetic screens. Deliberate manipulation of community membership combined with administration of retrievable artificial food particles, consisting of paramagnetic microscopic beads coated with dietary polysaccharides, disclosed the contributions of targeted species to fiber degradation. Our approach, including the use of bead-based biosensors, defines nutrient-harvesting strategies that underlie, as well as alleviate, competition between Bacteroides and control the selectivity of MDF components.
Assuntos
Bacteroides/genética , Fibras na Dieta/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Vida Livre de Germes/fisiologia , Interações Microbianas/efeitos dos fármacos , Polissacarídeos/farmacologia , Proteômica/métodos , Animais , Dieta/métodos , Fibras na Dieta/metabolismo , Fezes/microbiologia , Microbioma Gastrointestinal/fisiologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Polissacarídeos/metabolismoRESUMO
Recent advances in fluorescence imaging permit large-scale recording of neural activity and dynamics of neurochemical release with unprecedented resolution in behaving animals. Calcium imaging with highly optimized genetically encoded indicators provides a mesoscopic view of neural activity from genetically defined populations at cellular and subcellular resolutions. Rigorously improved voltage sensors and microscopy allow for robust spike imaging of populational neurons in various brain regions. In addition, recent protein engineering efforts in the past few years have led to the development of sensors for neurotransmitters and neuromodulators. Here, we discuss the development and applications of these genetically encoded fluorescent indicators in reporting neural activity in response to various behaviors in different biological systems as well as in drug discovery. We also report a simple model to guide sensor selection and optimization.
Assuntos
Neurônios , Receptores de Droga , Animais , Encéfalo/metabolismo , Neurônios/fisiologia , Neurotransmissores/metabolismo , Imagem Óptica , Receptores de Droga/metabolismoRESUMO
G-protein-coupled receptors (GPCRs), also known as seven transmembrane receptors (7TMRs), typically interact with two distinct signal-transducers, i.e., G proteins and ß-arrestins (ßarrs). Interestingly, there are some non-canonical 7TMRs that lack G protein coupling but interact with ßarrs, although an understanding of their transducer coupling preference, downstream signaling, and structural mechanism remains elusive. Here, we characterize two such non-canonical 7TMRs, namely, the decoy D6 receptor (D6R) and the complement C5a receptor subtype 2 (C5aR2), in parallel with their canonical GPCR counterparts. We discover that D6R and C5aR2 efficiently couple to ßarrs, exhibit distinct engagement of GPCR kinases (GRKs), and activate non-canonical downstream signaling pathways. We also observe that ßarrs adopt distinct conformations for D6R and C5aR2, compared to their canonical GPCR counterparts, in response to common natural agonists. Our study establishes D6R and C5aR2 as ßarr-coupled 7TMRs and provides key insights into their regulation and signaling with direct implication for biased agonism.
Assuntos
Membrana Celular/metabolismo , Conformação Proteica , Transdução de Sinais , beta-Arrestinas/química , Animais , Proteínas de Ligação ao GTP/química , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Transporte Proteico , Receptor da Anafilatoxina C5a/metabolismoRESUMO
Osmotic stress conditions occur at multiple stages of plant life. Changes in water availability caused by osmotic stress induce alterations in the mechanical properties of the plasma membrane, its interaction with the cell wall, and the concentration of macromolecules in the cytoplasm. We summarize the reported players involved in the sensing mechanisms of osmotic stress in plants. We discuss how changes in macromolecular crowding are perceived intracellularly by intrinsically disordered regions (IDRs) in proteins. Finally, we review methods for dynamically monitoring macromolecular crowding in living cells and discuss why their implementation is required for the discovery of new plant osmosensors. Elucidating the osmosensing mechanisms will be essential for designing strategies to improve plant productivity in the face of climate change.
Assuntos
Pressão Osmótica , Plantas , Plantas/metabolismo , Substâncias Macromoleculares/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/químicaRESUMO
Intrinsically disordered proteins and protein regions (IDRs) are abundant in eukaryotic proteomes and play a wide variety of essential roles. Instead of folding into a stable structure, IDRs exist in an ensemble of interconverting conformations whose structure is biased by sequence-dependent interactions. The absence of a stable 3D structure, combined with high solvent accessibility, means that IDR conformational biases are inherently sensitive to changes in their environment. Here, we argue that IDRs are ideally poised to act as sensors and actuators of cellular physicochemistry. We review the physical principles that underlie IDR sensitivity, the molecular mechanisms that translate this sensitivity to function, and recent studies where environmental sensing by IDRs may play a key role in their downstream function.
Assuntos
Proteínas Intrinsicamente Desordenadas , Conformação Proteica , Proteínas Intrinsicamente Desordenadas/metabolismo , Domínios ProteicosRESUMO
The tumor microenvironment (TME) is a dynamic pseudoorgan that shapes the development and progression of cancers. It is a complex ecosystem shaped by interactions between tumor and stromal cells. Although the traditional focus has been on the paracrine communication mediated by protein messengers, recent attention has turned to the metabolic secretome in tumors. Metabolic enzymes, together with exchanged substrates and products, have emerged as potential biomarkers and therapeutic targets. However, traditional techniques for profiling secreted metabolites in complex cellular contexts are limited. Surface-enhanced Raman scattering (SERS) has emerged as a promising alternative due to its nontargeted nature and simplicity of operation. Although SERS has demonstrated its potential for detecting metabolites in biological settings, its application in deciphering metabolic interactions within multicellular systems like the TME remains underexplored. In this study, we introduce a SERS-based strategy to investigate the secreted purine metabolites of tumor cells lacking methylthioadenosine phosphorylase (MTAP), a common genetic event associated with poor prognosis in various cancers. Our SERS analysis reveals that MTAP-deficient cancer cells selectively produce methylthioadenosine (MTA), which is taken up and metabolized by fibroblasts. Fibroblasts exposed to MTA exhibit: i) molecular reprogramming compatible with cancer aggressiveness, ii) a significant production of purine derivatives that could be readily recycled by cancer cells, and iii) the capacity to secrete purine derivatives that induce macrophage polarization. Our study supports the potential of SERS for cancer metabolism research and reveals an unprecedented paracrine crosstalk that explains TME reprogramming in MTAP-deleted cancers.
Assuntos
Ecossistema , Neoplasias , Humanos , Neoplasias/tratamento farmacológico , Purinas/metabolismo , Purina-Núcleosídeo Fosforilase/genética , Microambiente TumoralRESUMO
Circular RNAs (circRNAs) are evolutionarily conserved noncoding RNAs with tissue-specific expression patterns, and exert unique cellular functions that have the potential to become biomarkers in therapeutic applications. Therefore, accurate and sensitive detection of circRNA with facile platforms is essential for better understanding of circRNA biological processes and circRNA-related disease diagnosis and prognosis; and precise regulation of circRNA through efficient delivery of circRNA or siRNA is critical for therapeutic purposes. Here, we reviewed the current development of circRNA identification methodologies, including overviewing the purification steps, summarizing the sequencing methods of circRNA, as well as comparing the advantages and disadvantages of traditional and new detection methods. Then, we discussed the delivery and manipulation strategies for circRNAs in both research and clinic treatment. Finally, the challenges and opportunities of analyzing circRNAs were addressed.
Assuntos
RNA Circular , RNA , RNA/genética , RNA/metabolismo , BiomarcadoresRESUMO
Neurodegenerative diseases are complex and progressive, posing challenges to their study and understanding. Recent advances in microscopy imaging technologies have enabled the exploration of neurons in three spatial dimensions (3D) over time (4D). When applied to 3D cultures, tissues, or animals, these technologies can provide valuable insights into the dynamic and spatial nature of neurodegenerative diseases. This review focuses on the use of imaging techniques and neurodegenerative disease models to study neurodegeneration in 4D. Imaging techniques such as confocal microscopy, two-photon microscopy, miniscope imaging, light sheet microscopy, and robotic microscopy offer powerful tools to visualize and analyze neuronal changes over time in 3D tissue. Application of these technologies to in vitro models of neurodegeneration such as mouse organotypic culture systems and human organoid models provide versatile platforms to study neurodegeneration in a physiologically relevant context. Additionally, use of 4D imaging in vivo, including in mouse and zebrafish models of neurodegenerative diseases, allows for the investigation of early dysfunction and behavioral changes associated with neurodegeneration. We propose that these studies have the power to overcome the limitations of two-dimensional monolayer neuronal cultures and pave the way for improved understanding of the dynamics of neurodegenerative diseases and the development of effective therapeutic strategies.
Assuntos
Imageamento Tridimensional , Doenças Neurodegenerativas , Animais , Humanos , Doenças Neurodegenerativas/diagnóstico por imagem , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Imageamento Tridimensional/métodos , Neurônios/patologia , Neurônios/metabolismo , Camundongos , Modelos Animais de Doenças , Peixe-ZebraRESUMO
Gibberellins (GAs) are major regulators of developmental and growth processes in plants. Using the degradation-based signaling mechanism of GAs, we have built transcriptional regulator (DELLA)-based, genetically encoded ratiometric biosensors as proxies for hormone quantification at high temporal resolution and sensitivity that allow dynamic, rapid and simple analysis in a plant cell system, i.e. Arabidopsis protoplasts. These ratiometric biosensors incorporate a DELLA protein as a degradation target fused to a firefly luciferase connected via a 2A peptide to a renilla luciferase as a co-expressed normalization element. We have implemented these biosensors for all five Arabidopsis DELLA proteins, GA-INSENSITIVE, GAI; REPRESSOR-of-ga1-3, RGA; RGA-like1, RGL1; RGL2 and RGL3, by applying a modular design. The sensors are highly sensitive (in the low pm range), specific and dynamic. As a proof of concept, we have tested the applicability in three domains: the study of substrate specificity and activity of putative GA-oxidases, the characterization of GA transporters, and the use as a discrimination platform coupled to a GA agonists' chemical screening. This work demonstrates the development of a genetically encoded quantitative biosensor complementary to existing tools that allow the visualization of GA in planta.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Técnicas Biossensoriais , Giberelinas , Protoplastos , Transdução de Sinais , Giberelinas/metabolismo , Técnicas Biossensoriais/métodos , Arabidopsis/metabolismo , Arabidopsis/genética , Protoplastos/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Reguladores de Crescimento de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Insertable biosensor systems are medical diagnostic devices with two primary components: an implantable biosensor within the body and a wearable monitor that can remotely interrogate the biosensor from outside the body. Because the biosensor does not require a physical connection to the electronic monitor, insertable biosensor systems promise improved patient comfort, reduced inflammation and infection risk, and extended operational lifetimes relative to established percutaneous biosensor systems. However, the lack of physical connection also presents technical challenges that have necessitated new innovations in developing sensing chemistries, transduction methods, and communication modalities. In this review, we discuss the key developments that have made insertables a promising option for longitudinal biometric monitoring and highlight the essential needs and existing development challenges to realizing the next generation of insertables for extended-use diagnostic and prognostic devices.
Assuntos
Técnicas Biossensoriais , Desenho de Equipamento , Dispositivos Eletrônicos Vestíveis , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Humanos , Próteses e Implantes , Monitorização Fisiológica/instrumentação , Monitorização Fisiológica/métodosRESUMO
Among the various types of enzyme-based biosensors, sensors utilizing enzymes capable of direct electron transfer (DET) are recognized as the most ideal. However, only a limited number of redox enzymes are capable of DET with electrodes, that is, dehydrogenases harboring a subunit or domain that functions specifically to accept electrons from the redox cofactor of the catalytic site and transfer the electrons to the external electron acceptor. Such subunits or domains act as built-in mediators for electron transfer between enzymes and electrodes; consequently, such enzymes enable direct electron transfer to electrodes and are designated as DET-type enzymes. DET-type enzymes fall into several categories, including redox cofactors of catalytic reactions, built-in mediators for DET with electrodes and by their protein hierarchic structures, DET-type oxidoreductases with oligomeric structures harboring electron transfer subunits, and monomeric DET-type oxidoreductases harboring electron transfer domains. In this review, we cover the science of DET-type oxidoreductases and their biomedical applications. First, we introduce the structural biology and current understanding of DET-type enzyme reactions. Next, we describe recent technological developments based on DET-type enzymes for biomedical applications, such as biosensors and biochemical energy harvesting for self-powered medical devices. Finally, after discussing how to further engineer and create DET-type enzymes, we address the future prospects for DET-type enzymes in biomedical engineering.
Assuntos
Técnicas Biossensoriais , Oxirredução , Oxirredutases , Transporte de Elétrons , Técnicas Biossensoriais/métodos , Humanos , Oxirredutases/química , Oxirredutases/metabolismo , Eletrodos , Elétrons , Animais , Domínio Catalítico , Engenharia Biomédica/métodosRESUMO
Neurotropic viruses, with their ability to invade the central nervous system, present a significant public health challenge, causing a spectrum of neurological diseases. Clinical manifestations of neurotropic viral infections vary widely, from mild to life-threatening conditions, such as HSV-induced encephalitis or poliovirus-induced poliomyelitis. Traditional diagnostic methods, including polymerase chain reaction, serological assays, and imaging techniques, though valuable, have limitations. To address these challenges, biosensor-based methods have emerged as a promising approach. These methods offer advantages such as rapid results, high sensitivity, specificity, and potential for point-of-care applications. By targeting specific biomarkers or genetic material, biosensors utilise technologies like surface plasmon resonance and microarrays, providing a direct and efficient means of diagnosing neurotropic infections. This review explores the evolving landscape of biosensor-based methods, highlighting their potential to enhance the diagnostic toolkit for neurotropic viruses.
Assuntos
Técnicas Biossensoriais , Doenças do Sistema Nervoso , Poliomielite , Vírus , Humanos , Vírus/genéticaRESUMO
Carbon dots (CD) are widely investigated particles with interesting fluorescent properties which are reported to be used for various purposes, as they are biocompatible, resistant to photobleaching and with tuneable properties depending on the specific CD surface chemistry. In this work, we report on the possibility to use opportunely designed CD to distinguish among isobaric peptides almost undistinguishable by mass spectrometry, as well as to monitor protein aggregation phenomena. Particularly, cell-penetrating peptides containing the carnosine moiety at different positions in the peptide chain produce sequence specific fluorescent signals. Analogously, different insulin oligomerization states can also be distinguished by the newly proposed experimental approach. The latter is here described in details and can be potentially applied to any kind of peptide or protein.
RESUMO
G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins and play a crucial role in regulating diverse cellular functions. They transmit their signaling via binding to intracellular signal transducers and effectors, such as G proteins, GPCR kinases, and ß-arrestins. To influence specific GPCR signaling behaviors, ß-arrestins recruit effectors to form larger signaling complexes. Intriguingly, they facilitate divergent functions for the binding to different receptors. Recent studies relying on advanced structural approaches, novel biosensors and interactome analyses bring us closer to understanding how this specificity is achieved. In this article, we share our hypothesis of how active GPCRs induce specific conformational rearrangements within ß-arrestins to reveal distinct binding interfaces, enabling the recruitment of a subset of effectors to foster specialized signaling complexes. Furthermore, we discuss methods of how to comprehensively assess ß-arrestin conformational states and present the current state of research regarding the functionality of these multifaceted scaffolding proteins.
Assuntos
Arrestinas , Receptores Acoplados a Proteínas G , beta-Arrestinas/metabolismo , Arrestinas/química , Arrestinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologiaRESUMO
SignificanceThe detection of low-abundance molecular biomarkers is key to the liquid-biopsy-based disease diagnosis. Existing methods are limited by the affinity and specificity of recognition probes and the mass transportation of analyte molecules onto the sensor surfaces, resulting in insufficient sensitivity and long assay time. This work establishes a rapid and ultrasensitive approach by actively tuning binding kinetics and accelerating the mass transportation via nanoparticle micromanipulations. This is significant because it permits extremely sensitive measurements within clinically acceptable assay time. It is incubation-free, washing-free, and compatible with low- and high-affinity probes.
Assuntos
Imagem Individual de Molécula/métodos , Sítios de Ligação , Biomarcadores/metabolismo , Cinética , Limite de Detecção , TermodinâmicaRESUMO
In conventional electrochemiluminescence (ECL) systems, the presence of the competitive cathodic hydrogen evolution reaction (HER) in aqueous electrolytes is typically considered to be a side reaction, leading to a reduced ECL efficiency and stability due to H2 generation and aggregation at the electrode surface. However, the significant role of adsorbed hydrogen (H*) as a key intermediate, formed during the Volmer reaction in the HER process, has been largely overlooked. In this study, employing the luminol-H2O2 system as a model, we for the first time demonstrate a novel H*-mediated coreactant activation mechanism, which remarkably enhances the ECL intensity. H* facilitates cleavage of the O-O bond in H2O2, selectively generating highly reactive hydroxyl radicals for efficient ECL reactions. Experimental investigations and theoretical calculations demonstrate that this H*-mediated mechanism achieves superior coreactant activation compared to the conventional direct electron transfer pathway, which unveils a new pathway for coreactant activation in the ECL systems.
RESUMO
Neurotransmitters are important signaling molecules in the brain and are relevant in many diseases. Measuring them with high spatial and temporal resolutions in biological systems is challenging. Here, we develop a ratiometric fluorescent sensor/probe for catecholamine neurotransmitters on the basis of near-infrared (NIR) semiconducting single wall carbon nanotubes (SWCNTs). Phenylboronic acid (PBA)-based quantum defects are incorporated into them to interact selectively with catechol moieties. These PBA-SWCNTs are further modified with poly(ethylene glycol) phospholipids (PEG-PL) for biocompatibility. Catecholamines, including dopamine, do not affect the intrinsic E11 fluorescence (990 nm) of these (PEG-PL-PBA-SWCNT) sensors. In contrast, the defect-related E11* emission (1130 nm) decreases by up to 35%. Furthermore, this dual functionalization allows tuning selectivity by changing the charge of the PEG polymer. These sensors are not taken up by cells, which is beneficial for extracellular imaging, and they are functional in brain slices. In summary, we use dual functionalization of SWCNTs to create a ratiometric biosensor for dopamine.
Assuntos
Catecolaminas , Nanotubos de Carbono , Dopamina , Fluorescência , NeurotransmissoresRESUMO
On-demand engineering of cell membrane receptors to nongenetically intervene in cellular behaviors is still a challenge. Herein, a membraneless enzyme biofuel cell-based self-powered biosensor (EBFC-SPB) was developed for autonomously and precisely releasing Zn2+ to initiate DNAzyme-based reprogramming of cell membrane receptors, which further mediates signal transduction to regulate cellular behaviors. The critical component of EBFC-SPB is a hydrogel film on a biocathode which is prepared using a Fe3+-cross-linked alginate hydrogel film loaded with Zn2+ ions. In the working mode in the presence of glucose/O2, the hydrogel is decomposed due to the reduction of Fe3+ to Fe2+, accompanied by rapid release of Zn2+ to specifically activate a Zn2+-responsive DNAzyme nanodevice on the cell surface, leading to the dimerization of homologous or nonhomologous receptors to promote or inhibit cell proliferation and migration. This EBFC-SPB platform provides a powerful "sensing-actuating-treating" tool for chemically regulating cellular behaviors, which holds great promise in precision biomedicine.