Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 120
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Dev Cell ; 56(18): 2592-2606.e7, 2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34508658

RESUMO

Membrane contact between intracellular organelles is important in mediating organelle communication. However, the assembly of molecular machinery at membrane contact site and its internal organization correlating with its functional activity remain unclear. Here, we demonstrate that a gel-like condensation of Cidec, a crucial protein for obesity development by facilitating lipid droplet (LD) fusion, occurs at the LD-LD contact site (LDCS) through phase separation. The homomeric interaction between the multivalent N terminus of Cidec is sufficient to promote its phase separation both in vivo and in vitro. Interestingly, Cidec condensation at LDCSs generates highly plastic and lipid-permeable fusion plates that are geometrically constrained by donor LDs. In addition, Cidec condensates are distributed unevenly in the fusion plate generating stochastic sub-compartments that may represent unique lipid passageways during LD fusion. We have thus uncovered the organization and functional significance of geometry-constrained Cidec phase separation in mediating LD fusion and lipid homeostasis.

2.
Nucleic Acids Res ; 49(10): 5832-5844, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34037793

RESUMO

By virtue of its chaperone activity, the capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements. However, the role of DENV2C during the interaction of RNA elements involved in stabilizing the 5'-3' panhandle structure of DENV RNA is still unclear. Therefore, we determined how DENV2C affects structural functionality of the capsid-coding region hairpin element (cHP) during annealing and strand displacement of the 9-nt cyclization sequence (5CS) and its complementary 3CS. cHP has two distinct functions: a role in translation start codon selection and a role in RNA synthesis. Our results showed that cHP impedes annealing between 5CS and 3CS. Although DENV2C does not modulate structural functionality of cHP, it accelerates annealing and specifically promotes strand displacement of 3CS during 5'-3' panhandle formation. Furthermore, DENV2C exerts its chaperone activity by favouring one of the active conformations of cHP. Based on our results, we propose mechanisms for annealing and strand displacement involving cHP. Thus, our results provide mechanistic insights into how DENV2C regulates RNA synthesis by modulating essential RNA elements in the capsid-coding region, that in turn allow for DENV replication.


Assuntos
Proteínas do Capsídeo/metabolismo , Vírus da Dengue/metabolismo , Chaperonas Moleculares/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Recombinação Genética/genética , Replicação Viral/genética , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Códon de Iniciação , Ciclização/genética , Vírus da Dengue/genética , Cinética , Chaperonas Moleculares/genética , Conformação de Ácido Nucleico , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier
3.
Nat Commun ; 12(1): 1748, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33741958

RESUMO

Super-resolution microscopy and single molecule fluorescence spectroscopy require mutually exclusive experimental strategies optimizing either temporal or spatial resolution. To achieve both, we implement a GPU-supported, camera-based measurement strategy that highly resolves spatial structures (~100 nm), temporal dynamics (~2 ms), and molecular brightness from the exact same data set. Simultaneous super-resolution of spatial and temporal details leads to an improved precision in estimating the diffusion coefficient of the actin binding polypeptide Lifeact and corrects structural artefacts. Multi-parametric analysis of epidermal growth factor receptor (EGFR) and Lifeact suggests that the domain partitioning of EGFR is primarily determined by EGFR-membrane interactions, possibly sub-resolution clustering and inter-EGFR interactions but is largely independent of EGFR-actin interactions. These results demonstrate that pixel-wise cross-correlation of parameters obtained from different techniques on the same data set enables robust physicochemical parameter estimation and provides biological knowledge that cannot be obtained from sequential measurements.


Assuntos
Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Actinas/metabolismo , Animais , Células CHO , Membrana Celular , Cricetulus , Difusão , Receptores ErbB/metabolismo , Fluorescência , Humanos , Espectrometria de Fluorescência/métodos
4.
J Biol Chem ; 296: 100359, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33539927

RESUMO

Prion diseases are a group of neurodegenerative disorders that infect animals and humans with proteinaceous particles called prions. Prions consist of scrapie prion protein (PrPSc), a misfolded version of the cellular prion protein (PrPC). During disease progression, PrPSc replicates by interacting with PrPC and inducing its conversion to PrPSc. Attachment of PrPC to cellular membranes via a glycosylphosphatidylinositol (GPI) anchor is critical for the conversion of PrPC into PrPSc. However, the mechanisms governing PrPC conversion and replication on the membrane remain largely unclear. Here, a site-selectively modified PrP variant equipped with a fluorescent GPI anchor mimic (PrP-GPI) was employed to directly observe PrP at the cellular membrane in neuronal SH-SY5Y cells. PrP-GPI exhibits a cholesterol-dependent membrane accumulation and a cytoskeleton-dependent mobility. More specifically, inhibition of actin polymerization reduced the diffusion of PrP-GPI indicating protein clustering, which resembles the initial step of PrP aggregation and conversion into its pathogenic isoform. An intact actin cytoskeleton might therefore prevent conversion of PrPC into PrPSc and offer new therapeutic angles.


Assuntos
Citoesqueleto/fisiologia , Proteínas de Membrana/metabolismo , Príons/metabolismo , Actinas/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Análise por Conglomerados , Citoesqueleto/metabolismo , Glicosilfosfatidilinositóis/química , Glicosilfosfatidilinositóis/metabolismo , Humanos , Neurônios/metabolismo , Proteínas PrPSc/metabolismo , Doenças Priônicas/metabolismo , Proteínas Priônicas/metabolismo , Isoformas de Proteínas/metabolismo , Scrapie/metabolismo
5.
RNA Biol ; 18(5): 718-731, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33406991

RESUMO

The capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements using chaperone activity. However, the role of DENV2C during the interaction of RNA elements in the conserved 5' untranslated region (5'UTR) to the 3' untranslated region (3'UTR) is still unclear. Thus, we investigated the effect of DENV2C on the annealing mechanism of two RNA hairpin elements from the 5'UTR to their complementary sequences during (+)/(-) ds-RNAformation and (+) RNA circularization. DENV2C was found to switch the annealing pathway for RNA elements involved in (+)/(-) ds-RNA formation, but not for RNA elements related to (+) RNA circularization. In addition, we also determined that DENV2C modulates intrinsic dynamics and reduces kinetically trapped unfavourable conformations of the 5'UTR sequence. Thus, our results provide mechanistic insights by which DENV2C chaperones the interactions between RNA elements at the 5' and 3' ends during genome recombination, a prerequisite for DENV replication.

6.
Hum Mol Genet ; 29(23): 3765-3780, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33276371

RESUMO

Neurexins are presynaptic transmembrane proteins that control synapse activity and are risk factors for autism spectrum disorder. Zebrafish, a popular model for behavioral studies, has six neurexin genes, but their functions in embryogenesis and behavior remain largely unknown. We have previously reported that nrxn2a is aberrantly spliced and specifically dysregulated in motor neurons (MNs) in models of spinal muscular atrophy. In this study, we generated nrxn2aa-/- mutants by CRISPR/Cas9 to understand nrxn2aa function at the zebrafish neuromuscular junction (NMJ) and to determine the effects of its deficiency on adult behavior. Homozygous mutant embryos derived from heterozygous parents did not show obvious defects in axon outgrowth or synaptogenesis of MNs. In contrast, maternal-zygotic (MZ) nrxn2aa-/- mutants displayed extensively branched axons and defective MNs, suggesting a cell-autonomous role for maternally provided nrxn2aa in MN development. Analysis of the NMJs revealed enlarged choice points in MNs of mutant larvae and reduced co-localization of pre- and post-synaptic terminals, indicating impaired synapse formation. Severe early NMJ defects partially recovered in late embryos when mutant transcripts became strongly upregulated. Ultimately, however, the induced defects resulted in muscular atrophy symptoms in adult MZ mutants. Zygotic homozygous mutants developed normally but displayed increased anxiety at adult stages. Together, our data demonstrate an essential role for maternal nrxn2aa in NMJ synapse establishment, while zygotic nrxn2aa expression appears dispensable for synapse maintenance. The viable nrxn2aa-/- mutant furthermore serves as a novel model to study how an increase in anxiety-like behaviors impacts other deficits.

8.
Elife ; 92020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33236989

RESUMO

Wnt3 proteins are lipidated and glycosylated signaling molecules that play an important role in zebrafish neural patterning and brain development. However, the transport mechanism of lipid-modified Wnts through the hydrophilic extracellular environment for long-range action remains unresolved. Here we determine how Wnt3 accomplishes long-range distribution in the zebrafish brain. First, we characterize the Wnt3-producing source and Wnt3-receiving target regions. Subsequently, we analyze Wnt3 mobility at different length scales by fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. We demonstrate that Wnt3 spreads extracellularly and interacts with heparan sulfate proteoglycans (HSPG). We then determine the binding affinity of Wnt3 to its receptor, Frizzled1 (Fzd1), using fluorescence cross-correlation spectroscopy and show that the co-receptor, low-density lipoprotein receptor-related protein 5 (Lrp5), is required for Wnt3-Fzd1 interaction. Our results are consistent with the extracellular distribution of Wnt3 by a diffusive mechanism that is modified by tissue morphology, interactions with HSPG, and Lrp5-mediated receptor binding, to regulate zebrafish brain development.


Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteína Wnt3/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/embriologia , Embrião não Mamífero , Recuperação de Fluorescência Após Fotodegradação , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Microscopia Confocal , Ligação Proteica , Proteína Wnt3/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
10.
Biochemistry ; 59(40): 3783-3795, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32956586

RESUMO

G-Protein-coupled receptors (GPCRs) are ubiquitous within eukaryotes, responsible for a wide array of physiological and pathological processes. Indeed, the fact that they are the most drugged target in the human genome is indicative of their importance. Despite the clear interest in GPCRs, most information regarding their activity has been so far obtained by analyzing the response from a "bulk medium". As such, this Perspective summarizes some of the common methods for this indirect observation. Nonetheless, by inspecting approaches applying super-resolution imaging, we argue that imaging is perfectly situated to obtain more detailed structural and spatial information, assisting in the development of new GPCR-targeted drugs and clinical strategies. The benefits of direct optical visualization of GPCRs are analyzed in the context of potential future directions in the field.


Assuntos
Descoberta de Drogas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Animais , Microscopia Crioeletrônica/métodos , Cristalografia por Raios X/métodos , Descoberta de Drogas/métodos , Humanos , Espectrometria de Massas/métodos , Microscopia de Fluorescência/métodos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica/efeitos dos fármacos , Receptores Acoplados a Proteínas G/ultraestrutura , Imagem Individual de Molécula/métodos , Espectrometria de Fluorescência/métodos , Ressonância de Plasmônio de Superfície/métodos
11.
mBio ; 11(5)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994320

RESUMO

Estrogen, a major female sex steroid hormone, has been shown to promote the selection of mucoid Pseudomonas aeruginosa in the airways of patients with chronic respiratory diseases, including cystic fibrosis. This results in long-term persistence, poorer clinical outcomes, and limited therapeutic options. In this study, we demonstrate that at physiological concentrations, sex steroids, including testosterone and estriol, induce membrane stress responses in P. aeruginosa This is characterized by increased virulence and consequent inflammation and release of proinflammatory outer membrane vesicles promoting in vivo persistence of the bacteria. The steroid-induced P. aeruginosa response correlates with the molecular polarity of the hormones and membrane fluidic properties of the bacteria. This novel mechanism of interaction between sex steroids and P. aeruginosa explicates the reported increased disease severity observed in females with cystic fibrosis and provides evidence for the therapeutic potential of the modulation of sex steroids to achieve better clinical outcomes in patients with hormone-responsive strains.IMPORTANCE Molecular mechanisms by which sex steroids interact with P. aeruginosa to modulate its virulence have yet to be reported. Our work provides the first characterization of a steroid-induced membrane stress mechanism promoting P. aeruginosa virulence, which includes the release of proinflammatory outer membrane vesicles, resulting in inflammation, host tissue damage, and reduced bacterial clearance. We further demonstrate that at nanomolar (physiological) concentrations, male and female sex steroids promote virulence in clinical strains of P. aeruginosa based on their dynamic membrane fluidic properties. This work provides, for the first-time, mechanistic insight to better understand and predict the P. aeruginosa related response to sex steroids and explain the interindividual patient variability observed in respiratory diseases such as cystic fibrosis that are complicated by gender differences and chronic P. aeruginosa infection.

12.
APL Bioeng ; 4(2): 020901, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32478279

RESUMO

Fluorescence spectroscopy has been a cornerstone of research in membrane dynamics and organization. Technological advances in fluorescence spectroscopy went hand in hand with discovery of various physicochemical properties of membranes at nanometric spatial and microsecond timescales. In this perspective, we discuss the various challenges associated with quantification of physicochemical properties of membranes and how various modes of fluorescence spectroscopy have overcome these challenges to shed light on the structure and organization of membranes. Finally, we discuss newer measurement strategies and data analysis tools to investigate the structure, dynamics, and organization of membranes.

13.
Biophys J ; 118(10): 2434-2447, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32333863

RESUMO

Diffusion obstacles in membranes have not been directly visualized because of fast membrane dynamics and the occurrence of subresolution molecular complexes. To understand the obstacle characteristics, mobility-based methods are often used as an indirect way of assessing the membrane structure. Molecular movement in biological plasma membranes is often characterized by anomalous diffusion, but the exact underlying mechanisms are still elusive. Imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) is a well-established mobility-based method that provides spatially resolved diffusion coefficient maps and is combined with FCS diffusion law analysis to examine subresolution membrane organization. In recent years, although FCS diffusion law analysis has been instrumental in providing new insights into the membrane structure below the optical diffraction limit, there are certain exceptions and anomalies that require further clarification. To this end, we correlate the membrane structural features imaged by atomic force microscopy (AFM) with the dynamics measured using ITIR-FCS. We perform ITIR-FCS measurements on supported lipid bilayers (SLBs) of various lipid compositions to characterize the anomalous diffusion of lipid molecules in distinct obstacle configurations, along with the high-resolution imaging of the membrane structures with AFM. Furthermore, we validate our experimental results by performing simulations on image grids with experimentally determined obstacle configurations. This study demonstrates that FCS diffusion law analysis is a powerful tool to determine membrane heterogeneities implied from dynamics measurements. Our results corroborate the commonly accepted interpretations of imaging FCS diffusion law analysis, and we show that exceptions happen when domains reach the percolation threshold in a biphasic membrane and a network of domains behaves rather like a meshwork, resulting in hop diffusion.


Assuntos
Bicamadas Lipídicas , Membrana Celular , Difusão , Microscopia de Força Atômica , Espectrometria de Fluorescência
14.
J Lipid Res ; 61(2): 252-266, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31857388

RESUMO

A fundamental feature of the eukaryotic cell membrane is the asymmetric arrangement of lipids in its two leaflets. A cell invests significant energy to maintain this asymmetry and uses it to regulate important biological processes, such as apoptosis and vesiculation. The dynamic coupling of the inner or cytoplasmic and outer or exofacial leaflets is a challenging open question in membrane biology. Here, we combined fluorescence lifetime imaging microscopy (FLIM) with imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) to differentiate the dynamics and organization of the two leaflets of live mammalian cells. We characterized the biophysical properties of fluorescent analogs of phosphatidylcholine, sphingomyelin, and phosphatidylserine in the plasma membrane of two mammalian cell lines (CHO-K1 and RBL-2H3). Because of their specific transverse membrane distribution, these probes allowed leaflet-specific investigation of the plasma membrane. We compared the results of the two methods having different temporal and spatial resolution. Fluorescence lifetimes of fluorescent lipid analogs were in ranges characteristic for the liquid ordered phase in the outer leaflet and for the liquid disordered phase in the inner leaflet. The observation of a more fluid inner leaflet was supported by free diffusion in the inner leaflet, with high average diffusion coefficients. The liquid ordered phase in the outer leaflet was accompanied by slower diffusion and diffusion with intermittent transient trapping. Our results show that the combination of FLIM and ITIR-FCS with specific fluorescent lipid analogs is a powerful tool for investigating lateral and transbilayer characteristics of plasma membrane in live cell lines.


Assuntos
Membrana Celular/metabolismo , Lipídeos/química , Microscopia de Fluorescência , Animais , Linhagem Celular , Membrana Celular/química , Estrutura Molecular , Espectrometria de Fluorescência , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo
15.
Biochim Biophys Acta Biomembr ; 1862(3): 183153, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31857071

RESUMO

The spatiotemporal dynamics of the plasma membrane is a consequence of fine-tuned interactions between membrane components. However, the precise identity of molecular factors that maintain this delicate balance, which is lost even in cell membrane derived mimics, remains elusive. Here, we use two cell lines, CHO-K1 and RBL-2H3, which show differences in outer membrane organization, dynamics, and cytoskeleton coupling, to investigate the underlying factors. To our surprise, knock-down of the cytoskeleton-interacting Immunoglobulin E receptor, which is abundant in RBL-2H3 but not in CHO-K1 cells, is not responsible for lipid confinement or cytoskeleton coupling. A subsequent lipidomic analysis of the two cell membranes revealed differences in total membrane ceramide content (C16 to C24). Analysis of the dynamics and organization of ceramide treated live cell membranes by imaging fluorescence correlation spectroscopy demonstrates that C24 and C16 saturated ceramides uniquely alter membrane dynamics by promoting the formation of cholesterol-independent domains and by elevating the inter-leaflet coupling.


Assuntos
Ceramidas/química , Bicamadas Lipídicas/química , Animais , Células CHO , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Ceramidas/metabolismo , Colesterol/química , Colesterol/metabolismo , Cricetulus , Citoesqueleto/metabolismo , Microdomínios da Membrana/química , Membranas/metabolismo , Ratos
16.
Front Cell Dev Biol ; 7: 281, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31803740

RESUMO

While the lateral organization of plasma membrane components has been shown to control binding of Wnt ligands to their receptors preferentially in the ordered membrane domains, the role of posttranslational lipid modification of Wnt on this selective binding is unknown. Here, we identify that the canonical Wnt is presumably acylated by palmitic acid, a saturated 16-carbon fatty acid, at a conserved serine residue. Acylation of Wnt3 is dispensable for its secretion and binding to Fz8 while it is essential for Wnt3's proper binding and domain-like diffusion in the ordered membrane domains. We further unravel that non-palmitoylated Wnt3 is unable to activate Wnt/ß-catenin signaling either in zebrafish embryos or in mammalian cells. Based on these results, we propose that the lipidation of canonical Wnt, presumably by a saturated fatty acid, determines its competence in interacting with the receptors in the appropriate domains of the plasma membrane, ultimately keeping the signaling activity under control.

17.
NPJ Biofilms Microbiomes ; 5(1): 35, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31728202

RESUMO

The influence of the biofilm matrix on molecular diffusion is commonly hypothesized to be responsible for emergent characteristics of biofilms such as nutrient trapping, signal accumulation and antibiotic tolerance. Hence quantifying the molecular diffusion coefficient is important to determine whether there is an influence of biofilm microenvironment on the mobility of molecules. Here, we use single plane illumination microscopy fluorescence correlation spectroscopy (SPIM-FCS) to obtain 3D diffusion coefficient maps with micrometre spatial and millisecond temporal resolution of entire Pseudomonas aeruginosa microcolonies. We probed how molecular properties such as size and charge as well as biofilm properties such as microcolony size and depth influence diffusion of fluorescently labelled dextrans inside biofilms. The 2 MDa dextran showed uneven penetration and a reduction in diffusion coefficient suggesting that the biofilm acts as a molecular sieve. Its diffusion coefficient was negatively correlated with the size of the microcolony. Positively charged dextran molecules and positively charged antibiotic tobramycin preferentially partitioned into the biofilm and remained mobile inside the microcolony, albeit with a reduced diffusion coefficient. Lastly, we measured changes of diffusion upon induction of dispersal and detected an increase in diffusion coefficient inside the biofilm before any loss of biomass. Thus, the change in diffusion is a proxy to detect early stages of dispersal. Our work shows that 3D diffusion maps are very sensitive to physiological changes in biofilms, viz. dispersal. However, this study also shows that diffusion, as mediated by the biofilm matrix, does not account for the high level of antibiotic tolerance associated with biofilms.


Assuntos
Antibacterianos/análise , Biofilmes/crescimento & desenvolvimento , Dextranos/análise , Difusão , Matriz Extracelular de Substâncias Poliméricas/química , Pseudomonas aeruginosa/crescimento & desenvolvimento , Tobramicina/análise , Imageamento Tridimensional , Análise Espaço-Temporal , Espectrometria de Fluorescência
18.
Biophys J ; 117(9): 1615-1625, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31590891

RESUMO

How nuclear proteins diffuse and find their targets remains a key question in the transcription field. Dynamic proteins in the nucleus are classically subdiffusive and undergo anomalous diffusion, yet the underlying physical mechanisms are still debated. In this study, we explore the contribution of interactions to the generation of anomalous diffusion by the means of fluorescence spectroscopy and simulation. Using interaction-deficient mutants, our study indicates that HEXIM1 interactions with both 7SK RNA and positive transcription elongation factor b are critical for HEXIM1 subdiffusion and thus provides evidence of the effects of protein-RNA interaction on molecular diffusion. Numerical simulations allowed us to establish that the proportions of distinct oligomeric HEXIM1 subpopulations define the apparent anomaly parameter of the whole population. Slight changes in the proportions of these oligomers can lead to significant shifts in the diffusive features and recapitulate the modifications observed in cells with the various interaction-deficient mutants. By combining simulations and experiments, our work opens new prospects in which the anomaly α coefficient in diffusion becomes a helpful tool to infer alterations in molecular interactions.


Assuntos
Núcleo Celular/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Simulação por Computador , Difusão , Humanos , Modelos Moleculares , Ligação Proteica , RNA Longo não Codificante/genética , Espectrometria de Fluorescência
20.
Anal Bioanal Chem ; 411(15): 3229-3240, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31025181

RESUMO

Copper is one of the most important transition metals in many organisms where it catalyzes a manifold of different processes. As a result of copper's redox activity, organisms have to avoid unbound ions, and a dysfunctional copper homeostasis may lead to multifarious pathological processes in cells with very severe ramifications for the affected organisms. In many neurodegenerative diseases, however, the exact role of copper ions is still not completely clarified. In this work, a high-affinity and highly selective copper probe molecule, based on the naturally occurring tetrapeptide DAHK is synthesized. The sensor (log KD = - 12.8 ± 0.1) is tagged with a fluorescent BODIPY dye whose fluorescence lifetime distinctly decreases from 5.8 ns ± 0.2 ns to 0.4 ns ± 0.1 ns on binding to copper(II) cations. It is shown by using fluorescence lifetime correlation spectroscopy that the concentration of both probe and probe-copper complex can be simultaneously measured even at nanomolar concentration levels. This work presents a possible starting point for a new type of probe and method for future in vivo studies to further reveal the exact role of copper ions in organisms. Graphical abstract.


Assuntos
Compostos de Boro/química , Cobre/análise , Corantes Fluorescentes/química , Oligopeptídeos/química , Espectrometria de Fluorescência/métodos , Compostos de Boro/síntese química , Cátions Bivalentes/análise , Fluorescência , Corantes Fluorescentes/síntese química , Humanos , Oligopeptídeos/síntese química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...