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1.
Nat Commun ; 11(1): 5998, 2020 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-33243988

RESUMO

Intratumoral heterogeneity is a common feature of many myeloid leukemias and a significant reason for treatment failure and relapse. Thus, identifying the cells responsible for residual disease and leukemia re-growth is critical to better understanding how they are regulated. Here, we show that a knock-in reporter mouse for the stem cell gene Musashi 2 (Msi2) allows identification of leukemia stem cells in aggressive myeloid malignancies, and provides a strategy for defining their core dependencies. Specifically, we carry out a high throughput screen using Msi2-reporter blast crisis chronic myeloid leukemia (bcCML) and identify several adhesion molecules that are preferentially expressed in therapy resistant bcCML cells and play a key role in bcCML. In particular, we focus on syndecan-1, whose deletion triggers defects in bcCML growth and propagation and markedly improves survival of transplanted mice. Further, live imaging reveals that the spatiotemporal dynamics of leukemia cells are critically dependent on syndecan signaling, as loss of this signal impairs their localization, migration and dissemination to distant sites. Finally, at a molecular level, syndecan loss directly impairs integrin ß7 function, suggesting that syndecan exerts its influence, at least in part, by coordinating integrin activity in bcCML. These data present a platform for delineating the biological underpinnings of leukemia stem cell function, and highlight the Sdc1-Itgß7 signaling axis as a key regulatory control point for bcCML growth and dissemination.


Assuntos
Crise Blástica/terapia , Leucemia Mieloide Aguda/terapia , Células-Tronco Neoplásicas/patologia , Proteínas de Ligação a RNA/genética , Sindecana-1/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Crise Blástica/genética , Crise Blástica/patologia , Quimiorradioterapia/métodos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Genes Reporter/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Ensaios de Triagem em Larga Escala , Humanos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Cadeias beta de Integrinas/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos Transgênicos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos da radiação , RNA-Seq , Transdução de Sinais/efeitos dos fármacos , Sindecana-1/genética , Sindecana-1/metabolismo
2.
PLoS Biol ; 18(11): e3000936, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33137097

RESUMO

Using mRNA sequencing and de novo transcriptome assembly, we identified, cloned, and characterized 9 previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from A. victoria green fluorescent protein (avGFP). Among these FPs are the brightest green fluorescent protein (GFP) homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including 2 that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.


Assuntos
Hidrozoários/genética , Hidrozoários/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Animais , Técnicas Biossensoriais , Cor , Cristalografia por Raios X , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hidrozoários/química , Proteínas Luminescentes/química , Modelos Moleculares , Imagem Óptica , Filogenia , Eletricidade Estática
3.
PLoS One ; 15(11): e0242599, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33227033

RESUMO

Sensorineural hearing loss is one of the most common disabilities worldwide. Such prevalence necessitates effective tools for studying the molecular workings of cochlear cells. One prominent and effective vector for expressing genes of interest in research models is adeno-associated virus (AAV). However, AAV efficacy in transducing cochlear cells can vary for a number of reasons including serotype, species, and methodology, and oftentimes requires high multiplicity of infection which can damage the sensory cells. Reports in other systems suggest multiple approaches can be used to enhance AAV transduction including self-complementary vector design and pharmacological inhibition of degradation. Here we produced AAV to drive green fluorescent protein (GFP) expression in explanted neonatal mouse cochleae. Treatment with eeyarestatin I, tyrphostin 23, or lipofectamine 2000 did not result in increased transduction, however, self-complementary vector design resulted in significantly more GFP positive cells when compared to single-stranded controls. Similarly, self-complementary AAV2 vectors demonstrated enhanced transduction efficiency compared to single stranded AAV2 when injected via the posterior semicircular canal, in vivo. Self-complementary vectors for AAV1, 8, and 9 serotypes also demonstrated robust GFP transduction in cochlear cells in vivo, though these were not directly compared to single stranded vectors. These findings suggest that second-strand synthesis may be a rate limiting step in AAV transduction of cochlear tissues and that self-complementary AAV can be used to effectively target large numbers of cochlear cells in vitro and in vivo.


Assuntos
Cóclea/metabolismo , Dependovirus/genética , Transdução Genética/métodos , Animais , Animais Recém-Nascidos , Linhagem Celular Tumoral , Cóclea/virologia , Feminino , Expressão Gênica , Engenharia Genética/métodos , Terapia Genética/métodos , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sorogrupo , Transgenes/genética
4.
PLoS Biol ; 18(11): e3000965, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33232322

RESUMO

Near-infrared (NIR) genetically encoded calcium ion (Ca2+) indicators (GECIs) can provide advantages over visible wavelength fluorescent GECIs in terms of reduced phototoxicity, minimal spectral cross talk with visible light excitable optogenetic tools and fluorescent probes, and decreased scattering and absorption in mammalian tissues. Our previously reported NIR GECI, NIR-GECO1, has these advantages but also has several disadvantages including lower brightness and limited fluorescence response compared to state-of-the-art visible wavelength GECIs, when used for imaging of neuronal activity. Here, we report 2 improved NIR GECI variants, designated NIR-GECO2 and NIR-GECO2G, derived from NIR-GECO1. We characterized the performance of the new NIR GECIs in cultured cells, acute mouse brain slices, and Caenorhabditis elegans and Xenopus laevis in vivo. Our results demonstrate that NIR-GECO2 and NIR-GECO2G provide substantial improvements over NIR-GECO1 for imaging of neuronal Ca2+ dynamics.


Assuntos
Cálcio/metabolismo , Imagem Óptica/métodos , Animais , Encéfalo/metabolismo , Caenorhabditis elegans/metabolismo , Corantes Fluorescentes , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Indicadores e Reagentes , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Camundongos , Miócitos Cardíacos/metabolismo , Neurônios/metabolismo , Optogenética , Engenharia de Proteínas , Espectroscopia de Luz Próxima ao Infravermelho , Xenopus laevis/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(36): 22122-22127, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32839332

RESUMO

Cnidarian fluorescent protein (FP) derivatives such as GFP, mCherry, and mEOS2 have been widely used to monitor gene expression and protein localization through biological imaging because they are considered functionally inert. We demonstrate that FPs specifically bind amyloid fibrils formed from many natural peptides and proteins. FPs do not bind other nonamyloid fibrillar structures such as microtubules or actin filaments and do not bind to amorphous aggregates. FPs can also bind small aggregates formed during the lag phase and early elongation phase of fibril formation and can inhibit amyloid fibril formation in a dose-dependent manner. These findings suggest caution should be taken in interpreting FP-fusion protein localization data when amyloid structures may be present. Given the pathological significance of amyloid-related species in some diseases, detection and inhibition of amyloid fibril formation using FPs can provide insights on developing diagnostic tools.


Assuntos
Proteínas Amiloidogênicas/química , Proteínas de Fluorescência Verde/química , Microscopia Confocal/métodos , Sequência de Aminoácidos , Humanos , Proteínas Luminescentes , Conformação Proteica
6.
Nat Chem Biol ; 16(10): 1143-1148, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32601486

RESUMO

Membraneless organelles formed by liquid-liquid phase separation of proteins or nucleic acids are involved in diverse biological processes in eukaryotes. However, such cellular compartments have yet to be discovered or created synthetically in prokaryotes. Here, we report the formation of liquid protein condensates inside the cells of prokaryotic Escherichia coli upon heterologous overexpression of intrinsically disordered proteins such as spider silk and resilin. In vitro reconstitution under conditions that mimic intracellular physiologically crowding environments of E. coli revealed that the condensates are formed via liquid-liquid phase separation. We also show functionalization of these condensates via targeted colocalization of cargo proteins to create functional membraneless compartments able to fluoresce and to catalyze biochemical reactions. The ability to form and functionalize membraneless compartments may serve as a versatile tool to develop artificial organelles with on-demand functions in prokaryotes for applications in synthetic biology.


Assuntos
Membrana Celular , Escherichia coli/fisiologia , Organelas , Citosol/química , Citosol/metabolismo , Difusão Dinâmica da Luz , Fibroínas/química , Regulação Bacteriana da Expressão Gênica , Proteínas de Fluorescência Verde/química , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência
7.
Sci Rep ; 10(1): 11049, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32632329

RESUMO

Fluorogen-activating proteins (FAPs) are innovative fluorescent probes combining advantages of genetically-encoded proteins such as green fluorescent protein and externally added fluorogens that allow for highly tunable and on demand fluorescent signaling. Previously, a panel of green- and red-emitting FAPs has been created from bacterial lipocalin Blc (named DiBs). Here we present a rational design as well as functional and structural characterization of the first self-assembling FAP split system, DiB-splits. This new system decreases the size of the FAP label to ~8-12 kDa while preserving DiBs' unique properties: strong increase in fluorescence intensity of the chromophore upon binding, binding affinities to the chromophore in nanomolar to low micromolar range, and high photostability of the protein-ligand complex. These properties allow for use of DiB-splits for wide-field, confocal, and super-resolution fluorescence microscopy. DiB-splits also represent an attractive starting point for further design of a protein-protein interaction detection system as well as novel FAP-based sensors.


Assuntos
Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/química , Cristalografia por Raios X , Fluorescência , Proteínas de Fluorescência Verde/genética , Células HEK293 , Células HeLa , Humanos , Lipocalinas/química , Lipocalinas/genética , Microscopia de Fluorescência , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Engenharia de Proteínas/métodos , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
8.
Science ; 368(6496): 1205-1210, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32527825

RESUMO

Cell migration is driven by local membrane protrusion through directed polymerization of F-actin at the front. However, F-actin next to the plasma membrane also tethers the membrane and thus resists outgoing protrusions. Here, we developed a fluorescent reporter to monitor changes in the density of membrane-proximal F-actin (MPA) during membrane protrusion and cell migration. Unlike the total F-actin concentration, which was high in the front of migrating cells, MPA density was low in the front and high in the back. Back-to-front MPA density gradients were controlled by higher cofilin-mediated turnover of F-actin in the front. Furthermore, nascent membrane protrusions selectively extended outward from areas where MPA density was reduced. Thus, locally low MPA density directs local membrane protrusions and stabilizes cell polarization during cell migration.


Assuntos
Actinas/metabolismo , Movimento Celular , Extensões da Superfície Celular , Actinas/química , Actinas/genética , Membrana Celular , Polaridade Celular , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos
9.
Phys Chem Chem Phys ; 22(26): 14704-14711, 2020 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-32573569

RESUMO

Green fluorescent protein (GFP) is a widely used fluorescent probe in the life sciences and biosciences due to its high quantum yield and extinction coefficient, and its ability to bind to biological systems of interest. This study measures the fluorescence lifetime of GFP in sucrose/water solutions of known molarity in order to determine the refractive index dependent lifetime of GFP. A range of refractive indices from 1.43-1.53 were probed by levitating micron sized droplets composed of water/sucrose/GFP in an optical trap under well-constrained conditions of relative humidity. This setup allows for the first reported measurements of the fluorescence lifetime of GFP at refractive indices greater than 1.46. The results obtained at refractive indices less than 1.46 show good agreement with previous studies. Further experiments that trapped droplets of deionised water containing GFP allowed the hygroscopic properties of GFP to be measured. GFP is found to be mildly hygroscopic by mass, but the high ratio of molecular masses of GFP to water (ca. 1500 : 1) signifies that water uptake is large on a per-mole basis. Hygroscopic properties are verified using brightfield microscope imaging, of GFP droplets at low and high relative humidity, by measuring the humidity dependent droplet size. In addition, this experiment allowed the refractive index of pure GFP to be estimated for the first time (1.72 ± 0.07). This work provides reference data for future experiments involving GFP, especially for those conducted in high refractive index media. The work also demonstrates that GFP can be used as a probe for aerosol studies, which require determination of the refractive index of the aerosol of any shape.


Assuntos
Proteínas de Fluorescência Verde/química , Fluorescência , Pinças Ópticas , Refratometria , Sacarose/química , Água/química , Molhabilidade
10.
Proc Natl Acad Sci U S A ; 117(27): 16019-16026, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32576684

RESUMO

The intracellular redox state is one of the key factors regulating various physiological phenomena in the cell. Monitoring this state is therefore important for understanding physiological homeostasis in cells. Various fluorescent sensor proteins have already been developed to monitor intracellular redox state. We also developed fluorescent redox sensor proteins named Oba-Q and Re-Q, the emissions of which are quenched under oxidized and reduced conditions, respectively. Although these sensors were useful to visualize the redox changes in the cell over time, they have the weakness that their emission signals are directly influenced by their in situ expression levels. To overcome this problem, we developed a redox sensor protein with a single excitation peak and dual variable emission peaks. This sensor protein shows green emission under oxidized conditions and blue emission under reduced conditions. We therefore named this sensor FROG/B, fluorescent protein with redox-dependent change in green/blue. By using this sensor, we successfully measured the changes in intracellular redox potentials in cyanobacterial cells quantitatively caused by light/dark transition just by calculating the ratio of emission between green and blue signals.


Assuntos
Técnicas Biossensoriais , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Anabaena , Glutationa/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Oxirredução
11.
Acta Biochim Biophys Sin (Shanghai) ; 52(9): 998-1006, 2020 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-32582951

RESUMO

Bimolecular fluorescence complementation (BiFC) is a popular method used to detect protein-protein interactions. For a BiFC assay, a fluorescent protein is usually split into two parts, and the fluorescence is recovered upon the interaction between the fused proteins of interest. As an elegant extension of BiFC, a tripartite superfold green fluorescent protein (sfGFP) system that has the advantages of low background fluorescence and small fusion tag size has been developed. However, the tripartite system exhibits a low fluorescence signal in some cases. To address this problem, we proposed to increase the affinity between the two parts, G1-9 and G11, of the tripartite system by adding affinity pairs. Among the three affinity pairs tested, LgBiT-HiBiT improved both the signal and signal-to-noise (S/N) ratio to the greatest extent. More strikingly, the direct covalent fusion of G11 to G1-9, which converted the tripartite system into a new bipartite system, enhanced the S/N ratio from 20 to 146, which is superior to the bipartite sfGFP system split at 157/158 or 173/174. Our results implied that the 10th ß-strand of sfGFP has a low affinity and a good recovery efficiency to construct a robust BiFC system, and this concept might be applied to other fluorescent proteins with similar structure to construct new BiFC systems.


Assuntos
Fluorescência , Proteínas de Fluorescência Verde , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Microscopia de Fluorescência
12.
Nat Commun ; 11(1): 2876, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32513994

RESUMO

Precise gene editing aims at generating single-nucleotide modifications to correct or model human disease. However, precision editing with nucleases such as CRIPSR-Cas9 has seen limited success due to poor efficiency and limited practicality. Here, we establish a fluorescent DNA repair assay in human induced pluripotent stem (iPS) cells to visualize and quantify the frequency of DNA repair outcomes during monoallelic and biallelic targeting. We found that modulating both DNA repair and cell cycle phase via defined culture conditions and small molecules synergistically enhanced the frequency of homology-directed repair (HDR). Notably, targeting in homozygous reporter cells results in high levels of editing with a vast majority of biallelic HDR outcomes. We then leverage efficient biallelic HDR with mixed ssODN repair templates to generate heterozygous mutations. Synergistic gene editing represents an effective strategy to generate precise genetic modifications in human iPS cells.


Assuntos
Ciclo Celular , Reparo do DNA , Edição de Genes , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Alelos , Sequência de Aminoácidos , Sequência de Bases , Resposta ao Choque Frio , Fluorescência , Loci Gênicos , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Homozigoto , Humanos , Mutação/genética
13.
Nat Commun ; 11(1): 2809, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499559

RESUMO

Formation of membrane-less organelles via liquid-liquid phase separation is one way cells meet the biological requirement for spatiotemporal regulation of cellular components and reactions. Recently, tau, a protein known for its involvement in Alzheimer's disease and other tauopathies, was found to undergo liquid-liquid phase separation making it one of several proteins associated with neurodegenerative diseases to do so. Here, we demonstrate that tau forms dynamic liquid droplets in vitro at physiological protein levels upon molecular crowding in buffers that resemble physiological conditions. Tau droplet formation is significantly enhanced by disease-associated modifications, including the AT8 phospho-epitope and the P301L tau mutation linked to an inherited tauopathy. Moreover, tau droplet dynamics are significantly reduced by these modified forms of tau. Extended phase separation promoted a time-dependent adoption of toxic conformations and oligomerization, but not filamentous aggregation. P301L tau protein showed the greatest oligomer formation following extended phase separation. These findings suggest that phase separation of tau may facilitate the formation of non-filamentous pathogenic tau conformations.


Assuntos
Extração Líquido-Líquido , Proteínas tau/química , Animais , Benzotiazóis/química , Encéfalo/metabolismo , Linhagem Celular , Epitopos/química , Proteínas de Fluorescência Verde/química , Humanos , Insetos , Mutação , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Análise de Regressão
14.
Nat Commun ; 11(1): 2429, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415067

RESUMO

Proton-linked monocarboxylate transporters (MCTs) must transport monocarboxylate efficiently to facilitate monocarboxylate efflux in glycolytically active cells, and transport monocarboxylate slowly or even shut down to maintain a physiological monocarboxylate concentration in glycolytically inactive cells. To discover how MCTs solve this fundamental aspect of intracellular monocarboxylate homeostasis in the context of multicellular organisms, we analyzed pyruvate transport activity of human monocarboxylate transporter 2 (MCT2). Here we show that MCT2 transport activity exhibits steep dependence on substrate concentration. This property allows MCTs to turn on almost like a switch, which is physiologically crucial to the operation of MCTs in the cellular context. We further determined the cryo-electron microscopy structure of the human MCT2, demonstrating that the concentration sensitivity of MCT2 arises from the strong inter-subunit cooperativity of the MCT2 dimer during transport. These data establish definitively a clear example of evolutionary optimization of protein function.


Assuntos
Transportadores de Ácidos Monocarboxílicos/metabolismo , Motivos de Aminoácidos , Microscopia Crioeletrônica , Proteínas de Fluorescência Verde/química , Células HEK293 , Homeostase , Humanos , Microscopia de Fluorescência , Simulação de Acoplamento Molecular , Ligação Proteica , Multimerização Proteica , Transporte Proteico
15.
PLoS One ; 15(5): e0226472, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379828

RESUMO

The ParB-parS partition complexes that bacterial replicons use to ensure their faithful inheritance also find employment in visualization of DNA loci, as less intrusive alternatives to fluorescent repressor-operator systems. The ability of ParB molecules to interact via their N-terminal domains and to bind to non-specific DNA enables expansion of the initial complex to a size both functional in partition and, via fusion to fluorescent peptides, visible by light microscopy. We have investigated whether it is possible to dispense with the need to insert parS in the genomic locus of interest, by determining whether ParB fused to proteins that bind specifically to natural DNA sequences can still assemble visible complexes. In yeast cells, coproduction of fusions of ParB to a fluorescent peptide and to a TALE protein targeting an endogenous sequence did not yield visible foci; nor did any of several variants of these components. In E.coli, coproduction of fusions of SopB (F plasmid ParB) to fluorescent peptide, and to dCas9 together with specific guide RNAs, likewise yielded no foci. The result of coproducing analogous fusions of SopB proteins with distinct binding specificities was also negative. Our observations imply that in order to assemble higher order partition complexes, ParB proteins need specific activation through binding to their cognate parS sites.


Assuntos
Proteínas de Bactérias/metabolismo , Centrômero/química , Centrômero/metabolismo , DNA Bacteriano/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Bases , Sítios de Ligação , Proteína 9 Associada à CRISPR , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Plasmídeos/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Simportadores/genética , Simportadores/metabolismo
16.
Sci Rep ; 10(1): 7410, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366988

RESUMO

Construction of small molecule ligand (SML) based delivery systems has been performed starting from a polyfunctionalized isoxazoline scaffold, whose αvß3 and α5ß1 integrins' potency has been already established. The synthesis of this novel class of ligands was obtained by conjugation of linkers to the heterocyclic core via Huisgen-click reaction, with the aim to use them as "shuttles" for selective delivery of diagnostic agents to cancer cells, exploring the effects of the side chains in the interaction with the target. Compounds 17b and 24 showed excellent potency towards α5ß1 integrin acting as selective antagonist and agonist respectively. Further investigations confirmed their effects on target receptor through the analysis of fibronectin-induced ERK1/2 phosphorylation. In addition, confocal microscopy analysis allowed us to follow the fate of EGFP conjugated α5ß1 integrin and 17b FITC-conjugated (compound 31) inside the cells. Moreover, the stability in water solution at different values of pH and in bovine serum confirmed the possible exploitation of these peptidomimetic molecules for pharmaceutical application.


Assuntos
Integrina alfa5beta1/química , Integrina alfaVbeta3/química , Isoxazóis/química , Oligopeptídeos/química , Peptidomiméticos , Animais , Bovinos , Adesão Celular , Fibronectinas/química , Proteínas de Fluorescência Verde/química , Humanos , Concentração de Íons de Hidrogênio , Células K562 , Ligantes , Sistema de Sinalização das MAP Quinases , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular
17.
J Mol Biol ; 432(13): 3749-3760, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32302608

RESUMO

Optically controlled receptor tyrosine kinases (opto-RTKs) allow regulation of RTK signaling using light. Until recently, the majority of opto-RTKs were activated with blue-green light. Fusing a photosensory core module of Deinococcus radiodurans bacterial phytochrome (DrBphP-PCM) to the kinase domains of neurotrophin receptors resulted in opto-RTKs controlled with light above 650 nm. To expand this engineering approach to RTKs of other families, here we combined the DrBpP-PCM with the cytoplasmic domains of EGFR and FGFR1. The resultant Dr-EGFR and Dr-FGFR1 opto-RTKs are rapidly activated with near-infrared and inactivated with far-red light. The opto-RTKs efficiently trigger ERK1/2, PI3K/Akt, and PLCγ signaling. Absence of spectral crosstalk between the opto-RTKs and green fluorescent protein-based biosensors enables simultaneous Dr-FGFR1 activation and detection of calcium transients. Action mechanism of the DrBphP-PCM-based opto-RTKs is considered using the available RTK structures. DrBphP-PCM represents a versatile scaffold for engineering of opto-RTKs that are reversibly regulated with far-red and near-infrared light.


Assuntos
Fitocromo/ultraestrutura , Receptores Proteína Tirosina Quinases/ultraestrutura , Engenharia Tecidual/métodos , Tecidos Suporte/química , Técnicas Biossensoriais , Deinococcus/química , Deinococcus/genética , Proteínas de Fluorescência Verde/química , Células HeLa , Humanos , Luz , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/genética , Fosfatidilinositol 3-Quinases/genética , Fitocromo/química , Fitocromo/genética , Conformação Proteica/efeitos da radiação , Proteínas Proto-Oncogênicas c-akt/genética , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptores de Fator de Crescimento Neural/química , Receptores de Fator de Crescimento Neural/genética , Transdução de Sinais/efeitos da radiação
18.
Mater Sci Eng C Mater Biol Appl ; 111: 110768, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32279782

RESUMO

Functional polymer-protein nanoparticles (NPs) have broad applications in biotechnology and nanotechnology. In principle, controllable and vigorous mixing is required to fabricate homogeneous NPs, which remains a challenge via conventional bulk synthetic methods. In this study, an electrokinetics (EK) based microfluidic reactor with fast mixing is explored to assemble functional proteins with polymers in an ethanol/water co-solvent system. The resultant NPs show significantly improved size distribution by comparison with the ones prepared using conventional bulk method, while the NPs size can be tuned by adjusting the mass ratio of polymer to protein. The functionalities of the assembled proteins are sustained upon the EK based microfluidic mixing, indicating the application potential of our method in the controlled assembly of different functional proteins.


Assuntos
Microfluídica/métodos , Nanopartículas/química , Polímeros/química , Proteínas/química , Condutividade Elétrica , Proteínas de Fluorescência Verde/química , Tamanho da Partícula , Solventes/química
19.
Chemistry ; 26(27): 5942-5945, 2020 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-32153063

RESUMO

To turn on the fluorescence of the native green fluorescence protein (GFP) chromophore, 4-hydroxybenzylidene-dimethylimidazolinone (HBDI), in an artificial supramolecular system has been a challenging task, because it requires high local environmental rigidity. This work shows that the formation of H-aggregates of an HBDI-containing organogelator results in two orders of magnitude fluorescence enhancement (Φf =2.9 vs. 0.02 %), in which the inter-HBDI OH⋅⋅⋅OH H-bonds play a crucial role. The aggregation-induced fluorescence enhancement of HBDI has important implications on the origin of the high fluorescence quantum efficiency of HBDI in the GFP ß-barrel and on the supramolecular strategy for a full fluorescence recovery of HBDI. These results reveal a new approach to designing rigid chromophore aggregates for high-performance optoelectronic properties.


Assuntos
Proteínas de Fluorescência Verde/química , Fluorescência , Ligação de Hidrogênio , Estrutura Molecular
20.
PLoS One ; 15(3): e0230344, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32214330

RESUMO

In age-related macular degeneration (AMD) or diabetic retinopathy (DR), hypoxia and inflammatory processes lead to an upregulation of the vascular endothelial growth factor (VEGF) expression and thereby to pathological neovascularisation with incorrectly formed vessels prone to damage, thus increasing the vascular permeability and the risk of bleeding and oedema in the retina. State of the art treatment is the repeated intraocular injection of anti-VEGF molecules. For developing improved individualized treatment approaches, a minimally invasive, repeatable method for in vivo quantification of VEGF in the eye is necessary. Therefore, we designed single molecule eBRET2 VEGF biosensors by directly fusing a Renilla luciferase mutant (Rluc8) N-terminal and a green fluorescent protein (GFP2) C-terminal to a VEGF binding domain. In total, 10 different VEGF biosensors (Re01- Re10) were generated based on either single domains or full length of VEGF receptor 1 or 2 extracellular regions as VEGF binding domains. Full length expression of the biosensors in HEK293-T cells was verified via Western Blot employing an anti-Rluc8-IgG. Expression of alternative splice variants was eliminated through the deletion of the donor splice site by introduction of a silent point mutation. In all ten biosensors the energy transfer from the Rluc8 to the GFP2 occurs and generates a measurable eBRET2 ratio. Four biosensors show a relevant change of the BRET ratio (ΔBR) after VEGF binding. Furthermore, each biosensor shows a unique detection range for VEGF quantification and especially Re06 and Re07 have a high sensitivity in the range of in vivo VEGF concentrations in the eye, previously measured by invasive methods. In conclusion, we generated several eBRET2 biosensors that are suitable for VEGF quantification in vitro and could identify two eBRET2 biosensors, which may be suitable for non-invasive in vivo VEGF quantification with an implantable device.


Assuntos
Técnicas Biossensoriais/instrumentação , Medições Luminescentes/instrumentação , Proteínas Recombinantes de Fusão/química , Fator A de Crescimento do Endotélio Vascular/análise , Animais , Córnea/patologia , Retinopatia Diabética/diagnóstico , Retinopatia Diabética/patologia , Transferência de Energia , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Luciferases de Renilla/química , Luciferases de Renilla/genética , Degeneração Macular/diagnóstico , Degeneração Macular/patologia , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Retina/patologia , Transfecção , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo
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