Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 22.032
Filtrar
2.
J Phys Chem Lett ; 12(34): 8263-8271, 2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34424693

RESUMO

The role of protonation states of the chromophore and its neighboring amino acid side chains of the reversibly switching fluorescent protein rsEGFP2 upon photoswitching is characterized by molecular modeling methods. Numerous conformations of the chromophore-binding site in computationally derived model systems are obtained using the quantum chemistry and QM/MM approaches. Excitation energies are computed using the extended multiconfigurational quasidegenerate perturbation theory (XMCQDPT2). The obtained structures and absorption spectra allow us to provide an interpretation of the observed structural and spectral properties of rsEGFP2 in the active ON and inactive OFF states. The results demonstrate that in addition to the dominating anionic and neutral forms of the chromophore, the cationic and zwitterionic forms may participate in the photoswitching of rsEGFP2. Conformations and protonation forms of the Glu223 and His149 side chains in the chromophore-binding site play an essential role in stabilizing specific protonation forms of the chromophore.


Assuntos
Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Prótons , Teoria Quântica , Sítios de Ligação , Modelos Moleculares , Conformação Proteica
3.
Nat Commun ; 12(1): 5073, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417467

RESUMO

The contents of numerous membrane lipids change upon ageing. However, it is unknown whether and how any of these changes are causally linked to lifespan regulation. Acyl chains contribute to the functional specificity of membrane lipids. In this study, working with C. elegans, we identified an acyl chain-specific sphingolipid, C22 glucosylceramide, as a longevity metabolite. Germline deficiency, a conserved lifespan-extending paradigm, induces somatic expression of the fatty acid elongase ELO-3, and behenic acid (22:0) generated by ELO-3 is incorporated into glucosylceramide for lifespan regulation. Mechanistically, C22 glucosylceramide is required for the membrane localization of clathrin, a protein that regulates membrane budding. The reduction in C22 glucosylceramide impairs the clathrin-dependent autophagic lysosome reformation, which subsequently leads to TOR activation and longevity suppression. These findings reveal a mechanistic link between membrane lipids and ageing and suggest a model of lifespan regulation by fatty acid-mediated membrane configuration.


Assuntos
Caenorhabditis elegans/fisiologia , Ácidos Graxos não Esterificados/metabolismo , Glicoesfingolipídeos/metabolismo , Homeostase , Longevidade/fisiologia , Lisossomos/metabolismo , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Ceramidas/metabolismo , Colesterol/metabolismo , Clatrina/metabolismo , Mutação em Linhagem Germinativa/genética , Proteínas de Fluorescência Verde/metabolismo , Larva/metabolismo , Modelos Biológicos , Interferência de RNA , Estresse Fisiológico
4.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361009

RESUMO

The parietal cortex of rodents participates in sensory and spatial processing, movement planning, and decision-making, but much less is known about its role in associative learning and memory formation. The present study aims to examine the involvement of the parietal association cortex (PtA) in associative fear memory acquisition and retrieval in mice. Using ex vivo c-Fos immunohistochemical mapping and in vivo Fos-EGFP two-photon imaging, we show that PtA neurons were specifically activated both during acquisition and retrieval of cued fear memory. Fos immunohistochemistry revealed specific activation of the PtA neurons during retrieval of the 1-day-old fear memory. In vivo two-photon Fos-EGFP imaging confirmed this result and in addition detected specific c-Fos responses of the PtA neurons during acquisition of cued fear memory. To allow a more detailed study of the long-term activity of such PtA engram neurons, we generated a Fos-Cre-GCaMP transgenic mouse line that employs the Targeted Recombination in Active Populations (TRAP) technique to detect calcium events specifically in cells that were Fos-active during conditioning. We show that gradual accumulation of GCaMP3 in the PtA neurons of Fos-Cre-GCaMP mice peaks at the 4th day after fear learning. We also describe calcium transients in the cell bodies and dendrites of the TRAPed neurons. This provides a proof-of-principle for TRAP-based calcium imaging of PtA functions during memory processes as well as in experimental models of fear- and anxiety-related psychiatric disorders and their specific therapies.


Assuntos
Medo , Memória , Lobo Parietal/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Animais , Aprendizagem por Associação , Sinalização do Cálcio , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Lobo Parietal/citologia , Lobo Parietal/fisiologia , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
5.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445257

RESUMO

The production of pancreatic ß cells is the most challenging step for curing diabetes using next-generation treatments. Adult pancreatic endocrine cells are thought to be maintained by the self-duplication of differentiated cells, and pancreatic endocrine neogenesis can only be observed when the tissue is severely damaged. Experimentally, this can be performed using a method named partial duct ligation (PDL). As the success rate of PDL surgery is low because of difficulties in identifying the pancreatic duct, we previously proposed a method for fluorescently labeling the duct in live animals. Using this method, we performed PDL on neurogenin3 (Ngn3)-GFP transgenic mice to determine the origin of endocrine precursor cells and evaluate their potential to differentiate into multiple cell types. Ngn3-activated cells, which were marked with GFP, appeared after PDL operation. Because some GFP-positive cells were aligned proximally to the duct, we hypothesized that Ngn3-positive cells arise from the pancreatic duct. Therefore, we next developed an in vitro pancreatic duct culture system using Ngn3-GFP mice and examined whether Ngn3-positive cells emerge from this duct. We observed GFP expressions in ductal organoid cultures. GFP expressions were correlated with Ngn3 expressions and endocrine cell lineage markers. Interestingly, tuft cell markers were also correlated with GFP expressions. Our results demonstrate that in adult mice, Ngn3-positive endocrine precursor cells arise from the pancreatic ducts both in vivo and in vitro experiments indicating that the pancreatic duct could be a potential donor for therapeutic use.


Assuntos
Antígenos de Diferenciação/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ductos Pancreáticos/metabolismo , Células-Tronco/metabolismo , Animais , Antígenos de Diferenciação/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células Secretoras de Insulina/citologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Organoides/citologia , Organoides/metabolismo , Ductos Pancreáticos/citologia , Células-Tronco/citologia
6.
Bioengineered ; 12(1): 4407-4419, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34436976

RESUMO

Widespread infection due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has led to a global pandemic. Currently, various approaches are being taken up to develop vaccines and therapeutics to treat SARS-CoV2 infection. Consequently, the S protein has become an important target protein for developing vaccines and therapeutics against SARS-CoV2. However, the highly infective nature of SARS-CoV2 restricts experimentation with the virus to highly secure BSL3 facilities. The availability of fusion-enabled, nonreplicating, and nonbiohazardous mimics of SARS-CoV2 virus fusion, containing the viral S or S and M protein in their native conformation on mammalian cells, can serve as a useful substitute for studying viral fusion for testing various inhibitors of viral fusion. This would avoid the use of the BSL3 facility for fusion studies required to develop therapeutics. In the present study, we have developed SARS-CoV2 virus fusion mimics (SCFMs) using mammalian cells transfected with constructs coding for S or S and M protein. The fusogenic property of the mimic(s) and their interaction with the functional human ACE2 receptors was confirmed experimentally. We have also shown that such mimics can easily be used in an inhibition assay. These mimic(s) can be easily prepared on a large scale, and such SCFMs can serve as an invaluable resource for viral fusion inhibition assays and in vitro screening of antiviral agents, which can be shared/handled between labs/facilities without worrying about any biohazard while working under routine laboratory conditions, avoiding the use of BSL3 laboratory.Abbreviations :SCFM: SARS-CoV2 Virus Fusion Mimic; ACE2: Angiotensin-Converting Enzyme 2; hACE2: Human Angiotensin-Converting enzyme 2; MEF: Mouse Embryonic Fibroblasts; HBSS: Hanks Balanced Salt Solution; FBS: Fetal Bovine Serum.


Assuntos
Anticorpos Neutralizantes/farmacologia , Contenção de Riscos Biológicos/métodos , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Proteínas da Matriz Viral/antagonistas & inibidores , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Chlorocebus aethiops , Embrião de Mamíferos , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Células MCF-7 , Camundongos , Mimetismo Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , Ligação Proteica , Receptores Virais/genética , Receptores Virais/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Transfecção , Células Vero , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
7.
Molecules ; 26(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34443374

RESUMO

The activation of NFAT (nuclear factor of activated T cells) transcription factors by calcium-dependent phosphatase calcineurin is a key step in controlling T cell activation and plays a vital role during carcinogenesis. NFATs are overexpressed in many cancers, including the most common primary brain tumor, gliomas. In the present study, we demonstrate the expression of NFATs and NFAT-driven transcription in several human glioma cells. We used a VIVIT peptide for interference in calcineurin binding to NFAT via a conserved PxIxIT motif. VIVIT was expressed as a fusion protein with a green fluorescent protein (VIVIT-GFP) or conjugated to cell-penetrating peptides (CPP), Sim-2 or 11R. We analyzed the NFAT expression, phosphorylation, subcellular localization and their transcriptional activity in cells treated with peptides. Overexpression of VIVIT-GFP decreased the NFAT-driven activity and inhibited the transcription of endogenous NFAT-target genes. These effects were not reproduced with synthetic peptides: Sim2-VIVIT did not show any activity, and 11R-VIVIT did not inhibit NFAT signaling in glioma cells. The presence of two calcineurin docking sites in NFATc3 might require dual-specificity blocking peptides. The cell-penetrating peptides Sim-2 or 11R linked to VIVIT did not improve its action making it unsuitable for evaluating NFAT dependent events in glioma cells with high expression of NFATc3.


Assuntos
Neoplasias Encefálicas/patologia , Calcineurina/metabolismo , Glioma/patologia , Fatores de Transcrição NFATC/metabolismo , Oligopeptídeos/farmacologia , Transdução de Sinais , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacologia , Glioma/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Fatores de Transcrição NFATC/química , Oligopeptídeos/química , Peptídeos/farmacologia , Transporte Proteico/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
8.
Methods Mol Biol ; 2312: 237-251, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228294

RESUMO

Chemical control of protein localization is a powerful approach for manipulating mammalian cellular processes. Self-localizing ligand-induced protein translocation (SLIPT) is an emerging platform that enables control of protein localization in living mammalian cells using synthetic self-localizing ligands (SLs). We recently established a chemogenetic SLIPT system, in which any protein of interest fused to an engineered variant of Escherichia coli dihydrofolate reductase, DHFRiK6, can be rapidly and specifically translocated from the cytoplasm to the inner leaflet of the plasma membrane (PM) using a trimethoprim (TMP)-based PM-targeting SL, mDcTMP. The mDcTMP-mediated PM recruitment of DHFRiK6-fusion proteins can be efficiently returned to the cytoplasm by subsequent addition of free TMP, enabling temporal and reversible control over the protein localization. Here we describe the use of this mDcTMP/DHFRiK6-based SLIPT system for inducing (1) reversible protein translocation and (2) synthetic activation of the Raf/ERK pathway. This system provides a simple and versatile tool in mammalian synthetic biology for temporally manipulating various signaling molecules and pathways at the PM.


Assuntos
Engenharia Celular , Proteínas de Escherichia coli/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas Genéticas , Biologia Sintética , Tetra-Hidrofolato Desidrogenase/genética , Trimetoprima/farmacologia , Técnicas de Cultura de Células , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Tetra-Hidrofolato Desidrogenase/metabolismo , Quinases raf/metabolismo
9.
Methods Mol Biol ; 2312: 277-285, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228296

RESUMO

There are increasing evidence and growing interest in the relationship between protein aggregates/phase separation and various human diseases, especially neurodegenerative diseases. However, we do not entirely comprehend how aggregates generate or the clearance network of chaperones, proteasomes, ubiquitin ligases, and other factors interact with aggregates. Here, we describe chemically controllable systems compose with a genetically engineered cell and a small drug that enables us to rapidly induce protein aggregates' formation by withdrawing the small molecule. This trigger does not activate global stress responses induced by stimuli, such as proteasome inhibitors or heat shock. This method can produce aggregates in a specific compartment and diverse experimental systems, including live animals.


Assuntos
Engenharia Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Engenharia de Proteínas , Proteínas de Ligação a Tacrolimo/genética , Animais , Técnicas de Cultura de Células , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Camundongos , Microscopia de Fluorescência , Mutação , Células NIH 3T3 , Agregados Proteicos , Estabilidade Proteica , Proteostase , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Transfecção
10.
Methods Mol Biol ; 2312: 309-320, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228299

RESUMO

Developments in genome-editing technology, especially CRISPR-Cas9, have revolutionized the way in which genetically engineered animals are generated. However, the process of generation includes microinjection to the one-cell stage embryo and the transfer of the microinjected embryo to the surrogate animals, which requires trained personnel. We recently reported the method includes introduction of CRISPR-Cas9 systems to the developing cerebral cortex via in utero electroporation thus generating gene-targeted neural stem cells in vivo. This technique is widely applicable for gene knockout, monitoring gene expression, and lineage analysis in developmental biology. In this chapter, the detailed protocol of EGFP (enhanced green fluorescent protein) knock-in method via in utero electroporation is described.


Assuntos
Encéfalo/metabolismo , Eletroporação , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Técnicas de Transferência de Genes , Animais , Encéfalo/embriologia , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Técnicas de Introdução de Genes , Genes Reporter , Idade Gestacional , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Gravidez , RNA Guia/genética , RNA Guia/metabolismo
11.
Commun Biol ; 4(1): 841, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34230602

RESUMO

Characterizing protein-protein interactions (PPIs) is an effective method to help explore protein function. Here, through integrating a newly identified split human Rhinovirus 3 C (HRV 3 C) protease, super-folder GFP (sfGFP), and ClpXP-SsrA protein degradation machinery, we developed a fluorescence-assisted single-cell methodology (split protease-E. coli ClpXP (SPEC)) to explore protein-protein interactions for both eukaryotic and prokaryotic species in E. coli cells. We firstly identified a highly efficient split HRV 3 C protease with high re-assembly ability and then incorporated it into the SPEC method. The SPEC method could convert the cellular protein-protein interaction to quantitative fluorescence signals through a split HRV 3 C protease-mediated proteolytic reaction with high efficiency and broad temperature adaptability. Using SPEC method, we explored the interactions among effectors of representative type I-E and I-F CRISPR/Cas complexes, which combining with subsequent studies of Cas3 mutations conferred further understanding of the functions and structures of CRISPR/Cas complexes.


Assuntos
Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Mapas de Interação de Proteínas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas , Endopeptidase Clp/genética , Enterovirus/enzimologia , Enterovirus/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina Quinase/genética , Histidina Quinase/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteólise , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
12.
Nat Commun ; 12(1): 4504, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301936

RESUMO

Genes are expressed in stochastic transcriptional bursts linked to alternating active and inactive promoter states. A major challenge in transcription is understanding how promoter composition dictates bursting, particularly in multicellular organisms. We investigate two key Drosophila developmental promoter motifs, the TATA box (TATA) and the Initiator (INR). Using live imaging in Drosophila embryos and new computational methods, we demonstrate that bursting occurs on multiple timescales ranging from seconds to minutes. TATA-containing promoters and INR-containing promoters exhibit distinct dynamics, with one or two separate rate-limiting steps respectively. A TATA box is associated with long active states, high rates of polymerase initiation, and short-lived, infrequent inactive states. In contrast, the INR motif leads to two inactive states, one of which relates to promoter-proximal polymerase pausing. Surprisingly, the model suggests pausing is not obligatory, but occurs stochastically for a subset of polymerases. Overall, our results provide a rationale for promoter switching during zygotic genome activation.


Assuntos
Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Regiões Promotoras Genéticas/genética , TATA Box/genética , Imagem com Lapso de Tempo/métodos , Transcrição Genética/genética , Algoritmos , Animais , Animais Geneticamente Modificados , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Embrião não Mamífero/embriologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Modelos Teóricos
13.
Nat Commun ; 12(1): 4511, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301939

RESUMO

Septins are cytoskeletal proteins that assemble into hetero-oligomeric complexes and sense micron-scale membrane curvature. During infection with Shigella flexneri, an invasive enteropathogen, septins restrict actin tail formation by entrapping bacteria in cage-like structures. Here, we reconstitute septin cages in vitro using purified recombinant septin complexes (SEPT2-SEPT6-SEPT7), and study how these recognize bacterial cells and assemble on their surface. We show that septin complexes recognize the pole of growing Shigella cells. An amphipathic helix domain in human SEPT6 enables septins to sense positively curved membranes and entrap bacterial cells. Shigella strains lacking lipopolysaccharide components are more efficiently entrapped in septin cages. Finally, cryo-electron tomography of in vitro cages reveals how septins assemble as filaments on the bacterial cell surface.


Assuntos
Actinas/metabolismo , Disenteria Bacilar/metabolismo , Proteínas Recombinantes/metabolismo , Septinas/metabolismo , Shigella flexneri/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Disenteria Bacilar/microbiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Lipopolissacarídeos/metabolismo , Microscopia de Fluorescência , Mutação , Ligação Proteica , Septinas/genética , Shigella flexneri/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
Int J Mol Sci ; 22(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208100

RESUMO

Stress granules (SGs) are dynamic RNA-protein complexes localized in the cytoplasm that rapidly form under stress conditions and disperse when normal conditions are restored. The formation of SGs depends on the Ras-GAP SH3 domain-binding protein (G3BP). Formations, interactions and functions of plant and human SGs are strikingly similar, suggesting a conserved mechanism. However, functional analyses of plant G3BPs are missing. Thus, members of the Arabidopsis thaliana G3BP (AtG3BP) protein family were investigated in a complementation assay in a human G3BP knock-out cell line. It was shown that two out of seven AtG3BPs were able to complement the function of their human homolog. GFP-AtG3BP fusion proteins co-localized with human SG marker proteins Caprin-1 and eIF4G1 and restored SG formation in G3BP double KO cells. Interaction between AtG3BP-1 and -7 and known human G3BP interaction partners such as Caprin-1 and USP10 was also demonstrated by co-immunoprecipitation. In addition, an RG/RGG domain exchange from Arabidopsis G3BP into the human G3BP background showed the ability for complementation. In summary, our results support a conserved mechanism of SG function over the kingdoms, which will help to further elucidate the biological function of the Arabidopsis G3BP protein family.


Assuntos
Arabidopsis/metabolismo , Grânulos Citoplasmáticos/metabolismo , Estresse Fisiológico , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde/metabolismo , Humanos , Fenótipo , Filogenia , Ligação Proteica , Domínios Proteicos
15.
J Phys Chem B ; 125(29): 7997-8009, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34259526

RESUMO

Fluorescent proteins (FPs) have been widely used to visualize biological processes in living cells. It is essential to understand the underlying fluorescence mechanism to develop novel FPs and to interpret imaging data appropriately. Enhanced yellow fluorescent protein (eYFP) is one of the most typical FPs; however, several reports to date have been limited to individual discussion, which is insufficient to understand the full picture of the dynamics involved. In this study, we focused on the fluorescence resonance energy transfer (FRET) and dimerization behavior and performed picosecond time-resolved fluorescence measurements of eYFP and its A206K mutant, which does not form a dimer. The combination of the dissociation constant and the acid dissociation constant rationally explains the mechanism of ultrafast homo-FRET and ultrafast hetero-FRET. It is also shown that structural relaxation occurs in the dimer after excited-state proton transfer. The formation efficiencies and quaternary structures of dimers consisting of different protonation states are shown to be different. Furthermore, under high-concentration conditions, "slow" homo-FRET with tens of nanoseconds timescale occurs between monomers and dimers. The findings from this study will be applied to other fluorescent proteins such as Aequorea victoria green FP and its mutants and various red FPs with longer conjugation lengths.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Dimerização , Polarização de Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo
16.
Nat Commun ; 12(1): 4559, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315896

RESUMO

Activating mutations in the BRAF-MAPK pathway have been reported in histiocytoses, hematological inflammatory neoplasms characterized by multi-organ dissemination of pro-inflammatory myeloid cells. Here, we generate a humanized mouse model of transplantation of human hematopoietic stem and progenitor cells (HSPCs) expressing the activated form of BRAF (BRAFV600E). All mice transplanted with BRAFV600E-expressing HSPCs succumb to bone marrow failure, displaying myeloid-restricted hematopoiesis and multi-organ dissemination of aberrant mononuclear phagocytes. At the basis of this aggressive phenotype, we uncover the engagement of a senescence program, characterized by DNA damage response activation and a senescence-associated secretory phenotype, which affects also non-mutated bystander cells. Mechanistically, we identify TNFα as a key determinant of paracrine senescence and myeloid-restricted hematopoiesis and show that its inhibition dampens inflammation, delays disease onset and rescues hematopoietic defects in bystander cells. Our work establishes that senescence in the human hematopoietic system links oncogene-activation to the systemic inflammation observed in histiocytic neoplasms.


Assuntos
Senescência Celular , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Histiocitose/patologia , Inflamação/patologia , Células Mieloides/patologia , Oncogenes , Animais , Medula Óssea/patologia , Pontos de Checagem do Ciclo Celular/genética , Senescência Celular/genética , Doença Crônica , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Histiocitose/complicações , Humanos , Inflamação/complicações , Lentivirus/genética , Camundongos , Mutação/genética , Comunicação Parácrina , Análise de Componente Principal , Proteínas Proto-Oncogênicas B-raf/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo
17.
Int J Mol Sci ; 22(14)2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34298920

RESUMO

Protein dimerization plays a crucial role in the regulation of numerous biological processes. However, detecting protein dimers in a cellular environment is still a challenge. Here we present a methodology to measure the extent of dimerization of GFP-tagged proteins in living cells, using a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis of single-color fluorescence fluctuation data. We named this analysis method brightness and diffusion global analysis (BDGA) and adapted it for biological purposes. Using cell lysates containing different ratios of GFP and tandem-dimer GFP (diGFP), we show that the average brightness per particle is proportional to the fraction of dimer present. We further adapted this methodology for its application in living cells, and we were able to distinguish GFP, diGFP, as well as ligand-induced dimerization of FKBP12 (FK506 binding protein 12)-GFP. While other analysis methods have only sporadically been used to study dimerization in living cells and may be prone to errors, this paper provides a robust approach for the investigation of any cytosolic protein using single-color fluorescence fluctuation spectroscopy.


Assuntos
Multimerização Proteica/fisiologia , Proteínas/metabolismo , Células Cultivadas , Citosol/metabolismo , Dictyostelium/metabolismo , Difusão , Dimerização , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Ligantes , Fótons , Espectrometria de Fluorescência/métodos
18.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34073107

RESUMO

Inhaled Aspergillus fumigatus spores can be internalized by alveolar type II cells. Cell lines stably expressing fluorescently labeled components of endocytic pathway enable investigations of intracellular organization during conidia internalization and measurement of the process kinetics. The goal of this report was to evaluate the methodological appliance of cell lines for studying fungal conidia internalization. We have generated A549 cell lines stably expressing fluorescently labeled actin (LifeAct-mRuby2) and late endosomal protein (LAMP1-NeonGreen) following an evaluation of cell-pathogen interactions in live and fixed cells. Our data show that the LAMP1-NeonGreen cell line can be used to visualize conidia co-localization with LAMP1 in live and fixed cells. However, caution is necessary when using LifeAct-mRuby2-cell lines as it may affect the conidia internalization dynamics.


Assuntos
Células Epiteliais Alveolares , Aspergilose/microbiologia , Aspergillus fumigatus , Interações Hospedeiro-Patógeno , Células A549 , Células Epiteliais Alveolares/microbiologia , Células Epiteliais Alveolares/ultraestrutura , Aspergillus fumigatus/patogenicidade , Aspergillus fumigatus/fisiologia , Aspergillus fumigatus/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Imagem Óptica , Fagocitose , Esporos Fúngicos/metabolismo
19.
Aging (Albany NY) ; 13(11): 15620-15637, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34106880

RESUMO

Amyloid ß (Aß) plays a major role in the neurodegeneration of Alzheimer's disease (AD). The accumulation of misfolded Aß causes oxidative stress and inflammatory damage leading to apoptotic cell death. Traditional Chinese herbal medicine (CHM) has been widely used in treating neurodegenerative diseases by reducing oxidative stress and neuroinflammation. We examined the neuroprotective effect of formulated CHM Shaoyao Gancao Tang (SG-Tang, made of Paeonia lactiflora and Glycyrrhiza uralensis at 1:1 ratio) in AD cell and mouse models. In Aß-GFP SH-SY5Y cells, SG-Tang reduced Aß aggregation and reactive oxygen species (ROS) production, as well as improved neurite outgrowth. When the Aß-GFP-expressing cells were stimulated with conditioned medium from interferon (IFN)-γ-activated HMC3 microglia, SG-Tang suppressed expressions of inducible nitric oxide synthase (iNOS), NLR family pyrin domain containing 1 (NLRP1) and 3 (NLRP3), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6, attenuated caspase-1 activity and ROS production, and promoted neurite outgrowth. In streptozocin-induced hyperglycemic APP/PS1/Tau triple transgenic (3×Tg-AD) mice, SG-Tang also reduced expressions of NLRP1, NLRP3, Aß and Tau in hippocampus and cortex, as well as improved working and spatial memories in Y maze and Morris water maze. Collectively, our results demonstrate the potential of SG-Tang in treating AD by moderating neuroinflammation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Cognição , Medicamentos de Ervas Chinesas/uso terapêutico , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neuroproteção , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Animais , Linhagem Celular , Cognição/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Interferon gama/metabolismo , Memória/efeitos dos fármacos , Transtornos da Memória/complicações , Transtornos da Memória/fisiopatologia , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Modelos Biológicos , Crescimento Neuronal/efeitos dos fármacos , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Agregados Proteicos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Aprendizagem Espacial/efeitos dos fármacos , Proteínas tau/metabolismo
20.
Methods Mol Biol ; 2275: 301-314, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34118046

RESUMO

Our group has previously established a strategy utilizing fluorescence lifetime probes to image membrane protein supercomplex (SC) formation in situ. We showed that a probe at the interface between individual mitochondrial respiratory complexes exhibits a decreased fluorescence lifetime when a supercomplex is formed. This is caused by electrostatic interactions with the adjacent proteins. Fluorescence lifetime imaging microscopy (FLIM) records the resulting decrease of the lifetime of the SC-probe. Here we present the details of our method for performing SC-FLIM, including the evaluation of fluorescence lifetimes from the FLIM images. To validate the feasibility of the technique for monitoring adaptive SC formation, we compare data obtained under different metabolic conditions. The results confirm that SC formation is dynamic.


Assuntos
Proteínas de Fluorescência Verde/química , Mitocôndrias/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Modelos Moleculares , Conformação Molecular , Multimerização Proteica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...