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1.
Ecotoxicol Environ Saf ; 181: 214-223, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31195230

RESUMO

In the aftermath of the Great East Japan Earthquake of March 11, 2011, marine fish in Kesennuma Bay, Japan, have been contaminated with heavy oil containing polycyclic aromatic hydrocarbons (PAHs). To estimate the risk of six PAHs (benzo[α]pyrene, dibenzothiophene, phenanthrene, 2,3,5-trimethylnaphthalene, acenaphthene, and 1-methylphenanthrene), which have been detected at high levels in the tissues of fish from Kesennuma Bay, we attempted to evaluate the effects of these PAHs on the fish aryl hydrocarbon receptor (AHR) signaling pathway. We initially measured PAH concentrations and cytochrome P4501A catalytic activities (EROD: ethoxyresorufin-O-deethylase and MROD: methoxyresorufin-O-demethylase) as markers of AHR activation in greenlings (Hexagrammos otakii) collected from Kesennuma Bay in 2014. The results showed that alkylated PAH concentrations and EROD/MROD activities were higher in sites close to the oil-spilled sites than in the control site, suggesting AHR activation by spilled alkylated PAHs. We then investigated AHR-mediated responses to these PAHs in the in vitro reporter gene assay system where red seabream (Pagrus major) AHR1 (rsAHR1) or rsAHR2 expression plasmids were transiently transfected into COS-7 cells. The in vitro assay showed rsAHR isoform-, PAH-, and dose-dependent transactivation potencies. The relative effective concentrations of benzo[α]pyrene, dibenzothiophene, phenanthrene, 2,3,5-trimethylnaphthalene, acenaphthene, and 1-methylphenanthrene that induce 20% of the maximum benzo[α]pyrene response (REC20-BaP) for rsAHR1 activation were 0.052, 38, 79, 88, 270 nM, and no response, respectively, and those for rsAHR2 activation were 0.0049, 32, 53, 88, 60 nM, and no response, respectively. The results showed that the REC20-BaP values of benzo[α]pyrene for both the rsAHR1 and rsAHR2 isoforms were lower than the concentrations (0.041-0.20 nM) detected in the muscle tissue of fish from Kesennuma Bay, while the REC20-BaP values of other PAHs were higher than their tissue concentrations. In silico rsAHR homology modeling and subsequent ligand docking simulation analyses indicated that the rsAHR activation potencies of PAHs could be predicted from a rsAHR2 model. This study shows that in vitro and in silico rsAHR analyses may be a useful tool for assessing the risks to fish contaminated with PAHs.


Assuntos
Peixes/metabolismo , Poluição por Petróleo , Hidrocarbonetos Policíclicos Aromáticos/análise , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Células COS , Cercopithecus aethiops , Simulação por Computador , Citocromo P-450 CYP1A1/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Genes Reporter , Japão , Perciformes/metabolismo , Petróleo , Hidrocarbonetos Policíclicos Aromáticos/química , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/genética , Medição de Risco , Dourada/genética
2.
Nat Protoc ; 14(6): 1772-1802, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31101905

RESUMO

Membrane curvatures are involved in essential cellular processes, such as endocytosis and exocytosis, in which they are believed to act as microdomains for protein interactions and intracellular signaling. These membrane curvatures appear and disappear dynamically, and their locations are difficult or impossible to predict. In addition, the size of these curvatures is usually below the diffraction limit of visible light, making it impossible to resolve their values using live-cell imaging. Therefore, precise manipulation of membrane curvature is important to understanding how membrane curvature is involved in intracellular processes. Recent studies show that membrane curvatures can be induced by surface topography when cells are in direct contact with engineered substrates. Here, we present detailed procedures for using nanoscale structures to manipulate membrane curvatures and probe curvature-induced phenomena in live cells. We first describe detailed procedures for the design of nanoscale structures and their fabrication using electron-beam (E-beam) lithography. The fabrication process takes 2 d, but the resultant chips can be cleaned and reused repeatedly over the course of 2 years. Then we describe how to use these nanostructures to manipulate local membrane curvatures and probe intracellular protein responses, discussing surface coating, cell plating, and fluorescence imaging in detail. Finally, we describe a procedure to characterize the nanostructure-cell membrane interface using focused ion beam and scanning electron microscopy (FIB-SEM). Nanotopography-based methods can induce stable membrane curvatures with well-defined curvature values and locations in live cells, which enables the generation of a library of curvatures for probing curvature-related intracellular processes.


Assuntos
Técnicas de Cultura de Células/instrumentação , Membrana Celular/metabolismo , Micromanipulação/instrumentação , Nanoestruturas , Animais , Células COS , Comunicação Celular , Linhagem Celular , Membrana Celular/ultraestrutura , Cercopithecus aethiops , Endocitose , Desenho de Equipamento , Humanos , Camundongos , Microscopia Eletrônica de Varredura , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Imagem Óptica , Proteínas/metabolismo , Propriedades de Superfície
3.
Int J Mol Sci ; 20(9)2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31083458

RESUMO

To appraise how evolutionary processes, such as gene duplication and loss, influence an organism's xenobiotic sensitivity is a critical question in toxicology. Of particular importance are gene families involved in the mediation of detoxification responses, such as members of the nuclear receptor subfamily 1 group I (NR1I), the pregnane X receptor (PXR), and the constitutive androstane receptor (CAR). While documented in multiple vertebrate genomes, PXR and CAR display an intriguing gene distribution. PXR is absent in birds and reptiles, while CAR shows a tetrapod-specific occurrence. More elusive is the presence of PXR and CAR gene orthologs in early branching and ecologically-important Chondrichthyes (chimaeras, sharks and rays). Therefore, we investigated various genome projects and use them to provide the first identification and functional characterization of a Chondrichthyan PXR from the chimaera elephant shark (Callorhinchus milii, Holocephali). Additionally, we substantiate the targeted PXR gene loss in Elasmobranchii (sharks and rays). Compared to other vertebrate groups, the chimaera PXR ortholog displays a diverse expression pattern (skin and gills) and a unique activation profile by classical xenobiotic ligands. Our findings provide insights into the molecular landscape of detoxification mechanisms and suggest lineage-specific adaptations in response to xenobiotics in gnathostome evolution.


Assuntos
Elasmobrânquios/classificação , Elasmobrânquios/genética , Evolução Molecular , Redes Reguladoras de Genes , Filogenia , Receptor de Pregnano X/genética , Animais , Células COS , Cercopithecus aethiops , Genes Reporter , Inativação Metabólica/genética , Luciferases/metabolismo , Receptor de Pregnano X/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Sintenia/genética , Ativação Transcricional/genética
4.
Gene ; 707: 239-245, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31102715

RESUMO

Phenylketonuria (PKU), caused by phenylalanine hydroxylase (PAH) gene variants, is a common autosomal inherited metabolic disease. So far, 1111 PAH variants have been revealed. The residual activity of the PAH variants is the key determinant of the metabolic phenotype and BH4 responsiveness in PKU patients. In this study, the spectrum of PAH variants in 1083 Chinese PKU patients was analyzed. Then 20 variants (p.L52F, p.R86P, p.L128P, p.L142P, p.D163N, p.C203G, p.E214G, p.F260L, p.M276T, p.L311R, p.P314A, p.L364F, p.Q375H, p.F382I, p.A395S, p.V412D, p.E108*, p.C203*, p.C284* and p.E353*) were expressed in COS-7 cells. The residual activities and protein expression levels were detected by isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and Western blotting, respectively. We compared the results of the phenotypic prediction based on APV and PAH activity respectively, and further explored the relationship between residual activity and phenotype in PKU patients. We reported 9 newly discovered PAH variants for the first time, thereby expanding the spectrum of PAH variants. Among the 20 variants in our assay, 8 variants showed mild impaired residual activities (48-92%) and approximately normal protein expression levels compared to the wild-type PAH. In contrast, 9 variants showed severely impaired residual activities (0-34%) and reduced protein expression. However, three variants (p.L52F, p.F260L and p.P314A) showed impaired residual activities (5%, 32% and 29%), although the proteins were well expressed. We assigned APV scores for 14 variants, in which the results of the phenotypic prediction were consistent for 12/14 (86%) variants based on APV and residual activity respectively, and the residual activity correctly predicted 17/22 (77%) of the patients. Our study helped to further understand the genotype-phenotype correlation in PKU patients.


Assuntos
Variação Genética , Fenilalanina Hidroxilase/genética , Fenilalanina Hidroxilase/metabolismo , Fenilcetonúrias/genética , Animais , Células COS , Cercopithecus aethiops , Cromatografia Líquida , Regulação para Baixo , Feminino , Estudos de Associação Genética , Humanos , Masculino , Fenilcetonúrias/metabolismo , Espectrometria de Massas por Ionização por Electrospray
5.
Nat Cell Biol ; 21(6): 768-777, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061466

RESUMO

Controlling cellular processes with light can help elucidate their underlying mechanisms. Here we present zapalog, a small-molecule dimerizer that undergoes photolysis when exposed to blue light. Zapalog dimerizes any two proteins tagged with the FKBP and DHFR domains until exposure to light causes its photolysis. Dimerization can be repeatedly restored with uncleaved zapalog. We implement this method to investigate mitochondrial motility and positioning in cultured neurons. Using zapalog, we tether mitochondria to constitutively active kinesin motors, forcing them down the axon towards microtubule (+) ends until their instantaneous release via blue light, which results in full restoration of their endogenous motility. We find that one-third of stationary mitochondria cannot be pulled away from their position and that these firmly anchored mitochondria preferentially localize to VGLUT1-positive presynapses. Furthermore, inhibition of actin polymerization with latrunculin A reduces this firmly anchored pool. On release from exogenous motors, mitochondria are preferentially recaptured at presynapses.


Assuntos
Axônios/metabolismo , Mitocôndrias/genética , Fotólise , Multimerização Proteica/efeitos da radiação , Actinas/antagonistas & inibidores , Animais , Axônios/química , Axônios/efeitos da radiação , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células COS , Cercopithecus aethiops , Cinesina/química , Luz , Microtúbulos/genética , Microtúbulos/efeitos da radiação , Mitocôndrias/química , Mitocôndrias/efeitos da radiação , Neurônios/química , Neurônios/efeitos da radiação , Polimerização/efeitos dos fármacos , Domínios Proteicos/genética , Domínios Proteicos/efeitos da radiação , Multimerização Proteica/genética , Sinapses/química , Sinapses/genética , Sinapses/efeitos da radiação , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/genética , Tiazolidinas/farmacologia , Proteína Vesicular 1 de Transporte de Glutamato/genética
6.
Arch Virol ; 164(6): 1597-1607, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30949813

RESUMO

Hazara virus (HAZV) is closely related to Crimean-Congo hemorrhagic fever virus (CCHFV), but differs in that it is non-pathogenic to humans. Since HAZV was isolated for the first time in 1954, the biological characteristics of this virus, particularly its behavior within culture cells, have not been well-studied, despite its importance as a surrogate model for CCHFV. Nucleoprotein (N) is the main component of viral nucleocapsid and is the most abundant virion protein, it is believed to play a pivotal role in the viral lifecycle. Generation of a series of anti-HAZV N monoclonal antibodies has enabled us to directly examine the involvement of this protein on viral growth. Observation of HAZV-infected cells revealed that this infection caused apoptosis, which was further characterized by DNA ladder and elevated caspase-3/7 activity. HAZV titers initially increased in cell culture, but after reaching the peak titer began to rapidly decline. HAZV particles were found to be very unstable in culture medium at 37 °C, and virus particles tend to lose infectivity at that point. HAZV N appears to inhibit apoptosis, thus can potentially support efficient viral propagation.


Assuntos
Anticorpos Monoclonais/farmacologia , Infecções por Bunyaviridae/virologia , Nairovirus/crescimento & desenvolvimento , Nucleoproteínas/antagonistas & inibidores , Carga Viral/efeitos dos fármacos , Células A549 , Animais , Anticorpos Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Infecções por Bunyaviridae/metabolismo , Células COS , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular , Cercopithecus aethiops , Cães , Humanos , Células Madin Darby de Rim Canino , Nairovirus/efeitos dos fármacos , Proteínas Virais/antagonistas & inibidores
7.
Nat Cell Biol ; 21(4): 452-461, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30936472

RESUMO

Particles that bud off from the cell surface, including viruses and microvesicles, typically have a unique membrane protein composition distinct from that of the originating plasma membrane. This selective protein composition enables viruses to evade the immune response and infect other cells. But how membrane proteins sort into budding viruses such as human immunodeficiency virus (HIV) remains unclear. Proteins could passively distribute into HIV-assembly-site membranes producing compositions resembling pre-existing plasma-membrane domains. Here, we demonstrate that proteins instead sort actively into HIV-assembly-site membranes, generating compositions enriched in cholesterol and sphingolipids that undergo continuous remodelling. Proteins are recruited into and removed from the HIV assembly site through lipid-based partitioning, initiated by oligomerization of the HIV structural protein Gag. Changes in membrane curvature at the assembly site further amplify this sorting process. Thus, a lipid-based sorting mechanism, aided by increasing membrane curvature, generates the unique membrane composition of the HIV surface.


Assuntos
HIV/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Vírion/metabolismo , Animais , Antígeno 2 do Estroma da Médula Óssea/metabolismo , Células COS , Membrana Celular/ultraestrutura , Cercopithecus aethiops , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Células HeLa , Humanos , Vírion/química
8.
Mol Pharmacol ; 95(5): 551-562, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30944207

RESUMO

UDP-Glucuronosyltransferase (UGT) plays an important role in the metabolism of endogenous and exogenous compounds. UGT is a type I membrane protein, and has a dilysine motif (KKXX/KXKXX) in its C-terminal cytoplasmic domain. Although a dilysine motif is defined as an endoplasmic reticulum (ER) retrieval signal, it remains a matter of debate whether this motif functions in the ER localization of UGT. To address this issue, we generated systematic deletion mutants of UGT2B7, a major human isoform, and compared their subcellular localizations with that of an ER marker protein calnexin (CNX), using subcellular fractionation and immunofluorescent microscopy. We found that although the dilysine motif functioned as the ER retention signal in a chimera that replaced the cytoplasmic domain of CD4 with that of UGT2B7, UGT2B7 truncated mutants lacking this motif extensively colocalized with CNX, indicating dilysine motif-independent ER retention of UGT2B7. Moreover, deletion of the C-terminal transmembrane and cytoplasmic domains did not affect ER localization of UGT2B7, suggesting that the signal necessary for ER retention of UGT2B7 is present in its luminal domain. Serial deletions of the luminal domain, however, did not affect the ER retention of the mutants. Further, a cytoplasmic and transmembrane domain-deleted mutant of UGT2B7 was localized to the ER without being secreted. These results suggest that UGT2B7 could localize to the ER without any retention signal, and lead to the conclusion that the static localization of UGT results from lack of a signal for export from the ER.


Assuntos
Retículo Endoplasmático/metabolismo , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Deleção de Sequência/genética , Animais , Células COS , Calnexina/metabolismo , Linhagem Celular , Cercopithecus aethiops , Citoplasma/metabolismo , Dipeptídeos/metabolismo , Humanos , Proteínas de Membrana , Células Sf9
9.
Nat Cell Biol ; 21(5): 603-613, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30988424

RESUMO

Mitochondrial fission involves the preconstriction of an organelle followed by scission by dynamin-related protein Drp1. Preconstriction is facilitated by actin and non-muscle myosin II through a mechanism that remains unclear, largely due to the unknown cytoskeletal ultrastructure at mitochondrial constrictions. Here, using platinum replica electron microscopy, we show that mitochondria in cells are embedded in an interstitial cytoskeletal network that contains abundant unbranched actin filaments. Both spontaneous and induced mitochondrial constrictions typically associate with a criss-cross array of long actin filaments that comprise part of this interstitial network. Non-muscle myosin II is found adjacent to mitochondria but is not specifically enriched at the constriction sites. During ionomycin-induced mitochondrial fission, F-actin clouds colocalize with mitochondrial constriction sites, whereas dynamic myosin II clouds are present in the vicinity of constrictions. We propose that myosin II promotes mitochondrial constriction by inducing stochastic deformations of the interstitial actin network, which applies pressure on the mitochondrial surface and thus initiates curvature-sensing mechanisms that complete mitochondrial constriction.


Assuntos
Actinas/genética , Citoesqueleto/ultraestrutura , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial/genética , Miosina Tipo II/genética , Citoesqueleto de Actina/química , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Animais , Células COS , Cercopithecus aethiops , Constrição , Citoesqueleto/metabolismo , Ionomicina/farmacologia , Mitocôndrias/genética , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Miosina Tipo II/química , Miosina Tipo II/metabolismo
10.
Mater Sci Eng C Mater Biol Appl ; 101: 487-498, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31029343

RESUMO

Wound dressing is distinctly important to wound healing, because it can not only protect wound from external disturbance, but also provide an ideal environment for wound closure. However, most of wound dressings need additional active ingredients to assist the repair process. In order to develop new dressings that can present spontaneous healing activity, herein, an injectable hydrogel consisted of collagen I and hyaluronic acid has been designed to mimic extracellular matrix for vascular cells growing and wound closure. The preparation of hydrogel (COL-HA) was realized through in situ coupling of phenol moieties of collagen I-hydroxybenzoic acid (COL-P) and hyaluronic-acid-tyramine (HA-Tyr) through horseradish peroxidase (HRP). The physical structure and properties were characterized, and the biological performances were analyzed. COL-HA hydrogel presented porous structure that contributed to the exchange of gas, medium and nutrition. Human microvascular endothelial cells (HMEC) and fibroblasts (COS-7) cultured within this hydrogel showed significant proliferation behaviors. More importantly, a certain level of vascular endothelial growth factor (VEGF) was observed in HMEC cultured hydrogel, which led to the possibility of vascular regeneration. For the full-thickness wound, the healing ratio and validity of wound treated with COL-HA hydrogel were higher than commercial drug and individual COL-P hydrogel, HA-Tyr hydrogel groups, since collagen and hyaluronic acid made joint efforts to improve wound repair.


Assuntos
Biomimética/métodos , Colágeno/química , Colágeno/farmacologia , Ácido Hialurônico/química , Ácido Hialurônico/farmacologia , Hidrogéis/química , Cicatrização/efeitos dos fármacos , Animais , Células COS , Linhagem Celular , Cercopithecus aethiops , Humanos
11.
Int J Mol Sci ; 20(8)2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-30995786

RESUMO

Mouse activating Nkrp1 proteins are commonly described as type II transmembrane receptors with disulfide-linked homodimeric structure. Their function and the manner in which Nkrp1 proteins of mouse strain (C57BL/6) oligomerize are still poorly understood. To assess the oligomerization state of Nkrp1 proteins, mouse activating EGFP-Nkrp1s were expressed in mammalian lymphoid cells and their oligomerization evaluated by Förster resonance energy transfer (FRET). Alternatively, Nkrp1s oligomers were detected by Western blotting to specify the ratio between monomeric and dimeric forms. We also performed structural characterization of recombinant ectodomains of activating Nkrp1 receptors. Nkrp1 isoforms c1, c2 and f were expressed prevalently as homodimers, whereas the Nkrp1a displays larger proportion of monomers on the cell surface. Cysteine-to-serine mutants revealed the importance of all stalk cysteines for protein dimerization in living cells with a major influence of cysteine at position 74 in two Nkrp1 protein isoforms. Our results represent a new insight into the oligomerization of Nkrp1 receptors on lymphoid cells, which will help to determine their function.


Assuntos
Antígenos Ly/análise , Subfamília B de Receptores Semelhantes a Lectina de Células NK/análise , Receptores Imunológicos/análise , Animais , Células COS , Cercopithecus aethiops , Transferência Ressonante de Energia de Fluorescência , Humanos , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Multimerização Proteica , Redobramento de Proteína
12.
Nat Commun ; 10(1): 1268, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894522

RESUMO

Super-resolution (SR) techniques have extended the optical resolution down to a few nanometers. However, quantitative treatment of SR data remains challenging due to its complex dependence on a manifold of experimental parameters. Among the different SR variants, DNA-PAINT is relatively straightforward to implement, since it achieves the necessary 'blinking' without the use of rather complex optical or chemical activation schemes. However, it still suffers from image and quantification artifacts caused by inhomogeneous optical excitation. Here we demonstrate that several experimental challenges can be alleviated by introducing a segment-wise analysis approach and ultimately overcome by implementing a flat-top illumination profile for TIRF microscopy using a commercially-available beam-shaping device. The improvements with regards to homogeneous spatial resolution and precise kinetic information over the whole field-of-view were quantitatively assayed using DNA origami and cell samples. Our findings open the door to high-throughput DNA-PAINT studies with thus far unprecedented accuracy for quantitative data interpretation.


Assuntos
DNA/ultraestrutura , Microscopia de Fluorescência/métodos , Microscopia de Interferência/métodos , Oligonucleotídeos/química , Animais , Artefatos , Células COS , Cercopithecus aethiops , Humanos , Iluminação , Microscopia de Fluorescência/instrumentação , Microscopia de Interferência/instrumentação
13.
Carbohydr Polym ; 212: 361-367, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30832868

RESUMO

Chitin is the second abundant polysaccharide after cellulose, and has been demonstrated to possess various applications in the fields of the biomaterials, water treatment, etc. Here, mechanically strong chitin films were successfully fabricated from chitin solution in NaOH/urea aqueous system with cooling by chemical crosslinking with epichlorohydrin (ECH). The results indicated that tensile strength and elongation at break of the chitin film prepared by using the weight ratio of ECH to chitin solution of 6:100 enhanced significantly to achieve 139 MPa and 43%, respectively, showing highly strong strength and elongation. The cell assay results confirmed that the chitin films were non-toxicity. Water vapor transmittance rate testing indicated that the chitin films had better breathable properties than commercial product. The robust chitin films with high transmittance, biocompatibility and breathability have great potentials in the wide applications such as biomedical, food packaging, wound healing, garments fields, etc.


Assuntos
Materiais Biocompatíveis/síntese química , Quitina/síntese química , Química Verde/métodos , Resistência à Tração , Animais , Materiais Biocompatíveis/metabolismo , Células COS , Cercopithecus aethiops , Quitina/metabolismo
14.
Proc Natl Acad Sci U S A ; 116(13): 6152-6161, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30850543

RESUMO

Kinesin motor proteins that drive intracellular transport share an overall architecture of two motor domain-containing subunits that dimerize through a coiled-coil stalk. Dimerization allows kinesins to be processive motors, taking many steps along the microtubule track before detaching. However, whether dimerization is required for intracellular transport remains unknown. Here, we address this issue using a combination of in vitro and cellular assays to directly compare dimeric motors across the kinesin-1, -2, and -3 families to their minimal monomeric forms. Surprisingly, we find that monomeric motors are able to work in teams to drive peroxisome dispersion in cells. However, peroxisome transport requires minimal force output, and we find that most monomeric motors are unable to disperse the Golgi complex, a high-load cargo. Strikingly, monomeric versions of the kinesin-2 family motors KIF3A and KIF3B are able to drive Golgi dispersion in cells, and teams of monomeric KIF3B motors can generate over 8 pN of force in an optical trap. We find that intracellular transport and force output by monomeric motors, but not dimeric motors, are significantly decreased by the addition of longer and more flexible motor-to-cargo linkers. Together, these results suggest that dimerization of kinesin motors is not required for intracellular transport; however, it enables motor-to-motor coordination and high force generation regardless of motor-to-cargo distance. Dimerization of kinesin motors is thus critical for cellular events that require an ability to generate or withstand high forces.


Assuntos
Cinesina/metabolismo , Animais , Transporte Biológico , Células COS , Cercopithecus aethiops , Dimerização , Complexo de Golgi/metabolismo , Peroxissomos/metabolismo
15.
Mar Drugs ; 17(3)2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30875751

RESUMO

Integrated venomics techniques have shown that variable processing of conotoxins from Conus marmoreus resulted in a dramatic expansion in the number of expressed conotoxins. One conotoxin from C. marmoreus, the χ-conotoxin MrIA, is a selective inhibitor of human norepinephrine transporters (hNET) and therefore a drug candidate for attenuating chronic neuropathic pain. It has been found that "messy" processing of the MrIA transcripts results in the expression of MrIA analogs with different truncations of the pro-peptide that contains portions of the MrIA molecule. The aim of this study was to investigate if variable processing of the expressed peptides results in modulation of the existing hNET pharmacology or creates new pharmacologies. To this end, a number of MrIA analogs found in C. marmoreus venom were synthesized and evaluated for their activity at hNET receptors. While several of the analogs exhibited norepinephrine transporter inhibitory activity comparable to that of MrIA, none significantly improved on the potency of conotoxin MrIA, and those analogs with disrupted pharmacophores produced greatly reduced NET inhibition, confirming previous structure-activity relationships seen on χ-class conopeptides. Additionally, analogs were screened for new activities on ion channels using calcium influx assays, although no major new pharmacology was revealed.


Assuntos
Conotoxinas/química , Conotoxinas/farmacologia , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/antagonistas & inibidores , Peptídeos/farmacologia , Sequência de Aminoácidos , Aminoácidos/química , Animais , Células COS , Linhagem Celular , Cercopithecus aethiops , Caramujo Conus/química , Fluorenos/química , Humanos , Venenos de Moluscos/química , Peptídeos/síntese química
16.
BMC Res Notes ; 12(1): 188, 2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30925931

RESUMO

OBJECTIVE: Prenylated Rab Acceptor 1 (PRA1) is a transmembrane protein localized to the early secretory pathway. It has been found to interact with an array of Rab GTPases, leading to its hypothesized function in the recycling of Rab GTPases. However, all previous strategies used to screen for novel interacting partners have utilized a classic yeast two-hybrid approach that requires both bait and its potential binding partners to be cytosolic proteins. In the split-ubiquitin yeast two-hybrid screen, a protein interaction leads to the re-constitution of ubiquitin, which is followed by proteolytic release of a transcription activator that migrates to the nucleus alone. This allows for bait and/or prey to be integral membrane protein(s). To better understand the in vivo function of PRA1, we took an unbiased approach that screened PRA1 against a normalized mouse neuronal cDNA library using this variant of the classic screening strategy. RESULTS: We report 41 previously unidentified potential PRA1 binding partners revealed by this screen and validate the screen by confirming three of these interactions using a bi-molecular fluorescence complementation assay in mammalian cells. The identified proteins reside throughout the secretory pathway and are both membrane-bound and cytosolic in their identity, suggesting alternative functions for PRA1.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Transporte Vesicular/metabolismo , Animais , Células COS , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Cercopithecus aethiops , Proteínas de Ligação ao GTP/genética , Biblioteca Gênica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Neurônios/metabolismo , Ligação Proteica , Ubiquitina/genética , Ubiquitina/metabolismo , Proteínas de Transporte Vesicular/genética
17.
Cell Mol Life Sci ; 76(15): 3019-3031, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30904951

RESUMO

Sumoylation is a reversible post-translational modification essential to the modulation of neuronal function, including neurotransmitter release and synaptic plasticity. A tightly regulated equilibrium between the sumoylation and desumoylation processes is critical to the brain function and its disruption has been associated with several neurological disorders. This sumoylation/desumoylation balance is governed by the activity of the sole SUMO-conjugating enzyme Ubc9 and a group of desumoylases called SENPs, respectively. We previously demonstrated that the activation of type 5 metabotropic glutamate receptors (mGlu5R) triggers the transient trapping of Ubc9 in dendritic spines, leading to a rapid increase in the overall synaptic sumoylation. However, the mechanisms balancing this increased synaptic sumoylation are still not known. Here, we examined the diffusion properties of the SENP1 enzyme using a combination of advanced biochemical approaches and restricted photobleaching/photoconversion of individual hippocampal spines. We demonstrated that the activation of mGlu5R leads to a time-dependent decrease in the exit rate of SENP1 from dendritic spines. The resulting post-synaptic accumulation of SENP1 restores synaptic sumoylation to initial levels. Altogether, our findings reveal the mGlu5R system as a central activity-dependent mechanism to maintaining the homeostasis of sumoylation at the mammalian synapse.


Assuntos
Receptor de Glutamato Metabotrópico 5/metabolismo , Sinapses/metabolismo , Animais , Células COS , Células Cultivadas , Cercopithecus aethiops , Cisteína Endopeptidases/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Humanos , Microscopia de Fluorescência , Neurônios/citologia , Neurônios/metabolismo , Ratos Wistar , Proteína SUMO-1/metabolismo , Sumoilação , Enzimas de Conjugação de Ubiquitina/metabolismo
18.
Photochem Photobiol Sci ; 18(5): 1212-1217, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30834414

RESUMO

Bioluminescence is widely used in biosensors. Firefly luciferase-based bioluminescent sensors are among the most popular ones. Firefly luciferases are pH-sensitive, displaying a large red shift at acidic pH, a property that has been considered undesirable for most applications. Currently, biosensors that can detect intracellular pH are in demand, and some fluorescent biosensors are available. However, pH sensors using bioluminescence have not been used yet. Thus, we decided to harness a firefly luciferase to measure the intracellular pH in mammalian cells. For this purpose, we engineered the luciferase derived from Macrolampis sp2 firefly to localize it on the cytosol or nucleus, in order to observe pH variation in these compartments during biological activities. We first calibrated the emission ratios (R = Igreen/Ired) at different pH values. As expected, we observed a red shift of light emission under acidic conditions when the cells were subjected to different pH conditions in the presence of the K+/H+ ionophore, nigericin. Based on these results, we concluded that this firefly luciferase can be used as a diagnostic tool for measuring the intracellular pH variation in pathogenic cells or in cells during apoptosis. This is the first example of real time-monitoring of pH change using color tuning luciferase.


Assuntos
Técnicas Biossensoriais , Luciferases de Vaga-Lume/metabolismo , Medições Luminescentes , Organelas/metabolismo , Animais , Células COS , Cercopithecus aethiops , Vaga-Lumes , Concentração de Íons de Hidrogênio , Organelas/química
19.
Science ; 363(6434)2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30923194

RESUMO

Nature regulates interference between cellular processes-allowing more complexity of life-by confining specific functions to organelles. Inspired by this concept, we designed an artificial organelle dedicated to protein engineering. We generated a membraneless organelle to translate only one type of messenger RNA-by recruiting an RNA-targeting system, stop codon-suppression machinery, and ribosomes-by means of phase separation and spatial targeting. This enables site-specific protein engineering with a tailored noncanonical function in response to one specific codon in the entire genome only in the protein of choice. Our results demonstrate a simple yet effective approach to the generation of artificial organelles that provides a route toward customized orthogonal translation and protein engineering in semisynthetic eukaryotic cells.


Assuntos
Códon/genética , Código Genético , Organelas/metabolismo , Organelas/ultraestrutura , Biossíntese de Proteínas/genética , Engenharia de Proteínas/métodos , RNA Mensageiro/genética , Animais , Células COS , Caenorhabditis elegans/genética , Membrana Celular , Cercopithecus aethiops , Células HEK293 , Humanos , Lisina/análogos & derivados , Lisina/genética , Methanosarcina , Organelas/química , RNA de Transferência/química , Ribossomos/química , Biologia Sintética
20.
Mol Biol Cell ; 30(10): 1198-1213, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30865555

RESUMO

Mitochondria are essential and dynamic organelles undergoing constant fission and fusion. The primary players in mitochondrial morphology (MFN1/2, OPA1, DRP1) have been identified, but their mechanism(s) of regulation are still being elucidated. ARL2 is a regulatory GTPase that has previously been shown to play a role in the regulation of mitochondrial morphology. Here we demonstrate that ELMOD2, an ARL2 GTPase-activating protein (GAP), is necessary for ARL2 to promote mitochondrial elongation. We show that loss of ELMOD2 causes mitochondrial fragmentation and a lower rate of mitochondrial fusion, while ELMOD2 overexpression promotes mitochondrial tubulation and increases the rate of fusion in a mitofusin-dependent manner. We also show that a mutant of ELMOD2 lacking GAP activity is capable of promoting fusion, suggesting that ELMOD2 does not need GAP activity to influence mitochondrial morphology. Finally, we show that ELMOD2, ARL2, Mitofusins 1 and 2, Miros 1 and 2, and mitochondrial phospholipase D (mitoPLD) all localize to discrete, regularly spaced puncta along mitochondria. These results suggest that ELMOD2 is functioning as an effector downstream of ARL2 and upstream of the mitofusins to promote mitochondrial fusion. Our data provide insights into the pathway by which mitochondrial fusion is regulated in the cell.


Assuntos
Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Dinâmica Mitocondrial/fisiologia , Animais , Células COS , Linhagem Celular , Cercopithecus aethiops , GTP Fosfo-Hidrolases/metabolismo , Técnicas de Inativação de Genes/métodos , Humanos , Fusão de Membrana/fisiologia , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fosfolipase D/genética , Fosfolipase D/metabolismo
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