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1.
Free Radic Biol Med ; 113: 1-15, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28916473

RESUMO

NADPH oxidases (NOX) have many biological roles, but their regulation to control production of potentially toxic ROS molecules remains unclear. A previously identified insertion sequence of 21 residues (called NIS) influences NOX activity, and its predicted flexibility makes it a good candidate for providing a dynamic switch controlling the NOX active site. We constructed NOX2 chimeras in which NIS had been deleted or exchanged with those from other NOXs (NIS1, 3 and 4). All contained functional heme and were expressed normally at the plasma membrane of differentiated PLB-985 cells. However, NOX2-ΔNIS and NOX2-NIS1 had neither NADPH-oxidase nor reductase activity and exhibited abnormal translocation of p47phox and p67phox to the phagosomal membrane. This suggested a functional role of NIS. Interestingly after activation, NOX2-NIS3 cells exhibited superoxide overproduction compared with wild-type cells. Paradoxically, the Vmax of purified unstimulated NOX2-NIS3 was only one-third of that of WT-NOX2. We therefore hypothesized that post-translational events regulate NOX2 activity and differ between NOX2-NIS3 and WT-NOX2. We demonstrated that Ser486, a phosphorylation target of ataxia telangiectasia mutated kinase (ATM kinase) located in the NIS of NOX2 (NOX2-NIS), was phosphorylated in purified cytochrome b558 after stimulation with phorbol 12-myristate-13-acetate (PMA). Moreover, ATM kinase inhibition and a NOX2 Ser486Ala mutation enhanced NOX activity whereas a Ser486Glu mutation inhibited it. Thus, the absence of Ser486 in NIS3 could explain the superoxide overproduction in the NOX2-NIS3 mutant. These results suggest that PMA-stimulated NOX2-NIS phosphorylation by ATM kinase causes a dynamic switch that deactivates NOX2 activity. We hypothesize that this downregulation is defective in NOX2-NIS3 mutant because of the absence of Ser486.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Regulação da Expressão Gênica , NADPH Oxidase 2/metabolismo , Fagócitos/metabolismo , Processamento de Proteína Pós-Traducional , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , NADPH Oxidase 2/genética , Fagócitos/enzimologia , Fosforilação , Transdução de Sinais
2.
Methods Mol Biol ; 1615: 65-74, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28667602

RESUMO

In recent years it has become evident that lipoproteins play crucial roles in the assembly of bacterial envelope-embedded nanomachineries and in the processes of protein export/secretion. In this chapter we describe a method to determine their precise localisation, for example inner versus outer membrane, in Gram-negative bacteria using human opportunistic pathogen Pseudomonas aeruginosa as a model. A fusion protein between a given putative lipoprotein and the red fluorescent protein mCherry must be created and expressed in a strain expressing cytoplasmic green fluorescent protein (GFP). Then the peripheral localisation of the fusion protein in the cell can be examined by treating cells with lysozyme to create spheroplasts and monitoring fluorescence under a confocal microscope. Mutants in the signal peptide can be engineered to study the association with the membrane and efficiency of transport. This protocol can be adapted to monitor lipoprotein localisation in other Gram-negative bacteria.


Assuntos
Lipoproteínas/metabolismo , Microscopia de Fluorescência , Transporte Proteico , Proteínas de Bactérias/metabolismo , Expressão Gênica , Genes Reporter , Microscopia Confocal , Microscopia de Fluorescência/métodos , Pseudomonas aeruginosa/metabolismo , Esferoplastos/metabolismo
3.
Hum Mutat ; 38(2): 152-159, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27790796

RESUMO

Dent-2 disease and Lowe syndrome are two pathologies caused by mutations in inositol polyphosphate 5-phosphatase OCRL gene. Both conditions share proximal tubulopathy evolving to chronic kidney failure. Lowe syndrome is in addition defined by a bilateral congenital cataract, intellectual disability, and hypotonia. The pathology evolves in two decades to a severe condition with renal complications and a fatal issue. We describe here a proof of principle for a targeted gene therapy on a mutation of the OCRL gene that is associated with Lowe syndrome. The affected patient bears a deep intronic mutation inducing a pseudo-exon inclusion in the mRNA, leading to a OCRL-1 protein loss. An exon-skipping strategy was designed to correct the effect of the mutation in cultured cells. We show that a recombinant U7-modified small RNA efficiently triggered the restoration of normal OCRL expression at mRNA and protein levels in patient's fibroblasts. Moreover, the PI(4,5)P2 accumulation and cellular alterations that are hallmark of OCRL-1 dysfunction were also rescued. Altogether, we provide evidence that the restoration of OCRL-1 protein, even at a reduced level, through RNA-based therapy represents a potential therapeutic approach for patients with OCRL splice mutations.


Assuntos
Íntrons , Mutação , Síndrome Oculocerebrorrenal/genética , Síndrome Oculocerebrorrenal/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Alelos , Processamento Alternativo , Substituição de Aminoácidos , Pré-Escolar , Ativação Enzimática , Éxons , Fibroblastos , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Imagem Molecular , Síndrome Oculocerebrorrenal/diagnóstico , Fenótipo
4.
Biores Open Access ; 3(6): 311-26, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25469316

RESUMO

Chronic granulomatous disease (CGD) is an inherited orphan disorder caused by mutations in one of the five genes encoding reduced nicotinamide-adenine-dinucleotide-phosphate oxidase subunits, which subsequently lead to impairment in the production of microbicidal reactive oxygen species (ROS). In order to offer several cell line models of CGD and therefore support research on pathophysiology and new therapeutic approaches, we optimized protocols to differentiate induced pluripotent stem cells (iPSCs) from wild-type, X(0)-, AR22(0)- and AR47(0)-CGD patient's fibroblasts into neutrophils and into macrophages. Aberrant genetic clones were discarded after chromosome karyotyping and array-comparative genomic hybridization analysis. All remaining iPSC lines showed human embryonic stem cell-like morphology, expressed all tested pluripotency markers and formed embryoid bodies that contained cells originating from all three primary germ layers. Furthermore, each CGD patient-specific iPSC line retained the gp91 (phox) , p47 (phox) , and p22 (phox) mutations found in the corresponding patient's neutrophils. The average production of CD34(+) progenitors was of 1.5×10(6) cells after 10 days of differentiation of 10×10(6) iPSCs. They were terminally differentiated into about 3×10(5) neutrophils or into 3×10(7) macrophages. Based on morphological, phenotypical, and functional criteria both phagocyte types were mature and indistinguishable from the native human neutrophils and macrophages. However, neutrophils and macrophages derived from X(0)-, AR22(0)-, and AR47(0)-CGD patient-specific iPSC lines lacked ROS production and the corresponding mutated proteins. To simplify the phagocytes' production upon request, progenitors can be cryopreserved. In conclusion, we describe a reproducible, simple, and efficient way to generate neutrophils and macrophages from iPSCs and provide a new cellular model for the AR22(0)-CGD genetic form that has not been described before.

5.
Biochem J ; 464(3): 425-37, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25252997

RESUMO

Cytochrome b558, the redox core of the NADPH oxidase (NOX) complex in phagocytes, is composed of NOX2 and p22phox, the synthesis of which is intimately connected but not fully understood. We reproduced 10 rare X-minus chronic granulomatous disease (CGD) mutations of highly conserved residues in NOX1-NOX4, in X0-CGD PLB-985 cells in order to analyse their impact on the synthesis of cytochrome b558. According to the impact of these mutations on the level of expression of NADPH oxidase 2 (NOX2) and its activity, mutants were categorized into group A (W18C, E309K, K315del and I325F), characterized by a linear relationship between NOX2 expression and NOX activity, and group B (H338Y, P339H, G389A and F656-F570del), showing an absence of NOX activity associated with variable levels of NOX2 expression. These last residues belong to the FAD-binding pocket of NOX2, suggesting that this functional domain also plays a role in the structural integrity of NOX2. Finally, we observed an abnormal accumulation of p65 (65-kDa monomer), the NOX2 precursor and p65-p22phox dissociation in the W18C, E309K, I325F and G389A mutants, pointing out a possible role of the first transmembrane domain (Trp18), and the region between the membrane and the dehydrogenase domain of NOX2 (Glu309, Ile325 and Gly389), in the binding with p22phox.


Assuntos
Grupo dos Citocromos b/biossíntese , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/biossíntese , NADPH Oxidases/química , NADPH Oxidases/metabolismo , Fagócitos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Sequência de Aminoácidos , Substituição de Aminoácidos , Linhagem Celular , Humanos , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Mutação , NADPH Oxidase 2 , NADPH Oxidases/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Homologia de Sequência de Aminoácidos
6.
Am J Hum Genet ; 94(1): 95-104, 2014 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-24360805

RESUMO

Ten to fifteen percent of couples are confronted with infertility and a male factor is involved in approximately half the cases. A genetic etiology is likely in most cases yet only few genes have been formally correlated with male infertility. Homozygosity mapping was carried out on a cohort of 20 North African individuals, including 18 index cases, presenting with primary infertility resulting from impaired sperm motility caused by a mosaic of multiple morphological abnormalities of the flagella (MMAF) including absent, short, coiled, bent, and irregular flagella. Five unrelated subjects out of 18 (28%) carried a homozygous variant in DNAH1, which encodes an inner dynein heavy chain and is expressed in testis. RT-PCR, immunostaining, and electronic microscopy were carried out on samples from one of the subjects with a mutation located on a donor splice site. Neither the transcript nor the protein was observed in this individual, confirming the pathogenicity of this variant. A general axonemal disorganization including mislocalization of the microtubule doublets and loss of the inner dynein arms was observed. Although DNAH1 is also expressed in other ciliated cells, infertility was the only symptom of primary ciliary dyskinesia observed in affected subjects, suggesting that DNAH1 function in cilium is not as critical as in sperm flagellum.


Assuntos
Dineínas do Axonema/genética , Infertilidade Masculina/genética , Mutação , Cauda do Espermatozoide/patologia , Axonema/genética , Axonema/patologia , Cílios/genética , Cílios/patologia , Flagelos/patologia , Variação Genética , Homozigoto , Humanos , Síndrome de Kartagener/genética , Masculino , Sítios de Splice de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Motilidade dos Espermatozoides , Testículo/citologia , Testículo/patologia
7.
Environ Microbiol ; 15(2): 471-86, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22765374

RESUMO

Pseudomonas aeruginosa is capable of injecting protein toxins into other bacterial cells through one of its three type VI secretion systems (T6SSs). The activity of this T6SS is tightly regulated on the posttranslational level by phosphorylation-dependent and -independent pathways. The phosphorylation-dependent pathway consists of a Threonine kinase/phosphatase pair (PpkA/PppA) that acts on a forkhead domain-containing protein, Fha1, and a periplasmic protein, TagR, that positively regulates PpkA. In the present work, we biochemically and functionally characterize three additional proteins of the phosphorylation-dependent regulatory cascade that controls T6S activation: TagT, TagS and TagQ. We show that similar to TagR, these proteins act upstream of the PpkA/PppA checkpoint and influence phosphorylation of Fha1 and, apparatus assembly and effector export. Localization studies demonstrate that TagQ is an outer membrane lipoprotein and TagR is associated with the outer membrane. Consistent with their homology to lipoprotein outer membrane localization (Lol) components, TagT and TagS form a stable inner membrane complex with ATPase activity. However, we find that outer membrane association of T6SS lipoproteins TagQ and TssJ1, and TagR, is unaltered in a ΔtagTS background. Notably, we found that TagQ is indispensible for anchoring of TagR to the outer membrane fraction. As T6S-dependent fitness of P. aeruginosa requires TagT, S, R and Q, we conclude that these proteins likely participate in a trans-membrane signalling pathway that promotes H1-T6SS activity under optimal environmental conditions.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Sistemas de Secreção Bacterianos/fisiologia , Pseudomonas aeruginosa/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Transporte Biológico , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Estudo de Associação Genômica Ampla , Dados de Sequência Molecular , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Pseudomonas aeruginosa/genética , Alinhamento de Sequência , Transdução de Sinais
8.
PLoS Pathog ; 8(11): e1003052, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23209420

RESUMO

Bacterial biofilm is considered as a particular lifestyle helping cells to survive hostile environments triggered by a variety of signals sensed and integrated through adequate regulatory pathways. Pseudomonas aeruginosa, a Gram-negative bacterium causing severe infections in humans, forms biofilms and is a fantastic example for fine-tuning of the transition between planktonic and community lifestyles through two-component systems (TCS). Here we decipher the regulon of the P. aeruginosa response regulator PprB of the TCS PprAB. We identified genes under the control of this TCS and once this pathway is activated, analyzed and dissected at the molecular level the PprB-dependent phenotypes in various models. The TCS PprAB triggers a hyper-biofilm phenotype with a unique adhesive signature made of BapA adhesin, a Type 1 secretion system (T1SS) substrate, CupE CU fimbriae, Flp Type IVb pili and eDNA without EPS involvement. This unique signature is associated with drug hyper-susceptibility, decreased virulence in acutely infected flies and cytotoxicity toward various cell types linked to decreased Type III secretion (T3SS). Moreover, once the PprB pathway is activated, decreased virulence in orally infected flies associated with enhanced biofilm formation and dissemination defect from the intestinal lumen toward the hemolymph compartment is reported. PprB may thus represent a key bacterial adaptation checkpoint of multicellular and aggregative behavior triggering the production of a unique matrix associated with peculiar antibiotic susceptibility and attenuated virulence, a particular interesting breach for therapeutic intervention to consider in view of possible eradication of P. aeruginosa biofilm-associated infections.


Assuntos
Adesinas Bacterianas/metabolismo , Sistemas de Secreção Bacterianos/fisiologia , Biofilmes/crescimento & desenvolvimento , Pseudomonas aeruginosa/fisiologia , Adesinas Bacterianas/genética , Animais , Linhagem Celular , Drosophila melanogaster , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo
9.
J Biol Chem ; 287(10): 7556-72, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22194609

RESUMO

Adherens junctions are required for vascular endothelium integrity. These structures are formed by the clustering of the homophilic adhesive protein VE-cadherin, which recruits intracellular partners, such as ß- and α-catenins, vinculin, and actin filaments. The dogma according to which α-catenin bridges cadherin·ß-catenin complexes to the actin cytoskeleton has been challenged during the past few years, and the link between the VE-cadherin·catenin complex and the actin cytoskeleton remains unclear. Recently, epithelial protein lost in neoplasm (EPLIN) has been proposed as a possible bond between the E-cadherin·catenin complex and actin in epithelial cells. Herein, we show that EPLIN is expressed at similar levels in endothelial and epithelial cells and is located at interendothelial junctions in confluent cells. Co-immunoprecipitation and GST pulldown experiments provided evidence that EPLIN interacts directly with α-catenin and tethers the VE-cadherin·catenin complex to the actin cytoskeleton. In the absence of EPLIN, vinculin was delocalized from the junctions. Furthermore, suppression of actomyosin tension using blebbistatin triggered a similar vinculin delocalization from the junctions. In a Matrigel assay, EPLIN-depleted endothelial cells exhibited a reduced capacity to form pseudocapillary networks because of numerous breakage events. In conclusion, we propose a model in which EPLIN establishes a link between the cadherin·catenin complex and actin that is independent of actomyosin tension. This link acts as a mechanotransmitter, allowing vinculin binding to α-catenin and formation of a secondary molecular bond between the adherens complex and the cytoskeleton through vinculin. In addition, we provide evidence that the EPLIN clutch is necessary for stabilization of capillary structures in an angiogenesis model.


Assuntos
Citoesqueleto de Actina/metabolismo , Capilares/metabolismo , Proteínas do Citoesqueleto/metabolismo , Células Endoteliais/metabolismo , Modelos Biológicos , Neovascularização Fisiológica/fisiologia , alfa Catenina/metabolismo , Citoesqueleto de Actina/genética , Junções Aderentes/genética , Junções Aderentes/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Células CACO-2 , Caderinas/genética , Caderinas/metabolismo , Capilares/citologia , Proteínas do Citoesqueleto/genética , Cães , Células Endoteliais/citologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Mecanotransdução Celular/fisiologia , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Vinculina/genética , Vinculina/metabolismo , alfa Catenina/genética
10.
J Biol Chem ; 286(32): 28357-69, 2011 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-21659519

RESUMO

Flavocytochrome b(558) (cytb) of phagocytes is a heterodimeric integral membrane protein composed of two subunits, p22(phox) and gp91(phox). The latter subunit, also known as Nox2, has a cytosolic C-terminal "dehydrogenase domain" containing FAD/NADPH-binding sites. The N-terminal half of Nox2 contains six predicted transmembrane α-helices coordinating two hemes. We studied the role of the second transmembrane α-helix, which contains a "hot spot" for mutations found in rare X(+) and X(-) chronic granulomatous disease. By site-directed mutagenesis and transfection in X-CGD PLB-985 cells, we examined the functional and structural impact of seven missense mutations affecting five residues. P56L and C59F mutations drastically influence the level of Nox2 expression indicating that these residues are important for the structural stability of Nox2. A53D, R54G, R54M, and R54S mutations do not affect spectral properties of oxidized/reduced cytb, oxidase complex assembly, FAD binding, nor iodonitrotetrazolium (INT) reductase (diaphorase) activity but inhibit superoxide production. This suggests that Ala-53 and Arg-54 are essential in control of electron transfer from FAD. Surprisingly, the A57E mutation partially inhibits FAD binding, diaphorase activity, and oxidase assembly and affects the affinity of immunopurified A57E cytochrome b(558) for p67(phox). By competition experiments, we demonstrated that the second transmembrane helix impacts on the function of the first intracytosolic B-loop in the control of diaphorase activity of Nox2. Finally, by comparing INT reductase activity of immunopurified mutated and wild type cytb under aerobiosis versus anaerobiosis, we showed that INT reduction reflects the electron transfer from NADPH to FAD only in the absence of superoxide production.


Assuntos
Flavina-Adenina Dinucleotídeo/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Substituição de Aminoácidos , Linhagem Celular , Grupo dos Citocromos b/genética , Grupo dos Citocromos b/metabolismo , Di-Hidrolipoamida Desidrogenase/genética , Di-Hidrolipoamida Desidrogenase/metabolismo , Transporte de Elétrons/fisiologia , Estabilidade Enzimática/genética , Flavina-Adenina Dinucleotídeo/genética , Humanos , Glicoproteínas de Membrana/genética , Mutação de Sentido Incorreto , NADPH Oxidase 2 , NADPH Oxidases/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Superóxidos/metabolismo
11.
PLoS One ; 5(12): e15249, 2010 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-21179207

RESUMO

BACKGROUND: Visual and ß-arrestins are scaffolding proteins involved in the regulation of receptor-dependent intracellular signaling and their trafficking. The arrestin superfamilly includes several arrestin domain-containing proteins and the structurally related protein Vps26. In Dictyostelium discoideum, the arrestin-domain containing proteins form a family of six members, namely AdcA to -F. In contrast to canonical arrestins, Dictyostelium Adc proteins show a more complex architecture, as they possess, in addition to the arrestin core, other domains, such as C2, FYVE, LIM, MIT and SAM, which potentially mediate selective interactions with either lipids or proteins. METHODOLOGY AND PRINCIPAL FINDINGS: A detailed analysis of AdcA has been performed. AdcA extends on both sides of the arrestin core, in particular by a FYVE domain which mediates selective interactions with PI(3)P, as disclosed by intrinsic fluorescence measurements and lipid overlay assays. Localization studies showed an enrichment of tagged- and endogenous AdcA on the rim of early macropinosomes and phagosomes. This vesicular distribution relies on a functional FYVE domain. Our data also show that the arrestin core binds the ADP-ribosylation factor ArfA, the unique amoebal Arf member, in its GDP-bound conformation. SIGNIFICANCE: This work describes one of the 6 arrestin domain-containing proteins of Dictyostelium, a novel and atypical member of the arrestin clan. It provides the basis for a better understanding of arrestin-related protein involvement in trafficking processes and for further studies on the expanding roles of arrestins in eukaryotes.


Assuntos
Arrestina/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Arrestina/isolamento & purificação , Dictyostelium , Endossomos/metabolismo , Proteínas de Fluorescência Verde/química , Lipídeos/química , Camundongos , Microscopia de Fluorescência/métodos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Coelhos , Homologia de Sequência de Aminoácidos , Frações Subcelulares
12.
J Biol Chem ; 285(43): 33197-33208, 2010 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-20724480

RESUMO

The X(+)-linked chronic granulomatous disease (X(+)-CGD) variants are natural mutants characterized by defective NADPH oxidase activity but with normal Nox2 expression. According to the three-dimensional model of the cytosolic Nox2 domain, most of the X(+)-CGD mutations are located in/or close to the FAD/NADPH binding regions. A structure/function study of this domain was conducted in X(+)-CGD PLB-985 cells exactly mimicking 10 human variants: T341K, C369R, G408E, G408R, P415H, P415L, Δ507QKT509-HIWAinsert, C537R, L546P, and E568K. Diaphorase activity is defective in all these mutants. NADPH oxidase assembly is normal for P415H/P415L and T341K mutants where mutation occurs in the consensus sequences of NADPH- and FAD-binding sites, respectively. This is in accordance with their buried position in the three-dimensional model of the cytosolic Nox2 domain. FAD incorporation is abolished only in the T341K mutant explaining its absence of diaphorase activity. This demonstrates that NADPH oxidase assembly can occur without FAD incorporation. In addition, a defect of NADPH binding is a plausible explanation for the diaphorase activity inhibition in the P415H, P415L, and C537R mutants. In contrast, Cys-369, Gly-408, Leu-546, and Glu-568 are essential for NADPH oxidase complex assembly. However, according to their position in the three-dimensional model of the cytosolic domain of Nox2, only Cys-369 could be in direct contact with cytosolic factors during oxidase assembly. In addition, the defect in oxidase assembly observed in the C369R, G408E, G408R, and E568K mutants correlates with the lack of FAD incorporation. Thus, the NADPH oxidase assembly process and FAD incorporation are closely related events essential for the diaphorase activity of Nox2.


Assuntos
Flavina-Adenina Dinucleotídeo/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , NADP/metabolismo , Fagócitos/enzimologia , Sítios de Ligação , Linhagem Celular , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/genética , Regulação Enzimológica da Expressão Gênica , Doença Granulomatosa Crônica/enzimologia , Doença Granulomatosa Crônica/genética , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Mutação de Sentido Incorreto , NADP/química , NADP/genética , NADPH Oxidase 2 , NADPH Oxidases/química , NADPH Oxidases/genética , Estrutura Terciária de Proteína
13.
PLoS One ; 5(6): e10977, 2010 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-20544018

RESUMO

BACKGROUND: Cellular cholesterol is a vital component of the cell membrane. Its concentration is tightly controlled by mechanisms that remain only partially characterized. In this study, we describe a late endosome/lysosomes-associated protein whose expression level affects cellular free cholesterol content. METHODOLOGY/PRINCIPAL FINDINGS: Using a restricted proteomic analysis of detergent-resistant membranes (DRMs), we have identified a protein encoded by gene C11orf59. It is mainly localized to late endosome/lysosome (LE/LY) compartment through N-terminal myristoylation and palmitoylation. We named it Pdro for protein associated with DRMs and endosomes. Very recently, three studies have reported on the same protein under two other names: the human p27RF-Rho that regulates RhoA activation and actin dynamics, and its rodent orthologue p18 that controls both LE/LY dynamics through the MERK-ERK pathway and the lysosomal activation of mammalian target of rapamycin complex 1 by amino acids. We found that, consistent with the presence of sterol-responsive element consensus sequences in the promoter region of C11orf59, Pdro mRNA and protein expression levels are regulated positively by cellular cholesterol depletion and negatively by cellular cholesterol loading. Conversely, Pdro is involved in the regulation of cholesterol homeostasis, since its depletion by siRNA increases cellular free cholesterol content that is accompanied by an increased cholesterol efflux from cells. On the other hand, cells stably overexpressing Pdro display reduced cellular free cholesterol content. Pdro depletion-mediated excess cholesterol results, at least in part, from a stimulated low-density lipoprotein (LDL) uptake and an increased cholesterol egress from LE/LY. CONCLUSIONS/SIGNIFICANCE: LDL-derived cholesterol release involves LE/LY motility that is linked to actin dynamics. Because Pdro regulates these two processes, we propose that modulation of Pdro expression in response to sterol levels regulates LDL-derived cholesterol through both LDL uptake and LE/LY dynamics, to ultimately control free cholesterol homeostasis.


Assuntos
Proteínas de Transporte/metabolismo , Colesterol/metabolismo , Endossomos/metabolismo , Homeostase , Lisossomos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Proteínas de Transporte/química , Proteínas de Transporte/genética , Primers do DNA , Citometria de Fluxo , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lipoproteínas LDL/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em Tandem
14.
J Biol Chem ; 285(26): 20224-33, 2010 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-20385549

RESUMO

The Epstein-Barr virus basic leucine zipper transcriptional activator ZEBRA was shown recently to cross the outer membrane of live cells and to accumulate in the nucleus of lymphocytes. We investigated the potential application of the Epstein-Barr virus trans-activator ZEBRA as a transporter protein to facilitate transduction of cargo proteins. Analysis of different truncated forms of ZEBRA revealed that the minimal domain (MD) required for internalization spans residues 170-220. MD efficiently transported reporter proteins such as enhanced green fluorescent protein (EGFP) and beta-galactosidase in several normal and tumor cell lines. Functionality of internalized cargo proteins was confirmed by beta-galactosidase activity in transduced cells, and no MD-associated cell toxicity was detected. Translocation of MD through the cell membrane required binding to cell surface-associated heparan sulfate proteoglycans as shown by strong inhibition of protein uptake in the presence of heparin. We found that internalization was blocked at 4 degrees C, whereas no ATP was required as shown by an only 25% decreased uptake efficiency in energy-depleted cells. Common endocytotic inhibitors such as nystatin, chlorpromazine, and wortmannin had no significant impact on MD-EGFP uptake. Only methyl-beta-cyclodextrin inhibited MD-EGFP uptake by 40%, implicating the lipid raft-mediated endocytotic pathway. These data suggest that MD-reporter protein transduction occurs mostly via direct translocation through the lipid bilayer and not by endocytosis. This mechanism of MD-mediated internalization is suitable for the efficient delivery of biologically active proteins and renders ZEBRA-MD a promising candidate for therapeutic protein delivery applications.


Assuntos
Proteínas de Fluorescência Verde/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/metabolismo , beta-Galactosidase/metabolismo , Sítios de Ligação/genética , Linhagem Celular Tumoral , Sondas de DNA/genética , Sondas de DNA/metabolismo , Endocitose/efeitos dos fármacos , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/farmacocinética , Células HeLa , Humanos , Imuno-Histoquímica , Cinética , Microscopia de Fluorescência , Mutação , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Transativadores/genética , beta-Ciclodextrinas/farmacologia , beta-Galactosidase/genética , beta-Galactosidase/farmacocinética
15.
Nat Methods ; 6(8): 600-2, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19633664

RESUMO

In Drosophila melanogaster, widely used mitotic recombination-based strategies generate mosaic flies with positive readout for only one daughter cell after division. To differentially label both daughter cells, we developed the twin spot generator (TSG) technique, which through mitotic recombination generates green and red twin spots that are detectable after the first cell division as single cells. We propose wide applications of TSG to lineage and genetic mosaic studies.


Assuntos
Linhagem da Célula , Drosophila melanogaster/genética , Genômica/métodos , Mitose , Recombinação Genética , Animais , Células Clonais , Drosophila melanogaster/citologia , Fluorometria , Genômica/instrumentação , Mutação
16.
J Cell Sci ; 121(Pt 20): 3325-34, 2008 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-18796536

RESUMO

Nonaspanins are characterised by a large N-terminal extracellular domain and nine putative transmembrane domains. This evolutionarily conserved family comprises three members in Dictyostelium discoideum (Phg1A, Phg1B and Phg1C) and Drosophila melanogaster, and four in mammals (TM9SF1-TM9SF4), the function of which is essentially unknown. Genetic studies in Dictyostelium demonstrated that Phg1A is required for cell adhesion and phagocytosis. We created Phg1A/TM9SF4-null mutant flies and showed that they were sensitive to pathogenic Gram-negative, but not Gram-positive, bacteria. This increased sensitivity was not due to impaired Toll or Imd signalling, but rather to a defective cellular immune response. TM9SF4-null larval macrophages phagocytosed Gram-negative E. coli inefficiently, although Gram-positive S. aureus were phagocytosed normally. Mutant larvae also had a decreased wasp egg encapsulation rate, a process requiring haemocyte-dependent adhesion to parasitoids. Defective cellular immunity was coupled to morphological and adhesion defects in mutant larval haemocytes, which had an abnormal actin cytoskeleton. TM9SF4, and its closest paralogue TM9SF2, were both required for bacterial internalisation in S2 cells, where they displayed partial redundancy. Our study highlights the contribution of phagocytes to host defence in an organism possessing a complex innate immune response and suggests an evolutionarily conserved function of TM9SF4 in eukaryotic phagocytes.


Assuntos
Escherichia coli/imunologia , Hemócitos/imunologia , Imunidade Inata/fisiologia , Proteínas de Membrana/imunologia , Fagocitose/imunologia , Transdução de Sinais/imunologia , Staphylococcus aureus/imunologia , Animais , Adesão Celular/genética , Adesão Celular/imunologia , Linhagem Celular , Dictyostelium/genética , Dictyostelium/imunologia , Drosophila melanogaster , Hemócitos/citologia , Larva/genética , Larva/imunologia , Larva/microbiologia , Mamíferos/genética , Mamíferos/imunologia , Proteínas de Membrana/genética , Mutação/genética , Mutação/imunologia , Fagócitos/citologia , Fagócitos/imunologia , Fagocitose/genética , Transdução de Sinais/genética
17.
Biochem J ; 409(1): 87-94, 2008 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17725544

RESUMO

FtsZ is a key protein involved in bacterial and organellar division. Bacteria have only one ftsZ gene, while chlorophytes (higher plants and green alga) have two distinct FtsZ gene families, named FtsZ1 and FtsZ2. This raises the question of why chloroplasts in these organisms need distinct FtsZ proteins to divide. In order to unravel new functions associated with FtsZ proteins, we have identified and characterized an Arabidopsis thaliana FtsZ1 loss-of-function mutant. ftsZ1-knockout mutants are impeded in chloroplast division, and division is restored when FtsZ1 is expressed at a low level. FtsZ1-overexpressing plants show a drastic inhibition of chloroplast division. Chloroplast morphology is altered in ftsZ1, with chloroplasts having abnormalities in the thylakoid membrane network. Overexpression of FtsZ1 also induced defects in thylakoid organization with an increased network of twisting thylakoids and larger grana. We show that FtsZ1, in addition to being present in the stroma, is tightly associated with the thylakoid fraction. This association is developmentally regulated since FtsZ1 is found in the thylakoid fraction of young developing plant leaves but not in mature and old plant leaves. Our results suggest that plastid division protein FtsZ1 may have a function during leaf development in thylakoid organization, thus highlighting new functions for green plastid FtsZ.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/fisiologia , Plastídeos/metabolismo , Tilacoides/metabolismo , Clorofila/metabolismo , Cloroplastos/metabolismo , Imuno-Histoquímica , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Organelas/metabolismo , Fenótipo , Proteínas de Plantas/genética , Células-Tronco/metabolismo
18.
J Biol Chem ; 282(40): 29482-92, 2007 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-17636260

RESUMO

Chloroplast envelope quinone oxidoreductase (ceQORH) is an inner plastid envelope protein that is synthesized without cleavable chloroplast transit sequence for import. In the present work, we studied the in vitro-import characteristics of Arabidopsis ceQORH. We demonstrate that ceQORH import requires ATP and is dependent on proteinaceous receptor components exposed at the outer plastid surface. Competition experiments using small subunit precursor of ribulose-bisphosphate carboxylase/oxygenase and precursor of ferredoxin, as well as antibody blocking experiments, revealed that ceQORH import does not involve the main receptor and translocation channel proteins Toc159 and Toc75, respectively, which operate in import of proteins into the chloroplast. Molecular dissection of the ceQORH amino acid sequence by site-directed mutagenesis and subsequent import experiments in planta and in vitro highlighted that ceQORH consists of different domains that act concertedly in regulating import. Collectively, our results provide unprecedented evidence for the existence of a specific import pathway for transit sequence-less inner plastid envelope membrane proteins into chloroplasts.


Assuntos
Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Precursores de Proteínas/metabolismo , Transporte Proteico , Arabidopsis/metabolismo , Ligação Competitiva , Reagentes de Ligações Cruzadas/farmacologia , Ferredoxinas/química , Hordeum/metabolismo , Mutagênese Sítio-Dirigida , Plastídeos/metabolismo , Estrutura Terciária de Proteína , Frações Subcelulares/metabolismo
19.
J Neurosci ; 27(17): 4716-24, 2007 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-17460084

RESUMO

In the germinative zone of the adult rodent brain, neural progenitors migrate into niches delimited by astrocyte processes and differentiate into neuronal precursors. In the present study, we report a modulating role for the scaffolding protein IQGAP1 in neural progenitor migration. We have identified IQGAP1 as a new marker of amplifying neural progenitor and neuronal precursor cells of the subventricular zone (SVZ) and the rostral migratory stream (RMS) in the adult mouse brain. To determine functions for IQGAP1 in neural progenitors, we compared the properties of neural progenitor cells from wild-type and Iqgap1-null mutant mice in vivo and in vitro. The in vivo studies reveal a delay in the transition of de novo neural progenitors into neuronal precursor cells in Iqgap1-null mice. In vitro, we demonstrated that IQGAP1 acts as a downstream effector in the vascular endothelial growth factor (VEGF)-dependent migratory response of neural progenitors that also impacts on their neuronal differentiation. The Rho-family GTPases cdc42/Rac1 and Lis1 are major partners of IQGAP1 in this migratory process. Finally, astrocytes of the neurogenic SVZ and RMS are shown to express VEGF. We propose that VEGF synthesized by astrocytes could be involved in the guidance of neural progenitors to neurogenic niches and that IQGAP1 is an effector of the VEGF-dependent migratory signal.


Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/fisiologia , Movimento Celular/fisiologia , Neurônios/citologia , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Ventrículos Cerebrais/citologia , Técnicas In Vitro , Camundongos , Camundongos Knockout , Neuropeptídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP , Proteínas Ativadoras de ras GTPase/genética
20.
J Leukoc Biol ; 81(1): 238-49, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17060362

RESUMO

The role of Leu505 of Nox2 on the NADPH oxidase activation process was investigated. An X-CGD PLB-985 cell line expressing the Leu505Arg Nox2 mutant was obtained, exactly mimicking the phenotype of a previously published X91+-CGD case. In a reconstituted cell-free system (CFS), NADPH oxidase and iodonitrotetrazolium (INT) reductase activities were partially maintained concomitantly with a partial cytosolic factors translocation to the plasma membrane. This suggests that assembly and electron transfer from NADPH occurred partially in the Leu505Arg Nox2 mutant. Moreover, in a simplified CFS using purified mutant cytochrome b558 and recombinant p67phox, p47phox, and Rac1proteins, we found that the Km for NADPH and for NADH was about three times higher than those of purified WT cytochrome b558, indicating that the Leu505Arg mutation induces a slight decrease of the affinity for NADPH and NADH. In addition, oxidase activity can be extended by increasing the amount of p67phox in the simplified CFS assay. However, the maximal reconstituted oxidase activity using WT purified cytochrome b558 could not be reached using mutant cytochrome b558. In a three-dimensional model of the C-terminal tail of Nox2, Leu505 appears to have a strategic position just at the entry of the NADPH binding site and at the end of the alpha-helical loop (residues 484-504), a potential cytosolic factor binding region. The Leu505Arg mutation seems to affect the oxidase complex activation process through alteration of cytosolic factors binding and more particularly the p67phox interaction with cytochrome b558, thus affecting NADPH access to its binding site.


Assuntos
Grupo dos Citocromos b/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Fagossomos/metabolismo , Fosfoproteínas/fisiologia , Sequência de Aminoácidos , Linhagem Celular Tumoral , Cromossomos Humanos X , Grupo dos Citocromos b/genética , Ativação Enzimática , Doença Granulomatosa Crônica/genética , Humanos , Leucina/química , Glicoproteínas de Membrana/química , Dados de Sequência Molecular , NADPH Oxidase 2 , NADPH Oxidases/química , NADPH Oxidases/genética , Neutrófilos/fisiologia , Oxirredutases/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Sais de Tetrazólio/metabolismo
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