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1.
Mol Ther ; 31(7): 2206-2219, 2023 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-37198883

RESUMEN

X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disease caused by a retrotransposon insertion in intron 32 of the TAF1 gene. This insertion causes mis-splicing of intron 32 (TAF1-32i) and reduced TAF1 levels. TAF1-32i transcript is unique to XDP patient cells and can be detected in their extracellular vesicles (EVs). We engrafted patient and control iPSC-derived neural progenitor cells (hNPCs) into the striatum of mice. To track TAF1-32i transcript spread by EVs, we transduced the brain-implanted hNPCs with a lentiviral construct called ENoMi, which consists of a re-engineered tetraspanin scaffold tagged with bioluminescent and fluorescent reporter proteins under an EF-1α promoter. Alongside this improved detection in ENoMi-hNPCs-derived EVs, their surface allows specific immunocapture purification, thereby facilitating TAF1-32i analysis. Using this ENoMi-labeling method, TAF1-32i was demonstrated in EVs released from XDP hNPCs implanted in mouse brains. Post-implantation of ENoMi-XDP hNPCs, TAF1-32i transcript was retrieved in EVs isolated from mouse brain and blood, and levels increased over time in plasma. We compared and combined our EV isolation technique to analyze XDP-derived TAF1-32i with other techniques, including size exclusion chromatography and Exodisc. Overall, our study demonstrates the successful engraftment of XDP patient-derived hNPCs in mice as a tool for monitoring disease markers with EVs.


Asunto(s)
Vesículas Extracelulares , Enfermedades Neurodegenerativas , Humanos , Ratones , Animales , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismo , Biomarcadores , Encéfalo/metabolismo , Vesículas Extracelulares/metabolismo
2.
Mov Disord ; 36(1): 206-215, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32975318

RESUMEN

BACKGROUND: X-linked dystonia-parkinsonism is a rare neurological disease endemic to the Philippines. Dystonic symptoms appear in males at the mean age of 40 years and progress to parkinsonism with degenerative pathology in the striatum. A retrotransposon inserted in intron 32 of the TAF1 gene leads to alternative splicing in the region and a reduction of the full-length mRNA transcript. OBJECTIVES: The objective of this study was to discover cell-based and biofluid-based biomarkers for X-linked dystonia-parkinsonism. METHODS: RNA from patient-derived neural progenitor cells and their secreted extracellular vesicles were used to screen for dysregulation of TAF1 expression. Droplet-digital polymerase chain reaction was used to quantify the expression of TAF1 mRNA fragments 5' and 3' to the retrotransposon insertion and the disease-specific splice variant TAF1-32i in whole-blood RNA. Plasma levels of neurofilament light chain were measured using single-molecule array. RESULTS: In neural progenitor cells and their extracellular vesicles, we confirmed that the TAF1-3'/5' ratio was lower in patient samples, whereas TAF1-32i expression is higher relative to controls. In whole-blood RNA, both TAF1-3'/5' ratio and TAF1-32i expression can differentiate patient (n = 44) from control samples (n = 18) with high accuracy. Neurofilament light chain plasma levels were significantly elevated in patients (n = 43) compared with both carriers (n = 16) and controls (n = 21), with area under the curve of 0.79. CONCLUSIONS: TAF1 dysregulation in blood serves as a disease-specific biomarker that could be used as a readout for monitoring therapies targeting TAF1 splicing. Neurofilament light chain could be used in monitoring neurodegeneration and disease progression in patients. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.


Asunto(s)
Enfermedades Genéticas Ligadas al Cromosoma X , Factores Asociados con la Proteína de Unión a TATA , Adulto , Biomarcadores , Trastornos Distónicos , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Histona Acetiltransferasas/genética , Humanos , Filamentos Intermedios , Masculino , Factores Asociados con la Proteína de Unión a TATA/genética , Factor de Transcripción TFIID/genética
3.
PLoS One ; 15(12): e0243655, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33315879

RESUMEN

X-linked Dystonia-Parkinsonism (XDP) is a neurodegenerative disease linked to an insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within an intron of TAF1. This SVA insertion induces aberrant TAF1 splicing and partial intron retention, thereby decreasing levels of the full-length transcript. Here we sought to determine if these altered transcriptional dynamics caused by the SVA are also accompanied by local changes in histone acetylation, given that these modifications influence gene expression. Because TAF1 protein may itself exhibit histone acetyltransferase activity, we also examined whether decreased TAF1 expression in XDP cell lines and post-mortem brain affects global levels of acetylated histone H3 (AcH3). The results demonstrate that total AcH3 are not altered in XDP post-mortem prefrontal cortex or cell lines. We also did not detect local differences in AcH3 associated with TAF1 exons or intronic sites flanking the SVA insertion. There was, however, a decrease in AcH3 association with the exon immediately proximal to the intronic SVA, and this decrease was normalized by CRISPR/Cas-excision of the SVA. Collectively, these data suggest that the SVA insertion alters histone status in this region, which may contribute to the dysregulation of TAF1 expression.


Asunto(s)
Trastornos Distónicos/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Histona Acetiltransferasas/genética , Histonas/metabolismo , Factores Asociados con la Proteína de Unión a TATA/genética , Factor de Transcripción TFIID/genética , Acetilación , Células Cultivadas , Trastornos Distónicos/metabolismo , Fibroblastos/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/metabolismo , Humanos , Intrones , Retroelementos
4.
Neurobiol Dis ; 144: 105032, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32739252

RESUMEN

Neuroinflammation plays a pathogenic role in neurodegenerative diseases and recent findings suggest that it may also be involved in X-linked Dystonia-Parkinsonism (XDP) pathogenesis. Previously, fibroblasts and neuronal stem cells derived from XDP patients demonstrated hypersensitivity to TNF-α, dysregulation in NFκB signaling, and an increase in several pro-inflammatory markers. However, the role of inflammatory processes in XDP patient brain remains unknown. Here we demonstrate that there is a significant increase in astrogliosis and microgliosis in human post-mortem XDP prefrontal cortex (PFC) compared to control. Furthermore, there is a significant increase in histone H3 citrullination (H3R2R8R17cit3) with a concomitant increase in peptidylarginine deaminase 2 (PAD2) and 4 (PAD4), the enzymes catalyzing citrullination, in XDP post-mortem PFC. While there is a significant increase in myeloperoxidase (MPO) levels in XDP PFC, neutrophil elastase (NE) levels are not altered, suggesting that MPO may be released by activated microglia or reactive astrocytes in the brain. Similarly, there was an increase in H3R2R8R17cit3, PAD2 and PAD4 levels in XDP-derived fibroblasts. Importantly, treatment of fibroblasts with Cl-amidine, a pan inhibitor of PAD enzymes, reduced histone H3 citrullination and pro-inflammatory chemokine expression, without affecting cell survival. Taken together, our results demonstrate that inflammation is increased in XDP post-mortem brain and fibroblasts and unveil a new epigenetic potential therapeutic target.


Asunto(s)
Citrulinación , Trastornos Distónicos/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/metabolismo , Histonas/metabolismo , Inflamación/metabolismo , Corteza Prefrontal/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Astrocitos/metabolismo , Astrocitos/patología , Autopsia , Supervivencia Celular , Quimiocinas/efectos de los fármacos , Quimiocinas/metabolismo , Citrulinación/efectos de los fármacos , Trastornos Distónicos/patología , Femenino , Fibroblastos/efectos de los fármacos , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Gliosis/metabolismo , Gliosis/patología , Histonas/efectos de los fármacos , Humanos , Inflamación/patología , Elastasa de Leucocito/metabolismo , Masculino , Microglía/metabolismo , Microglía/patología , Persona de Mediana Edad , Ornitina/análogos & derivados , Ornitina/farmacología , Peroxidasa/metabolismo , Corteza Prefrontal/patología , Arginina Deiminasa Proteína-Tipo 2/metabolismo , Arginina Deiminasa Proteína-Tipo 4/metabolismo
5.
Cell ; 172(5): 897-909.e21, 2018 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-29474918

RESUMEN

X-linked Dystonia-Parkinsonism (XDP) is a Mendelian neurodegenerative disease that is endemic to the Philippines and is associated with a founder haplotype. We integrated multiple genome and transcriptome assembly technologies to narrow the causal mutation to the TAF1 locus, which included a SINE-VNTR-Alu (SVA) retrotransposition into intron 32 of the gene. Transcriptome analyses identified decreased expression of the canonical cTAF1 transcript among XDP probands, and de novo assembly across multiple pluripotent stem-cell-derived neuronal lineages discovered aberrant TAF1 transcription that involved alternative splicing and intron retention (IR) in proximity to the SVA that was anti-correlated with overall TAF1 expression. CRISPR/Cas9 excision of the SVA rescued this XDP-specific transcriptional signature and normalized TAF1 expression in probands. These data suggest an SVA-mediated aberrant transcriptional mechanism associated with XDP and may provide a roadmap for layered technologies and integrated assembly-based analyses for other unsolved Mendelian disorders.


Asunto(s)
Trastornos Distónicos/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Genoma Humano , Transcriptoma/genética , Empalme Alternativo/genética , Elementos Alu/genética , Secuencia de Bases , Sistemas CRISPR-Cas/genética , Estudios de Cohortes , Familia , Femenino , Sitios Genéticos , Haplotipos/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Intrones/genética , Masculino , Repeticiones de Minisatélite/genética , Modelos Genéticos , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Elementos de Nucleótido Esparcido Corto , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/metabolismo
6.
Proc Natl Acad Sci U S A ; 114(51): E11020-E11028, 2017 12 19.
Artículo en Inglés | MEDLINE | ID: mdl-29229810

RESUMEN

X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disease associated with an antisense insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within an intron of TAF1 This unique insertion coincides with six additional noncoding sequence changes in TAF1, the gene that encodes TATA-binding protein-associated factor-1, which appear to be inherited together as an identical haplotype in all reported cases. Here we examined the sequence of this SVA in XDP patients (n = 140) and detected polymorphic variation in the length of a hexanucleotide repeat domain, (CCCTCT)n The number of repeats in these cases ranged from 35 to 52 and showed a highly significant inverse correlation with age at disease onset. Because other SVAs exhibit intrinsic promoter activity that depends in part on the hexameric domain, we assayed the transcriptional regulatory effects of varying hexameric lengths found in the unique XDP SVA retrotransposon using luciferase reporter constructs. When inserted sense or antisense to the luciferase reading frame, the XDP variants repressed or enhanced transcription, respectively, to an extent that appeared to vary with length of the hexamer. Further in silico analysis of this SVA sequence revealed multiple motifs predicted to form G-quadruplexes, with the greatest potential detected for the hexameric repeat domain. These data directly link sequence variation within the XDP-specific SVA sequence to phenotypic variability in clinical disease manifestation and provide insight into potential mechanisms by which this intronic retroelement may induce transcriptional interference in TAF1 expression.


Asunto(s)
Expansión de las Repeticiones de ADN , Trastornos Distónicos/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Histona Acetiltransferasas/genética , Retroelementos , Elementos de Nucleótido Esparcido Corto , Factores Asociados con la Proteína de Unión a TATA/genética , Factor de Transcripción TFIID/genética , Orden Génico , Estudios de Asociación Genética , Sitios Genéticos , Humanos , Masculino , Modelos Biológicos , Linaje , Fenotipo , Regiones Promotoras Genéticas , Activación Transcripcional
7.
Neurobiol Dis ; 100: 108-118, 2017 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-28017799

RESUMEN

X-linked Dystonia-Parkinsonism (XDP) is a progressive neurodegenerative disease involving the loss of medium spiny neurons within the striatum. An XDP-specific haplotype has been identified, consisting of seven sequence variants which cluster around the human TAF1 gene, but a direct relationship between any of these variants and disease pathogenesis has not yet been demonstrated. Because the pathogenic gene lesion remains unclear, it has been difficult to predict cellular pathways which are affected in XDP cells. To address that issue, we assayed expression of defined gene sets in XDP vs. control fibroblasts to identify networks of functionally-related transcripts which may be dysregulated in XDP patient cells. That analysis derived a 51-gene signature distinguishing XDP vs. control fibroblasts which mapped strongly to nuclear factor-kappa B (NFκB), a transcription factor pathway also implicated in the pathogenesis of other neurodegenerative diseases, including Parkinson's (PD) and Huntington's disease (HD). Constitutive and TNFα-evoked NFκB signaling was further evaluated in XDP vs. control fibroblasts based on luciferase reporter activity, DNA binding of NFκB subunits, and endogenous target gene transcription. Compared to control cells, XDP fibroblasts exhibited decreased basal NFκB activity and decreased levels of the active NFκB p50 subunit, but increased target gene expression in response to TNFα. NFκB signaling was further examined in neural stem cells differentiated from XDP and control induced pluripotent stem cell (iPSC) lines, revealing a similar pattern of increased TNFα responses in the patient lines compared to controls. These data indicate that an NFκB signaling phenotype is present in both patient fibroblasts and neural stem cells, suggesting this pathway as a site of dysfunction in XDP.


Asunto(s)
Núcleo Celular/metabolismo , Trastornos Distónicos/genética , Fibroblastos/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/genética , FN-kappa B/metabolismo , Células-Madre Neurales/metabolismo , Transducción de Señal , Cuerpo Estriado/metabolismo , Distonía/genética , Distonía/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/patología , Fenotipo , Factor de Transcripción TFIID/genética
8.
Dis Model Mech ; 9(4): 451-62, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26769797

RESUMEN

X-linked dystonia-parkinsonism (XDP) is a hereditary neurodegenerative disorder involving a progressive loss of striatal medium spiny neurons. The mechanisms underlying neurodegeneration are not known, in part because there have been few cellular models available for studying the disease. The XDP haplotype consists of multiple sequence variations in a region of the X chromosome containingTAF1, a large gene with at least 38 exons, and a multiple transcript system (MTS) composed of five unconventional exons. A previous study identified an XDP-specific insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon in intron 32 ofTAF1, as well as a neural-specific TAF1 isoform, N-TAF1, which showed decreased expression in post-mortem XDP brain compared with control tissue. Here, we generated XDP patient and control fibroblasts and induced pluripotent stem cells (iPSCs) in order to further probe cellular defects associated with this disease. As initial validation of the model, we compared expression ofTAF1and MTS transcripts in XDP versus control fibroblasts and iPSC-derived neural stem cells (NSCs). Compared with control cells, XDP fibroblasts exhibited decreased expression ofTAF1transcript fragments derived from exons 32-36, a region spanning the SVA insertion site. N-TAF1, which incorporates an alternative exon (exon 34'), was not expressed in fibroblasts, but was detectable in iPSC-differentiated NSCs at levels that were ∼threefold lower in XDP cells than in controls. These results support the previous findings that N-TAF1 expression is impaired in XDP, but additionally indicate that this aberrant transcription might occur in neural cells at relatively early stages of development that precede neurodegeneration.


Asunto(s)
Trastornos Distónicos/genética , Trastornos Distónicos/patología , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Histona Acetiltransferasas/genética , Células-Madre Neurales/metabolismo , Factores Asociados con la Proteína de Unión a TATA/genética , Factor de Transcripción TFIID/genética , Adulto , Anciano , Anciano de 80 o más Años , Empalme Alternativo/genética , Secuencia de Aminoácidos , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular Tumoral , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Marcadores Genéticos , Genotipo , Haplotipos/genética , Histona Acetiltransferasas/química , Histona Acetiltransferasas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Persona de Mediana Edad , Células-Madre Neurales/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores Asociados con la Proteína de Unión a TATA/química , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factor de Transcripción TFIID/química , Factor de Transcripción TFIID/metabolismo
9.
Adv Immunol ; 121: 191-211, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24388216

RESUMEN

The CD200:CD200R1 inhibitory signaling pathway has been implicated in playing a prominent role in limiting inflammation in a wide range of inflammatory diseases. CD200R1 signaling inhibits the expression of proinflammatory molecules including tumor necrosis factor, interferons, and inducible nitric oxide synthase in response to selected stimuli. Unsurprisingly, due to the regulatory role that CD200R1 plays in multiple inflammatory pathways, an increasing number of parasitic, bacterial, and viral pathogens exploit this pathway to suppress host defenses. A complete understanding of the pathways regulated by CD200R1 signaling and the diverse mechanisms that pathogens have evolved to manipulate the CD200:CD200R1 pathway can help identify clinical situations where targeting this interaction can be of therapeutic benefit. In this review, we compare CD200R1 to other pathogen-targeted inhibitory receptors and highlight how this signaling pathway is utilized by a diverse number of pathogens and, therefore, may represent a novel targeting strategy for the treatment of infectious diseases.


Asunto(s)
Antígenos CD/fisiología , Antígenos de Superficie/fisiología , Interacciones Huésped-Patógeno/inmunología , Receptores de Superficie Celular/fisiología , Transducción de Señal/inmunología , Animales , Líquido Extracelular/inmunología , Líquido Extracelular/microbiología , Líquido Extracelular/virología , Interacciones Huésped-Patógeno/genética , Humanos , Inmunoglobulinas/fisiología , Inflamación/genética , Inflamación/microbiología , Inflamación/virología , Gripe Humana/genética , Gripe Humana/inmunología , Gripe Humana/virología , Lectinas Tipo C/fisiología , Ratones , Receptores de Orexina , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/inmunología , Receptores de Superficie Celular/deficiencia , Receptores KIR/administración & dosificación , Receptores KIR/genética , Transducción de Señal/genética
10.
J Immunol ; 190(7): 3525-32, 2013 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-23427254

RESUMEN

Polymeric microparticles have been widely investigated as platforms for delivery of drugs, vaccines, and imaging contrast agents and are increasingly used in a variety of clinical applications. Microparticles activate the inflammasome complex and induce the processing and secretion of IL-1ß, a key innate immune cytokine. Recent work suggests that although receptors are clearly important for particle phagocytosis, other physical characteristics, especially shape, play an important role in the way microparticles activate cells. We examined the role of particle surface texturing not only on uptake efficiency but also on the subsequent immune cell activation of the inflammasome. Using a method based on emulsion processing of amphiphilic block copolymers, we prepared microparticles with similar overall sizes and surface chemistries but having either smooth or highly microtextured surfaces. In vivo, textured (budding) particles induced more rapid neutrophil recruitment to the injection site. In vitro, budding particles were more readily phagocytosed than smooth particles and induced more lipid raft recruitment to the phagosome. Remarkably, budding particles also induced stronger IL-1ß secretion than smooth particles through activation of the NLRP3 inflammasome. These findings demonstrate a pronounced role of particle surface topography in immune cell activation, suggesting that shape is a major determinant of inflammasome activation.


Asunto(s)
Inmunidad Innata , Inflamasomas/inmunología , Polímeros , Animales , Proteínas Portadoras/metabolismo , Humanos , Interleucina-1/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/inmunología , Microdominios de Membrana/metabolismo , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Neutrófilos/inmunología , Neutrófilos/metabolismo , Tamaño de la Partícula , Fagocitosis/inmunología , Polímeros/química , Transducción de Señal , Propiedades de Superficie
11.
PLoS One ; 7(10): e47740, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23082204

RESUMEN

The CD200R1:CD200 axis is traditionally considered to limit tissue inflammation by down-regulating pro-inflammatory signaling in myeloid cells bearing the receptor. We generated CD200R1(-/-) mice and employed them to explore both the role of CD200R1 in regulating macrophage signaling via TLR2 as well as the host response to an in vivo, TLR2-dependent model, herpes simplex virus 1 (HSV-1) infection. CD200R1(-/-) peritoneal macrophages demonstrated a 70-75% decrease in the generation of IL-6 and CCL5 (Rantes) in response to the TLR2 agonist Pam(2)CSK(4) and to HSV-1. CD200R1(-/-) macrophages could neither up-regulate the expression of TLR2, nor assemble a functional inflammasome in response to HSV-1. CD200R1(-/-) mice were protected from HSV-1 infection and exhibited dysfunctional TLR2 signaling. Finally, both CD200R1(-/-) mice and CD200R1(-/-) fibroblasts and macrophages showed a markedly reduced ability to support HSV-1 replication. In summary, our data demonstrate an unanticipated and novel requirement for CD200R1 in "licensing" pro-inflammatory functions of TLR2 and in limiting viral replication that are supported by ex vivo and in vivo evidence.


Asunto(s)
Antígenos de Superficie/metabolismo , Herpesvirus Humano 1/fisiología , Inflamación/inmunología , Receptores de Superficie Celular/metabolismo , Transducción de Señal/inmunología , Receptor Toll-Like 2/metabolismo , Replicación Viral/fisiología , Animales , Encéfalo/inmunología , Encéfalo/patología , Encéfalo/virología , Embrión de Mamíferos/citología , Encefalitis/inmunología , Encefalitis/patología , Encefalitis/virología , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/virología , Marcación de Gen , Inflamación/patología , Interferón Tipo I/biosíntesis , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/patología , Macrófagos Peritoneales/virología , Ratones , Receptores de Orexina , Receptores de Superficie Celular/deficiencia , Carga Viral/inmunología
12.
Mol Biol Cell ; 23(22): 4456-64, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23015755

RESUMEN

Leukotrienes (LTs) are lipid-signaling molecules derived from arachidonic acid (AA) that initiate and amplify inflammation. To initiate LT formation, the 5-lipoxygenase (5-LO) enzyme translocates to nuclear membranes, where it associates with its scaffold protein, 5-lipoxygenase-activating protein (FLAP), to form the core of the multiprotein LT synthetic complex. FLAP is considered to function by binding free AA and facilitating its use as a substrate by 5-LO to form the initial LT, LTA(4). We used a combination of fluorescence lifetime imaging microscopy, cell biology, and biochemistry to identify discrete AA-dependent and AA-independent steps that occur on nuclear membranes to control the assembly of the LT synthetic complex in polymorphonuclear leukocytes. The association of AA with FLAP changes the configuration of the scaffold protein, enhances recruitment of membrane-associated 5-LO to form complexes with FLAP, and controls the closeness of this association. Granulocyte monocyte colony-stimulating factor provides a second AA-independent signal that controls the closeness of 5-LO and FLAP within complexes but not the number of complexes that are assembled. Our results demonstrate that the LT synthetic complex is a signal integrator that transduces extracellular signals to modulate the interaction of 5-LO and FLAP.


Asunto(s)
Leucotrienos/metabolismo , Membrana Nuclear/metabolismo , Proteínas Activadoras de la 5-Lipooxigenasa/metabolismo , Animales , Araquidonato 5-Lipooxigenasa/metabolismo , Ácido Araquidónico/metabolismo , Leucotrienos/fisiología , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Transducción de Señal
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