RESUMEN

Oropouche fever, a debilitating illness common in South America, is caused by Oropouche virus (OROV), an arbovirus. OROV belongs to the Peribunyaviridae family, a large group of RNA viruses. Little is known about the biology of Peribunyaviridae in host cells, especially assembly and egress processes. Our research reveals that the small GTPase Rab27a mediates intracellular transport of OROV induced compartments and viral release from infected cells. We show that Rab27a interacts with OROV glycoproteins and colocalizes with OROV during late phases of the infection cycle. Moreover, Rab27a activity is required for OROV trafficking to the cell periphery and efficient release of infectious particles. Consistently, depleting Rab27a's downstream effector, Myosin Va, or inhibiting actin polymerization also hinders OROV compartments targeting to the cell periphery and infectious viral particle egress. These data indicate that OROV hijacks Rab27a activity for intracellular transport and cell externalization. Understanding these crucial mechanisms of OROV's replication cycle may offer potential targets for therapeutic interventions and aid in controlling the spread of Oropouche fever.

Asunto(s)

Cadenas Pesadas de Miosina , Miosina Tipo V , Liberación del Virus , Proteínas rab27 de Unión a GTP , Proteínas rab27 de Unión a GTP/metabolismo , Humanos , Liberación del Virus/fisiología , Miosina Tipo V/metabolismo , Miosina Tipo V/genética , Cadenas Pesadas de Miosina/metabolismo , Infecciones por Bunyaviridae/metabolismo , Infecciones por Bunyaviridae/virología , Orthobunyavirus/metabolismo , Orthobunyavirus/fisiología , Replicación Viral/fisiología , Animales , Interacciones Huésped-Patógeno

RESUMEN

[Figure: see text].

Asunto(s)

Angiopoyetina 2/metabolismo , Exocitosis , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Proteínas de la Membrana/metabolismo , Neovascularización Fisiológica , Cuerpos de Weibel-Palade/metabolismo , Proteínas de Pez Cebra/metabolismo , Angiopoyetina 2/genética , Animales , Animales Modificados Genéticamente , Células Cultivadas , Humanos , Proteínas de la Membrana/genética , Receptor TIE-2/metabolismo , Transducción de Señal , Cuerpos de Weibel-Palade/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas rab27 de Unión a GTP/metabolismo

RESUMEN

Sperm must undergo the regulated exocytosis of its dense core granule (the acrosome reaction, AR) to fertilize the egg. We have previously described that Rabs3 and 27 are organized in a RabGEF cascade within the signaling pathway elicited by exocytosis stimuli in human sperm. Here, we report the identity and the role of two molecules that link these secretory Rabs in the RabGEF cascade: Rabphilin3a and GRAB. Like Rab3 and Rab27, GRAB and Rabphilin3a are present, localize to the acrosomal region and are required for calcium-triggered exocytosis in human sperm. Sequestration of either protein with specific antibodies introduced into streptolysin O-permeabilized sperm impairs the activation of Rab3 in the acrosomal region elicited by calcium, but not that of Rab27. Biochemical and functional assays indicate that Rabphilin3a behaves as a Rab27 effector during the AR and that GRAB exhibits GEF activity toward Rab3A. Recombinant, active Rab27A pulls down Rabphilin3a and GRAB from human sperm extracts. Conversely, immobilized Rabphilin3a recruits Rab27 and GRAB; the latter promotes Rab3A activation. The enzymatic activity of GRAB toward Rab3A was also suggested by in silico and in vitro assays with purified proteins. In summary, we describe here a signaling module where Rab27A-GTP interacts with Rabphilin3a, which in turn recruits a guanine nucleotide-exchange activity toward Rab3A. This is the first description of the interaction of Rabphilin3a with a GEF. Because the machinery that drives exocytosis is highly conserved, it is tempting to hypothesize that the RabGEF cascade unveiled here might be part of the molecular mechanisms that drive exocytosis in other secretory systems.

Asunto(s)

Reacción Acrosómica , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Espermatozoides/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab27 de Unión a GTP/metabolismo , Proteína de Unión al GTP rab3A/metabolismo , Acrosoma/metabolismo , Exocitosis , Humanos , Masculino , Proteína de Unión al GTP rab3A/química , Rabfilina-3A

RESUMEN

The acrosome reaction is the regulated exocytosis of mammalian sperm's single secretory granule, essential for fertilization. It relies on small GTPases, the cAMP binding protein Epac, and the SNARE complex, among other components. Here, we describe a novel tool to investigate Rab27-related signaling pathways: a hybrid recombinant protein consisting of human Rab27A fused to TAT, a cell penetrating peptide. With this tool, we aimed to unravel the connection between Rab3, Rab27 and Rap1 in sperm exocytosis and to deepen our understanding about how isoprenylation and guanine nucleotides influence the behaviour of Rab27 in exocytosis. Our results show that TAT-Rab27A-GTP-γ-S permeated into live sperm and triggered acrosomal exocytosis per se when geraylgeranylated but inhibited it when not lipid-modified. Likewise, an impermeant version of Rab27A elicited exocytosis in streptolysin O-permeabilized - but not in non-permeabilized - cells when geranylgeranylated and active. When GDP-ß-S substituted for GTP-γ-S, isoprenylated TAT-Rab27A inhibited the acrosome reaction triggered by progesterone and an Epac-selective cAMP analogue, whereas the non-isoprenylated protein did not. Geranylgeranylated TAT-Rab27A-GTP-γ-S promoted the exchange of GDP for GTP on Rab3 and Rap1 detected by far-immunofluorescence with Rab3-GTP and Rap1-GTP binding cassettes. In contrast, TAT-Rab27A lacking isoprenylation or loaded with GDP-ß-S prevented the activation of Rab3 and Rap1 elicited by progesterone. Challenging streptolysin O-permeabilized human sperm with calcium increased the population of sperm with Rap1-GTP, Rab3-GTP and Rab27-GTP in the acrosomal region; pretreatment with anti-Rab27 antibodies prevented the activation of all three. The novel findings reported here include: the description of membrane permeant TAT-Rab27A as a trustworthy tool to unveil the regulation of the human sperm acrosome reaction by Rab27 under physiological conditions; that the activation of endogenous Rab27 is required for that of Rab3 and Rap1; and the connection between Epac and Rab27 and between Rab27 and the configuration of the SNARE complex. Moreover, we present direct evidence that Rab27A's lipid modification, and activation/inactivation status correlate with its stimulatory or inhibitory roles in exocytosis.

Asunto(s)

Reacción Acrosómica , Exocitosis , Nucleótidos de Guanina/metabolismo , Prenilación , Proteínas de Unión a Telómeros/metabolismo , Proteínas rab27 de Unión a GTP/metabolismo , Proteínas de Unión al GTP rab3/metabolismo , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Péptidos de Penetración Celular/genética , Péptidos de Penetración Celular/metabolismo , Factores de Intercambio de Guanina Nucleótido , Humanos , Masculino , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Prenilación de Proteína , Proteínas Recombinantes/genética , Proteínas SNARE/metabolismo , Complejo Shelterina , Transducción de Señal , Proteínas rab27 de Unión a GTP/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo

RESUMEN

The role and regulators of extracellular vesicle (EV) secretion in hepatic ischemia/reperfusion (IR) injury have not been defined. Rab27a is a guanosine triphosphatase known to control EV release. Interferon regulatory factor 1 (IRF-1) is a transcription factor that plays an important role in liver IR and regulates certain guanosine triphosphatases. However, the relationships among IRF-1, Rab27a, and EV secretion are largely unknown. Here, we show induction of IRF-1 and Rab27a both in vitro in hypoxic hepatocytes and in vivo in warm IR and orthotopic liver transplantation livers. Interferon γ stimulation, IRF-1 transduction, or IR promoted Rab27a expression and EV secretion. Meanwhile, silencing of IRF-1 decreased Rab27a expression and EV secretion. Rab27a silencing decreased EV secretion and liver IR injury. Ten putative IRF-1 binding motifs in the 1,692-bp Rab27a promoter region were identified. Chromatin immunoprecipitation and electrophoretic mobility shift assay verified five functional IRF-1 binding motifs, which were confirmed by a Rab27a promoter luciferase assay. IR-induced EVs contained higher oxidized phospholipids (OxPL). OxPLs on the EV surface activated neutrophils through the toll-like receptor 4 pathway. OxPL-neutralizing E06 antibody blocked the effect of EVs and decreased liver IR injury. CONCLUSION: These findings provide a novel mechanism by which IRF-1 regulates Rab27a transcription and EV secretion, leading to OxPL activation of neutrophils and subsequent hepatic IR injury. (Hepatology 2018;67:1056-1070).

Asunto(s)

Vesículas Extracelulares/metabolismo , Factor 1 Regulador del Interferón/metabolismo , Hígado/patología , Daño por Reperfusión/metabolismo , Proteínas rab27 de Unión a GTP/metabolismo , Animales , Técnicas de Cultivo de Célula , Regulación de la Expresión Génica , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Trasplante de Hígado , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados

RESUMEN

Exocytosis is a fundamental process used by eukaryotic cells to release biological compounds and to insert lipids and proteins in the plasma membrane. Specialized secretory cells undergo regulated exocytosis in response to physiological signals. Sperm exocytosis or acrosome reaction (AR) is essentially a regulated secretion with special characteristics. We will focus here on some of these unique features, covering the topology, kinetics, and molecular mechanisms that prepare, drive, and regulate membrane fusion during the AR. Last, we will compare acrosomal release with exocytosis in other model systems.

Asunto(s)

Reacción Acrosómica/fisiología , Acrosoma/metabolismo , Membrana Celular/metabolismo , Exocitosis/fisiología , Acrosoma/química , Animales , Calcio/metabolismo , Membrana Celular/química , Regulación de la Expresión Génica , Cinética , Masculino , Fusión de Membrana/fisiología , Ratones , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Transducción de Señal , Sinaptotagminas/genética , Sinaptotagminas/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas rab27 de Unión a GTP , Proteínas de Unión al GTP rab3/genética , Proteínas de Unión al GTP rab3/metabolismo

RESUMEN

During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55(Gag) is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4(+) T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P2, therefore controlling Pr55(Gag) membrane association. Rab27a also controls PI(4,5)P2 levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55(Gag) with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P2 production and, consequently, HIV-1 replication.

Asunto(s)

Membrana Celular/metabolismo , VIH-1/fisiología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Ensamble de Virus/fisiología , Replicación Viral/fisiología , Proteínas de Unión al GTP rab/metabolismo , Transporte Biológico Activo/genética , Membrana Celular/genética , Membrana Celular/virología , Endosomas/genética , Endosomas/metabolismo , Endosomas/virología , Humanos , Células Jurkat , Macrófagos/metabolismo , Macrófagos/virología , Proteínas de la Membrana/metabolismo , Antígenos de Histocompatibilidad Menor , Fosfatidilinositol 4,5-Difosfato/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas rab27 de Unión a GTP

RESUMEN

Two so-called "secretory Rabs," Rab3 and Rab27, regulate late steps during dense-core vesicle exocytosis in neuroendocrine cells. Sperm contain a single large dense-core granule that is released by regulated exocytosis (termed the acrosome reaction) during fertilization or on exposure to inducers in vitro. Sperm exocytosis uses the same fusion machinery as neurons and neuroendocrine cells, with an additional requirement for active Rab3. Here we show that Rab27 is also required for the acrosome reaction, as demonstrated by the inability of inducers to elicit exocytosis when streptolysin O-permeabilized human sperm were loaded with inhibitory anti-Rab27 antibodies or the Rab27-GTP binding domain of the effector Slac2-b. The levels of GTP-bound Rab27 increased on initiation of exocytosis, as did the proportion of GTP-bound Rab3A. We have developed a fluorescence microscopy-based method for detecting endogenous Rab3A-GTP and Rab27-GTP in the acrosomal region of human sperm. Challenge with an inducer increased the population of cells exhibiting GTP-bound Rabs in this subcellular domain. Interestingly, introducing recombinant Rab27A loaded with GTP-γ-S into sperm elicited a remarkable increase in the number of cells evincing GTP-bound Rab3A. In the converse condition, recombinant Rab3A did not modify the percentage of Rab27-GTP-containing cells. Furthermore, Rab27A-GTP recruited a Rab3 GDP/GTP exchange factor (GEF) activity. Our findings suggest that Rab27/Rab3A constitutes a Rab-GEF cascade in dense-core vesicle exocytosis.

Asunto(s)

Reacción Acrosómica/fisiología , Acrosoma/fisiología , Exocitosis/fisiología , Vesículas Secretoras/fisiología , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab3/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Bacterianas , Western Blotting , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente Indirecta , Glutatión Transferasa , Guanosina Trifosfato/metabolismo , Humanos , Masculino , Microscopía Fluorescente , Prenilación , Proteínas Recombinantes/metabolismo , Vesículas Secretoras/metabolismo , Sefarosa , Estreptolisinas , Proteínas rab27 de Unión a GTP

RESUMEN

Griscelli syndrome (GS) is a rare autosomal recessive disorder caused by mutation in the MYO5A (GS1, Elejalde), RAB27A (GS2) or MLPH (GS3) genes. Typical features of all three subtypes of this disease include pigmentary dilution of the hair and skin and silvery-gray hair. Whereas the GS3 phenotype is restricted to the pigmentation dysfunction, GS1 patients also show primary neurological impairment and GS2 patients have severe immunological deficiencies that lead to recurrent infections and hemophagocytic syndrome. We report here the diagnosis of GS2 in 3-year-old twin siblings, with silvery-gray hair, immunodeficiency, hepatosplenomegaly and secondary severe neurological symptoms that culminated in multiple organ failure and death. Light microscopy examination of the hair showed large, irregular clumps of pigments characteristic of GS. A homozygous nonsense mutation, C-T transition (c.550C>T), in the coding region of the RAB27A gene, which leads to a premature stop codon and prediction of a truncated protein (R184X), was found. In patient mononuclear cells, RAB27A mRNA levels were the same as in cells from the parents, but no protein was detected. In addition to the case report, we also present an updated summary on the exon/intron organization of the human RAB27A gene, a literature review of GS2 cases, and a complete list of the human mutations currently reported in this gene. Finally, we propose a flow chart to guide the early diagnosis of the GS subtypes and Chédiak-Higashi syndrome.

Asunto(s)

Enfermedades en Gemelos/genética , Color del Cabello/genética , Linfohistiocitosis Hemofagocítica/genética , Mutación/genética , Trastornos de la Pigmentación/genética , Proteínas de Unión al GTP rab/genética , Preescolar , Enfermedades en Gemelos/diagnóstico , Resultado Fatal , Humanos , Linfohistiocitosis Hemofagocítica/diagnóstico , Masculino , Trastornos de la Pigmentación/diagnóstico , Síndrome , Proteínas rab27 de Unión a GTP

RESUMEN

Griscelli syndrome (GS) is caused by mutations in the MYO5A (GS1), RAB27A (GS2), or MLPH (GS3) genes, all of which lead to a similar pigmentary dilution. In addition, GS1 patients show primary neurological impairment, whereas GS2 patients present immunodeficiency and periods of lymphocyte proliferation and activation, leading to their infiltration in many organs, such as the nervous system, causing secondary neurological damage. We report the diagnosis of GS2 in a 4-year-old child with haemophagocytic syndrome, immunodeficiency, and secondary neurological disorders. Typical melanosome accumulation was found in skin melanocytes and pigment clumps were observed in hair shafts. Two heterozygous mutant alleles of the RAB27A gene were found, a C-T transition (C352T) that leads to Q118stop and a G-C transversion on the exon 5 splicing donor site (G467+1C). Functional assays showed increased cellular activation and decreased cytotoxic activity of NK and CD8+ T cells, associated with defective lytic granules release. Myosin-Va expression and localization in the patient lymphocytes were also analyzed. Most importantly, we show that cytotoxic activity of the patient's CD8+ T lymphocytes can be rescued in vitro by RAB27A gene transfer mediated by a recombinant retroviral vector, a first step towards a potential treatment of the acute phase of GS2 by RAB27A transduced lymphocytes.

Asunto(s)

Terapia Genética/métodos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/terapia , Mutación , Linfocitos T Citotóxicos/efectos de los fármacos , Proteínas de Unión al GTP rab/administración & dosificación , Linfocitos T CD8-positivos/inmunología , Preescolar , Análisis Mutacional de ADN , Humanos , Síndromes de Inmunodeficiencia/diagnóstico , Células Asesinas Naturales/inmunología , Masculino , Enfermedades del Sistema Nervioso , Linaje , Piebaldismo/genética , Piebaldismo/terapia , Retroviridae/genética , Linfocitos T Citotóxicos/patología , Resultado del Tratamiento , Proteínas de Unión al GTP rab/genética , Proteínas rab27 de Unión a GTP