RESUMEN
Positive-strand RNA viruses such as hepatitis C virus (HCV) hijack key factors of lipid metabolism of infected cells and extensively modify intracellular membranes to support the viral lifecycle. While lipid metabolism plays key roles in viral particle assembly and maturation, viral RNA synthesis is closely linked to the remodeling of intracellular membranes. The formation of viral replication factories requires a number of interactions between virus proteins and host factors including lipids. The structure-function relationship of those proteins is influenced by their lipid environments and lipids that selectively modulate protein function. Here, we review our current understanding on the roles of phospholipids in HCV replication and of lipid-protein interactions in the structure-function relationship of the NS5A protein. NS5A is a key factor in membrane remodeling in HCV-infected cells and is known to recruit phosphatidylinositol 4-kinase III alpha to generate phosphatidylinositol 4-phosphate at the sites of replication. The dynamic interplay between lipids and viral proteins within intracellular membranes is likely key towards understanding basic mechanisms in the pathobiology of virus diseases, the mode of action of specific antiviral agents and related drug resistance mechanisms.
RESUMEN
Ferlins are multiple-C2-domain proteins involved in Ca2+-triggered membrane dynamics within the secretory, endocytic and lysosomal pathways. In bony vertebrates there are six ferlin genes encoding, in humans, dysferlin, otoferlin, myoferlin, Fer1L5 and 6 and the long noncoding RNA Fer1L4. Mutations in DYSF (dysferlin) can cause a range of muscle diseases with various clinical manifestations collectively known as dysferlinopathies, including limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. A mutation in MYOF (myoferlin) was linked to a muscular dystrophy accompanied by cardiomyopathy. Mutations in OTOF (otoferlin) can be the cause of nonsyndromic deafness DFNB9. Dysregulated expression of any human ferlin may be associated with development of cancer. This review provides a detailed description of functions of the vertebrate ferlins with a focus on muscle ferlins and discusses the mechanisms leading to disease development.
Asunto(s)
Distrofia Muscular de Cinturas/metabolismo , Vertebrados/metabolismo , Animales , HumanosRESUMEN
Cochlear inner hair cells (IHCs) use Ca(2+)-dependent exocytosis of glutamate to signal sound information. Otoferlin (Otof), a C(2) domain protein essential for IHC exocytosis and hearing, may serve as a Ca(2+) sensor in vesicle fusion in IHCs that seem to lack the classical neuronal Ca(2+) sensors synaptotagmin 1 (Syt1) and Syt2. Support for the Ca(2+) sensor of fusion hypothesis for otoferlin function comes from biochemical experiments, but additional roles in late exocytosis upstream of fusion have been indicated by physiological studies. Here, we tested the functional equivalence of otoferlin and Syt1 in three neurosecretory model systems: auditory IHCs, adrenal chromaffin cells, and hippocampal neurons. Long-term and short-term ectopic expression of Syt1 in IHCs of Otof (-/-) mice by viral gene transfer in the embryonic inner ear and organotypic culture failed to rescue their Ca(2+) influx-triggered exocytosis. Conversely, virally mediated overexpression of otoferlin did not restore phasic exocytosis in Syt1-deficient chromaffin cells or neurons but enhanced asynchronous release in the latter. We further tested exocytosis in Otof (-/-) hippocampal neurons and in Syt1(-/-) IHCs but found no deficits in vesicle fusion. Expression analysis of different synaptotagmin isoforms indicated that Syt1 and Syt2 are absent from mature IHCs. Our data argue against a simple functional equivalence of the two C(2) domain proteins in exocytosis of IHC ribbon synapses, chromaffin cells, and hippocampal synapses.
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Exocitosis/fisiología , Proteínas de la Membrana/fisiología , Sinaptotagmina I/fisiología , Estimulación Acústica/métodos , Animales , Animales Recién Nacidos , Potenciales Evocados Auditivos del Tronco Encefálico/genética , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Exocitosis/genética , Hipocampo/citología , Hipocampo/fisiología , Fusión de Membrana/genética , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Inhibición Neural/genética , Neuronas/metabolismo , Técnicas de Cultivo de Órganos , Sinapsis/genética , Sinapsis/fisiología , Sinaptotagmina I/deficiencia , Sinaptotagmina I/genéticaRESUMEN
SNARE proteins mediate membrane fusion. Neurosecretion depends on neuronal soluble NSF attachment protein receptors (SNAREs; SNAP-25, syntaxin-1, and synaptobrevin-1 or synaptobrevin-2) and is blocked by neurotoxin-mediated cleavage or genetic ablation. We found that exocytosis in mouse inner hair cells (IHCs) was insensitive to neurotoxins and genetic ablation of neuronal SNAREs. mRNA, but no synaptically localized protein, of neuronal SNAREs was present in IHCs. Thus, IHC exocytosis is unconventional and may operate independently of neuronal SNAREs.
Asunto(s)
Exocitosis/fisiología , Células Ciliadas Auditivas Internas/metabolismo , Proteínas SNARE/genética , Sinapsis/metabolismo , Animales , Bovinos , Células Cultivadas , Exocitosis/efectos de los fármacos , Células Ciliadas Auditivas Internas/efectos de los fármacos , Células Ciliadas Auditivas Internas/ultraestructura , Ratones , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Proteínas SNARE/deficiencia , Sinapsis/efectos de los fármacos , Sinapsis/ultraestructuraRESUMEN
Lipid droplets are intracellular organelles enriched in adipose tissue that govern the body fat stores of animals. In mammals, members of the evolutionarily conserved PERILIPIN protein family are associated with the lipid droplet surface and participate in lipid homeostasis. Here, we show that Drosophila mutants lacking the PERILIPIN PLIN1 are hyperphagic and suffer from adult-onset obesity. PLIN1 is a central and Janus-faced component of fat metabolism. It provides barrier function to storage lipid breakdown and acts as a key factor of stimulated lipolysis by modulating the access of proteins to the lipid droplet surface. It also shapes lipid droplet structure, transforming unilocular into multilocular fat cells. We generated flies devoid of all PERILIPIN family members and show that they exhibit impaired yet functional body fat regulation. Our data reveal the existence of a basal and possibly ancient lipid homeostasis system.
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Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Grasas/metabolismo , Metabolismo de los Lípidos , Animales , Drosophila/genética , Proteínas de Drosophila/genética , Mutación , Obesidad/genética , Obesidad/metabolismoRESUMEN
Hearing relies on Ca(2+) influx-triggered exocytosis in cochlear inner hair cells (IHCs). Here we studied the role of the Ca(2+) channel subunit Ca(V)beta(2) in hearing. Of the Ca(V)beta(1-4) mRNAs, IHCs predominantly contained Ca(V)beta(2). Hearing was severely impaired in mice lacking Ca(V)beta(2) in extracardiac tissues (Ca(V)beta(2)(-/-)). This involved deficits in cochlear amplification and sound encoding. Otoacoustic emissions were reduced or absent in Ca(V)beta(2)(-/-) mice, which showed strongly elevated auditory thresholds in single neuron recordings and auditory brainstem response measurements. Ca(V)beta(2)(-/-) IHCs showed greatly reduced exocytosis (by 68%). This was mostly attributable to a decreased number of membrane-standing Ca(V)1.3 channels. Confocal Ca(2+) imaging revealed presynaptic Ca(2+) microdomains albeit with much lower amplitudes, indicating synaptic clustering of fewer Ca(V)1.3 channels. The coupling of the remaining Ca(2+) influx to IHC exocytosis appeared unaffected. Extracellular recordings of sound-evoked spiking in the cochlear nucleus and auditory nerve revealed reduced spike rates in the Ca(V)beta(2)(-/-) mice. Still, sizable onset and adapted spike rates were found during suprathreshold stimulation in Ca(V)beta(2)(-/-) mice. This indicated that residual synaptic sound encoding occurred, although the number of presynaptic Ca(V)1.3 channels and exocytosis were reduced to one-third. The normal developmental upregulation, clustering, and gating of large-conductance Ca(2+) activated potassium channels in IHCs were impaired in the absence of Ca(V)beta(2). Moreover, we found the developmental efferent innervation to persist in Ca(V)beta(2)-deficient IHCs. In summary, Ca(V)beta(2) has an essential role in regulating the abundance and properties of Ca(V)1.3 channels in IHCs and, thereby, is critical for IHC development and synaptic encoding of sound.
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Canales de Calcio Tipo L/fisiología , Células Ciliadas Auditivas Internas/fisiología , Estimulación Acústica/métodos , Factores de Edad , Animales , Animales Recién Nacidos , Apamina/farmacología , Calcio/metabolismo , Canales de Calcio Tipo L/deficiencia , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/genética , Cesio/farmacología , Quelantes/farmacología , Cloruros/farmacología , Ácido Egtácico/farmacología , Estimulación Eléctrica/métodos , Potenciales Evocados Auditivos del Tronco Encefálico/genética , Exocitosis/efectos de los fármacos , Células Ciliadas Auditivas Internas/efectos de los fármacos , Técnicas In Vitro , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Emisiones Otoacústicas Espontáneas/genética , Parvalbúminas/metabolismo , Técnicas de Placa-Clamp/métodosRESUMEN
Complexins (CPXs I-IV) presumably act as regulators of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, but their function in the intact mammalian nervous system is not well established. Here, we explored the role of CPXs in the mouse auditory system. Hearing was impaired in CPX I knock-out mice but normal in knock-out mice for CPXs II, III, IV, and III/IV as measured by auditory brainstem responses. Complexins were not detectable in cochlear hair cells but CPX I was expressed in spiral ganglion neurons (SGNs) that give rise to the auditory nerve. Ca(2+)-dependent exocytosis of inner hair cells and sound encoding by SGNs were unaffected in CPX I knock-out mice. In the absence of CPX I, the resting release probability in the endbulb of Held synapses of the auditory nerve fibers with bushy cells in the cochlear nucleus was reduced. As predicted by computational modeling, bushy cells had decreased spike rates at sound onset as well as longer and more variable first spike latencies explaining the abnormal auditory brainstem responses. In addition, we found synaptic transmission to outlast the stimulus at many endbulb of Held synapses in vitro and in vivo, suggesting impaired synchronization of release to stimulus offset. Although sound encoding in the cochlea proceeds in the absence of complexins, CPX I is required for faithful processing of sound onset and offset in the cochlear nucleus.
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Vías Auditivas/fisiología , Núcleo Coclear/metabolismo , Audición/fisiología , Proteínas del Tejido Nervioso/deficiencia , Neuronas/metabolismo , Transmisión Sináptica/fisiología , Proteínas Adaptadoras del Transporte Vesicular , Animales , Nervio Coclear/fisiología , Células Ciliadas Auditivas Internas/metabolismo , Inmunohistoquímica , Ratones , Ratones Noqueados , Microscopía Confocal , Proteínas del Tejido Nervioso/genética , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sinapsis/metabolismoRESUMEN
TIP47 (tail-interacting protein of 47 kD) was characterized as a cargo selection device for mannose 6-phosphate receptors (MPRs), directing their transport from endosomes to the trans-Golgi network. In contrast, our current analysis shows that cytosolic TIP47 is not recruited to organelles of the biosynthetic and endocytic pathways. Knockdown of TIP47 expression had no effect on MPR distribution or trafficking and did not affect lysosomal enzyme sorting. Therefore, our data argue against a function of TIP47 as a sorting device. Instead, TIP47 is recruited to lipid droplets (LDs) by an amino-terminal sequence comprising 11-mer repeats. We show that TIP47 has apolipoprotein-like properties and reorganizes liposomes into small lipid discs. Suppression of TIP47 blocked LD maturation and decreased the incorporation of triacylglycerol into LDs. We conclude that TIP47 functions in the biogenesis of LDs.
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Proteínas de Unión al ADN/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Metabolismo de los Lípidos , Lípidos , Proteínas Gestacionales/fisiología , Secuencia de Aminoácidos , Apolipoproteínas , Línea Celular , Proteínas de Unión al ADN/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Liposomas/metabolismo , Perilipina-3 , Proteínas Gestacionales/metabolismo , Transporte de Proteínas , Receptor IGF Tipo 2 , Triglicéridos , Proteínas de Transporte VesicularRESUMEN
The protein-tyrosine phosphatase PTPMEG2 is located on the cytoplasmic face of the enclosing membrane of secretory vesicles, where it regulates vesicle size by promoting homotypic vesicle fusion by dephosphorylating N-ethylmaleimide-sensitive factor, a key regulator of vesicle fusion. Here we address the question of how PTPMEG2 is targeted to this subcellular location. Using a series of deletion mutants, we pinpointed the N-terminal Sec14p homology (SEC14) domain of PTPMEG2, residues 1-261, as the region containing the secretory vesicle targeting signal. This domain, alone or appended to a heterologous protein, was localized to intracellular vesicle membranes. Yeast two-hybrid screening identified a number of secretory vesicle proteins that interacted directly with the SEC14 domain of PTPMEG2, providing a mechanism for PTPMEG2 targeting to secretory vesicles. Two such proteins, mannose 6-phosphate receptor-interacting protein TIP47 and Arfaptin2, were found to alter PTPMEG2 localization when overexpressed, and elimination of TIP47 resulted in loss of PTPMEG2 function. We conclude that the N terminus of PTPMEG2 is necessary for the targeting of this phosphatase to the secretory vesicle compartment by association with other proteins involved in intracellular transport.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Gestacionales/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas R-SNARE/metabolismo , Vesículas Secretoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Transporte Biológico Activo/fisiología , Proteínas Portadoras/genética , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Perilipina-3 , Proteínas Gestacionales/genética , Estructura Terciaria de Proteína/genética , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas no Receptoras , Proteínas R-SNARE/genética , Vesículas Secretoras/genética , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas de Transporte VesicularRESUMEN
Ribbon synapses of inner hair cells (IHCs) undergo developmental maturation until after the onset of hearing. Here, we studied whether IHC synaptogenesis is regulated by thyroid hormone (TH). We performed perforated patch-clamp recordings of Ca2+ currents and exocytic membrane capacitance changes in IHCs of athyroid and TH-substituted Pax8-/- mice during postnatal development. Ca2+ currents remained elevated in athyroid IHCs at the end of the second postnatal week, when it had developmentally declined in wild-type and TH-rescued mutant IHCs. The efficiency of Ca2+ influx in triggering exocytosis of the readily releasable vesicle pool was reduced in athyroid IHCs. Ribbon synapses were formed despite the TH deficiency. However, different from wild type, in which synapse elimination takes place at approximately the onset of hearing, the number of ribbon synapses remained elevated in 2-week-old athyroid IHCs. Moreover, the ultrastructure of these synapses appeared immature. Using quantitative reverse transcription-PCR, we found a TH-dependent developmental upregulation of the mRNAs for the neuronal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, SNAP25 (synaptosomal-associated protein of 25 kDa) and synaptobrevin 1, in the organ of Corti. These molecular changes probably contribute to the improvement of exocytosis efficiency in mature IHCs. IHCs of 2-week-old athyroid Pax8-/- mice maintained the normally temporary efferent innervation. Moreover, they lacked large-conductance Ca2+-activated K+ channels and KCNQ4 channels. This together with the persistently increased Ca2+ influx permitted continued action potential generation. We conclude that TH regulates IHC differentiation and is essential for morphological and functional maturation of their ribbon synapses. We suggest that presynaptic dysfunction of IHCs is a mechanism in congenital hypothyroid deafness.
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Células Ciliadas Auditivas Internas/crecimiento & desarrollo , Células Ciliadas Auditivas Internas/metabolismo , Sinapsis/fisiología , Hormonas Tiroideas/fisiología , Animales , Femenino , Células Ciliadas Auditivas Internas/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor de Transcripción PAX8 , Factores de Transcripción Paired Box/deficiencia , Factores de Transcripción Paired Box/genética , Hormonas Tiroideas/genéticaRESUMEN
Cysteine string protein (CSP) alpha is an abundant synaptic vesicle protein that contains a DNA-J domain characteristic of Hsp40-type cochaperones. Previous studies showed that deletion of CSPalpha in mice leads to massive lethal neurodegeneration but did not clarify how the neurodegeneration affects specific subpopulations of neurons. Here, we analyzed the effects of the CSPalpha deficiency on tonically active ribbon synapses of the retina and the inner ear. We show that CSPalpha-deficient photoreceptor terminals undergo dramatic and rapidly progressive neurodegeneration that starts before eye opening and initially does not affect other retinal synapses. These changes are associated with progressive blindness. In contrast, ribbon synapses of auditory hair cells did not exhibit presynaptic impairments in CSPalpha-deficient mice. Hair cells, but not photoreceptor cells or central neurons, express CSPbeta, thereby accounting for the lack of a hair-cell phenotype in CSPalpha knockout mice. Our data demonstrate that tonically active ribbon synapses in retina are particularly sensitive to the deletion of CSPalpha and that expression of at least one CSP isoform is essential to protect such tonically active synapses from neurodegeneration.