RESUMEN
The amyloid precursor protein (APP) is anterogradely transported by conventional kinesin in a distinct transport vesicle, but both the biochemical composition of such a vesicle and the specific kinesin-1 motor responsible for transport are poorly defined. APP may be sequentially cleaved by beta- and gamma-secretases leading to accumulation of beta-amyloid (Abeta) peptides in brains of Alzheimer's disease patients, whereas cleavage of APP by alpha-secretases prevents Abeta generation. Here, we demonstrate by time-lapse analysis and immunoisolations that APP is a cargo of a vesicle containing the kinesin heavy chain isoform kinesin-1C, the small GTPase Rab3A, and a specific subset of presynaptic protein components. Moreover, we report that assembly of kinesin-1C and APP in this vesicle type requires Rab3A GTPase activity. Finally, we show cleavage of APP in transport vesicles by alpha-secretase activity, likely mediated by ADAM10. Together, these data indicate that maturation of APP transport vesicles, including recruitment of conventional kinesin, requires Rab3 GTPase activity.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Vesículas Transportadoras/metabolismo , Proteína de Unión al GTP rab3A/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Animales , Secuencia de Bases , Línea Celular Tumoral , Activación Enzimática/genética , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Cinesinas/química , Cinesinas/metabolismo , Cinesinas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Transporte de Proteínas/fisiología , Vesículas Transportadoras/química , Vesículas Transportadoras/genética , Proteína de Unión al GTP rab3A/química , Proteína de Unión al GTP rab3A/genéticaRESUMEN
The cellular uptake of vitamin A from its RBP4-bound circulating form (holo-RBP4) is a homeostatic process that evidently depends on the multidomain membrane protein STRA6. In humans, mutations in STRA6 are associated with Matthew-Wood syndrome, manifested by multisystem developmental malformations. Here we addressed the metabolic basis of this inherited disease. STRA6-dependent transfer of retinol from RBP4 into cultured NIH 3T3 fibroblasts was enhanced by lecithin:retinol acyltransferase (LRAT). The retinol transfer was bidirectional, strongly suggesting that STRA6 acts as a retinol channel/transporter. Loss-of-function analysis in zebrafish embryos revealed that Stra6 deficiency caused vitamin A deprivation of the developing eyes. We provide evidence that, in the absence of Stra6, holo-Rbp4 provokes nonspecific vitamin A excess in several embryonic tissues, impairing retinoic acid receptor signaling and gene regulation. These fatal consequences of Stra6 deficiency, including craniofacial and cardiac defects and microphthalmia, were largely alleviated by reducing embryonic Rbp4 levels by morpholino oligonucleotide or pharmacological treatments.
Asunto(s)
Anomalías Múltiples/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas Plasmáticas de Unión al Retinol/metabolismo , Vitamina A/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Anomalías Múltiples/genética , Aciltransferasas/metabolismo , Animales , Anomalías Cardiovasculares/embriología , Anomalías Cardiovasculares/metabolismo , Anomalías Craneofaciales/embriología , Anomalías Craneofaciales/metabolismo , Modelos Animales de Enfermedad , Ojo/embriología , Ojo/enzimología , Ojo/metabolismo , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Homeostasis , Humanos , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Ratones , Morfolinas/metabolismo , Células 3T3 NIH , Oligonucleótidos Antisentido/metabolismo , Proteínas Plasmáticas de Unión al Retinol/genética , Síndrome , Factores de Tiempo , Transducción Genética , Tretinoina/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
In vertebrate retinal photoreceptors, the absorption of light by rhodopsin leads to photoisomerization of 11-cis-retinal to its all-trans isomer. To sustain vision, a metabolic system evolved that recycles all-trans-retinal back to 11-cis-retinal. The importance of this visual (retinoid) cycle is underscored by the fact that mutations in genes encoding visual cycle components induce a wide spectrum of diseases characterized by abnormal levels of specific retinoid cycle intermediates. In addition, intense illumination can produce retinoid cycle by-products that are toxic to the retina. Thus, inhibition of the retinoid cycle has therapeutic potential in physiological and pathological states. Four classes of inhibitors that include retinoid and nonretinoid compounds have been identified. We investigated the modes of action of these inhibitors by using purified visual cycle components and in vivo systems. We report that retinylamine was the most potent and specific inhibitor of the retinoid cycle among the tested compounds and that it targets the retinoid isomerase, RPE65. Hydrophobic primary amines like farnesylamine also showed inhibitory potency but a short duration of action, probably due to rapid metabolism. These compounds also are reactive nucleophiles with potentially high cellular toxicity. We also evaluated the role of a specific protein-mediated mechanism on retinoid cycle inhibitor uptake by the eye. Our results show that retinylamine is transported to and taken up by the eye by retinol-binding protein-independent and retinoic acid-responsive gene product 6-independent mechanisms. Finally, we provide evidence for a crucial role of lecithin: retinol acyltransferase activity in mediating tissue specific absorption and long lasting therapeutic effects of retinoid-based visual cycle inhibitors.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Diterpenos/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas del Ojo/antagonistas & inhibidores , Farnesol/análogos & derivados , Células Fotorreceptoras de Vertebrados/enzimología , Retinaldehído/metabolismo , Visión Ocular/efectos de los fármacos , cis-trans-Isomerasas/antagonistas & inhibidores , Absorción , Animales , Transporte Biológico/efectos de los fármacos , Proteínas Portadoras/metabolismo , Proteínas del Ojo/metabolismo , Farnesol/farmacología , Ratones , Ratones Noqueados , Células 3T3 NIH , Especificidad de Órganos/efectos de los fármacos , Rodopsina/metabolismo , Trastornos de la Visión/tratamiento farmacológico , Trastornos de la Visión/enzimología , cis-trans-Isomerasas/metabolismoRESUMEN
Carotenoids are currently investigated regarding their potential to lower the risk of chronic disease and to combat vitamin A deficiency in humans. These plant-derived compounds must be cleaved and metabolically converted by intrinsic carotenoid oxygenases to support the panoply of vitamin A-dependent physiological processes. Two different carotenoid-cleaving enzymes were identified in mammals, the classical carotenoid-15,15'-oxygenase (CMO1) and a putative carotenoid-9',10'-oxygenase (CMO2). To analyze the role of CMO1 in mammalian physiology, here we disrupted the corresponding gene by targeted homologous recombination in mice. On a diet providing beta-carotene as major vitamin A precursor, vitamin A levels fell dramatically in several tissues examined. Instead, this mouse mutant accumulated the provitamin in large quantities (e.g. as seen by an orange coloring of adipose tissues). Besides impairments in beta-carotene metabolism, CMO1 deficiency more generally interfered with lipid homeostasis. Even on a vitamin A-sufficient chow, CMO1(-/-) mice developed a fatty liver and displayed altered serum lipid levels with elevated serum unesterified fatty acids. Additionally, this mouse mutant was more susceptible to high fat diet-induced impairments in fatty acid metabolism. Quantitative reverse transcription-PCR analysis revealed that the expression of peroxisome proliferator-activated receptor gamma-regulated marker genes related to adipogenesis was elevated in visceral adipose tissues. Thus, our study identifies CMO1 as the key enzyme for vitamin A production and provides evidence for a role of carotenoids as more general regulators of lipid metabolism.