RESUMEN
MiR-204-5p, as a tumour suppressor, has been found in several cancers. However, whether miR-204-5p is involved in papillary thyroid carcinoma (PTC) has not yet been investigated. In this study, we identified miR-204-5p as a down-regulated miRNA in PTC tissues, unveiling that the levels of miR-204-5p in serum of patients with PTC were linked to PTC risk, and that the expression in patients concomitant with both PTC and benign lesions was much lower than that in patients only with PTC. Furthermore, we documented that miR-204-5p inhibited proliferation, migration, invasion, and cell cycle progression and triggered apoptosis of PTC cells via cell biology experiments. Finally, we identified that AP1S2 was a target of miR-204-5p using RNA-seq, iTRAQ, and bioinformatics prediction. Overall, miR-204-5p functions as a suppressor for PTC pathogenesis via the miR-204-5p/AP1S2 axis.
Asunto(s)
Subunidades sigma de Complejo de Proteína Adaptadora , MicroARNs , Neoplasias de la Tiroides , Humanos , Cáncer Papilar Tiroideo/patología , Neoplasias de la Tiroides/patología , Línea Celular Tumoral , Proliferación Celular/genética , MicroARNs/genética , MicroARNs/metabolismo , Regulación Neoplásica de la Expresión Génica , Subunidades sigma de Complejo de Proteína Adaptadora/genética , Subunidades sigma de Complejo de Proteína Adaptadora/metabolismoRESUMEN
AP2S1 is the sigma 2 subunit of adaptor protein 2 (AP2) that is essential for endocytosis. In this study, we investigated the potential role of AP2S1 in intracellular processing of amyloid precursor protein (APP), which contributes to the pathogenesis of Alzheimer disease (AD) by generating the toxic ß-amyloid peptide (Aß). We found that knockdown or overexpression of AP2S1 decreased or increased the protein levels of APP and Aß in cells stably expressing human full-length APP695, respectively. This effect was unrelated to endocytosis but involved lysosomal degradation. Morphological studies revealed that silencing of AP2S1 promoted the translocalization of APP from RAB9-positive late endosomes (LE) to LAMP1-positive lysosomes, which was paralleled by the enhanced LE-lysosome fusion. In support, silencing of vacuolar protein sorting-associated protein 41 (VPS41) that is implicated in LE-lyso fusion prevented AP2S1-mediated regulation of APP degradation and translocalization. In APP/PS1 mice, an animal model of AD, AAV-mediated delivery of AP2S1 shRNA in the hippocampus significantly reduced the protein levels of APP and Aß, with the concomitant APP translocalization, LE-lyso fusion and the improved cognitive functions. Taken together, these data uncover a LE-lyso fusion mechanism in APP degradation and suggest a novel role for AP2S1 in the pathophysiology of AD.
Asunto(s)
Subunidades sigma de Complejo de Proteína Adaptadora , Enfermedad de Alzheimer , Ratones , Humanos , Animales , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/metabolismo , Endosomas/metabolismo , Lisosomas/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Complejo 2 de Proteína Adaptadora/metabolismo , Subunidades sigma de Complejo de Proteína Adaptadora/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
A 45-year-old man was referred to endocrine for the evaluation of hypercalcaemia. The calcium was elevated, vitamin D was low with a normal parathyroid hormone. Dual-energy X-ray absorptiometry scan revealed osteoporosis at the lumbar spine and femoral neck. A 24-hour urine collection revealed low urinary calcium, which was believed to be secondary to vitamin D deficiency. A diagnosis of primary hyperparathyroidism was made. The patient underwent a four-gland parathyroid exploration surgery in which three of his parathyroid glands were removed. The pathology was consistent with benign parathyroid tissue. Post surgery, the patient had persistently elevated calcium levels. He was then started on bisphosphonate and cinacalcet for osteoporosis and hypercalcaemia, respectively. Genetic analysis of familial hypocalciuric hypercalcaemia (FHH) showed a p.arg15cys mutation in the AP2S1 gene, confirming the diagnosis of FHH type 3.
Asunto(s)
Complejo 2 de Proteína Adaptadora/genética , Subunidades sigma de Complejo de Proteína Adaptadora/genética , ADN/genética , Hipercalcemia/congénito , Mutación Missense , Complejo 2 de Proteína Adaptadora/metabolismo , Subunidades sigma de Complejo de Proteína Adaptadora/metabolismo , Análisis Mutacional de ADN , Diagnóstico Diferencial , Humanos , Hipercalcemia/diagnóstico , Hipercalcemia/genética , Hipercalcemia/metabolismo , Masculino , Persona de Mediana Edad , Tomografía Computarizada por Rayos X , UltrasonografíaRESUMEN
Ring finger protein 41 (RNF41) is an E3 ubiquitin ligase involved in the ubiquitination and degradation of many proteins including ErbB3 receptors, BIRC6, and parkin. Next to this, RNF41 regulates the intracellular trafficking of certain JAK2-associated cytokine receptors by ubiquitinating and suppressing USP8, which, in turn, destabilizes the ESCRT-0 complex. To further elucidate the function of RNF41 we used different orthogonal approaches to reveal the RNF41 protein complex: affinity purification-mass spectrometry, BioID, and Virotrap. We combined these results with known data sets for RNF41 obtained with microarray MAPPIT and Y2H screens. This way, we establish a comprehensive high-resolution interactome network comprising 175 candidate protein partners. To remove potential methodological artifacts from this network, we distilled the data into a high-confidence interactome map by retaining a total of 19 protein hits identified in two or more of the orthogonal methods. AP2S1, a novel RNF41 interaction partner, was selected from this high-confidence interactome for further functional validation. We reveal a role for AP2S1 in leptin and LIF receptor signaling and show that RNF41 stabilizes and relocates AP2S1.
Asunto(s)
Mapas de Interacción de Proteínas , Ubiquitina-Proteína Ligasas/metabolismo , Complejo 2 de Proteína Adaptadora/metabolismo , Subunidades sigma de Complejo de Proteína Adaptadora/metabolismo , Humanos , Leptina/metabolismo , Métodos , Unión Proteica , Receptores OSM-LIF/metabolismo , Transducción de SeñalRESUMEN
Mutations of the sigma subunit of the heterotetrameric adaptor-related protein complex 2 (AP2σ) impair signalling of the calcium-sensing receptor (CaSR), and cause familial hypocalciuric hypercalcaemia type 3 (FHH3). To date, FHH3-associated AP2σ mutations have only been identified at one residue, Arg15. We hypothesized that additional rare AP2σ variants may also be associated with altered CaSR function and hypercalcaemia, and sought for these by analysing >111 995 exomes (>60 706 from ExAc and dbSNP, and 51 289 from the Geisinger Health System-Regeneron DiscovEHR dataset, which also contains clinical data). This identified 11 individuals to have 9 non-synonymous AP2σ variants (Arg3His, Arg15His (x3), Ala44Thr, Phe52Tyr, Arg61His, Thr112Met, Met117Ile, Glu122Gly and Glu142Lys) with 3 of the 4 individuals who had Arg15His and Met117Ile AP2σ variants having mild hypercalcaemia, thereby indicating a prevalence of FHH3-associated AP2σ mutations of â¼7.8 per 100 000 individuals. Structural modelling of the novel eight AP2σ variants (Arg3His, Ala44Thr, Phe52Tyr, Arg61His, Thr112Met, Met117Ile, Glu122Gly and Glu142Lys) predicted that the Arg3His, Thr112Met, Glu122Gly and Glu142Lys AP2σ variants would disrupt polar contacts within the AP2σ subunit or affect the interface between the AP2σ and AP2α subunits. Functional analyses of all eight AP2σ variants in CaSR-expressing cells demonstrated that the Thr112Met, Met117Ile and Glu142Lys variants, located in the AP2σ α4-α5 helical region that forms an interface with AP2α, impaired CaSR-mediated intracellular calcium (Cai2+) signalling, consistent with a loss of function, and this was rectified by treatment with the CaSR positive allosteric modulator cinacalcet. Thus, our studies demonstrate another potential class of FHH3-causing AP2σ mutations located at the AP2σ-AP2α interface.
Asunto(s)
Subunidades alfa de Complejo de Proteína Adaptadora/metabolismo , Subunidades sigma de Complejo de Proteína Adaptadora/genética , Mutación , Receptores Sensibles al Calcio/metabolismo , Complejo 2 de Proteína Adaptadora/genética , Complejo 2 de Proteína Adaptadora/metabolismo , Subunidades sigma de Complejo de Proteína Adaptadora/metabolismo , Cinacalcet/farmacología , Bases de Datos Genéticas , Exoma , Femenino , Humanos , Hipercalcemia/tratamiento farmacológico , Hipercalcemia/genética , Masculino , Persona de Mediana Edad , Modelos Moleculares , Conformación Proteica , Transducción de Señal , Secuenciación del ExomaRESUMEN
γ2 adaptin is homologous to γ1, but is only expressed in vertebrates while γ1 is found in all eukaryotes. We know little about γ2 functions and their relation to γ1. γ1 is an adaptin of the heterotetrameric AP-1 complexes, which sort proteins in and do form clathrin-coated transport vesicles and they also regulate maturation of early endosomes. γ1 knockout mice develop only to blastocysts and thus γ2 does not compensate γ1-deficiency in development. γ2 has not been classified as a clathrin-coated vesicle adaptor protein in proteome analyses and functions for monomeric γ2 in endosomal protein sorting have been proposed, but adaptin interaction studies suggested formation of heterotetrameric AP-1/γ2 complexes. We detected γ2 at the trans-Golgi network, on peripheral vesicles and identified γ2 clathrin-coated vesicles in mice. Ubiquitous σ1A and tissue-specific σ1B adaptins bind γ2 and γ1. σ1B knockout in mice does not effect γ1/σ1A AP-1 levels, but γ2/σ1A AP-1 levels are increased in brain and adipocytes. Also γ2 is essential in development. In zebrafish AP-1/γ2 and AP-1/γ1 fulfill different, essential functions in brain and the vascular system.
Asunto(s)
Subunidades gamma de Complejo de Proteína Adaptadora/metabolismo , Subunidades sigma de Complejo de Proteína Adaptadora/metabolismo , Encéfalo/metabolismo , Clatrina/metabolismo , Vesículas Transportadoras/metabolismo , Red trans-Golgi/metabolismo , Subunidades gamma de Complejo de Proteína Adaptadora/química , Subunidades gamma de Complejo de Proteína Adaptadora/genética , Subunidades sigma de Complejo de Proteína Adaptadora/química , Subunidades sigma de Complejo de Proteína Adaptadora/genética , Adipocitos/citología , Adipocitos/metabolismo , Animales , Vasos Sanguíneos/crecimiento & desarrollo , Vasos Sanguíneos/metabolismo , Encéfalo/crecimiento & desarrollo , Línea Celular , Clatrina/genética , Embrión de Mamíferos , Embrión no Mamífero , Endosomas/metabolismo , Endosomas/ultraestructura , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Noqueados , Modelos Moleculares , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Estructura Secundaria de Proteína , Transporte de Proteínas/genética , Transducción de Señal , Vesículas Transportadoras/ultraestructura , Pez Cebra , Red trans-Golgi/ultraestructuraRESUMEN
Despite increasing amounts of experimental evidence depicting the involvement of non-coding RNAs in cancer, the study of BRAFV600E-regulated genes has thus far focused mainly on protein-coding ones. Here, we identify and study the microRNAs that BRAFV600E regulates through the ERK pathway.By performing small RNA sequencing on A375 melanoma cells and a vemurafenib-resistant clone that was taken as negative control, we discover miR-204 and miR-211 as the miRNAs most induced by vemurafenib. We also demonstrate that, although belonging to the same family, these two miRNAs have distinctive features. miR-204 is under the control of STAT3 and its expression is induced in amelanotic melanoma cells, where it acts as an effector of vemurafenib's anti-motility activity by targeting AP1S2. Conversely, miR-211, a known transcriptional target of MITF, is induced in melanotic melanoma cells, where it targets EDEM1 and consequently impairs the degradation of TYROSINASE (TYR) through the ER-associated degradation (ERAD) pathway. In doing so, miR-211 serves as an effector of vemurafenib's pro-pigmentation activity. We also show that such an increase in pigmentation in turn represents an adaptive response that needs to be overcome using appropriate inhibitors in order to increase the efficacy of vemurafenib.In summary, we unveil the distinct and context-dependent activities exerted by miR-204 family members in melanoma cells. Our work challenges the widely accepted "same miRNA family = same function" rule and provides a rationale for a novel treatment strategy for melanotic melanomas that is based on the combination of ERK pathway inhibitors with pigmentation inhibitors.
Asunto(s)
Melanoma Amelanótico/genética , Melanoma/genética , MicroARNs/genética , Neoplasias Cutáneas/genética , Subunidades sigma de Complejo de Proteína Adaptadora/genética , Subunidades sigma de Complejo de Proteína Adaptadora/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Indoles/farmacología , Sistema de Señalización de MAP Quinasas , Melanoma/metabolismo , Melanoma/patología , Melanoma Amelanótico/tratamiento farmacológico , Melanoma Amelanótico/metabolismo , Melanoma Amelanótico/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Sulfonamidas/farmacología , Transfección , VemurafenibRESUMEN
The σ1 subunit of the AP-1 clathrin-coated-vesicle adaptor-protein complex is expressed as three isoforms. Tissues express σ1A and one of the σ1B and σ1C isoforms. Brain is the tissue with the highest σ1A and σ1B expression. σ1B-deficiency leads to severe mental retardation, accumulation of early endosomes in synapses and fewer synaptic vesicles, whose recycling is slowed down. AP-1/σ1A and AP-1/σ1B regulate maturation of these early endosomes into multivesicular body late endosomes, thereby controlling synaptic vesicle protein transport into a degradative pathway. σ1A binds ArfGAP1, and with higher affinity brain-specific ArfGAP1, which bind Rabex-5. AP-1/σ1A-ArfGAP1-Rabex-5 complex formation leads to more endosomal Rabex-5 and enhanced, Rab5(GTP)-stimulated Vps34 PI3-kinase activity, which is essential for multivesicular body endosome formation. Formation of AP-1/σ1A-ArfGAP1-Rabex-5 complexes is prevented by σ1B binding of Rabex-5 and the amount of endosomal Rabex-5 is reduced. AP-1 complexes differentially regulate endosome maturation and coordinate protein recycling and degradation, revealing a novel molecular mechanism by which they regulate protein transport besides their established function in clathrin-coated-vesicle formation.
Asunto(s)
Subunidades sigma de Complejo de Proteína Adaptadora/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Endosomas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Neuronas/metabolismo , Transducción de Señal , Subunidades sigma de Complejo de Proteína Adaptadora/deficiencia , Animales , Encéfalo/metabolismo , Endosomas/ultraestructura , Proteínas Activadoras de GTPasa/metabolismo , Ratones Noqueados , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Sinaptosomas/metabolismoRESUMEN
Hepatitis C virus (HCV) infects 130 million people worldwide and is a leading cause of liver cirrhosis, end-stage liver disease and hepatocellular carcinoma. The interactions between viral elements and host factors play critical role on HCV invade, replication and release. Here, we identified adaptor protein complex 1 sigma 3 subunit (AP1S3) as a dependency factor for the efficient HCV infection in hepatoma cells. AP1S3 silencing in cultivated Huh7.5.1 cells significantly reduced the production of HCV progeny particles. Immunoprecipitation analysis revealed that AP1S3 interacted with the HCV E2 protein. With this interaction, AP1S3 could protect HCV E2 from ubiquitin-mediated proteasomal degradation. Using in vivo ubiquitylation assay, we identified that E6-Associated Protein (E6AP) was associated with HCV E2. In addition, treatment with synthetic peptide that contains the AP1S3-recognized motif inhibited HCV infection in Huh7.5.1 cells. Our data reveal AP1 as a novel host network that is required by viruses during infection and provides a potential target for developing broad-spectrum anti-virus strategies.
Asunto(s)
Subunidades sigma de Complejo de Proteína Adaptadora/metabolismo , Hepacivirus/fisiología , Interacciones Huésped-Patógeno , Proteínas del Envoltorio Viral/metabolismo , Subunidades sigma de Complejo de Proteína Adaptadora/deficiencia , Subunidades sigma de Complejo de Proteína Adaptadora/genética , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Silenciador del Gen , Hepacivirus/efectos de los fármacos , Humanos , Inmunoprecipitación , Neoplasias Hepáticas/virología , Péptidos/síntesis química , Péptidos/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Viral , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ensamble de VirusRESUMEN
The functional requirement of adapter protein 2 (AP2) complex in synaptic membrane retrieval by clathrin-mediated endocytosis is not fully understood. Here we isolated and functionally characterized a mutation that dramatically altered synaptic development. Based on the aberrant neuromuscular junction (NMJ) synapse, we named this mutation angur (a Hindi word meaning "grapes"). Loss-of-function alleles of angur show more than twofold overgrowth in bouton numbers and a dramatic decrease in bouton size. We mapped the angur mutation to σ2-adaptin, the smallest subunit of the AP2 complex. Reducing the neuronal level of any of the subunits of the AP2 complex or disrupting AP2 complex assembly in neurons phenocopied the σ2-adaptin mutation. Genetic perturbation of σ2-adaptin in neurons leads to a reversible temperature-sensitive paralysis at 38°. Electrophysiological analysis of the mutants revealed reduced evoked junction potentials and quantal content. Interestingly, high-frequency nerve stimulation caused prolonged synaptic fatigue at the NMJs. The synaptic levels of subunits of the AP2 complex and clathrin, but not other endocytic proteins, were reduced in the mutants. Moreover, bone morphogenetic protein (BMP)/transforming growth factor ß (TGFß) signaling was altered in these mutants and was restored by normalizing σ2-adaptin in neurons. Thus, our data suggest that (1) while σ2-adaptin facilitates synaptic vesicle (SV) recycling for basal synaptic transmission, its activity is also required for regenerating SVs during high-frequency nerve stimulation, and (2) σ2-adaptin regulates NMJ morphology by attenuating TGFß signaling.
Asunto(s)
Subunidades sigma de Complejo de Proteína Adaptadora/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Unión Neuromuscular/metabolismo , Transmisión Sináptica , Subunidades sigma de Complejo de Proteína Adaptadora/genética , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Clatrina/metabolismo , Drosophila/genética , Drosophila/fisiología , Proteínas de Drosophila/genética , Potenciales Evocados , Mutación , Unión Neuromuscular/fisiología , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Neurons are highly polarized cells having distinct somatodendritic and axonal domains. Here we report that polarized sorting of the Cu(2+) transporter ATP7B and the vesicle-SNARE VAMP4 to the somatodendritic domain of rat hippocampal neurons is mediated by recognition of dileucine-based signals in the cytosolic domains of the proteins by the σ1 subunit of the clathrin adaptor AP-1. Under basal Cu(2+) conditions, ATP7B was localized to the trans-Golgi network (TGN) and the plasma membrane of the soma and dendrites but not the axon. Mutation of a dileucine-based signal in ATP7B or overexpression of a dominant-negative σ1 mutant resulted in nonpolarized distribution of ATP7B between the somatodendritic and axonal domains. Furthermore, addition of high Cu(2+) concentrations, previously shown to reduce ATP7B incorporation into AP-1-containing clathrin-coated vesicles, caused loss of TGN localization and somatodendritic polarity of ATP7B. These findings support the notion of AP-1 as an effector of polarized sorting in neurons and suggest that altered polarity of ATP7B in polarized cell types might contribute to abnormal copper metabolism in the MEDNIK syndrome, a neurocutaneous disorder caused by mutations in the σ1A subunit isoform of AP-1.
Asunto(s)
Complejo 1 de Proteína Adaptadora/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Leucina/metabolismo , Neuronas/metabolismo , Complejo 1 de Proteína Adaptadora/química , Complejo 1 de Proteína Adaptadora/genética , Subunidades sigma de Complejo de Proteína Adaptadora/química , Subunidades sigma de Complejo de Proteína Adaptadora/genética , Subunidades sigma de Complejo de Proteína Adaptadora/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Membrana Celular/metabolismo , Polaridad Celular , Células Cultivadas , Cobre/metabolismo , Cobre/farmacología , ATPasas Transportadoras de Cobre , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Leucina/química , Leucina/genética , Microscopía Confocal , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Neuronas/efectos de los fármacos , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Proteínas R-SNARE/química , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Ratas , Red trans-Golgi/metabolismoRESUMEN
Here, we describe altered sorting of sortilin in adipocytes deficient for the σ1B-containing AP-1 complex, leading to the inhibition of adipogenesis. The AP-1 complex mediates protein sorting between the trans-Golgi network and endosomes. Vertebrates express three AP1 σ1 subunit isoforms - σ1A, σ1B and σ1C (also known as AP1S1, AP1S2 and AP1S3, respectively). σ1B-deficient mice display impaired recycling of synaptic vesicles and lipodystrophy. Here, we show that sortilin is overexpressed in adipose tissue from σ1B(-/-) mice, and that its overexpression in wild-type cells is sufficient to suppress adipogenesis. σ1B-specific binding of sortilin requires the sortilin DxxD-x12-DSxxxL motif. σ1B deficiency does not lead to a block of sortilin transport out of a specific organelle, but the fraction that reaches lysosomes is reduced. Sortilin binds to the receptor DLK1, an inhibitor of adipocyte differentiation, and the overexpression of sortilin prevents DLK1 downregulation, leading to enhanced inhibition of adipogenesis. DLK1 and sortilin expression are not increased in the brain tissue of σ1B(-/-) mice, although this is the tissue with the highest expression of σ1B and sortilin. Thus, adipose-tissue-specific and σ1B-dependent routes for the transport of sortilin exist and are involved in the regulation of adipogenesis and adipose-tissue mass.
Asunto(s)
Complejo 1 de Proteína Adaptadora/metabolismo , Subunidades sigma de Complejo de Proteína Adaptadora/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Adipocitos/metabolismo , Adipogénesis , Tejido Adiposo/metabolismo , Complejo 1 de Proteína Adaptadora/genética , Subunidades sigma de Complejo de Proteína Adaptadora/genética , Proteínas Adaptadoras del Transporte Vesicular/genética , Adipocitos/citología , Tejido Adiposo/citología , Animales , Femenino , Masculino , Ratones , Ratones Noqueados , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de ProteínasRESUMEN
Clathrin-mediated endocytosis (CME) is the major pathway for concentrative uptake of receptors and receptor-ligand complexes (cargo). Although constitutively internalized cargos are known to accumulate into maturing clathrin-coated pits (CCPs), whether and how cargo recruitment affects the initiation and maturation of CCPs is not fully understood. Previous studies have addressed these issues by analyzing the global effects of receptor overexpression on CME or CCP dynamics. Here, we exploit a refined approach using expression of a biotinylated transferrin receptor (bTfnR) and controlling its local clustering using mono- or multivalent streptavidin. We show that local clustering of bTfnR increased CCP initiation. By tracking cargo loading in individual CCPs, we found that bTfnR clustering preceded clathrin assembly and confirmed that bTfnR-containing CCPs mature more efficiently than bTfnR-free CCPs. Although neither the clustering nor the related changes in cargo loading altered the rate of CCP maturation, bTfnR-containing CCPs exhibited significantly longer lifetimes than other CCPs within the same cell. Together these results demonstrate that cargo composition is a key source of the differential dynamics of CCPs.
Asunto(s)
Invaginaciones Cubiertas de la Membrana Celular/fisiología , Agregación de Receptores/fisiología , Receptores de Transferrina/metabolismo , Complejo 2 de Proteína Adaptadora/genética , Complejo 2 de Proteína Adaptadora/metabolismo , Subunidades sigma de Complejo de Proteína Adaptadora/genética , Subunidades sigma de Complejo de Proteína Adaptadora/metabolismo , Animales , Biotina/metabolismo , Biotinilación/genética , Biotinilación/métodos , Ligasas de Carbono-Nitrógeno/genética , Línea Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Cadenas Ligeras de Clatrina/genética , Cadenas Ligeras de Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Endocitosis/fisiología , Células Epiteliales/citología , Células Epiteliales/fisiología , Proteínas de Escherichia coli/genética , Humanos , Cinética , Unión Proteica/fisiología , Ratas , Receptores de Transferrina/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Estreptavidina/genética , Estreptavidina/metabolismo , Transducción GenéticaRESUMEN
Adaptins are subunits of the heterotetrameric (beta/mu/gamma/sigma) adaptor protein (AP) complexes that are involved in clathrin-mediated membrane trafficking. Here, we show that in Schizosaccharomyces pombe the deletion strains of each individual subunit of the AP-1 complex [Apl2 (beta), Apl4 (gamma), Apm1 (mu) and Aps1 (sigma)] caused distinct phenotypes on growth sensitivity to temperature or drugs. We also show that the Deltaapm1 and Deltaapl2 mutants displayed similar but more severe phenotypes than those of Deltaaps1 or Deltaapl4 mutants. Furthermore, the Deltaapl2Deltaaps1 and Deltaapl2Deltaapl4 double mutants displayed synthetic growth defects, whereas the Deltaaps1Deltaapl4 and Deltaapl2Deltaapm1 double mutants did not. In pull-down assay, Apm1 binds Apl2 even in the absence of Aps1 and Apl4, and Apl4 binds Aps1 even in the absence of Apm1 and Apl2. Consistently, the deletion of any subunit generally caused the disassociation of the heterotetrameric complex from endosomes, although some subunits weakly localized to endosomes. In addition, the deletion of individual subunits caused similar endosomal accumulation of v-SNARE synaptobrevin Syb1. Altogether, results suggest that the four subunits are all essential for the heterotetrameric complex formation and for the AP-1 function in exit transport from endosomes.
Asunto(s)
Complejo 1 de Proteína Adaptadora/metabolismo , Subunidades del Complejo de Proteínas Adaptadoras/genética , Subunidades del Complejo de Proteínas Adaptadoras/metabolismo , Eliminación de Gen , Schizosaccharomyces/crecimiento & desarrollo , Complejo 1 de Proteína Adaptadora/genética , Subunidades beta de Complejo de Proteína Adaptadora/genética , Subunidades beta de Complejo de Proteína Adaptadora/metabolismo , Subunidades delta de Complexo de Proteína Adaptadora/genética , Subunidades delta de Complexo de Proteína Adaptadora/metabolismo , Subunidades mu de Complejo de Proteína Adaptadora/genética , Subunidades mu de Complejo de Proteína Adaptadora/metabolismo , Subunidades sigma de Complejo de Proteína Adaptadora/genética , Subunidades sigma de Complejo de Proteína Adaptadora/metabolismo , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Datos de Secuencia Molecular , Fenotipo , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Temperatura , Ácido Valproico/farmacologíaRESUMEN
Adaptor protein (AP) complexes regulate clathrin-coated vesicle assembly, protein cargo sorting, and vesicular trafficking between organelles in eukaryotic cells. Because disruption of the various subunits of the AP complexes is embryonic lethal in the majority of cases, characterization of their function in vivo is still lacking. Here, we describe the first mutation in the human AP1S1 gene, encoding the small subunit sigma1A of the AP-1 complex. This founder splice mutation, which leads to a premature stop codon, was found in four families with a unique syndrome characterized by mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratodermia (MEDNIK). To validate the pathogenic effect of the mutation, we knocked down Ap1s1 expression in zebrafish using selective antisens morpholino oligonucleotides (AMO). The knockdown phenotype consisted of perturbation in skin formation, reduced pigmentation, and severe motility deficits due to impaired neural network development. Both neural and skin defects were rescued by co-injection of AMO with wild-type (WT) human AP1S1 mRNA, but not by co-injecting the truncated form of AP1S1, consistent with a loss-of-function effect of this mutation. Together, these results confirm AP1S1 as the gene responsible for MEDNIK syndrome and demonstrate a critical role of AP1S1 in development of the skin and spinal cord.
Asunto(s)
Complejo 1 de Proteína Adaptadora/genética , Subunidades sigma de Complejo de Proteína Adaptadora/genética , Técnicas de Silenciamiento del Gen , Síndromes Neurocutáneos/genética , Piel/crecimiento & desarrollo , Médula Espinal/crecimiento & desarrollo , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Complejo 1 de Proteína Adaptadora/metabolismo , Subunidades sigma de Complejo de Proteína Adaptadora/metabolismo , Animales , Células Cultivadas , Femenino , Humanos , Masculino , Mutación , Síndromes Neurocutáneos/metabolismo , Linaje , Fenotipo , Piel/metabolismo , Médula Espinal/metabolismo , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
In a systematic sequencing screen of the coding exons of the X chromosome in 250 families with X-linked mental retardation (XLMR), we identified two nonsense mutations and one consensus splice-site mutation in the AP1S2 gene on Xp22 in three families. Affected individuals in these families showed mild-to-profound mental retardation. Other features included hypotonia early in life and delay in walking. AP1S2 encodes an adaptin protein that constitutes part of the adaptor protein complex found at the cytoplasmic face of coated vesicles located at the Golgi complex. The complex mediates the recruitment of clathrin to the vesicle membrane. Aberrant endocytic processing through disruption of adaptor protein complexes is likely to result from the AP1S2 mutations identified in the three XLMR-affected families, and such defects may plausibly cause abnormal synaptic development and function. AP1S2 is the first reported XLMR gene that encodes a protein directly involved in the assembly of endocytic vesicles.