RESUMEN
Postsynaptic density protein 95 (PSD95) and synapse-associated protein 97 (SAP97) are homologous scaffold proteins with different N-terminal domains, possessing either a palmitoylation site (PSD95) or an L27 domain (SAP97). Here, we measured PSD95 and SAP97 conformation in vitro and in postsynaptic densities (PSDs) using FRET and EM, and examined how conformation regulated interactions with AMPA-type and NMDA-type glutamate receptors (AMPARs/NMDARs). Palmitoylation of PSD95 changed its conformation from a compact to an extended configuration. PSD95 associated with AMPARs (via transmembrane AMPAR regulatory protein subunits) or NMDARs [via glutamate ionotropic receptor NMDA-type subunit 2B (GluN2B) subunits] only in its palmitoylated and extended conformation. In contrast, in its extended conformation, SAP97 associates with NMDARs, but not with AMPARs. Within PSDs, PSD95 and SAP97 were largely in the extended conformation, but had different orientations. PSD95 oriented perpendicular to the PSD membrane, with its palmitoylated, N-terminal domain at the membrane. SAP97 oriented parallel to the PSD membrane, likely as a dimer through interactions of its N-terminal L27 domain. Changing PSD95 palmitoylation in PSDs altered PSD95 and AMPAR levels but did not affect NMDAR levels. These results indicate that in PSDs, PSD95 palmitoylation, conformation, and its interactions are dynamic when associated with AMPARs and more stable when associated with NMDARs. Altogether, our results are consistent with differential regulation of PSD95 palmitoylation in PSDs resulting from the clustering of palmitoylating and depalmitoylating enzymes into AMPAR nanodomains segregated away from NMDAR nanodomains.
Asunto(s)
Homólogo 4 de la Proteína Discs Large/metabolismo , Lipoilación , Densidad Postsináptica , Receptores de Glutamato/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Homólogo 1 de la Proteína Discs Large , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Hipocampo/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Mutación , Neuronas/metabolismo , Dominios Proteicos , Multimerización de Proteína , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Palmitoylation regulates diverse aspects of neuronal protein trafficking and function. Here a global characterization of rat neural palmitoyl-proteomes identifies most of the known neural palmitoyl proteins-68 in total, plus more than 200 new palmitoyl-protein candidates, with further testing confirming palmitoylation for 21 of these candidates. The new palmitoyl proteins include neurotransmitter receptors, transporters, adhesion molecules, scaffolding proteins, as well as SNAREs and other vesicular trafficking proteins. Of particular interest is the finding of palmitoylation for a brain-specific Cdc42 splice variant. The palmitoylated Cdc42 isoform (Cdc42-palm) differs from the canonical, prenylated form (Cdc42-prenyl), both with regard to localization and function: Cdc42-palm concentrates in dendritic spines and has a special role in inducing these post-synaptic structures. Furthermore, assessing palmitoylation dynamics in drug-induced activity models identifies rapidly induced changes for Cdc42 as well as for other synaptic palmitoyl proteins, suggesting that palmitoylation may participate broadly in the activity-driven changes that shape synapse morphology and function.
Asunto(s)
Lipoilación , Neuronas/metabolismo , Proteómica , Sinapsis/metabolismo , Empalme Alternativo/genética , Animales , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/embriología , Dendritas/metabolismo , Modelos Neurológicos , Especificidad de Órganos , Proteoma/metabolismo , Ratas , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Huntingtin interacting protein 14 (HIP14, ZDHHC17) is a huntingtin (HTT) interacting protein with palmitoyl transferase activity. In order to interrogate the function of Hip14, we generated mice with disruption in their Hip14 gene. Hip14-/- mice displayed behavioral, biochemical and neuropathological defects that are reminiscent of Huntington disease (HD). Palmitoylation of other HIP14 substrates, but not Htt, was reduced in the Hip14-/- mice. Hip14 is dysfunctional in the presence of mutant htt in the YAC128 mouse model of HD, suggesting that altered palmitoylation mediated by HIP14 may contribute to HD.
Asunto(s)
Aciltransferasas/deficiencia , Enfermedad de Huntington/etiología , Lipoilación/genética , Proteínas del Tejido Nervioso/deficiencia , Aciltransferasas/genética , Aciltransferasas/metabolismo , Animales , Muerte Celular/genética , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Encefalinas/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Ratones , Ratones Noqueados , Actividad Motora/genética , Proteínas Mutantes/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuronas/patología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Sinapsis/metabolismoRESUMEN
ATP-binding cassette transporter (ABC)A1 lipidates apolipoprotein A-I both directly at the plasma membrane and also uses lipids from the late endosomal or lysosomal compartment in the internal lipidation of apolipoprotein A-I. However, how ABCA1 targeting to these specific membranes is regulated remains unknown. Palmitoylation is a dynamically regulated lipid modification that targets many proteins to specific membrane domains. We hypothesized that palmitoylation may also regulate ABCA1 transport and function. Indeed, ABCA1 is robustly palmitoylated at cysteines 3, -23, -1110, and -1111. Abrogation of palmitoylation of ABCA1 by mutation of the cysteines results in a reduction of ABCA1 localization at the plasma membranes and a reduction in the ability of ABCA1 to efflux lipids to apolipoprotein A-I. ABCA1 is palmitoylated by the palmitoyl transferase DHHC8, and increasing DHHC8 protein results in increased ABCA1-mediated lipid efflux. Thus, palmitoylation regulates ABCA1 localization at the plasma membrane, and regulates its lipid efflux ability.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Procesamiento Proteico-Postraduccional , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Aciltransferasas/genética , Aciltransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Apolipoproteína A-I/metabolismo , Transporte Biológico , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Colesterol/metabolismo , Cisteína , Humanos , Lipoilación , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Palmitatos/metabolismo , Fosfolípidos/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión , Relación Estructura-Actividad , TransfecciónRESUMEN
The synaptic insertion of GluR1-containing AMPA-type glutamate receptors (AMPARs) is critical for synaptic plasticity. However, mechanisms responsible for GluR1 insertion and retention at the synapse are unclear. The synapse-associated protein SAP97 directly binds GluR1 and participates in its forward trafficking from the Golgi network to the plasma membrane. Whether SAP97 also plays a role in scaffolding GluR1 at the postsynaptic membrane is controversial, attributable to its expression as a collection of alternatively spliced isoforms with ill-defined spatial and temporal distributions. In the present study, we have used live imaging and electrophysiology to demonstrate that two postsynaptic, N-terminal isoforms of SAP97 directly modulate the levels, dynamics, and function of synaptic GluR1-containing AMPARs. Specifically, the unique N-terminal domains confer distinct subsynaptic localizations onto SAP97, targeting the palmitoylated alpha-isoform to the postsynaptic density (PSD) and the L27 domain-containing beta-isoform primarily to non-PSD, perisynaptic regions. Consequently, alpha- and betaSAP97 differentially influence the subsynaptic localization and dynamics of AMPARs by creating binding sites for GluR1-containing receptors within their respective subdomains. These results indicate that N-terminal splicing of SAP97 can control synaptic strength by regulating the distribution of AMPARs and, hence, their responsiveness to presynaptically released glutamate.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Ácido Glutámico/fisiología , Proteínas de la Membrana/fisiología , Terminales Presinápticos/fisiología , Receptores AMPA/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Células Cultivadas , Hipocampo/química , Hipocampo/fisiología , Proteínas de la Membrana/genética , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/fisiología , Terminales Presinápticos/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Ratas , Ratas Sprague-Dawley , Receptores AMPA/genética , Receptores AMPA/metabolismo , Sinapsis/química , Sinapsis/genética , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/metabolismoRESUMEN
Post-translational modification by the lipid palmitate is crucial for the correct targeting and function of many proteins. Here we show that huntingtin (htt) is normally palmitoylated at cysteine 214, which is essential for its trafficking and function. The palmitoylation and distribution of htt are regulated by the palmitoyl transferase huntingtin interacting protein 14 (HIP14). Expansion of the polyglutamine tract of htt, which causes Huntington disease, results in reduced interaction between mutant htt and HIP14 and consequently in a marked reduction in palmitoylation. Mutation of the palmitoylation site of htt, making it palmitoylation resistant, accelerates inclusion formation and increases neuronal toxicity. Downregulation of HIP14 in mouse neurons expressing wild-type and mutant htt increases inclusion formation, whereas overexpression of HIP14 substantially reduces inclusions. These results suggest that the expansion of the polyglutamine tract in htt results in decreased palmitoylation, which contributes to the formation of inclusion bodies and enhanced neuronal toxicity.
Asunto(s)
Proteínas Portadoras/metabolismo , Corteza Cerebral/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Ácido Palmítico/metabolismo , Aciltransferasas , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos/fisiología , Animales , Animales Recién Nacidos , Células COS , Proteínas Portadoras/genética , Células Cultivadas , Corteza Cerebral/citología , Chlorocebus aethiops , Cisteína/metabolismo , Regulación hacia Abajo/genética , Humanos , Proteína Huntingtina , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Ratones , Ratones Transgénicos , Mutación/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Proteínas Nucleares/química , Proteínas Nucleares/genética , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Transporte de Proteínas/fisiología , Ratas , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
Neuronal alpha-bungarotoxin receptors (BgtRs) are nicotinic receptors that require as yet unidentified post-translational modifications to achieve functional expression. In this study, we examined the role of protein palmitoylation in BgtR expression. BgtR alpha7 subunits are highly palmitoylated in neurons from brain and other cells capable of BgtR expression, such as pheochromocytoma 12 (PC12) cells. In PC12 cells, alpha7 subunits are palmitoylated with a stoichiometry of approximately one palmitate per subunit, and inhibition of palmitoylation blocks BgtR expression. In cells incapable of BgtR expression, such as human embryonic kidney cells, alpha7 subunits are not significantly palmitoylated. However, in these same cells, chimeric subunits with the N-terminal half of alpha7 fused to the C-terminal half of serotonin-3A receptor (alpha7/5-HT3A) subunits form functional BgtRs that are palmitoylated to an extent similar to that of BgtRalpha7 subunits in PC12 cells. Palmitoylation of PC12 and alpha7/5-HT3A BgtRs occurred during assembly in the endoplasmic reticulum (ER). In conclusion, our data indicate a function for protein palmitoylation in which palmitoylation of assembling alpha7 subunits in the ER has a role in the formation of functional BgtRs.
Asunto(s)
Bungarotoxinas/metabolismo , Ácido Palmítico/metabolismo , Procesamiento Proteico-Postraduccional , Receptores Nicotínicos/metabolismo , Animales , Encéfalo/metabolismo , Línea Celular , Retículo Endoplásmico/metabolismo , Humanos , Palmitatos/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Subunidades de Proteína/metabolismo , Ratas , Receptores Nicotínicos/biosíntesis , Receptores Nicotínicos/aislamiento & purificación , Receptores de Serotonina 5-HT3/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
As a reversible posttranslational modification, protein palmitoylation has the potential to regulate the trafficking and function of a variety of proteins. However, the extent, function, and dynamic nature of palmitoylation are poorly resolved because of limitations in assay methods. Here, we introduce methods where hydroxylamine-mediated cleavage of the palmitoyl-thioester bond generates a free sulfhydryl, which can then be specifically labeled with sulfhydryl-reactive reagents. This methodology is more sensitive and allows for quantitative estimates of palmitoylation. Unlike other techniques used to assay posttranslational modifications, the techniques we have developed can label all sites of modification with a variety of probes, radiolabeled or nonradioactive, and can be used to assay the palmitoylation of proteins expressed in vivo in brain or other tissues.
Asunto(s)
Palmitatos/metabolismo , Proteínas/metabolismo , Coloración y Etiquetado/métodos , Animales , Sitios de Unión , Biotinilación , Western Blotting/métodos , Línea Celular , Pollos , Etilmaleimida/metabolismo , Vectores Genéticos , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Proteínas del Tejido Nervioso/metabolismo , Palmitatos/análisis , Unión Proteica , Proteínas/análisis , Proteína 25 Asociada a Sinaptosomas , Transfección , Tritio/metabolismoRESUMEN
Proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases generates beta-amyloid (Abeta) peptides, which accumulate in the brains of individuals affected by Alzheimer disease. Detergent-resistant membrane microdomains (DRM) rich in cholesterol and sphingolipid, termed lipid rafts, have been implicated in Abeta production. Previously, we and others reported that the four integral subunits of the gamma-secretase associate with DRM. In this study we investigated the mechanisms underlying DRM association of gamma-secretase subunits. We report that in cultured cells and in brain the gamma-secretase subunits nicastrin and APH-1 undergo S-palmitoylation, the post-translational covalent attachment of the long chain fatty acid palmitate common in lipid raft-associated proteins. By mutagenesis we show that nicastrin is S-palmitoylated at Cys(689), and APH-1 is S-palmitoylated at Cys(182) and Cys(245). S-Palmitoylation-defective nicastrin and APH-1 form stable gamma-secretase complexes when expressed in knock-out fibroblasts lacking wild type subunits, suggesting that S-palmitoylation is not essential for gamma-secretase assembly. Nevertheless, fractionation studies show that S-palmitoylation contributes to DRM association of nicastrin and APH-1. Moreover, pulse-chase analyses reveal that S-palmitoylation is important for nascent polypeptide stability of both proteins. Co-expression of S-palmitoylation-deficient nicastrin and APH-1 in cultured cells neither affects Abeta40, Abeta42, and AICD production, nor intramembrane processing of Notch and N-cadherin. Our findings suggest that S-palmitoylation plays a role in stability and raft localization of nicastrin and APH-1, but does not directly modulate gamma-secretase processing of APP and other substrates.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Lipoilación/fisiología , Glicoproteínas de Membrana/metabolismo , Microdominios de Membrana/enzimología , Proteínas de la Membrana/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular , Endopeptidasas , Estabilidad de Enzimas/fisiología , Humanos , Glicoproteínas de Membrana/genética , Lípidos de la Membrana/genética , Lípidos de la Membrana/metabolismo , Microdominios de Membrana/genética , Proteínas de la Membrana/genética , Ratones , Péptido Hidrolasas , Receptores Notch/genética , Receptores Notch/metabolismoRESUMEN
Epibatidine and mecamylamine are ligands used widely in the study of nicotinic acetylcholine receptors (nAChRs) in the central and peripheral nervous systems. In the present study, we find that nicotine blocks only 75% of (125)I-epibatidine binding to rat brain membranes, whereas ligands specific for serotonin type 3 receptors (5-HT(3)Rs) block the remaining 25%. (125)I-Epibatidine binds with a high affinity to native 5-HT(3)Rs of N1E-115 cells and to receptors composed of only 5-HT(3A) subunits expressed in HEK cells. In these cells, serotonin, the 5-HT(3)R-specific antagonist MDL72222, and the 5-HT(3)R agonist chlorophenylbiguanide readily competed with (125)I-epibatidine binding to 5-HT(3)Rs. Nicotine was a poor competitor for (125)I-epibatidine binding to 5-HT(3)Rs. However, the noncompetitive nAChR antagonist mecamylamine acted as a potent competitive inhibitor of (125)I-epibatidine binding to 5-HT(3)Rs. Epibatidine inhibited serotonin-induced currents mediated by endogenous 5-HT(3)Rs in neuroblastoma cell lines and 5-HT(3A)Rs expressed in HEK cells in a competitive manner. Our results demonstrate that 5-HT(3)Rs are previously uncharacterized high affinity epibatidine binding sites in the brain and indicate that epibatidine and mecamylamine act as 5-HT(3)R antagonists. Previous studies that depended on epibatidine and mecamylamine as nAChR-specific ligands, in particular studies of analgesic properties of epibatidine, may need to be reinterpreted with respect to the potential role of 5-HT(3)Rs.
Asunto(s)
Encéfalo/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Agonistas Nicotínicos/farmacología , Piridinas/farmacología , Antagonistas del Receptor de Serotonina 5-HT3 , Animales , Química Encefálica/efectos de los fármacos , Línea Celular Tumoral , Humanos , Mecamilamina/farmacología , Ratones , Nicotina/metabolismo , Nicotina/farmacología , Antagonistas Nicotínicos/farmacología , Unión Proteica , Ratas , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Receptores de Serotonina 5-HT3/genética , Receptores de Serotonina 5-HT3/metabolismoRESUMEN
Protein palmitoylation plays an important role in the structure and function of a wide array of proteins. Unlike other lipid modifications, protein palmitoylation is highly dynamic and cycles of palmitoylation and depalmitoylation can regulate protein function and localization. The dynamic nature of palmitoylation is poorly resolved because of limitations in assay methods. Here, we discuss various methods that can be used to measure protein palmitoylation and identify sites of palmitoylation. We describe new methodology based on "fatty acyl exchange labeling" in which palmitate is removed via hydroxylamine-mediated cleavage of the palmitoyl-thioester bond and then exchanged with a sulfhydryl-specific labeling compound. The techniques are highly sensitive and allow for quantitative estimates of palmitoylation. Unlike other techniques used to assay posttranslational modifications, the techniques we have developed can label all sites of modification with a variety of probes, radiolabeled or non-radioactive, and can be used to assay the palmitoylation of proteins from tissue samples.