RESUMEN
Imbalances in the amounts of amyloid-ß peptides (Aß) generated by the membrane proteases ß- and γ-secretase are considered as a trigger of Alzheimer's disease (AD). Cell-free studies of γ-secretase have shown that increasing membrane thickness modulates Aß generation but it has remained unclear if these effects are translatable to cells. Here we show that the very long-chain fatty acid erucic acid (EA) triggers acyl chain remodeling in AD cell models, resulting in substantial lipidome alterations which included increased esterification of EA in membrane lipids. Membrane remodeling enhanced γ-secretase processivity, resulting in the increased production of the potentially beneficial Aß37 and/or Aß38 species in multiple cell lines. Unexpectedly, we found that the membrane remodeling stimulated total Aß secretion by cells expressing WT γ-secretase but lowered it for cells expressing an aggressive familial AD mutant γ-secretase. We conclude that EA-mediated modulation of membrane composition is accompanied by complex lipid homeostatic changes that can impact amyloidogenic processing in different ways and elicit distinct γ-secretase responses, providing critical implications for lipid-based AD treatment strategies.
Asunto(s)
Enfermedad de Alzheimer , Secretasas de la Proteína Precursora del Amiloide , Humanos , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Lípidos de la Membrana/metabolismo , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Línea Celular , Precursor de Proteína beta-Amiloide/metabolismo , Presenilina-1/metabolismoRESUMEN
Previous studies have identified a crucial role of the gut microbiome in modifying Alzheimer's disease (AD) progression. However, the mechanisms of microbiome-brain interaction in AD were so far unknown. Here, we identify microbiota-derived short chain fatty acids (SCFA) as microbial metabolites which promote Aß deposition. Germ-free (GF) AD mice exhibit a substantially reduced Aß plaque load and markedly reduced SCFA plasma concentrations; conversely, SCFA supplementation to GF AD mice increased the Aß plaque load to levels of conventionally colonized (specific pathogen-free [SPF]) animals and SCFA supplementation to SPF mice even further exacerbated plaque load. This was accompanied by the pronounced alterations in microglial transcriptomic profile, including upregulation of ApoE. Despite increased microglial recruitment to Aß plaques upon SCFA supplementation, microglia contained less intracellular Aß. Taken together, our results demonstrate that microbiota-derived SCFA are critical mediators along the gut-brain axis which promote Aß deposition likely via modulation of the microglial phenotype.
Asunto(s)
Ácidos Grasos Volátiles/metabolismo , Microbioma Gastrointestinal , Microglía/metabolismo , Placa Amiloide/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Femenino , Masculino , Ratones , Organismos Libres de Patógenos EspecíficosRESUMEN
Steroids play an important role in cell regulation and homeostasis. Many diseases like Alzheimer's disease or Smith-Lemli-Opitz syndrome are known to be associated with deviations in the steroid profile. Most published methods only allow the analysis of small subgroups of steroids and cannot give an overview of the total steroid profile. We developed and validated a method that allows the analysis of free neutral steroids, including intermediates of cholesterol biosynthesis, free oxysterols, C19 and C21 steroids, free steroid acids, including bile acids, and sterol sulfates using gas chromatography-mass spectrometry. Samples were analyzed in scan mode for screening purposes and in dynamic multiple reaction monitoring mode for highly sensitive quantitative analysis. The method was validated for mouse brain and liver tissue and consists of sample homogenization, lipid extraction, steroid group separation, deconjugation, derivatization and gas chromatography-mass spectrometry analysis. We applied the method on brain and liver samples of mice (10 months and 3 weeks old) and cultured N2a cells and report the endogenous concentrations of 29 physiological steroids.
Asunto(s)
Encéfalo/metabolismo , Cromatografía de Gases y Espectrometría de Masas/métodos , Hígado/metabolismo , Esteroides/análisis , Sulfatos/análisis , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Esteroides/metabolismo , Sulfatos/metabolismoRESUMEN
The biophysical properties and biological functions of membranes are highly dependent on lipid composition. Supplementing cellular membranes with very long chain fatty acids (vlcFAs) is notoriously difficult given the extreme insolubility of vlcFAs in aqueous solution. Herein, we report a solvent-free, photochemical approach to enrich target membranes with vlcFA. To prevent aggregation of vlcFA, we created light-sensitive micelles composed exclusively of poly-ethylene-glycol-nervonic acid amphiphiles (NA-PEG), which spontaneously disassemble in the presence of lipid bilayers. Once embedded within a membrane, UV light is used to cleave off PEG, leaving free nervonic acid (NA, i.e. FA24:1) in the target membrane. When applied to living cells, free NA was processed by the cell to generate various species of membrane and other lipids with incorporated vlcFAs. In this way, we were able to alter the membrane lipid composition of cellular membranes and modulate the enzymatic activity of γ-secretase, an intramembrane protease whose dysfunction has been implicated in the onset and progression of Alzheimer's disease.
Asunto(s)
Membrana Celular/química , Ácidos Grasos/química , Membrana Dobles de Lípidos/química , Enfermedad de Alzheimer , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Membrana Celular/metabolismo , Ácidos Grasos Monoinsaturados/química , Humanos , Membrana Dobles de Lípidos/aislamiento & purificación , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Membranas/metabolismo , Micelas , Procesos Fotoquímicos , Polietilenglicoles/químicaRESUMEN
The uncoupling protein (UCP1) is a proton (H+) transporter in the mitochondrial inner membrane. By dissipating the electrochemical H+ gradient, UCP1 uncouples respiration from ATP synthesis, which drives an increase in substrate oxidation via the TCA cycle flux that generates more heat. The mitochondrial uncoupling-mediated non-shivering thermogenesis in brown adipose tissue is vital primarily to mammals, such as rodents and new-born humans, but more recently additional functions in adult humans have been described. UCP1 is regulated by ß-adrenergic receptors through the sympathetic nervous system and at the molecular activity level by nucleotides and fatty acid to meet thermogenesis needs. The discovery of novel UCP homologs has greatly contributed to the understanding of human diseases, such as obesity and diabetes. In this article, we review the progress made towards the molecular mechanism and function of the UCPs, in particular focusing on the influential contributions from Martin Klingenberg's laboratory. Because all members of the UCP family are potentially promising drug targets, we also present and discuss possible approaches and methods for UCP-related drug discovery.
Asunto(s)
Proteínas Desacopladoras Mitocondriales/química , Proteínas Desacopladoras Mitocondriales/metabolismo , Adenosina Trifosfato/metabolismo , Tejido Adiposo Pardo/metabolismo , Animales , Sitios de Unión , Ácidos Grasos no Esterificados/metabolismo , Humanos , Unión Proteica , Termogénesis/fisiologíaRESUMEN
Bexarotene is a pleiotropic molecule that has been proposed as an amyloid-ß (Aß)-lowering drug for the treatment of Alzheimer's disease (AD). It acts by upregulation of an apolipoprotein E (apoE)-mediated Aß clearance mechanism. However, whether bexarotene induces removal of Aß plaques in mouse models of AD has been controversial. Here, we show by NMR and CD spectroscopy that bexarotene directly interacts with and stabilizes the transmembrane domain α-helix of the amyloid precursor protein (APP) in a region where cholesterol binds. This effect is not mediated by changes in membrane lipid packing, as bexarotene does not share with cholesterol the property of inducing phospholipid condensation. Bexarotene inhibited the intramembrane cleavage by γ-secretase of the APP C-terminal fragment C99 to release Aß in cell-free assays of the reconstituted enzyme in liposomes, but not in cells, and only at very high micromolar concentrations. Surprisingly, in vitro, bexarotene also inhibited the cleavage of Notch1, another major γ-secretase substrate, demonstrating that its inhibition of γ-secretase is not substrate specific and not mediated by acting via the cholesterol binding site of C99. Our data suggest that bexarotene is a pleiotropic molecule that interfere with Aß metabolism through multiple mechanisms.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/metabolismo , Bexaroteno/farmacología , Liposomas/metabolismo , Fármacos Neuroprotectores/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Bexaroteno/química , Colesterol/metabolismo , Células HEK293 , Humanos , Estructura Molecular , Fármacos Neuroprotectores/química , Fosfatidilcolinas/química , Conformación Proteica en Hélice alfa , Dominios Proteicos , Receptor Notch1/metabolismo , Electricidad EstáticaRESUMEN
The amyloid precursor protein (APP) is a single-span integral membrane protein whose C-terminal fragment C99 is cleaved within the transmembrane helix by γ-secretase. Cleavage produces various Aß peptides that are linked to the etiology of Alzheimer's disease. The transmembrane helix is known to homodimerize in a sequence-specific manner, and considerable controversy about whether the homodimeric form of C99 is cleaved by γ-secretase exists. Here, we generated various covalent C99 homodimers via cross-linking at engineered cysteine residues. None of the homodimers was cleaved in vitro by purified γ-secretase, strongly suggesting that homodimerization protects C99 from cleavage.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Péptidos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/química , Cisteína/análisis , Cisteína/metabolismo , Humanos , Multimerización de ProteínaRESUMEN
Intramembrane proteolysis has emerged as a key mechanism required for membrane proteostasis and cellular signaling. One of the intramembrane-cleaving proteases (I-CLiPs), γ-secretase, is also intimately implicated in Alzheimer's disease, a major neurodegenerative disease and leading cause of dementia. High-resolution crystal structural analyses have revealed that I-CLiPs harbor their active sites buried deeply in the membrane bilayer. Surprisingly, however, the key kinetic constants of these proteases, turnover number kcat and catalytic efficiency kcat/KM, are largely unknown. By investigating the kinetics of intramembrane cleavage of the Alzheimer's disease-associated ß-amyloid precursor protein in vitro and in human embryonic kidney cells, we show that γ-secretase is a very slow protease with a kcat value similar to those determined recently for rhomboid-type I-CLiPs. Our results indicate that low turnover numbers may be a general feature of I-CLiPs.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Proteolisis , Células HEK293 , Humanos , Membranas Intracelulares/química , CinéticaRESUMEN
Pathogenic generation of amyloid ß-peptide (Aß) by sequential cleavage of ß-amyloid precursor protein (APP) by ß- and γ-secretases is widely believed to causally underlie Alzheimer disease (AD). ß-Secretase initially cleaves APP thereby generating a membrane-bound APP C-terminal fragment, from which γ-secretase subsequently liberates 37-43-amino acid long Aß species. Although the latter cleavages are intramembranous and although lipid alterations have been implicated in AD, little is known of how the γ-secretase-mediated release of the various Aß species, in particular that of the pathogenic longer variants Aß(42) and Aß(43), is affected by the lipid environment. Using a cell-free system, we have directly and systematically investigated the activity of γ-secretase reconstituted in defined model membranes of different thicknesses. We found that bilayer thickness is a critical parameter affecting both total activity as well as cleavage specificity of γ-secretase. Whereas the generation of the pathogenic Aß(42/43) species was markedly attenuated in thick membranes, that of the major and rather benign Aß(40) species was enhanced. Moreover, the increased production of Aß(42/43) by familial AD mutants of presenilin 1, the catalytic subunit of γ-secretase, could be substantially lowered in thick membranes. Our data demonstrate an effective modulation of γ-secretase activity by membrane thickness, which may provide an approach to lower the generation of the pathogenic Aß(42/43) species.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Membrana Celular/metabolismo , Lípidos de la Membrana/metabolismo , Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/genética , Péptidos beta-Amiloides/genética , Membrana Celular/patología , Células HEK293 , Humanos , Lípidos de la Membrana/genética , MutaciónRESUMEN
The two proteases beta-secretase and gamma-secretase generate the amyloid beta peptide and are drug targets for Alzheimer's disease. Here we tested the possibility of targeting the cellular environment of beta-secretase cleavage instead of the beta-secretase enzyme itself. beta-Secretase has an acidic pH optimum and cleaves the amyloid precursor protein in the acidic endosomes. We identified two drugs, bepridil and amiodarone, that are weak bases and are in clinical use as calcium antagonists. Independently of their calcium-blocking activity, both compounds mildly raised the membrane-proximal, endosomal pH and inhibited beta-secretase cleavage at therapeutically achievable concentrations in cultured cells, in primary neurons, and in vivo in guinea pigs. This shows that an alkalinization of the cellular environment could be a novel therapeutic strategy to inhibit beta-secretase. Surprisingly, bepridil and amiodarone also modulated gamma-secretase cleavage independently of endosomal alkalinization. Thus, both compounds act as dual modulators that simultaneously target beta- and gamma-secretase through distinct molecular mechanisms. In addition to Alzheimer's disease, compounds with dual properties may also be useful for drug development targeting other membrane proteins.
Asunto(s)
Amiodarona/farmacología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Bepridil/farmacología , Inhibidores Enzimáticos/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , Amiodarona/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/sangre , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Bepridil/química , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Encéfalo/metabolismo , Línea Celular , Células Cultivadas , Inhibidores Enzimáticos/química , Femenino , Cobayas , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/metabolismo , Nexinas de Proteasas , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismoRESUMEN
CK1 and gamma-secretase are interesting targets for therapeutic intervention in the treatment of cancer and Alzheimer's disease. The CK1 inhibitor IC261 was reported to inhibit gamma-secretase activity. The question is: Does CK1 inhibition directly influence gamma-secretase activity? Therefore we analyzed the SAR of 15 analogues and their impact on gamma-secretase activity. The most active compounds were investigated on CK1delta activity. These findings exclude a direct influence of CK1delta on gamma-secretase, because any change in the substitution pattern of IC261 diminished CK1 inhibition, whereas gamma-secretase inhibition is still exerted by several analogues.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Quinasa de la Caseína I/antagonistas & inhibidores , Quinasa Idelta de la Caseína/antagonistas & inhibidores , Indoles/química , Floroglucinol/análogos & derivados , Inhibidores de Proteasas/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Sitios de Unión , Quinasa de la Caseína I/metabolismo , Quinasa Idelta de la Caseína/metabolismo , Indoles/farmacología , Floroglucinol/química , Floroglucinol/farmacología , Inhibidores de Proteasas/farmacología , Relación Estructura-ActividadRESUMEN
Pathogenic generation of the 42-amino acid variant of the amyloid beta-peptide (Abeta) by beta- and gamma-secretase cleavage of the beta-amyloid precursor protein (APP) is believed to be causative for Alzheimer disease (AD). Lowering of Abeta(42) production by gamma-secretase modulators (GSMs) is a hopeful approach toward AD treatment. The mechanism of GSM action is not fully understood. Moreover, whether GSMs target the Abeta domain is controversial. To further our understanding of the mode of action of GSMs and the cleavage mechanism of gamma-secretase, we analyzed mutations located at different positions of the APP transmembrane domain around or within the Abeta domain regarding their response to GSMs. We found that Abeta(42)-increasing familial AD mutations of the gamma-secretase cleavage site domain responded robustly to Abeta(42)-lowering GSMs, especially to the potent compound GSM-1, irrespective of the amount of Abeta(42) produced. We thus expect that familial AD patients carrying mutations at the gamma-secretase cleavage sites of APP should respond to GSM-based therapeutic approaches. Systematic phenylalanine-scanning mutagenesis of this region revealed a high permissiveness to GSM-1 and demonstrated a complex mechanism of GSM action as other Abeta species (Abeta(41), Abeta(39)) could also be lowered besides Abeta(42). Moreover, certain mutations simultaneously increased Abeta(42) and the shorter peptide Abeta(38), arguing that the proposed precursor-product relationship of these Abeta species is not general. Finally, mutations of residues in the proposed GSM-binding site implicated in Abeta(42) generation (Gly-29, Gly-33) and potentially in GSM-binding (Lys-28) were also responsive to GSMs, a finding that may question APP substrate targeting of GSMs.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/fisiología , Enfermedad de Alzheimer/metabolismo , Secuencias de Aminoácidos , Antiinflamatorios no Esteroideos/farmacología , Línea Celular , Sistema Libre de Células , Humanos , Inmunoensayo , Modelos Biológicos , Mutación , Péptido Hidrolasas/química , Péptidos/química , Fenilalanina/genética , Unión ProteicaRESUMEN
Gamma-secretase is a unique intramembrane-cleaving protease complex, which cleaves the Alzheimer's disease-associated beta-amyloid precursor protein (APP) and a number of other type I membrane proteins. Human gamma-secretase consists of the catalytic subunit presenilin (PS) (PS1 or PS2), the substrate receptor nicastrin, APH-1 (APH-1a or APH-1b), and PEN-2. To facilitate in-depth biochemical analysis of gamma-secretase, we developed a fast and convenient multistep purification procedure for the endogenous enzyme. The enzyme was purified from HEK293 cells in an active form and had a molecular mass of approximately 500 kDa. Purified gamma-secretase was capable of producing the major amyloid-beta peptide (Abeta) species, such as Abeta40 and Abeta42, from a recombinant APP substrate in physiological ratios. Abeta generation could be modulated by pharmacological gamma-secretase modulators. Moreover, the Abeta42/Abeta40 ratio was strongly increased by purified PS1 L166P, an aggressive familial Alzheimer's disease mutant. Tandem mass spectrometry analysis revealed the consistent coisolation of several proteins with the known gamma-secretase core subunits. Among these were the previously described gamma-secretase interactors CD147 and TMP21 as well as other known interactors of these. Interestingly, the Niemann-Pick type C1 protein, a cholesterol transporter previously implicated in gamma-secretase-mediated processing of APP, was identified as a major copurifying protein. Affinity capture experiments using a biotinylated transition-state analogue inhibitor of gamma-secretase showed that these proteins are absent from active gamma-secretase complexes. Taken together, we provide an effective procedure for isolating endogenous gamma-secretase in considerably high grade, thus aiding further characterization of this pivotal enzyme. In addition, we provide evidence that the copurifying proteins identified are unlikely to be part of the active gamma-secretase enzyme.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/aislamiento & purificación , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Inhibidores Enzimáticos/farmacología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/química , Biotinilación/efectos de los fármacos , Línea Celular , Cromatografía de Afinidad , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Humanos , Peso Molecular , Complejos Multiproteicos/metabolismo , Proteínas Mutantes/metabolismo , Unión Proteica/efectos de los fármacos , Subunidades de Proteína/metabolismo , Especificidad por Sustrato/efectos de los fármacos , Espectrometría de Masas en TándemRESUMEN
Gamma-secretase is an intramembrane cleaving aspartyl protease complex intimately implicated in Alzheimer disease pathogenesis. The protease is composed of the catalytic subunit presenilin (PS1 or PS2), the substrate receptor nicastrin (NCT), and two additional subunits, APH-1 (APH-1a, as long and short splice forms (APH-1aL, APH-1aS), or APH-1b) and PEN-2. Apart from the Alzheimer disease-associated beta-amyloid precursor protein, gamma-secretase has been shown to cleave a large number of other type I membrane proteins. Despite the progress in elucidating gamma-secretase function, basic questions concerning the precise organization of its subunits, their molecular interactions, and their exact stoichiometry in the complex are largely unresolved. Here we isolated endogenous human gamma-secretase from human embryonic kidney 293 cells and investigated the subunit architecture of the gamma-secretase complex formed by PS1, NCT, APH-1aL, and PEN-2 by chemical cross-linking. Using this approach, we provide evidence for the close neighborhood of the PS1 N- and C-terminal fragments (NTF and CTF, respectively), the PS1 NTF and PEN-2, the PS1 CTF and APH-1aL, and NCT and APH-1aL. We thus identify a previously unrecognized PS1 CTF/APH-1aL interaction, verify subunit interactions deduced previously from indirect approaches, and provide a model of the gamma-secretase complex subunit architecture. Finally, we further show that, like the PS1 CTF, the PS2 CTF also interacts with APH-1aL, and we provide evidence that these interactions also occur with the other APH-1 variants, suggesting similar subunit architectures of all gamma-secretase complexes.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas de la Membrana/química , Presenilinas/química , Línea Celular , Cromatografía por Intercambio Iónico , Reactivos de Enlaces Cruzados/farmacología , Dimerización , Electroforesis en Gel Bidimensional , Endopeptidasas , Humanos , Péptido Hidrolasas , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
More than 150 familial Alzheimer disease (FAD)-associated missense mutations in presenilins (PS1 and PS2), the catalytic subunit of the gamma-secretase complex, cause aberrant amyloid beta-peptide (Abeta) production, by increasing the relative production of the highly amyloidogenic 42-amino acid variant. The molecular mechanism behind this pathological activity is unclear, and different possibilities ranging from a gain of function to a loss of function have been discussed. gamma-Secretase, signal peptide peptidase (SPP) and SPP-like proteases (SPPLs) belong to the same family of GXGD-type intramembrane cleaving aspartyl proteases and share several functional similarities. We have introduced the FAD-associated PS1 G384A mutation, which occurs within the highly conserved GXGD motif of PS1 right next to the catalytically critical aspartate residue, into the corresponding GXGD motif of the signal peptide peptidase-like 2b (SPPL2b). Compared with wild-type SPPL2b, mutant SPPL2b slowed intramembrane proteolysis of tumor necrosis factor alpha and caused a relative increase of longer intracellular cleavage products. Because the N termini of the secreted counterparts remain unchanged, the mutation selectively affects the liberation of the intracellular processing products. In vitro experiments demonstrate that the apparent accumulation of longer intracellular cleavage products is the result of slowed sequential intramembrane cleavage. The longer cleavage products are still converted to shorter peptides, however only after prolonged incubation time. This suggests that FAD-associated PS mutation may also result in reduced intramembrane cleavage of beta-amyloid precursor protein (betaAPP). Indeed, in vitro experiments demonstrate slowed intramembrane proteolysis by gamma-secretase containing PS1 with the G384A mutation. As compared with wild-type PS1, the mutation selectively slowed Abeta40 production, whereas Abeta42 generation remained unaffected. Thus, the PS1 G384A mutation causes a selective loss of function by slowing the processing pathway leading to the benign Abeta40.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Ácido Aspártico Endopeptidasas/genética , Mutación , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidasas/química , Catálisis , Línea Celular , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Péptidos/química , Estructura Terciaria de Proteína , Temperatura , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
gamma-Secretase is an intramembrane cleaving protease involved in Alzheimer's disease. gamma-Secretase occurs as a high molecular weight complex composed of presenilin (PS1/2), nicastrin (NCT), anterior pharynx-defective phenotype 1 and PS enhancer 2. Little is known about the cellular mechanisms of gamma-secretase assembly. Here we demonstrate that the cytoplasmic tail of PS1 fulfills several functions required for complex formation, retention of unincorporated PS1 and gamma-secretase activity. The very C-terminus interacts with the transmembrane domain of NCT and may penetrate into the membrane. Deletion of the last amino acid is sufficient to completely block gamma-secretase assembly and release of PS1 from the endoplasmic reticulum (ER). This suggests that unincorporated PS1 is actively retained within the ER. We identified a hydrophobic stretch of amino acids within the cytoplasmic tail of PS1 distinct from the NCT-binding site, which is required to retain unincorporated PS1 within the ER. Deletion of the retention signal results in the release of PS1 from the ER and the assembly of a nonfunctional gamma-secretase complex, suggesting that at least a part of the retention motif may also be required for the function of PS1.
Asunto(s)
Endopeptidasas/metabolismo , Retículo Endoplásmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Animales , Células COS , Chlorocebus aethiops , Glicoproteínas de Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Presenilina-1 , Unión Proteica , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de SecuenciaRESUMEN
gamma-Secretase is a membrane protein complex with an unusual aspartyl protease activity that catalyses the regulated intramembranous cleavage of the beta-amyloid precursor protein (APP) to release the Alzheimer's disease (AD)-associated amyloid beta-peptide (Abeta) and the APP intracellular domain (AICD). Here we show the reconstitution of gamma-secretase activity in the yeast Saccharomyces cerevisiae, which lacks endogenous gamma-secretase activity. Reconstituted gamma-secretase activity depends on the presence of four complex components including presenilin (PS), nicastrin (Nct), APH-1 (refs 3-6) and PEN-2 (refs 4, 7), is associated with endoproteolysis of PS, and produces Abeta and AICD in vitro. Thus, the biological activity of gamma-secretase is reconstituted by the co-expression of human PS, Nct, APH-1 and PEN-2 in yeast.
Asunto(s)
Membrana Celular/enzimología , Endopeptidasas/metabolismo , Células Eucariotas/enzimología , Saccharomyces cerevisiae/enzimología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides/biosíntesis , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidasas , Células Cultivadas , Regulación Enzimológica de la Expresión Génica/genética , Regulación Fúngica de la Expresión Génica/genética , Genes Reporteros/genética , Humanos , Sustancias Macromoleculares , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Péptido Hidrolasas , Presenilina-1 , Proteínas Recombinantes de FusiónRESUMEN
The Alzheimer disease-associated presenilin (PS) proteins apparently provide the active site of gamma-secretase, an unusual intramembrane-cleaving aspartyl protease. PSs principally occur as high molecular weight protein complexes that contain nicastrin (Nct) and additional so far unidentified components. Recently, PEN-2 has been implicated in gamma-secretase function. Here we identify PEN-2 as a critical component of PS1/gamma-secretase and PS2/gamma-secretase complexes. Strikingly, in the absence of PS1 and PS1/PS2, PEN-2 levels are strongly reduced. Similarly, PEN-2 levels are reduced upon RNA interference-mediated down-regulation of Nct. On the other side, down-regulation of PEN-2 by RNA interference is associated with reduced PS levels, impaired Nct maturation, and deficient gamma-secretase complex formation. We conclude that PEN-2 is an integral gamma-secretase complex component and that gamma-secretase complex components are expressed in a coordinated manner.
Asunto(s)
Endopeptidasas/metabolismo , Glicoproteínas de Membrana/biosíntesis , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas , Línea Celular , ADN Complementario/metabolismo , Regulación hacia Abajo , Electroforesis en Gel de Poliacrilamida , Epítopos , Humanos , Ratones , Ratones Noqueados , Pruebas de Precipitina , Presenilina-1 , Presenilina-2 , Unión Proteica , Estructura Terciaria de Proteína , ARN/metabolismoRESUMEN
The Alzheimer's disease (AD)-associated presenilin (PS) proteins are required for the gamma-secretase cleavages of the beta-amyloid precursor protein and the site 3 (S3) protease cleavage of Notch. These intramembrane cleavages release amyloid-beta peptide (Abeta), including the pathogenic 42-aa variant (Abeta(42)), as well as the beta-amyloid precursor protein and the Notch intracellular domains (AICD, NICD). Whereas Abeta is generated by endoproteolysis in the middle of the transmembrane domain, AICD and NICD are generated by cleavages at analogous positions close to the cytoplasmic border of the transmembrane domain. Numerous mutations causing familial AD (FAD) that all cause increased production of Abeta(42) have been found in the PS1 gene. Here we have investigated the previously uncharacterized, very aggressive FAD mutation L166P that causes onset of AD in adolescence. Strikingly, the PS1 L166P mutation not only induces an exceptionally high increase of Abeta(42) production but also impairs NICD production and Notch signaling, as well as AICD generation. Thus, FAD-associated PS mutants cannot only affect the generation of NICD, but also that of AICD. Moreover, further analysis with artificial L166 mutants revealed that the gamma-secretase cleavage at position 40/42 and the S3-like gamma-secretase cleavage at position 49 of the Abeta domain are both differentially affected by PS1 L166 mutants. Finally, we show that PS1 L166 mutants affect the generation of NICD and AICD in a similar manner, supporting the concept that S3 protease and S3-like gamma-secretase cleavages are mediated by identical proteolytic activities.
Asunto(s)
Enfermedad de Alzheimer/genética , Sustitución de Aminoácidos , Péptidos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/metabolismo , Leucina , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Fragmentos de Péptidos/genética , Precursor de Proteína beta-Amiloide/química , Línea Celular , Fibroblastos , Humanos , Riñón , Proteínas de la Membrana/química , Proteínas de la Membrana/deficiencia , Presenilina-1 , Receptores Notch , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Amyloid beta-peptide (Abeta) is generated by the consecutive cuts of two membrane-bound proteases. Beta-secretase cuts at the N terminus of the Abeta domain, whereas gamma-secretase mediates the C-terminal cut. Recent evidence suggests that the presenilin (PS) proteins, PS1 and PS2, may be gamma-secretases. Because PSs principally exist as high molecular weight protein complexes, biologically active gamma-secretases likely require other cofactors such as nicastrin (Nct) for their activities. Here we show that preferentially mature Nct forms a stable complex with PSs. Furthermore, we have down-regulated Nct levels by using a highly specific and efficient RNA interference approach. Very similar to a loss of PS function, down-regulation of Nct levels leads to a massive accumulation of the C-terminal fragments of the beta-amyloid precursor protein. In addition, Abeta production was markedly reduced. Strikingly, down-regulation of Nct destabilized PS and strongly lowered levels of the high molecular weight PS1 complex. Interestingly, absence of the PS1 complex in PS1(-/-) cells was associated with a strong down-regulation of the levels of mature Nct, suggesting that binding to PS is required for trafficking of Nct through the secretory pathway. Based on these findings we conclude that Nct and PS regulate each other and determine gamma-secretase function via complex formation.