RESUMO
γ-Secretase is a multisubunit membrane protein complex containing presenilin (PS1) as a catalytic subunit. Familial Alzheimer disease (FAD) mutations within PS1 were analyzed in yeast cells artificially expressing membrane-bound substrate, amyloid precursor protein, or Notch fused to Gal4 transcriptional activator. The FAD mutations, L166P and G384A (Leu-166 to Pro and Gly-384 to Ala substitution, respectively), were loss-of-function in yeast. We identified five amino acid substitutions that suppress the FAD mutations. The cleavage of amyloid precursor protein or Notch was recovered by the secondary mutations. We also found that secondary mutations alone activated the γ-secretase activity. FAD mutants with suppressor mutations, L432M or S438P within TMD9 together with a missense mutation in the second or sixth loops, regained γ-secretase activity when introduced into presenilin null mouse fibroblasts. Notably, the cells with suppressor mutants produced a decreased amount of Aß42, which is responsible for Alzheimer disease. These results indicate that the yeast system is useful to screen for mutations and chemicals that modulate γ-secretase activity.
Assuntos
Doença de Alzheimer/enzimologia , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Mutação/genética , Presenilina-1/genética , Supressão Genética , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Embrião de Mamíferos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Camundongos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Presenilina-1/química , Estrutura Terciária de Proteína , Receptores Notch/metabolismo , Supressão Genética/efeitos dos fármacos , beta-Galactosidase/metabolismoRESUMO
γ-Secretase is an intramembrane-cleaving protease responsible for the generation of amyloid-ß (Aß) peptides. Recently, a series of compounds called γ-secretase modulators (GSMs) has been shown to decrease the levels of long toxic Aß species (i.e., Aß42), with a concomitant elevation of the production of shorter Aß species. In this study, we show that a phenylimidazole-type GSM allosterically induces conformational changes in the catalytic site of γ-secretase to augment the proteolytic activity. Analyses using the photoaffinity labeling technique and systematic mutational studies revealed that the phenylimidazole-type GSM targets a previously unidentified extracellular binding pocket within the N-terminal fragment of presenilin (PS). Collectively, we provide a model for the mechanism of action of the phenylimidazole-type GSM in which binding at the luminal side of PS induces a conformational change in the catalytic center of γ-secretase to modulate Aß production.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Imidazóis/farmacologia , Regulação Alostérica/efeitos dos fármacos , Aminoácidos/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Domínio Catalítico , Ativação Enzimática/efeitos dos fármacos , Fluorescência , Humanos , Imidazóis/química , Modelos Moleculares , Mutação/genética , Peptídeos/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato/efeitos dos fármacosRESUMO
γ-Secretase is a multisubunit protease complex that is responsible for generating amyloid-ß peptides, which are associated with Alzheimer disease. The catalytic subunit of γ-secretase is presenilin 1 (PS1), which contains an initial substrate-binding site that is distinct from the catalytic site. Processive cleavage is suggested in the intramembrane-cleaving mechanism of γ-secretase. However, it largely remains unknown as to how γ-secretase recognizes its substrate during proteolysis. Here, we identified that the α-helical structural region of hydrophilic loop 1 (HL1) and the C-terminal region of human PS1 are distinct substrate-binding sites. Mutational analyses revealed that substrate binding to the HL1 region is critical for both ε- and γ-cleavage, whereas binding to the C-terminal region hampers γ-cleavage. Moreover, we propose that substrate binding triggers conformational changes in PS1, rendering it suitable for catalysis. Our data provide new insights into the complicated catalytic mechanism of PS1.
Assuntos
Secretases da Proteína Precursora do Amiloide/fisiologia , Presenilina-1/fisiologia , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/genética , Animais , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Presenilina-1/química , Presenilina-1/genética , Estrutura Secundária de Proteína , Especificidade por Substrato , TransfecçãoRESUMO
Amyloid-ß peptide ending at the 42nd residue (Aß42) is implicated in the pathogenesis of Alzheimer's disease (AD). Small compounds that exhibit selective lowering effects on Aß42 production are termed γ-secretase modulators (GSMs) and are deemed as promising therapeutic agents against AD, although the molecular target as well as the mechanism of action remains controversial. Here, we show that a phenylpiperidine-type compound GSM-1 directly targets the transmembrane domain (TMD) 1 of presenilin 1 (PS1) by photoaffinity labelling experiments combined with limited digestion. Binding of GSM-1 affected the structure of the initial substrate binding and the catalytic sites of the γ-secretase, thereby decreasing production of Aß42, possibly by enhancing its conversion to Aß38. These data indicate an allosteric action of GSM-1 by directly binding to the TMD1 of PS1, pinpointing the target structure of the phenylpiperidine-type GSMs.
Assuntos
Regulação Alostérica/efeitos dos fármacos , Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Piperidinas/farmacologia , Presenilina-1 , Conformação Proteica/efeitos dos fármacos , Doença de Alzheimer/fisiopatologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/análise , Animais , Sítios de Ligação/efeitos dos fármacos , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/análise , Piperidinas/síntese química , Presenilina-1/química , Presenilina-1/metabolismo , Ligação Proteica/efeitos dos fármacosRESUMO
Amyloid ß-peptide (Aß) pathology is an invariant feature of Alzheimer disease, preceding any detectable clinical symptoms by more than a decade. To this end, we seek to identify agents that can reduce Aß levels in the brain via novel mechanisms. We found that (20S)-Rg3, a triterpene natural compound known as ginsenoside, reduced Aß levels in cultured primary neurons and in the brains of a mouse model of Alzheimer disease. The (20S)-Rg3 treatment induced a decrease in the association of presenilin 1 (PS1) fragments with lipid rafts where catalytic components of the γ-secretase complex are enriched. The Aß-lowering activity of (20S)-Rg3 directly correlated with increased activity of phosphatidylinositol 4-kinase IIα (PI4KIIα), a lipid kinase that mediates the rate-limiting step in phosphatidylinositol 4,5-bisphosphate synthesis. PI4KIIα overexpression recapitulated the effects of (20S)-Rg3, whereas reduced expression of PI4KIIα abolished the Aß-reducing activity of (20S)-Rg3 in neurons. Our results substantiate an important role for PI4KIIα and phosphoinositide modulation in γ-secretase activity and Aß biogenesis.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ginsenosídeos/farmacologia , Microdomínios da Membrana/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Presenilina-1/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Ativação Enzimática/efeitos dos fármacos , Ginsenosídeos/química , Humanos , Microdomínios da Membrana/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Receptores de Fator de Crescimento Neural/química , Receptores de Fator de Crescimento Neural/metabolismo , Receptores Notch/química , Receptores Notch/metabolismo , Relação Estrutura-AtividadeRESUMO
Presenilin 1 (PS1) comprises a catalytic subunit of γ-secretase, which is an intramembrane-cleaving protease responsible for generation of amyloid-ß peptides as well as Notch cleavage, the latter being implicated in cancer. We have shown that transmembrane domains (TMDs) 1, 6, 7, and 9 of PS1 form the "catalytic pore" structure within the membrane for intramembrane proteolysis. Here we report a novel monoclonal antibody 9D11, which directly recognizes the TMD1-proximal residues in the hydrophilic loop region. Intriguingly, 9D11 inhibited the γ-secretase activity irrespective of the binding of known γ-secretase inhibitors and abolished Notch signaling-dependent cancer cell viability. Our data suggest that the juxtamembrane region of TMD1 of PS1 is a novel molecular target for the mechanism-based inhibition of γ-secretase and the development of the anticancer drug.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Presenilina-1/imunologia , Animais , Domínio Catalítico , Linhagem Celular , Humanos , Interações Hidrofóbicas e Hidrofílicas , Presenilina-1/química , Estrutura Terciária de Proteína , RatosRESUMO
Sphingosine kinase (SphK) 1 and 2 phosphorylate sphingosine to generate sphingosine-1-phosphate (S1P), a pluripotent lipophilic mediator implicated in a variety of cellular events. Here we show that the activity of ß-site APP cleaving enzyme-1 (BACE1), the rate-limiting enzyme for amyloid-ß peptide (Aß) production, is modulated by S1P in mouse neurons. Treatment by SphK inhibitor, RNA interference knockdown of SphK, or overexpression of S1P degrading enzymes decreased BACE1 activity, which reduced Aß production. S1P specifically bound to full-length BACE1 and increased its proteolytic activity, suggesting that cellular S1P directly modulates BACE1 activity. Notably, the relative activity of SphK2 was upregulated in the brains of patients with Alzheimer's disease. The unique modulatory effect of cellular S1P on BACE1 activity is a novel potential therapeutic target for Alzheimer's disease.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Lisofosfolipídeos/metabolismo , Neurônios/metabolismo , Esfingosina/análogos & derivados , Peptídeos beta-Amiloides/metabolismo , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Humanos , Camundongos , Neurônios/citologia , Interferência de RNA , Esfingosina/metabolismoRESUMO
Signal peptide peptidase (SPP) is an atypical aspartic protease that hydrolyzes peptide bonds within the transmembrane domain of substrates and is implicated in several biological and pathological functions. Here, we analyzed the structure of human SPP by electron microscopy and reconstructed the three-dimensional structure at a resolution of 22 Å. Enzymatically active SPP forms a slender, bullet-shaped homotetramer with dimensions of 85 × 85 × 130 Å. The SPP complex has four concaves on the rhombus-like sides, connected to a large chamber inside the molecule. Intriguingly, the N-terminal region of SPP is sufficient for the tetrameric assembly. Moreover, overexpression of the N-terminal region inhibited the formation of the endogenous SPP tetramer and the proteolytic activity within cells. These data suggest that the homotetramer is the functional unit of SPP and that its N-terminal region, which works as the structural scaffold, has a novel modulatory function for the intramembrane-cleaving activity of SPP.
Assuntos
Ácido Aspártico Endopeptidases/química , Animais , Ácido Aspártico Endopeptidases/metabolismo , Células HEK293 , Humanos , Microscopia Eletrônica , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de ProteínaRESUMO
Gamma-secretase is a membrane protein complex that catalyzes intramembrane proteolysis of a variety of substrates including the amyloid beta precursor protein of Alzheimer disease. Nicastrin (NCT), a single-pass membrane glycoprotein that harbors a large extracellular domain, is an essential component of the gamma-secretase complex. Here we report that overexpression of a single chain variable fragment (scFv) against NCT as an intrabody suppressed the gamma-secretase activity. Biochemical analyses revealed that the scFv disrupted the proper folding and the appropriate glycosyl maturation of the endogenous NCT, which are required for the stability of the gamma-secretase complex and the intrinsic proteolytic activity, respectively, implicating the dual role of NCT in the gamma-secretase complex. Our results also highlight the importance of the calnexin cycle in the functional maturation of the gamma-secretase complex. The engineered intrabodies may serve as rationally designed, molecular targeting tools for the discovery of novel actions of the membrane proteins.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Anticorpos/imunologia , Região Variável de Imunoglobulina/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/imunologia , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/imunologia , Animais , Anticorpos/genética , Linhagem Celular , Inibidores Enzimáticos/metabolismo , Humanos , Região Variável de Imunoglobulina/genética , Indolizinas/metabolismo , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Screening of our library of peroxisome proliferator-activated receptor (PPAR) agonists yielded several phenylpropanoic acid-derived gamma-secretase inhibitors (GSIs). Structure-activity relationship studies indicated that (R)-configuration of alpha-substituted phenylpropanoic acid structure and cinnamic acid structure is favorable to prepare Notch-sparing GSIs.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , PPAR gama/agonistas , Receptores Notch/metabolismo , Relação Estrutura-AtividadeRESUMO
BACKGROUND: The presenilin-1 protein (PS1) is the catalytic unit of γ-secretase implicated in the production of abnormally long forms of amyloid-ß (Aß), including Aß42, proteins thought critical in the pathogenesis of Alzheimer's disease (AD). In AD of autosomal dominant inheritance, the majority of pathogenic mutations have been found in the PSEN1 gene within which the location of the mutation can provide clues as to the mechanism of pathogenesis. OBJECTIVE: To describe clinical features of two novel mutations in the transmembrane portion 1 (TMD-1) of PSEN1 as well as biochemical features in one and neuropathological findings in the other. METHODS: Two index patients with young onset AD with an autosomal dominant pattern of inheritance underwent clinical and imaging assessments, as well as PSEN1 sequencing. Postmortem examination was completed in one patient. An artificial construct in which the P88L mutation was introduced was created to examine its effects on γ-secretase cleavage. RESULTS: Two novel variants in TMD-1 (P88L and V89L) were identified in affected probands. The neuropathological findings of AD were confirmed in the V89L mutation. Both patients presented around age 40 with early short-term memory deficits followed by seizures and corticospinal tract signs. The P88L mutation additionally featured early myoclonus followed by Parkinsonism. The causal role of the P88L mutation is supported by demonstration that this mutation dramatically increased Aß42 and decreased APP and Notch intracellular domain production in vitro. CONCLUSION: Changes in a single amino acid in codons 88 and 89 of TMD-1 can result in young-onset AD. The TMD-1 of PS1 is a region important for the γ-secretase cleavage of Aß.
Assuntos
Doença de Alzheimer/genética , Mutação/genética , Presenilina-1/genética , Idoso , Doença de Alzheimer/diagnóstico por imagem , Precursor de Proteína beta-Amiloide/genética , Autopsia , Progressão da Doença , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: The recommended dose of erlotinib is 150 mg daily either 1 h before a meal (complete fasting) or 2 h after a meal (2 h post-meal), because of the food effect. METHODS: We conducted a cross-over pharmacokinetic study to compare the fed bioequivalence in the two conditions. RESULTS: Twenty-three patients with non-small cell lung cancer were included in the analysis. AUC0-24 and C max in the 2-h post-meal status were significantly higher than in the complete fasting status (GMR = 1.33, P < 0.001; GMR = 1.44, P < 0.001, respectively). However, because the concentration of erlotinib did not reach the steady state within 7 days in the complete fasting state, we conducted analyses only on day 14, which showed no significant difference in AUC0-24 or C max between the two conditions. The more rapid increase in AUC0-24 and C min did not produce any earlier and more severe toxic events. CONCLUSION: The AUC0-24 increased significantly faster (48-53 % greater) in the 2-h post-meal status than in complete fasting status, which suggested that the two gastric emptying states might differ in their absorption. However, there was no clinically significant difference in bioavailability or toxicity between the two clinically used fed conditions at least in 14 days.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Interações Alimento-Droga , Neoplasias Pulmonares/metabolismo , Período Pós-Prandial , Inibidores de Proteínas Quinases/farmacocinética , Quinazolinas/farmacocinética , Adulto , Idoso , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Estudos de Coortes , Estudos Cross-Over , Cloridrato de Erlotinib , Jejum/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Quinazolinas/administração & dosagemRESUMO
BACKGROUND: Amyloid-ß peptide ending at 42nd residue (Aß42) is believed as a pathogenic peptide for Alzheimer disease. Although γ-secretase is a responsible protease to generate Aß through a processive cleavage, the proteolytic mechanism of γ-secretase at molecular level is poorly understood. RESULTS: We found that the transmembrane domain (TMD) 1 of presenilin (PS) 1, a catalytic subunit for the γ-secretase, as a key modulatory domain for Aß42 production. Aß42-lowering and -raising γ-secretase modulators (GSMs) directly targeted TMD1 of PS1 and affected its structure. A point mutation in TMD1 caused an aberrant secretion of longer Aß species including Aß45 that are the precursor of Aß42. We further found that the helical surface of TMD1 is involved in the binding of Aß45/48 and that the binding was altered by GSMs as well as TMD1 mutation. CONCLUSIONS: Binding between PS1 TMD1 and longer Aß is critical for Aß42 production.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Presenilina-1/metabolismo , Peptídeos beta-Amiloides/química , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estrutura Quaternária de Proteína , TransfecçãoRESUMO
Substrate-selective inhibition or modulation of the activity of γ-secretase, which is responsible for the generation of amyloid-ß peptides, might be an effective strategy for prevention and treatment of Alzheimer's disease. We have shown that helical ß-peptide foldamers are potent and specific inhibitors of γ-secretase. Here we report identification of target site of the foldamers by using a photoaffinity probe. The photoprobe directly and specifically labeled the N-terminal fragment of presenilin 1, in which the initial substrate docking site is predicted to be located. We also optimized the foldamer structure by preparing a variety of derivatives and obtained two highly potent foldamers by incorporation of a hydrophilic and neutral functional group into the parent structure. The class of side chain functional group and the position of incorporation were both important for γ-secretase-inhibitory activity. The substrate selectivity of the inhibitory activity was also quite sensitive to the class of side chain group incorporated.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/química , Oligopeptídeos/química , Ciclopentanos/síntese química , Ciclopentanos/química , Ciclopentanos/farmacologia , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mimetismo Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Prolina/análogos & derivados , Prolina/síntese química , Prolina/química , Prolina/farmacologia , Estrutura Secundária de Proteína , Estereoisomerismo , Especificidade por SubstratoRESUMO
Sphingosine-1-phosphate (S1P) is a pluripotent lipophilic mediator working as a ligand for G-protein coupled S1P receptors (S1PR), which is currently highlighted as a therapeutic target for autoimmune diseases including relapsing forms of multiple sclerosis. Sphingosine related compounds, FTY720 and KRP203 known as S1PR modulators, are phosphorylated by sphingosine kinase 2 (SphK2) to yield the active metabolites FTY720-P and KRP203-P, which work as functional antagonists for S1PRs. Here we report that FTY720 and KRP203 decreased production of Amyloid-ß peptide (Aß), a pathogenic proteins causative for Alzheimer disease (AD), in cultured neuronal cells. Pharmacological analyses suggested that the mechanism of FTY720-mediated Aß decrease in cells was independent of known downstream signaling pathways of S1PRs. Unexpectedly, 6-days treatment of APP transgenic mice with FTY720 resulted in a decrease in Aß40, but an increase in Aß42 levels in brains. These results suggest that S1PR modulators are novel type of regulators for Aß metabolisms that are active in vitro and in vivo.
Assuntos
Peptídeos beta-Amiloides/biossíntese , Neurônios/metabolismo , Propilenoglicóis/farmacologia , Esfingosina/análogos & derivados , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Cloridrato de Fingolimode , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Propilenoglicóis/química , Transdução de Sinais/efeitos dos fármacos , Esfingosina/química , Esfingosina/farmacologia , Compostos de Sulfidrila/química , Compostos de Sulfidrila/farmacologiaRESUMO
Neuroligin (NLG), a postsynaptic adhesion molecule, is involved in the formation of synapses by binding to a cognate presynaptic ligand, neurexin. Here we report that neuroligin-1 (NLG1) undergoes ectodomain shedding at the juxtamembrane stalk region to generate a secreted form of NLG1 and a membrane-tethered C-terminal fragment (CTF) in adult rat brains in vivo as well as in neuronal cultures. Pharmacological and genetic studies identified ADAM10 as the major protease responsible for NLG1 shedding, the latter being augmented by synaptic NMDA receptor activation or interaction with soluble neurexin ligands. NLG1-CTF was subsequently cleaved by presenilin/γ-secretase. Secretion of soluble NLG1 was significantly upregulated under a prolonged epileptic seizure condition, and inhibition of NLG1 shedding led to an increase in numbers of dendritic spines in neuronal cultures. Collectively, neuronal activity-dependent proteolytic processing of NLG1 may negatively regulate the remodeling of spines at excitatory synapses.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteólise , Proteínas ADAM/deficiência , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/deficiência , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Animais Recém-Nascidos , Biotinilação , Proteínas de Ligação ao Cálcio , Moléculas de Adesão Celular Neuronais/genética , Células Cultivadas , Cerebelo/metabolismo , Espinhas Dendríticas/metabolismo , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Humanos , Masculino , Proteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Agonistas Muscarínicos/toxicidade , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurônios/ultraestrutura , Técnicas de Cultura de Órgãos , Pilocarpina/toxicidade , Interferência de RNA/fisiologia , Ratos , Ratos Wistar , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/metabolismo , Estado Epiléptico/patologia , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , TransfecçãoRESUMO
gamma-Secretase is an aspartic protease that hydrolyzes type I membrane proteins within the hydrophobic environment of the lipid bilayer. Using the CHAPSO-solubilized gamma-secretase assay system, we previously found that gamma-secretase activity was sensitive to the concentrations of detergent and phosphatidylcholine. This strongly suggests that the composition of the lipid bilayer has a significant impact on the activity of gamma-secretase. Recently, level of secreted beta-amyloid protein was reported to be attenuated by increasing levels of phosphatidylinositol 4,5-diphosphate (PI(4,5)P2) in cultured cells. However, it is not clear whether PI(4,5)P2 has a direct effect on gamma-secretase activity. In this study, we found that phosphoinositides directly inhibited CHAPSO-solubilized gamma-secretase activity. Interestingly, neither phosphatidylinositol nor inositol triphosphate altered gamma-secretase activity. PI(4,5)P2 was also found to inhibit gamma-secretase activity in CHAPSO-insoluble membrane microdomains (rafts). Kinetic analysis of beta-amyloid protein production in the presence of PI(4,5)P2 suggested a competitive inhibition. Even though phosphoinositides are minor phospholipids of the membrane, the concentration of PI(4,5)P2 within the intact membrane has been reported to be in the range of 4-8 mm. The presence of PI(4,5)P2-rich rafts in the membrane has been reported in a range of cell types. Furthermore, gamma-secretase is enriched in rafts. Taking these data together, we propose that phosphoinositides potentially regulate gamma-secretase activity by suppressing its association with the substrate.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Ácidos Cólicos/química , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/enzimologia , Fosfatidilcolinas/metabolismo , Secretases da Proteína Precursora do Amiloide/química , Peptídeos beta-Amiloides/química , Animais , Células CHO , Cricetinae , Cricetulus , Bicamadas Lipídicas/química , Microdomínios da Membrana/química , Fosfatidilcolinas/química , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismoRESUMO
We showed previously that cells expressing wild-type (WT) beta-amyloid precursor protein (APP) or coexpressing WTAPP and WT presenilin (PS) 1/2 produced APP intracellular domains (AICD) 49-99 and 50-99, with the latter predominating. On the other hand, the cells expressing mutant (MT) APP or coexpressing WTAPP and MTPS1/2 produced a greater proportion of AICD-(49-99) than AICD-(50-99). In addition, the expression of amyloid beta-protein (Abeta) 49 in cells resulted in predominant production of Abeta40 and that of Abeta48 leads to preferential production of Abeta42. These observations suggest that epsilon-cleavage and gamma-cleavage are interrelated. To determine the stoichiometry between Abeta and AICD, we have established a 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized gamma-secretase assay system that exhibits high specific activity. By using this assay system, we have shown that equal amounts of Abeta and AICD are produced from beta-carboxyl-terminal fragment (C99) by gamma-secretase, irrespective of WT or MTAPP and PS1/2. Although various Abeta species, including Abeta40, Abeta42, Abeta43, Abeta45, Abeta48, and Abeta49, are generated, only two species of AICD, AICD-(49-99) and AICD-(50-99), are detected. We also have found that M233T MTPS1 produced only one species of AICD, AICD-(49-99), and only one for its counterpart, Abeta48, in contrast to WT and other MTPS1s. These strongly suggest that epsilon-cleavage is the primary event, and the produced Abeta48 and Abeta49 rapidly undergo gamma-cleavage, resulting in generation of various Abeta species.