RESUMO
A complex composed of presenilin (PS), nicastrin, PEN-2, and APH-1 is absolutely required for γ-secretase activity in vivo. Evidence has emerged to suggest a role for PS as the catalytic subunit of γ-secretase, but it has not been established that PS is catalytically active in the absence of associated subunits. We now report that bacterially synthesized, recombinant PS (rPS) reconstituted into liposomes exhibits γ-secretase activity. Moreover, an rPS mutant that lacks a catalytic aspartate residue neither exhibits reconstituted γ-secretase activity nor interacts with a transition-state γ-secretase inhibitor. Importantly, we demonstrate that rPS harboring mutations that cause early onset familial Alzheimer's disease (FAD) lead to elevations in the ratio of Aß42 to Aß40 peptides produced from a wild-type APP substrate and that rPS enhances the Aß42/Aß40 peptide ratio from FAD-linked mutant APP substrates, findings that are entirely consistent with the results obtained in in vivo settings. Thus, γ-secretase cleavage specificity is an inherent property of the polypeptide. Finally, we demonstrate that PEN2 is sufficient to promote the endoproteolysis of PS1 to generate the active form of γ-secretase. Thus, we conclusively establish that activated PS is catalytically competent and the bimolecular interaction of PS1 and PEN2 can convert the PS1 zymogen to an active protease.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Presenilina-1/metabolismo , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/metabolismo , Doença de Alzheimer/enzimologia , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Ativação Enzimática , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Presenilina-1/genética , Subunidades Proteicas/genética , Proteolipídeos/química , Proteínas Recombinantes/genéticaRESUMO
gamma-Secretase cleaves multiple substrates within the transmembrane domain that include the amyloid precursor protein as well as the Notch family of receptors. These substrates are associated with Alzheimer disease and cancer. Despite extensive investigation of this protease, little is known regarding the regulation of gamma-secretase specificity. To discover selective inhibitors for drug development and for probing the mechanisms of gamma-secretase specificity, we screened chemical libraries and consequently developed a di-coumarin family of inhibitors that preferentially inhibit gamma-secretase-mediated production of Abeta42 over other cleavage activities. These coumarin dimer-based compounds interact with gamma-secretase by binding to an allosteric site. By developing a multiple photo-affinity probe approach, we demonstrate that this allosteric binding causes a conformational change within the active site of gamma-secretase at the S2 and S1 sub-sites that leads to selective inhibition of Abeta42. In conclusion, by using these di-coumarin compounds, we reveal a mechanism by which gamma-secretase specificity is regulated and provide insights into the molecular basis by which familial presenilin mutations may affect the active site and specificity of gamma-secretase. Furthermore, this class of selective inhibitors provides the basis for development of Alzheimer disease therapeutic agents.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Cumarínicos/farmacologia , Conformação Proteica , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Cumarínicos/metabolismo , Descoberta de Drogas , Humanos , Cinética , Mutação/genética , Marcadores de Fotoafinidade , Presenilinas/genética , Ligação Proteica , Bibliotecas de Moléculas Pequenas , Especificidade por SubstratoRESUMO
gamma-Secretase is an aspartyl protease that cleaves multiple substrates including the amyloid precursor protein (APP) and the Notch proteins. Abnormal proteolysis of APP is involved in the pathogenesis of Alzheimer's disease (AD) and overactive Notch signaling plays an oncogenic role in a variety of cancers. gamma-Secretase has emerged as a promising target for drug development in the treatment of AD and cancer. Assays with increased capacity for high-throughput screening would allow for quicker screening of chemical libraries and facilitate inhibitor development. We have developed a homogeneous time-resolved fluorescence (HTRF)-based assay that makes use of a novel biotinylated recombinant APP substrate and solubilized membrane preparation as the source of the gamma-secretase enzyme. The assay was miniaturized to a 1536-well format and validated in a pilot screen against a library of approximately 3,000 compounds. The overall assay performance was robust due to a calculated Z' factor of 0.74 and its demonstrated ability to identify known gamma-secretase inhibitors such as pepstatin A. This validated assay can readily be used for primary screening against large chemical libraries searching for novel inhibitors of gamma-secretase activity that may represent potential therapeutics for AD and a variety of neoplasms.
Assuntos
Secretases da Proteína Precursora do Amiloide/análise , Avaliação Pré-Clínica de Medicamentos/instrumentação , Secretases da Proteína Precursora do Amiloide/metabolismo , Automação , Análise Custo-Benefício , Relação Dose-Resposta a Droga , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos/métodos , Células HeLa , Humanos , Indicadores e Reagentes , Luminescência , MiniaturizaçãoRESUMO
gamma-Secretase is an aspartyl protease that cleaves multiple substrates that are involved in broad biological processes ranging from stem cell development to neurodegeneration. The investigation of gamma-secretase has been limited by currently available assays that require genetic or biochemical manipulation in the form of substrate transfection or membrane preparation. Here we report an exo-cell assay that is capable of characterizing gamma-secretase activity in any cellular system without limitation. Using a highly active, recombinant substrate this assay can quickly and easily ascertain the status of gamma-secretase activity in cell systems and patient samples. We have applied this method to determine the activity of gamma-secretase in primary cell samples where transfection and/or membrane isolation are not viable options. Importantly, it allows for the detection of real time gamma-secretase activity after inhibitor or drug treatment. The application of this assay to determine the role of gamma-secretase in physiological and pathological conditions will greatly facilitate our characterization of this complex protease and help in the development and evaluation of gamma-secretase-targeted therapies in Alzheimer's disease or a variety of neoplasms.
RESUMO
Because Notch signaling is implicated in colon cancer tumorigenesis and protects cells from apoptosis by inducing prosurvival targets, it was hypothesized that inhibition of Notch signaling with gamma-secretase inhibitors (GSI) may enhance the chemosensitivity of colon cancer cells. We first show that the Notch-1 receptor, as well as its downstream target Hes-1, is up-regulated with colon cancer progression, similar to other genes involved in chemoresistance. We then report that chemotherapy induces Notch-1, as oxaliplatin, 5-fluorouracil (5-FU), or SN-38 (the active metabolite of irinotecan) induced Notch-1 intracellular domain (NICD) protein and activated Hes-1. Induction of NICD by oxaliplatin was caused by an increase in the activity and expression of gamma-secretase complex, as suppression of the protein subunit nicastrin with small interfering RNA (siRNA) prevented NICD induction after oxaliplatin. Subsequent inhibition of Notch-1 signaling with a sulfonamide GSI (GSI34) prevented the induction of NICD by chemotherapy and blunted Hes-1 activation. Blocking the activation of Notch signaling with GSI34 sensitized cells to chemotherapy and was synergistic with oxaliplatin, 5-FU, and SN-38. This chemosensitization was mediated by Notch-1, as inhibition of Notch-1 with siRNA enhanced chemosensitivity whereas overexpression of NICD increased chemoresistance. Down-regulation of Notch signaling also prevented the induction of prosurvival pathways, most notably phosphoinositide kinase-3/Akt, after oxaliplatin. In summary, colon cancer cells may up-regulate Notch-1 as a protective mechanism in response to chemotherapy. Therefore, combining GSIs with chemotherapy may represent a novel approach for treating metastatic colon cancers by mitigating the development of chemoresistance.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Antineoplásicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Compostos Organoplatínicos/farmacologia , Receptor Notch1/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/enzimologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/enzimologia , Ativação Enzimática , Células HCT116 , Células HT29 , Humanos , Oxaliplatina , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Notch1/biossíntese , Receptor Notch1/genética , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologiaRESUMO
The stereoselective synthesis of novel photoreactive gamma-secretase inhibitors 2 and 3 has been achieved. Key steps of the strategy involve preparation of alpha-N-Boc-epoxide 8 and formation of lactone 14 in a practical and stereo-controlled fashion. Compounds 2 and 3 are potent gamma-secretase inhibitors and directly interact with presenilin-1, a catalytic subunit of gamma-secretase.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Biotinilação , Cristalografia por Raios X/métodos , Dipeptídeos/química , Desenho de Fármacos , Compostos de Epóxi/química , Células HeLa , Humanos , Concentração Inibidora 50 , Lactonas/química , Modelos Químicos , Fotoquímica/métodos , Presenilina-1/químicaRESUMO
In the present study, we investigated whether anandamide produces its behavioral effects through a cannabinoid CB(1) receptor mechanism of action. The behavioral effects of anandamide were evaluated in mice that lacked both fatty acid amide hydrolase (FAAH) and cannabinoid CB(1) receptors (DKO) as compared to FAAH (-/-), cannabinoid CB(1) (-/-), and wild type mice. Anandamide produced analgesia, catalepsy, and hypothermia in FAAH (-/-) mice, but failed to elicit any of these effects in the other three genotypes. In contrast, anandamide decreased locomotor behavior regardless of genotype, suggesting the involvement of multiple mechanisms of action, including its products of degradation. These findings indicate that the cannabinoid CB(1) receptor is the predominant target mediating anandamide's behavioral effects.
Assuntos
Analgésicos/farmacologia , Ácidos Araquidônicos/farmacologia , Moduladores de Receptores de Canabinoides/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Receptor CB1 de Canabinoide/metabolismo , Amidoidrolases/deficiência , Amidoidrolases/genética , Animais , Catalepsia/induzido quimicamente , Endocanabinoides , Feminino , Hipotermia/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Limiar da Dor/efeitos dos fármacos , Receptor CB1 de Canabinoide/deficiência , Receptor CB1 de Canabinoide/genéticaRESUMO
Reactive oxygen species (ROS) are key components of postreceptor intracellular signaling pathways; however, the role of ROS in signal initiation is uncertain. We discovered that receptor-ligand interaction caused the generation of hydrogen peroxide (H2O2). Using members of the hematopoietin receptor superfamily, as well as EGF receptor, we show that H2O2 is generated by specific receptor-ligand interaction in cells and in cell-free systems. With cognate ligand, the extracellular domain of the receptor was sufficient for H2O2 generation. We also found that production of H2O2 was diminished in a granulocyte-macrophage colony-stimulating factor receptor mutant unable to bind ligand. Exogenously added H2O2 induced signaling in the absence of ligand, whereas catalase and a membrane-bound peroxiredoxin inhibited ligand-dependent signaling. Our results suggest that H2O2 produced by receptor-ligand interaction is involved as a chemical mediator that facilitates cell signaling.
Assuntos
Peróxido de Hidrogênio/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Sequência de Bases , Catalase/farmacologia , Linhagem Celular , DNA Complementar/genética , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Líquido Extracelular/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Técnicas In Vitro , Ligantes , Modelos Biológicos , Mutação , Peroxidases/genética , Peroxidases/metabolismo , Peroxirredoxinas , Subunidades Proteicas , Espécies Reativas de Oxigênio/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/química , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Fatty acid amide hydrolase (FAAH) is the primary catabolic regulator of several bioactive lipid amides in vivo, including the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. Inhibitors of FAAH are considered a potential therapeutic approach for the treatment of several nervous system disorders, including pain, anxiety, and insomnia. However, for FAAH inhibitors to achieve clinical utility, they must not only display efficacy in vivo but also selectivity for this enzyme relative to the numerous other serine hydrolases present in mammalian proteomes. Here, we report a general strategy for evaluating the pharmacological activity and target specificity of FAAH inhibitors and its implementation to develop the first class of selective reversible inhibitors of this enzyme that are highly efficacious in vivo. Using a series of functional proteomics, analytical chemistry, and behavioral pharmacology assays, we have identified a class of alpha-keto-heterocycles that show unprecedented selectivity for FAAH relative to other mammalian hydrolases, and, when administered to rodents, raise central nervous system levels of anandamide and promote cannabinoid receptor 1-dependent analgesia in several assays of pain sensation. These studies provide further evidence that FAAH may represent an attractive therapeutic target and describe a general route by which inhibitors of this enzyme can be optimized to achieve exceptional potency, selectivity, and efficacy in vivo.
Assuntos
Amidoidrolases/antagonistas & inibidores , Analgesia , Ácidos Araquidônicos , Inibidores Enzimáticos/farmacologia , Piridinas , Animais , Sinergismo Farmacológico , Endocanabinoides , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Alcamidas Poli-Insaturadas , Proteômica , Receptor CB1 de Canabinoide/fisiologiaRESUMO
Although the N-arachidonoyl ethanolamine (anandamide) binds to cannabinoid receptors and has been implicated in the suppression of pain, its rapid catabolism in vivo by fatty acid amide hydrolase (FAAH) has presented a challenge in investigating the physiological functions of this endogenous cannabinoid. In order to test whether anandamide and other non-cannabinoid fatty amides modulate nociception, we compared FAAH (+/+) and (-/-) mice in the tail immersion, hot plate, and formalin tests, as well as for thermal hyperalgesia in the carrageenan and the chronic constriction injury (CCI) models. FAAH (-/-) mice exhibited a CB1 receptor-mediated phenotypic hypoalgesia in thermal nociceptive tests. These mice also exhibited CB1 receptor-mediated hypoalgesia in both phases of the formalin test accompanied with a phenotypic anti-edema effect, which was not blocked by either CB1 or CB2 antagonists. Additionally, FAAH (-/-) mice displayed thermal anti-hyperalgesic and anti-inflammatory effects in the carrageenan model that were mediated, in part, by CB2, but not CB1 receptors. In contrast, no genotype differences in pain behavior were evident following CCI, which was instead found to obliterate the phenotypic hypoalgesia displayed by FAAH (-/-) mice in the tail immersion and hot plate tests, suggesting that nerve injury may promote adaptive changes in these animals. Collectively, these findings demonstrate a cannabinoid receptor-mediated analgesic phenotype in FAAH (-/-) mice. In more general terms, these findings suggest that selective inhibitors of FAAH might represent a viable pharmacological approach for the clinical treatment of pain disorders.