RESUMEN
SAMHD1 is a dNTP triphosphohydrolase governing nucleotide pool homeostasis and can detoxify chemotherapy metabolites controlling their clinical responses. To understand SAMHD1 biology and investigate the potential of targeting SAMHD1 as neoadjuvant to current chemotherapies, we set out to discover selective small-molecule inhibitors. Here, we report a discovery pipeline encompassing a biochemical screening campaign and a set of complementary biochemical, biophysical, and cell-based readouts for rigorous characterization of the screen output. The identified small molecules, TH6342 and analogs, accompanied by inactive control TH7126, demonstrated specific, low µM potency against both physiological and oncology-drug-derived substrates. By coupling kinetic studies with thermal shift assays, we reveal the inhibitory mechanism of TH6342 and analogs, which engage pre-tetrameric SAMHD1 and deter oligomerization and allosteric activation without occupying nucleotide-binding pockets. Altogether, our study diversifies inhibitory modes against SAMHD1, and the discovery pipeline reported herein represents a thorough framework for future SAMHD1 inhibitor development.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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With a diverse network of substrates, NUDIX hydrolases have emerged as a key family of nucleotide-metabolizing enzymes. NUDT5 (also called NUDIX5) has been implicated in ADP-ribose and 8-oxo-guanine metabolism and was recently identified as a rheostat of hormone-dependent gene regulation and proliferation in breast cancer cells. Here, we further elucidate the physiological relevance of known NUDT5 substrates and underscore the biological requirement for NUDT5 in gene regulation and proliferation of breast cancer cells. We confirm the involvement of NUDT5 in ADP-ribose metabolism and dissociate a relationship to oxidized nucleotide sanitation. Furthermore, we identify potent NUDT5 inhibitors, which are optimized to promote maximal NUDT5 cellular target engagement by CETSA. Lead compound, TH5427, blocks progestin-dependent, PAR-derived nuclear ATP synthesis and subsequent chromatin remodeling, gene regulation and proliferation in breast cancer cells. We herein present TH5427 as a promising, targeted inhibitor that can be used to further study NUDT5 activity and ADP-ribose metabolism.
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Inhibidores Enzimáticos/farmacología , Progestinas/metabolismo , Pirofosfatasas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Adenosina Difosfato Ribosa/metabolismo , Adenosina Trifosfato/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Femenino , Células HL-60 , Humanos , Estructura Molecular , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Interferencia de ARN , Especificidad por SustratoRESUMEN
A major hallmark of Alzheimer's disease (AD) is the deposition of amyloid-ß (Aß) peptides in amyloid plaques. Aß peptides are produced by sequential cleavage of the amyloid precursor protein by the ß amyloid cleaving enzyme (BACE) and the γ-secretase (γ-sec) complex. Pharmacological treatments that decrease brain levels of in particular the toxic Aß42 peptide are thought to be promising approaches for AD disease modification. Potent and selective BACE1 inhibitors as well as γ-sec modulators (GSMs) have been designed. Pharmacological intervention of secretase function is not without risks of either on- or off-target adverse effects. One way of improving the therapeutic window could be to combine treatment on multiple targets, using smaller individual doses and thereby minimizing adverse effect liability. We show that combined treatment of primary cortical neurons with a BACE1 inhibitor and a GSM gives an additive effect on Aß42 level change compared with the individual treatments. We extend this finding to C57BL/6 mice, where the combined treatment results in reduction of brain Aß42 levels reflecting the sum of the individual treatment efficacies. These results show that pharmacological targeting of two amyloid precursor protein processing steps is feasible without negatively interfering with the mechanism of action on individual targets. We conclude that targeting Aß production by combining a BACE inhibitor and a GSM could be a viable approach for therapeutic intervention in AD modification.
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Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/biosíntesis , Animales , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Sinergismo Farmacológico , Femenino , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/metabolismo , Inhibidores de Proteasas/administración & dosificación , Inhibidores de Proteasas/farmacología , Piranos/administración & dosificación , Piranos/farmacología , Pirimidinas/administración & dosificación , Pirimidinas/farmacologíaRESUMEN
Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.
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Enzimas Reparadoras del ADN/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Nucleótidos/metabolismo , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Animales , Dominio Catalítico , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cristalización , Daño del ADN , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/metabolismo , Nucleótidos de Desoxiguanina/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Masculino , Ratones , Modelos Moleculares , Conformación Molecular , Terapia Molecular Dirigida , Neoplasias/patología , Oxidación-Reducción/efectos de los fármacos , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/metabolismo , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Pirofosfatasas/antagonistas & inhibidores , Reproducibilidad de los Resultados , Ensayos Antitumor por Modelo de Xenoinjerto , Hidrolasas NudixRESUMEN
Activated RAS GTPase signalling is a critical driver of oncogenic transformation and malignant disease. Cellular models of RAS-dependent cancers have been used to identify experimental small molecules, such as SCH51344, but their molecular mechanism of action remains generally unknown. Here, using a chemical proteomic approach, we identify the target of SCH51344 as the human mutT homologue MTH1 (also known as NUDT1), a nucleotide pool sanitizing enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells, whereas MTH1 overexpression mitigated sensitivity towards SCH51344. Searching for more drug-like inhibitors, we identified the kinase inhibitor crizotinib as a nanomolar suppressor of MTH1 activity. Surprisingly, the clinically used (R)-enantiomer of the drug was inactive, whereas the (S)-enantiomer selectively inhibited MTH1 catalytic activity. Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys and MTH1 co-crystal structures of both enantiomers provide a rationale for this remarkable stereospecificity. Disruption of nucleotide pool homeostasis via MTH1 inhibition by (S)-crizotinib induced an increase in DNA single-strand breaks, activated DNA repair in human colon carcinoma cells, and effectively suppressed tumour growth in animal models. Our results propose (S)-crizotinib as an attractive chemical entity for further pre-clinical evaluation, and small-molecule inhibitors of MTH1 in general as a promising novel class of anticancer agents.
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Antineoplásicos/farmacología , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/metabolismo , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Aminoquinolinas/farmacología , Animales , Antineoplásicos/química , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Crizotinib , Cristalización , Roturas del ADN de Cadena Simple/efectos de los fármacos , Reparación del ADN , Enzimas Reparadoras del ADN/biosíntesis , Enzimas Reparadoras del ADN/química , Modelos Animales de Enfermedad , Femenino , Homeostasis/efectos de los fármacos , Humanos , Ratones , Ratones SCID , Modelos Moleculares , Nucleótidos/metabolismo , Monoéster Fosfórico Hidrolasas/biosíntesis , Monoéster Fosfórico Hidrolasas/química , Conformación Proteica , Inhibidores de Proteínas Quinasas/química , Proteómica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras) , Pirazoles/química , Piridinas/química , Especificidad por Sustrato , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/genéticaRESUMEN
Alzheimer's disease (AD) is a slowly progressing disease and the evaluation of clinical effects of candidate drugs requires large clinical cohorts as well as long treatment trials. There is a great need for central biomarkers and translatable pre-clinical models to provide early indication of treatment effects. We set out to evaluate the guinea pig as a clinically translatable model looking at Aß peptides. Our data demonstrate homology between ß-amyloid (Aß) peptide pattern in cerebrospinal fluid (CSF) from human and guinea pig. To further evaluate the model a novel γ-secretase modulator was used. Dose and time response studies confirm the modulatory properties with a statistically significant decrease in relative levels of Aß1-40, Aß1-42 and increase in Aß1-37 already one hour after administration. We suggest that the guinea pig is a compelling pre-clinical model for evaluating and translating central effects on Aß peptides in CSF after treatment. Further quantitative data are needed to confirm our data together with data from clinical trials in order to back translate and validate our findings.
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Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide , Animales , Modelos Animales de Enfermedad , Cobayas , Humanos , MasculinoRESUMEN
BACKGROUND: The first-line treatment in chronic myeloid leukemia (CML), imatinib, has been shown to decrease the production of amyloid-ß (Aß) in vitro and in animal studies. However, whether imatinib has this effect in humans is not known. METHODS: Plasma levels of Aß42 were analyzed in sequential samples from CML patients treated with imatinib (n=51). The effect of imatinib on Aß production was also investigated in human embryonic kidney 293 (HEK293) cells overexpressing the amyloid precursor protein (APP) with the Swedish mutation, in mouse primary cortical neurons and in human Down syndrome embryonic stem-cell-derived cortical neurons. RESULTS: Twelve months of imatinib treatment did not lower plasma Aß42 levels in CML patients, and imatinib treatment did not lead to less Aß42 production in any of the in vitro models whereas ß- and γ-secretase inhibitors did. CONCLUSION: These data question the previously described role of imatinib in inhibiting amyloidogenic APP processing and as a drug candidate for AD.
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Péptidos beta-Amiloides/metabolismo , Benzamidas/farmacología , Fragmentos de Péptidos/metabolismo , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Anciano , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/fisiopatología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Benzamidas/uso terapéutico , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/fisiopatología , Síndrome de Down/tratamiento farmacológico , Síndrome de Down/fisiopatología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/fisiología , Femenino , Células HEK293 , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/sangre , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Masculino , Ratones , Neuronas/efectos de los fármacos , Neuronas/fisiología , Piperazinas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéuticoRESUMEN
Recent reports have implicated tau-tubulin kinase 1 (TTBK1) in the pathological phosphorylation of tau that occurs in Alzheimer's disease (AD). The present study was undertaken to provide an extensive characterization of TTBK1 mRNA and protein expression in human brain from AD cases and non-demented controls so as to better understand the disease relevance of this novel kinase. In situ hybridization and immunohistochemistry revealed abundant expression of TTBK1 in the somatodendritic compartment of cortical and hippocampal neurons of both AD cases and controls. TTBK1 immunoreactivity appeared to vary with the level of phospho-tau staining, and was strong in the somatodendritic compartment of apparently healthy hippocampal neurons as well as in pre-tangle neurons where it co-localized with diffuse phospho-Ser422 tau staining. Ser422 was confirmed as a TTBK1 substrate in vitro, and an antibody towards the site, in addition to labeling AT8-positive neurofibrillary tangles (NFTs), neuritic plaques and neuropil threads, also labeled a small population of neurons that were unlabeled with AT8. These data suggest a role for TTBK1 in pre-tangle formation prior to the formation of fibrillar tau and strengthen the idea that tau is phosphorylated at Ser422 at an early/intermediate stage in NFT formation.
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Enfermedad de Alzheimer/patología , Encéfalo/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Serina/metabolismo , Proteínas tau/metabolismo , Anciano , Anciano de 80 o más Años , Encéfalo/patología , Dendritas/metabolismo , Dendritas/patología , Femenino , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Masculino , Ovillos Neurofibrilares/metabolismo , Ovillos Neurofibrilares/patología , Neuronas/metabolismo , Neuronas/patología , Fosforilación/genética , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/metabolismo , Transfección , Proteínas tau/genéticaRESUMEN
Amino-2H-imidazoles are described as a new class of BACE-1 inhibitors for the treatment of Alzheimer's disease. Synthetic methods, crystal structures, and structure-activity relationships for target activity, permeability, and hERG activity are reported and discussed. Compound (S)-1m was one of the most promising compounds in this report, with high potency in the cellular assay and a good overall profile. When guinea pigs were treated with compound (S)-1m, a concentration and time dependent decrease in Aß40 and Aß42 levels in plasma, brain, and CSF was observed. The maximum reduction of brain Aß was 40-50%, 1.5 h after oral dosing (100 µmol/kg). The results presented highlight the potential of this new class of BACE-1 inhibitors with good target potency and with low effect on hERG, in combination with a fair CNS exposure in vivo.
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Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Encéfalo/efectos de los fármacos , Imidazoles/síntesis química , Fragmentos de Péptidos/metabolismo , Secretasas de la Proteína Precursora del Amiloide/química , Péptidos beta-Amiloides/sangre , Péptidos beta-Amiloides/líquido cefalorraquídeo , Animales , Ácido Aspártico Endopeptidasas/química , Encéfalo/metabolismo , Línea Celular , Cristalografía por Rayos X , Perros , Femenino , Cobayas , Humanos , Imidazoles/química , Imidazoles/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Estructura Molecular , Fragmentos de Péptidos/sangre , Fragmentos de Péptidos/líquido cefalorraquídeo , Permeabilidad , Estereoisomerismo , Relación Estructura-Actividad , Distribución TisularRESUMEN
The γ-secretase complex is an appealing drug target when the therapeutic strategy is to alter amyloid-ß peptide (Aß) aggregation in Alzheimer disease. γ-Secretase is directly involved in Aß formation and determines the pathogenic potential of Aß by generating the aggregation-prone Aß42 peptide. Because γ-secretase mediates cleavage of many substrates involved in cell signaling, such as the Notch receptor, it is crucial to sustain these pathways while altering the Aß secretion. A way of avoiding interference with the physiological function of γ-secretase is to use γ-secretase modulators (GSMs) instead of inhibitors of the enzyme. GSMs modify the Aß formation from producing the amyloid-prone Aß42 variant to shorter and less amyloidogenic Aß species. The modes of action of GSMs are not fully understood, and even though the pharmacology of GSMs has been thoroughly studied regarding Aß generation, knowledge is lacking about their effects on other substrates, such as Notch. Here, using immunoprecipitation followed by MALDI-TOF MS analysis, we found that two novel, second generation GSMs modulate both Notch ß and Aß production. Moreover, by correlating S3-specific Val-1744 cleavage of Notch intracellular domain (Notch intracellular domain) to total Notch intracellular domain levels using immunocytochemistry, we also demonstrated that Notch intracellular domain is not modulated by the compounds. Interestingly, two well characterized, nonsteroidal anti-inflammatory drugs (nonsteroidal anti-inflammatory drug), R-flurbiprofen and sulindac sulfide, affect only Aß and not Notch ß formation, indicating that second generation GSMs and nonsteroidal anti-inflammatory drug-based GSMs have different modes of action regarding Notch processing.
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Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Amiloide/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Flurbiprofeno/farmacología , Receptores Notch/metabolismo , Sulindac/análogos & derivados , Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Femenino , Células HEK293 , Humanos , Ratones , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/genética , Estructura Terciaria de Proteína , Receptores Notch/genética , Sulindac/farmacologíaRESUMEN
γ-Secretase-mediated cleavage of amyloid precursor protein (APP) results in the production of Alzheimer disease-related amyloid-ß (Aß) peptides. The Aß42 peptide in particular plays a pivotal role in Alzheimer disease pathogenesis and represents a major drug target. Several γ-secretase modulators (GSMs), such as the nonsteroidal anti-inflammatory drugs (R)-flurbiprofen and sulindac sulfide, have been suggested to modulate the Alzheimer-related Aß production by targeting the APP. Here, we describe novel GSMs that are selective for Aß modulation and do not impair processing of Notch, EphB2, or EphA4. The GSMs modulate Aß both in cell and cell-free systems as well as lower amyloidogenic Aß42 levels in the mouse brain. Both radioligand binding and cellular cross-competition experiments reveal a competitive relationship between the AstraZeneca (AZ) GSMs and the established second generation GSM, E2012, but a noncompetitive interaction between AZ GSMs and the first generation GSMs (R)-flurbiprofen and sulindac sulfide. The binding of a (3)H-labeled AZ GSM analog does not co-localize with APP but overlaps anatomically with a γ-secretase targeting inhibitor in rodent brains. Combined, these data provide compelling evidence of a growing class of in vivo active GSMs, which are selective for Aß modulation and have a different mechanism of action compared with the original class of GSMs described.
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Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/biosíntesis , Precursor de Proteína beta-Amiloide/metabolismo , Azepinas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Piranos/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Azepinas/química , Unión Competitiva , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Carbamatos/farmacología , Sistema Libre de Células , Dibenzazepinas/farmacología , Dipéptidos/farmacología , Interacciones Farmacológicas , Femenino , Flurbiprofeno/farmacología , Cobayas , Células HEK293 , Humanos , Imidazoles/farmacología , Ratones , Ratones Endogámicos C57BL , Piperidinas/farmacología , Unión Proteica , Piranos/química , Piridinas/química , Pirimidinas/química , Ratas , Receptor EphA4/metabolismo , Receptor EphB2/metabolismo , Receptores Notch/metabolismo , Sulfonamidas/farmacología , Sulindac/análogos & derivados , Sulindac/farmacologíaRESUMEN
The gamma-secretase complex processes substrate proteins within membranes and consists of four proteins: presenilin (PS), nicastrin, Aph-1 and Pen-2. PS harbours the enzymatic activity of the complex, and there are two mammalian PS homologues: PS1 and PS2. PS undergoes endoproteolysis, generating the N- and C-terminal fragments, NTF and CTF, which represent the active species of PS. To characterize the functional similarity between complexes of various PS composition, we analysed PS1, PS2, and chimeric PS composed of the NTF from PS1 and CTF from PS2, or vice versa, in assembly and function of the gamma-secretase complex. Chimeric PSs, like PS1 and PS2, undergo normal endoproteolysis when introduced into cells devoid of endogenous PS. Furthermore, PS2 CTF can, at least partially, restore processing in a truncated PS1, which cannot undergo endoproteolysis. All PS forms enable maturation of nicastrin and cleave full length Notch receptors, indicating that both PS1 and PS2 are present at the cell surface. Finally, when co-introduced as separate molecules, NTF and CTF of different PS origin reconstitute gamma-secretase activity. In conclusion, these data show that endoproteolysis, NTF-CTF interactions, and the assembly and activity of gamma-secretase complexes are very conserved between PS1 and PS2.
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Endopeptidasas/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Ácido Aspártico Endopeptidasas , Línea Celular , Endopeptidasas/química , Endopeptidasas/metabolismo , Activación Enzimática/fisiología , Regulación Enzimológica de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Presenilina-1 , Presenilina-2 , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The activity of the gamma-secretase complex is critical for the processing of a number of transmembrane proteins, including Notch. Functional gamma-secretase activity can be reconstituted from four proteins--presenilin, nicastrin, Pen-2 and Aph-1--but the role of the individual proteins remains unclear. In this report we describe the cellular localization and protein interactions of Aph-1, with particular regard to Notch receptor processing. We found that Aph-1 is present at the cell surface, where it interacts with Pen-2, the mature forms of presenilin and nicastrin, and full-length Notch. Aph-1 also interacts with a truncated form of Notch, which is a direct substrate for gamma-secretase, but not with the Notch intracellular domain. Immunoprecipitation data for Notch and Aph-1 showed that the Notch-containing gamma-secretase complexes most likely form a small subset of the total number of gamma-secretase complexes. In conclusion, these data demonstrate that Aph-1 is present at the cell surface, presumably in active gamma-secretase complexes, and interacts with the Notch receptor, both before and after ligand activation.