RESUMO
Mutations in the presenilins that cause familial Alzheimer's disease alter the activity of these proteases to increase generation of an aggregation-prone isoform of the amyloid ß-peptide (Aß). How these mutations do so has been unclear. Sannerud et al. now show that regulation of subcellular localization plays a central role, advancing our understanding of the cell biology of Alzheimer's disease.
Assuntos
Doença de Alzheimer/genética , Presenilinas/genética , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Humanos , Transporte ProteicoRESUMO
The intramembrane protease γ-secretase is a key player in signaling and Alzheimer's disease, but its structural features have remained obscure. A structure reported recently reveals a horseshoe-shaped arrangement of 19 transmembrane helices and an extracellular domain positioned for substrate recognition. This advance bodes well for a finer resolution before long.
Assuntos
Secretases da Proteína Precursora do Amiloide/química , Modelos Moleculares , HumanosRESUMO
A dual goal for treating Alzheimer's disease (AD) is to decrease deposition of neurotoxic amyloid beta-peptide in the brain and to boost repair of damaged neurons. Donmez et al. (2010) now show that SIRT1 may mediate both processes by deacetylating the transcription factor retinoic acid receptor beta, a potential new therapeutic target for AD.
RESUMO
Presenilin (PSEN)/γ-secretase is a protease complex responsible for the proteolytic processing of numerous substrates. These substrates include the amyloid precursor protein (APP), the cleavage of which by γ-secretase results in the production of ß-amyloid (Aß) peptides. However, exactly where within the neuron γ-secretase processes APP C99 to generate Aß and APP intracellular domain (AICD) is still not fully understood. Here, we employ novel Förster resonance energy transfer (FRET)-based multiplexed imaging assays to directly "visualize" the subcellular compartment(s) in which γ-secretase primarily cleaves C99 in mouse cortex primary neurons (from both male and female embryos). Our results demonstrate that γ-secretase processes C99 mainly in LysoTracker-positive low-pH compartments. Using a new immunostaining protocol which distinguishes Aß from C99, we also show that intracellular Aß is significantly accumulated in the same subcellular loci. Furthermore, we found functional correlation between the endo-lysosomal pH and cellular γ-secretase activity. Taken together, our findings are consistent with Aß being produced from C99 by γ-secretase within acidic compartments such as lysosomes and late endosomes in living neurons.SIGNIFICANCE STATEMENT Alzheimer's disease (AD) genetics and histopathology highlight the importance of amyloid precursor protein (APP) processing by γ-secretase in pathogenesis. For the first time, this study has enabled us to directly "visualize" that γ-secretase processes C99 mainly in acidic compartments such as late endosomes and lysosomes in live neurons. Furthermore, we uncovered that intracellular ß-amyloid (Aß) is significantly accumulated in the same subcellular loci. Emerging evidence proposes the great importance of the endo-lysosomal pathway in mechanisms of misfolded proteins propagation (e.g., Tau, α-Syn). Therefore, the predominant processing of C99 and enrichment of Aß in late endosomes and lysosomes may be critical events in the molecular cascade leading to AD.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Neurônios/metabolismo , Presenilinas/metabolismo , Animais , Feminino , Masculino , CamundongosRESUMO
This work reports substrate-selective inhibition of a protease with broad substrate specificity based on direct binding of a small-molecule inhibitor to the substrate. The target for these studies was γ-secretase protease, which cleaves dozens of different single-span membrane protein substrates, including both the C99 domain of the human amyloid precursor protein and the Notch receptor. Substrate-specific inhibition of C99 cleavage is desirable to reduce production of the amyloid-ß polypeptide without inhibiting Notch cleavage, a major source of toxicity associated with broad specificity γ-secretase inhibitors. In order to identify a C99-selective inhibitors of the human γ-secretase, we conducted an NMR-based screen of FDA-approved drugs against C99 in model membranes. From this screen, we identified the small-molecule verteporfin with these properties. We observed that verteporfin formed a direct 1:1 complex with C99, with a KD of 15-47 µM (depending on the membrane mimetic used), and that it did not bind the transmembrane domain of the Notch-1 receptor. Biochemical assays showed that direct binding of verteporfin to C99 inhibits γ-secretase cleavage of C99 with IC50 values in the range of 15-164 µM, while Notch-1 cleavage was inhibited only at higher concentrations, and likely via a mechanism that does not involve binding to Notch-1. This work documents a robust NMR-based approach to discovery of small-molecule binders to single-span membrane proteins and confirmed that it is possible to inhibit γ-secretase in a substrate-specific manner.
Assuntos
Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide , Verteporfina , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Proteínas de Membrana/metabolismo , Domínios Proteicos , Receptores Notch/metabolismo , Verteporfina/metabolismo , Verteporfina/farmacologiaRESUMO
INTRODUCTION: Identifying CSF-based biomarkers for the ß-amyloidosis that initiates Alzheimer's disease (AD) could provide inexpensive and dynamic tests to distinguish AD from normal aging and predict future cognitive decline. METHODS: We developed immunoassays specifically detecting all C-terminal variants of secreted amyloid ß-protein and identified a novel biomarker, the Aß 37/42 ratio, that outperforms the canonical Aß42/40 ratio as a means to evaluate the γ-secretase activity and brain Aß accumulation. RESULTS: We show that Aß 37/42 can distinguish physiological and pathological status in (1) presenilin-1 mutant vs wild-type cultured cells, (2) AD vs control brain tissue, and (3) AD versus cognitively normal (CN) subjects in CSF, where 37/42 (AUC 0.9622) outperformed 42/40 (AUC 0.8651) in distinguishing CN from AD. DISCUSSION: We conclude that the Aß 37/42 ratio sensitively detects presenilin/γ-secretase dysfunction and better distinguishes CN from AD than Aß42/40 in CSF. Measuring this novel ratio alongside promising phospho-tau analytes may provide highly discriminatory fluid biomarkers for AD.
Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Humanos , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides , Secretases da Proteína Precursora do Amiloide , Proteínas tau , Fragmentos de Peptídeos , Disfunção Cognitiva/diagnóstico , BiomarcadoresRESUMO
γ-Secretase was initially defined as a proteolytic activity that cleaves within the transmembrane of the amyloid precursor protein (APP) to produce the amyloid ß-peptide of Alzheimer's disease. The discovery of mutations in APP and the presenilins associated with familial Alzheimer's disease and their effects on APP processing dovetailed with pharmacological studies on γ-secretase, leading to the revelation that presenilins are unprecedented membrane-embedded aspartyl proteases. Other members of what became known as the γ-secretase complex were subsequently identified. In parallel with these advances, connections between presenilins and Notch receptors essential to metazoan development became evident, resulting in the concurrent realization that γ-secretase also carries out intramembrane proteolysis of Notch as part of its signaling mechanism. Substantial progress has been made toward elucidating how γ-secretase carries out complex processing of transmembrane domains, how it goes awry in familial Alzheimer's disease, the scope of its substrates, and the atomic details of its structure. Critical questions remain for future study, toward further unraveling the complexity of this unique membrane-embedded proteolytic machine and its roles in biology and disease.
Assuntos
Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , HumanosRESUMO
Production of amyloid ß-protein (Aß) is carried out by the membrane-embedded γ-secretase complex. Mutations in the transmembrane domain of amyloid ß-protein precursor (APP) associated with early-onset familial Alzheimer's disease (FAD) can alter the ratio of aggregation-prone 42-residue Aß (Aß42) to 40-residue Aß (Aß40). However, APP substrate is proteolyzed processively by γ-secretase along two pathways: Aß49âAß46âAß43âAß40 and Aß48âAß45âAß42âAß38. Effects of FAD mutations on each proteolytic step are unknown, largely due to difficulties in detecting and quantifying longer Aß peptides. To address this, we carried out systematic and quantitative analyses of all tri- and tetrapeptide coproducts from proteolysis of wild-type and 14 FAD-mutant APP substrates by purified γ-secretase. These small peptides, including FAD-mutant forms, were detected by tandem mass spectrometry and quantified by establishing concentration curves for each of 32 standards. APP intracellular domain (AICD) coproducts were quantified by immunoblot, and the ratio of AICD products corresponding to Aß48 and Aß49 was determined by mass spectrometry. Levels of individual Aß peptides were determined by subtracting levels of peptide coproducts associated with degradation from those associated with production. This method was validated for Aß40 and Aß42 by specific ELISAs and production of equimolar levels of Aß and AICD. Not all mutant substrates led to increased Aß42/40. However, all 14 disease-causing mutations led to inefficient processing of longer forms of Aß ≥ 45 residues. In addition, the effects of certain mutations provided insight into the mechanism of processive proteolysis: intermediate Aß peptides apparently remain bound for subsequent trimming and are not released and reassociated.
Assuntos
Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Mutação/genética , Domínios Proteicos/genética , ProteóliseRESUMO
γ-Secretase is responsible for the proteolysis of amyloid precursor protein (APP) into amyloid-beta (Aß) peptides, which are centrally implicated in the pathogenesis of Alzheimer's disease (AD). The biochemical mechanism of how processing by γ-secretase is regulated, especially as regards the interaction between enzyme and substrate, remains largely unknown. Here, mutagenesis reveals that the hydrophilic loop-1 (HL-1) of presenilin-1 (PS1) is critical for both γ-secretase step-wise cleavages (processivity) and its allosteric modulation by heterocyclic γ-modulatory compounds. Systematic mutagenesis of HL-1, including all of its familial AD mutations and additional engineered variants, and quantification of the resultant Aß products show that HL-1 is necessary for proper sequential γ-secretase processivity. We identify Y106, L113, and Y115 in HL-1 as key targets for heterocyclic γ-secretase modulators (GSMs) to stimulate processing of pathogenic Aß peptides. Further, we confirm that the GxxxG domain in the APP transmembrane region functions as a critical substrate motif for γ-secretase processivity: a G29A substitution in APP-C99 mimics the beneficial effects of GSMs. Together, these findings provide a molecular basis for the structural regulation of γ-processivity by enzyme and substrate, facilitating the rational design of new GSMs that lower AD-initiating amyloidogenic Aß peptides.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Presenilina-1/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Células Cultivadas , Predisposição Genética para Doença , Humanos , Mutação , Presenilina-1/química , Presenilina-1/genética , Domínios Proteicos , Proteólise , Especificidade por SubstratoRESUMO
The membrane-embedded γ-secretase complex processively cleaves within the transmembrane domain of amyloid precursor protein (APP) to produce 37-to-43-residue amyloid ß-peptides (Aß) of Alzheimer's disease (AD). Despite its importance in pathogenesis, the mechanism of processive proteolysis by γ-secretase remains poorly understood. Here, mass spectrometry and Western blotting were used to quantify the efficiency of tripeptide trimming of wild-type (WT) and familial AD (FAD) mutant Aß49. In comparison to WT Aß49, the efficiency of tripeptide trimming was similar for the I45F, A42T, and V46F Aß49 FAD mutants but substantially diminished for the I45T and T48P mutants. In parallel with biochemical experiments, all-atom simulations using a novel peptide Gaussian accelerated molecular dynamics (Pep-GaMD) method were applied to investigate the tripeptide trimming of Aß49 by γ-secretase. The starting structure was the active γ-secretase bound to Aß49 and APP intracellular domain (AICD), as generated from our previous study that captured the activation of γ-secretase for the initial endoproteolytic cleavage of APP (Bhattarai, A., ACS Cent. Sci. 2020, 6, 969-983). Pep-GaMD simulations captured remarkable structural rearrangements of both the enzyme and substrate, in which hydrogen-bonded catalytic aspartates and water became poised for tripeptide trimming of Aß49 to Aß46. These structural changes required a positively charged N-terminus of endoproteolytic coproduct AICD, which could dissociate during conformational rearrangements of the protease and Aß49. The simulation findings were highly consistent with biochemical experimental data. Taken together, our complementary biochemical experiments and Pep-GaMD simulations have enabled elucidation of the mechanism of tripeptide trimming of Aß49 by γ-secretase.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Humanos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismoRESUMO
A series of analogs based on a prototype aryl aminothiazole γ-secretase modulator (GSM) were synthesized and tested for their effects on the profile of 37-to-42-residue amyloid ß-peptides (Aß), generated through processive proteolysis of precursor protein substrate by γ-secretase. Certain substitutions on the terminal aryl D ring resulted in an altered profile of Aß production compared to that seen with the parent molecule. Small structural changes led to concentration-dependent increases in Aß37 and Aß38 production without parallel decreases in their precursors Aß40 and Aß42, respectively. The new compounds therefore apparently also stimulate carboxypeptidase trimming of Aß peptides ≥ 43 residues, providing novel chemical tools for mechanistic studies of processive proteolysis by γ-secretase.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/biossíntese , Descoberta de Drogas , Tiazóis/farmacologia , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Tiazóis/químicaRESUMO
The membrane-embedded γ-secretase complex carries out hydrolysis within the lipid bilayer in proteolyzing nearly 150 different membrane protein substrates. Among these substrates, the amyloid precursor protein (APP) has been the most studied, as generation of aggregation-prone amyloid ß-protein (Aß) is a defining feature of Alzheimer's disease (AD). Mutations in APP and in presenilin, the catalytic component of γ-secretase, cause familial AD, strong evidence for a pathogenic role of Aß. Substrate-based chemical probes-synthetic peptides and peptidomimetics-have been critical to unraveling the complexity of γ-secretase, and small drug-like inhibitors and modulators of γ-secretase activity have been essential for exploring the potential of the protease as a therapeutic target for Alzheimer's disease. Such chemical probes and therapeutic prototypes will be reviewed here, with concluding commentary on the future directions in the study of this biologically important protease complex and the translation of basic findings into therapeutics.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fármacos Neuroprotetores/farmacologia , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Inibidores Enzimáticos/química , Humanos , Conformação Molecular , Fármacos Neuroprotetores/químicaRESUMO
Familial Alzheimer's disease (FAD) is a rare early-onset genetic form of a common dementia of old age. Striking in middle age, FAD is caused by missense mutations in three genes: APP (encoding the amyloid precursor protein) and PSEN1 and PSEN2 (encoding presenilin-1 and presenilin-2). APP is proteolytically processed successively by ß-secretase and γ-secretase to produce the amyloid ß-peptide (Aß). Presenilin is the catalytic component of γ-secretase, a membrane-embedded aspartyl protease complex that cleaves APP within its single transmembrane domain to produce Aß of varying lengths. Thus, all FAD mutations are found in the substrate and the enzyme that produce Aß. The 42-residue variant Aß42 has been the primary focus of Alzheimer drug discovery for over two decades, as this particular peptide is highly prone to aggregation, is the major protein deposited in the characteristic cerebral plaques of Alzheimer's disease, and is proportionately elevated in FAD. Despite extensive efforts, all agents targeting Aß and Aß42 have failed in the clinic, including γ-secretase inhibitors, leading to questioning of the amyloid hypothesis of Alzheimer pathogenesis. However, processing of the APP transmembrane domain by γ-secretase is complex, involving initial endoproteolysis followed by successive carboxypeptidase trimming steps to secreted Aß peptides such as Aß42. Recent findings reveal that FAD mutations in PSEN1 and in APP result in deficient trimming of initially formed long Aß peptides. A logical drug discovery strategy for FAD could therefore involve the search for compounds that rescue this deficient carboxypeptidase activity. The rare early-onset FAD arguably presents a simpler path to developing effective therapeutics compared to the much more complex heterogeneous sporadic Alzheimer's disease.
RESUMO
γ-Secretase is a membrane-embedded aspartyl protease complex central in biology and medicine. How this enzyme recognizes transmembrane substrates and catalyzes hydrolysis in the lipid bilayer is unclear. Inhibitors that mimic the entire substrate transmembrane domain and engage the active site should provide important tools for structural biology, yielding insight into substrate gating and trapping the protease in the active state. Here, we report transmembrane peptidomimetic inhibitors of the γ-secretase complex that contain an N-terminal helical peptide region that engages a substrate docking exosite and a C-terminal transition-state analog moiety targeted to the active site. Both regions are required for stoichiometric inhibition of γ-secretase. Moreover, enzyme inhibition kinetics and photoaffinity probe displacement experiments demonstrate that both the docking exosite and the active site are engaged by the bipartite inhibitors. The solution conformations of these potent transmembrane-mimetic inhibitors are similar to those of bound natural substrates, suggesting these probes are preorganized for high-affinity binding and should allow visualization of the active γ-secretase complex, poised for intramembrane proteolysis, by cryo-electron microscopy.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptidomiméticos/química , Inibidores de Proteases/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Domínio Catalítico , Células HEK293 , Humanos , Cinética , Simulação de Acoplamento Molecular , Peptidomiméticos/metabolismo , Inibidores de Proteases/metabolismo , Ligação Proteica , Conformação Proteica em alfa-HéliceRESUMO
γ-Secretase is a membrane-embedded protease complex, with presenilin as the catalytic component containing two transmembrane aspartates in the active site. With more than 90 known substrates, the γ-secretase complex is considered "the proteasome of the membrane", with central roles in biology and medicine. The protease carries out hydrolysis within the lipid bilayer to cleave the transmembrane domain of the substrate multiple times before releasing secreted products. For many years, elucidation of γ-secretase structure and function largely relied on small-molecule probes and mutagenesis. Recently, however, advances in cryo-electron microscopy have led to the first detailed structures of the protease complex. Two new reports of structures of γ-secretase bound to membrane protein substrates provide great insight into the nature of substrate recognition and how Alzheimer's disease-causing mutations in presenilin might alter substrate binding and processing. These new structures offer a powerful platform for elucidating enzyme mechanisms, deciphering effects of disease-causing mutations, and advancing Alzheimer's disease drug discovery.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Membrana Celular/genética , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Humanos , Hidrólise , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Mutagênese , Presenilina-1/química , Presenilina-1/genética , Presenilina-1/metabolismo , Conformação ProteicaRESUMO
γ-Secretase is a membrane-embedded aspartyl protease complex with presenilin as the catalytic component that cleaves within the transmembrane domain (TMD) of >90 known substrates, including the amyloid precursor protein (APP) of Alzheimer's disease. Processing by γ-secretase of the APP TMD produces the amyloid ß-peptide (Aß), including the 42-residue variant (Aß42) that pathologically deposits in the Alzheimer brain. Complex proteolysis of APP substrate by γ-secretase involves initial endoproteolysis and subsequent carboxypeptidase trimming, resulting in two pathways of Aß production: Aß49 â Aß46 â Aß43 â Aß40 and Aß48 â Aß45 â Aß42 â Aß38. Dominant mutations in APP and presenilin cause early onset familial Alzheimer's disease (FAD). Understanding how γ-secretase processing of APP is altered in FAD is essential for elucidating pathogenic mechanisms in FAD and developing effective therapeutics. To improve our understanding, we designed synthetic APP-based TMD substrates as convenient functional probes for γ-secretase. Installation of the helix-inducing residue α-aminoisobutyric acid provided full TMD helical substrates while also facilitating their synthesis and increasing the solubility of these highly hydrophobic peptides. Through mass spectrometric analysis of proteolytic products, synthetic substrates were identified that were processed in a manner that reproduced physiological processing of APP substrates. Validation of these substrates was accomplished through mutational variants, including the installation of two natural APP FAD mutations. These FAD mutations also resulted in increased levels of formation of Aß-like peptides corresponding to Aß45 and longer, raising the question of whether the levels of such long Aß peptides are indeed increased and might contribute to FAD pathogenesis.
Assuntos
Secretases da Proteína Precursora do Amiloide/química , Precursor de Proteína beta-Amiloide/química , Fragmentos de Peptídeos/química , Doença de Alzheimer/genética , Sequência de Aminoácidos , Ácidos Aminoisobutíricos/química , Precursor de Proteína beta-Amiloide/síntese química , Precursor de Proteína beta-Amiloide/genética , Espectrometria de Massas , Mutação , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/genética , Conformação Proteica em alfa-Hélice , ProteóliseRESUMO
The biological underpinnings linking stress to Alzheimer's disease (AD) risk are poorly understood. We investigated how corticotrophin releasing factor (CRF), a critical stress response mediator, influences amyloid-ß (Aß) production. In cells, CRF treatment increases Aß production and triggers CRF receptor 1 (CRFR1) and γ-secretase internalization. Co-immunoprecipitation studies establish that γ-secretase associates with CRFR1; this is mediated by ß-arrestin binding motifs. Additionally, CRFR1 and γ-secretase co-localize in lipid raft fractions, with increased γ-secretase accumulation upon CRF treatment. CRF treatment also increases γ-secretase activity in vitro, revealing a second, receptor-independent mechanism of action. CRF is the first endogenous neuropeptide that can be shown to directly modulate γ-secretase activity. Unexpectedly, CRFR1 antagonists also increased Aß. These data collectively link CRF to increased Aß through γ-secretase and provide mechanistic insight into how stress may increase AD risk. They also suggest that direct targeting of CRF might be necessary to effectively modulate this pathway for therapeutic benefit in AD, as CRFR1 antagonists increase Aß and in some cases preferentially increase Aß42 via complex effects on γ-secretase.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/biossíntese , Hormônio Liberador da Corticotropina/metabolismo , Modelos Biológicos , Estresse Fisiológico/fisiologia , Doença de Alzheimer/etiologia , Análise de Variância , Animais , Western Blotting , AMP Cíclico/metabolismo , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , Sistema Hipotálamo-Hipofisário/fisiologia , Imunoprecipitação , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Sistema Hipófise-Suprarrenal/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Hormônio Liberador da Corticotropina/metabolismoRESUMO
Genetics strongly implicate the amyloid ß-peptide (Aß) in the pathogenesis of Alzheimer's disease. Dominant missense mutation in the presenilins and the amyloid precursor protein (APP) cause early-onset familial Alzheimer's disease (FAD). As presenilin is the catalytic component of the γ-secretase protease complex that produces Aß from APP, mutation of the enzyme or substrate that produce Aß leads to FAD. However, the mechanism by which presenilin mutations cause FAD has been controversial, with gain of function and loss of function offered as binary choices. This overview will instead present the case that presenilins are dysfunctional in FAD. γ-Secretase is a multi-functional enzyme that proteolyzes the APP transmembrane domain in a complex and processive manner. Reduction in a specific function-the carboxypeptidase trimming of initially formed long Aß peptides containing most of the transmembrane domain to shorter secreted forms-is an emerging common feature of FAD-mutant γ-secretase complexes.
Assuntos
Doença de Alzheimer/enzimologia , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , HumanosRESUMO
γ-Secretase is an intramembrane-cleaving protease that processes many type-I integral membrane proteins within the lipid bilayer, an event preceded by shedding of most of the substrate's ectodomain by α- or ß-secretases. The mechanism by which γ-secretase selectively recognizes and recruits ectodomain-shed substrates for catalysis remains unclear. In contrast to previous reports that substrate is actively recruited for catalysis when its remaining short ectodomain interacts with the nicastrin component of γ-secretase, we find that substrate ectodomain is entirely dispensable for cleavage. Instead, γ-secretase-substrate binding is driven by an apparent tight-binding interaction derived from substrate transmembrane domain, a mechanism in stark contrast to rhomboid--another family of intramembrane-cleaving proteases. Disruption of the nicastrin fold allows for more efficient cleavage of substrates retaining longer ectodomains, indicating that nicastrin actively excludes larger substrates through steric hindrance, thus serving as a molecular gatekeeper for substrate binding and catalysis.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Glicoproteínas de Membrana/fisiologia , Secretases da Proteína Precursora do Amiloide/fisiologia , Bicamadas Lipídicas , Especificidade por SubstratoRESUMO
INTRODUCTION: There is keen interest in elucidating the biological mechanisms underlying recent failures of ß-site amyloid precursor protein-cleaving enzyme-1 (BACE1) inhibitors in Alzheimer's disease trials. METHODS: We developed a highly sensitive and specific immunoassay for BACE1 in cell lines and iPSC-derived human neurons to systematically analyze the effects of eight clinically relevant BACE1 inhibitors. RESULTS: Seven of 8 inhibitors elevated BACE1 protein levels. Among protease inhibitors tested, the elevation was specific to BACE1 inhibitors. The inhibitors did not increase BACE1 transcription but extended the protein's half-life. BACE1 became elevated at concentrations below the IC50 for amyloid ß (Aß). DISCUSSION: Elevation of BACE1 by 7 of 8 BACE1 inhibitors raises new concerns about advancing such ß-secretase inhibitors for AD. Chronic elevation could lead to intermittently uninhibited BACE1 when orally dosed inhibitors reach trough levels, abnormally increasing substrate processing. Compounds such as roburic acid that lower Aß by dissociating ß/γ secretase complexes are better candidates because they neither inhibit ß- and γ-secretase nor increase BACE1 levels.