RESUMO
BACKGROUND: The formation of amyloid-ß (Aß) plaques is one of the key neuropathological hallmarks of Alzheimer's disease (AD). Near-infrared (NIR) probes show great potential for imaging of Aß plaques in vivo and in vitro. Dicyanoisophorone (DCIP) based Aß probes have attracted considerable attention due to their exceptional properties. However, DCIP probes still has some drawbacks, such as short emission wavelength (<650 nm) and low fluorescence intensity after binding to Aß. It is clear that further modification is needed to improve their luminescence efficiency and sensitivity. RESULTS: We designed and synthesize four novel pyrrolidine-alkylamino-substituted DCIP derivatives (6a-d) as imaging agents for ß-amyloid (Aß) aggregates. Compound 6c responds better to Aß aggregates than the other three compounds (6a, 6b and 6d) and its precursor DCIP. The calculated detection limit is to be as low as 0.23 µM. Compound 6c shows no cytotoxicity in the tested concentration for SH-SY5Y and HL-7702 cells. Additionally, compound 6c is successfully applied to monitor Aß aggregates in live SH-SY5Y cells and APP/PS1 transgenic mice. The retention time in the transgenic mice brain is much longer than that of age-matched wild-type mice. SIGNIFICANCE: The results indicates that compound 6c had an excellent ability to penetrate the blood-brain barrier and it could effectively distinguish APP/PS1 transgenic mice and wide-type mice. This represents its promising applications for Aß detection in basic and biomedical research.
Assuntos
Cicloexanonas , Humanos , Linhagem Celular , Precursor de Proteína beta-Amiloide/análise , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Pirrolidinas/química , Cicloexanonas/síntese química , Cicloexanonas/química , Cicloexanonas/farmacologia , Espectroscopia de Luz Próxima ao Infravermelho , Estrutura Molecular , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Espectrometria de Fluorescência , Modelos Moleculares , Estrutura Terciária de Proteína , Simulação de Acoplamento Molecular , Sobrevivência Celular/efeitos dos fármacos , Animais , Camundongos , Masculino , Camundongos Endogâmicos C57BL , Encéfalo/metabolismo , Aminação , AlquilaçãoRESUMO
Amyloid precursor protein (APP) plays a pivotal role in the pathology of Alzheimer's disease (AD). Since the fragmentation of the membrane-bound APP that results in the production of amyloid-ß peptides is the starting point for amyloid toxicity in AD, it is important to investigate the structure and dynamics of APP in a near-native lipid-bilayer environment. However, the reconstitution of APP into a stable and suitable membrane-mimicking lipid environment is a challenging task. In this study, the 99-residue C-terminal domain of APP is successfully reconstituted into polymer nanodiscs and characterized using size-exclusion chromatography, mass spectrometry, solution NMR, and magic-angle spinning solid-state NMR. In addition, the feasibility of using lipid-solubilizing polymers for isolating and characterizing APP in the native Escherichia. coli membrane environment is demonstrated.
Assuntos
Precursor de Proteína beta-Amiloide , Nanoestruturas , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Nanoestruturas/química , Escherichia coli , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Ressonância Magnética Nuclear BiomolecularRESUMO
Successive cleavages of amyloid precursor protein C-terminal fragment with 99 residues (APP-C99) by γ-secretase result in amyloid-ß (Aß) peptides of varying lengths. Most cleavages have a step size of three residues. To elucidate the underlying mechanism, we determined the atomic structures of human γ-secretase bound individually to APP-C99, Aß49, Aß46, and Aß43. In all cases, the substrate displays the same structural features: a transmembrane α-helix, a three-residue linker, and a ß-strand that forms a hybrid ß-sheet with presenilin 1 (PS1). Proteolytic cleavage occurs just ahead of the substrate ß-strand. Each cleavage is followed by unwinding and translocation of the substrate α-helix by one turn and the formation of a new ß-strand. This mechanism is consistent with existing biochemical data and may explain the cleavages of other substrates by γ-secretase.
Assuntos
Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides , Precursor de Proteína beta-Amiloide , Presenilina-1 , Humanos , Peptídeos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/química , Secretases da Proteína Precursora do Amiloide/química , Cristalografia por Raios X , Modelos Moleculares , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/química , Presenilina-1/química , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Proteólise , Especificidade por SubstratoRESUMO
TDP-43 is a ubiquitously expressed, multidomain functional protein that is distinctively known to form aggregates in many fatal neurodegenerative disorders. However, the information for arresting TDP-43 aggregation is missing due to a lack of understanding of the molecular mechanism of the aggregation and structural properties of TDP-43. TDP-43 is inherently prone to aggregation and has minimal protein solubility. Multiple studies have been performed on the smaller parts of TDP-43 or the full-length protein attached to a large solubilization tag. However, the presence of co-solutes or solubilization tags is observed to interfere with the molecular properties and aggregation mechanism of full-length TDP-43. Notably, this study populated and characterized the native, dimeric state of TDP-43 without the interference of co-solutes or protein modifications. We observed that the electrostatics of the local environment is capable of the partial unfolding and monomerization of the native dimeric state of TDP-43 into an amyloidogenic molten globule. By employing the tools of thermodynamics and kinetics, we reveal the structural characteristics and temporal order of the early intermediates and transition states during the transition of the molten globule to ß-rich, amyloid-like aggregates of TDP-43, which is governed by the electrostatics of the environment. The current advanced understanding of the nature of native and early aggregation-prone intermediates, early steps, and the influence of electrostatics in TDP-43 aggregation is essential for drug design.
Assuntos
Proteínas de Ligação a DNA , Agregados Proteicos , Eletricidade Estática , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Cinética , Termodinâmica , Multimerização Proteica , Amiloide/química , Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismoRESUMO
Deposition of amyloid-ß (Aß) peptides in the brain is a hallmark of Alzheimer's disease. Aßs are generated through sequential proteolysis of the amyloid precursor protein by the γ-secretase complexes (GSECs). Aß peptide length, modulated by the Presenilin (PSEN) and APH-1 subunits of GSEC, is critical for Alzheimer's pathogenesis. Despite high relevance, mechanistic understanding of the proteolysis of Aß, and its modulation by APH-1, remain incomplete. Here, we report cryo-EM structures of human GSEC (PSEN1/APH-1B) reconstituted into lipid nanodiscs in apo form and in complex with the intermediate Aß46 substrate without cross-linking. We find that three non-conserved and structurally divergent APH-1 regions establish contacts with PSEN1, and that substrate-binding induces concerted rearrangements in one of the identified PSEN1/APH-1 interfaces, providing structural basis for APH-1 allosteric-like effects. In addition, the GSEC-Aß46 structure reveals an interaction between Aß46 and loop 1PSEN1, and identifies three other H-bonding interactions that, according to functional validation, are required for substrate recognition and efficient sequential catalysis.
Assuntos
Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides , Microscopia Crioeletrônica , Proteínas de Membrana , Presenilina-1 , Humanos , Secretases da Proteína Precursora do Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/química , Presenilina-1/metabolismo , Presenilina-1/química , Presenilina-1/genética , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/química , Endopeptidases/metabolismo , Endopeptidases/química , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Ligação Proteica , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/química , Doença de Alzheimer/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/química , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/química , Modelos Moleculares , ProteóliseRESUMO
The accumulation of amyloid beta (Aß1-42) results in neurotoxicity and is strongly related to neurodegenerative disorders, especially Alzheimer's disease (AD), but the underlying molecular mechanism is still poorly understood. Therefore, there is an urgent need for researchers to discover the proteins that interact with Aß1-42 to determine the molecular basis. Previously, we developed peptide-ligand-induced changes in the abundance of proTeinS (PACTS)-assisted thermal proteome profiling (TPP) to identify proteins that interact with peptide ligands. In the present study, we applied this technique to analyze clinical samples to identify Aß1-42-interacting proteins. We detected 115 proteins that interact with Aß1-42 in human frontal lobe tissue. Pathway enrichment analysis revealed that the differentially expressed proteins were involved mainly in neurodegenerative diseases. Further orthogonal validation revealed that Aß1-42 interacted with the AD-associated protein mitogen-activated protein kinase 3 (MAPK3), and knockdown of the Aß1-42 amyloid precursor protein (APP) inhibited the MAPK signaling pathway, suggesting potential functional roles for Aß1-42 in interacting with MAPK3. Overall, this study demonstrated the application of the PACTS-TPP in clinical samples and provided a valuable data source for research on neurodegenerative diseases.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Fragmentos de Peptídeos , Proteômica , Humanos , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/análise , Proteômica/métodos , Doença de Alzheimer/metabolismo , Proteoma/análise , Proteoma/metabolismo , Lobo Frontal/metabolismo , Lobo Frontal/química , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Ligação ProteicaRESUMO
BACKGROUND: Alzheimer's disease (AD) is characterized by the deposition of amyloid-ß peptide (Aß) in the brain. Aß is produced by sequential ß- and γ-secretase cleavages of amyloid precursor protein (APP). Clinical trials targeting ß- and γ-secretases have all failed, partly because of the strong side effects. The aims of this work were to determine if the direct cleavage of APP by γ-secretase inhibits Aß production, and to identify γ-cleavage-inhibiting signals within APP that can be targeted to prevent Aß generation without inhibiting any enzyme. METHODS: An APP mutant mimicking secreted APPγ was overexpressed in cells to test ß-cleavage and Aß production. APP deletion and truncation mutants were overexpressed in cells to identify the γ-secretase-inhibiting domain. The intracellular transport of the mutants was examined using immunofluorescence. Co-immunoprecipitation was performed to investigate the molecular mechanisms. RESULTS: The APP N-terminal fragment mimicking the direct γ-cleavage product was not cleaved by beta-secretase 1 to produce detectable Aß. However, in cells, the C-terminal fragments of APP longer than the last 116 residues could not be cleaved by γ-secretase in cells. No deletion mutant was cleaved by γ-secretase. C99, the direct precursor of Aß, was no longer a γ-secretase substrate when green fluorescent protein was fused to its N-terminus. The large ectodomains prevented access to γ-secretase. CONCLUSIONS: Enabling the direct γ-cleavage of APP is a new and valid strategy to reduce Aß. However, APP does not inhibit γ-cleavage via a specific inhibitory sequence in the ectodomain. Other methods to fulfill the strategy may benefit AD prevention and therapy.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Humanos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/prevenção & controle , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismoRESUMO
Cleavage of the transmembrane domain (TMD) of amyloid-ß precursor protein (APP) by γ-secretase, an intramembrane aspartyl protease, generates Aß peptides of various lengths that form plaques in the brains of Alzheimer's disease patients. Although the debate has not been finally resolved whether these plaques trigger the onset of Alzheimer's or are side products, disease-related mutations suggest their implication in the etiology of the dementia. These occur both in presenilin, the catalytic subunit of γ-secretase, and in the TMD of APP. Despite two seminal cryo-electron microscopy structures that show the complex of γ-secretase with its substrates APP and Notch, the mechanism of γ-secretase is not yet fully understood. Especially on which basis it selects its substrates is still an enigma. The presenilin homolog PSH from the archaeon Methanoculleus marisnigri JR1 (MCMJR1) is catalytically active without accessory proteins in contrast to γ-secretase making it an excellent model for studies of the basic cleavage process. We here focused on the cell-free expression of PSH screening a range of conditions. Cleavage assays to verify the activity show that not only the yield, but mainly the activity of the protease depends on the careful selection of expression conditions. Optimal results were found for a cell-free expression at relatively low temperature, 20 °C, employing cell lysates prepared from E. coli Rosetta cells. To speed up protein preparation for immediate functional assays, a crude purification protocol was developed. This allows to produce ready-made PSH in a fast and efficient manner in less than two days.
Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Humanos , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Presenilinas/química , Presenilinas/metabolismo , Doença de Alzheimer/genética , Peptídeos beta-AmiloidesRESUMO
Recent advances have driven the development of immunotherapies that act by either promoting or suppressing a patient's immune system to treat inflammation, autoimmune disease, cardiovascular disease, infectious diseases, and several cancers. In addition, research conducted over the past 25 years has identified therapeutic targets and indicated that immunotherapy could be used to treat Alzheimer's disease (AD). Despite a number of setbacks, this approach has now led to the development of the first disease-modifying treatments for this devastating disease. A key neuropathological feature of AD is the accumulation of a ~40-amino acid peptide known as amyloid ß (Aß) in the brain and cerebrovasculature. Our detection of an Aß precursor protein mutation that caused early-onset AD in a Swedish family by enhancing Aß protofibril formation sharpened the focus on soluble Aß aggregates (oligomers and protofibrils) as viable therapeutic targets. Initial studies developed and tested a mouse monoclonal antibody (mAb158) with specific conformation-dependent binding to these soluble Aß aggregates. Treatment with mAb158 selectively reduced Aß protofibrils in the brain and cerebrospinal fluid of a transgenic mouse model of AD. A humanized version of mAb158 (lecanemab) subsequently entered clinical trials. Based on promising Phase 2 data showing plaque clearance and reduced cognitive decline, a Phase 3 trial found that lecanemab slowed decline on the primary cognitive endpoint by 27% over 18 months and also produced positive effects on secondary clinical endpoints and key biomarkers. In July 2023, the FDA granted lecanemab a full approval, and this therapeutic antibody will be marketed as Leqembi®. This represents a significant advance for patients with AD, although many challenges remain. In particular, it is now more important than ever to identify individuals who are vulnerable to AD, so that treatment can be initiated at an early stage in the disease process.
Assuntos
Doença de Alzheimer , Camundongos , Humanos , Animais , Doença de Alzheimer/genética , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Camundongos Transgênicos , MutaçãoRESUMO
A primary pathology of Alzheimer's disease (AD) is amyloid ß (Aß) deposition in brain parenchyma and blood vessels, the latter being called cerebral amyloid angiopathy (CAA). Parenchymal amyloid plaques presumably originate from neuronal Aß precursor protein (APP). Although vascular amyloid deposits' origins remain unclear, endothelial APP expression in APP knock-in mice was recently shown to expand CAA pathology, highlighting endothelial APP's importance. Furthermore, two types of endothelial APP-highly O-glycosylated APP and hypo-O-glycosylated APP-have been biochemically identified, but only the former is cleaved for Aß production, indicating the critical relationship between APP O-glycosylation and processing. Here, we analyzed APP glycosylation and its intracellular trafficking in neurons and endothelial cells. Although protein glycosylation is generally believed to precede cell surface trafficking, which was true for neuronal APP, we unexpectedly observed that hypo-O-glycosylated APP is externalized to the endothelial cell surface and transported back to the Golgi apparatus, where it then acquires additional O-glycans. Knockdown of genes encoding enzymes initiating APP O-glycosylation significantly reduced Aß production, suggesting this non-classical glycosylation pathway contributes to CAA pathology and is a novel therapeutic target.
Assuntos
Acetilgalactosamina , Doença de Alzheimer , Peptídeos beta-Amiloides , Precursor de Proteína beta-Amiloide , Angiopatia Amiloide Cerebral , Glicosilação , Animais , Camundongos , Doença de Alzheimer/complicações , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/biossíntese , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Angiopatia Amiloide Cerebral/complicações , Angiopatia Amiloide Cerebral/metabolismo , Angiopatia Amiloide Cerebral/patologia , Células Endoteliais/metabolismo , Transporte Proteico , Neurônios/metabolismo , Complexo de Golgi/metabolismo , Acetilgalactosamina/metabolismoRESUMO
Amyloid precursor protein (APP) is the biological precursor of ß-amyloids, a known histopathological hallmark associated with Alzheimer's disease (AD). The function of APP is of great interest yet remains elusive. One of the extracellular domains of APP, the E2 domain, has been proposed to possess ferroxidase activity and affect neuronal iron homeostasis. However, contradicting evidence has been reported, and its precise role remains inconclusive. Here, we studied the Cu-binding site of the E2 domain using extended X-ray absorption fine structure (EXAFS), UV-vis, and electron paramagnetic resonance (EPR) and discovered that a new labile water ligand coordinates to the Cu(II) cofactor in addition to the four known histidines. We explored the proposed ferroxidase activity of the Cu(II)-E2 domain through reactions with ferrous iron and observed single-turnover ferrous oxidation activity with a rate up to 1.0 × 102 M-1 s-1. Cu(I)-E2 reacted with molecular oxygen at a rate of only 5.3 M-1 s-1, which would restrict any potential multiturnover ferroxidase activity to this slow rate and prevents observation of activity under multiturnover conditions. The positive electrostatic potential surface of the protein indicates possible reactivity with negatively charged small substrates such as superoxide radicals (O2â¢-) and peroxynitrite (ONOO-) that are major contributors to the oxidative stress prevalent in the extracellular environment. Our assays showed that Cu(I)-E2 can remove O2â¢- at a rate of 1.6 × 105 M-1 s-1, which is slower than the rates of native SODs. However, the reaction between Cu(I)-E2 and ONOO- achieved a rate of 1.1 × 105 M-1 s-1, comparable to native ONOO- scavenger peroxiredoxins (105-107 M-1 s-1). Therefore, the E2 domain of APP can serve as an enzymatic site that may function as a ferroxidase under substrate-limiting conditions, a supplemental O2â¢- scavenger, and an ONOO- remover in the vicinity of the cellular iron efflux channel and protect neuron cells from reactive oxygen species (ROS) and reactive nitrogen species (RNS) damage.
Assuntos
Precursor de Proteína beta-Amiloide , Ceruloplasmina , Ceruloplasmina/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Superóxidos , Ácido Peroxinitroso/metabolismo , Ferro/metabolismoRESUMO
The multiple-step cleavage of amyloid precursor protein (APP) generates amyloid-ß peptides (Aß), highly toxic molecules causing Alzheimer's disease (AD). The nonspecific cleavage between the transmembrane region of APP (APPTM) and γ-secretase is the key step of Aß generation. Reconstituting APPTM under physiologically-relevant conditions is crucial to investigate how it interacts with γ-secretase and for future AD drug discovery. Although producing recombinant APPTM was reported before, the large scale purification was hindered by the use of biological protease in the presence of membrane protein. Here, we expressed recombinant APPTM in Escherichia coli using the pMM-LR6 vector and recovered the fusion protein from inclusion bodies. By combining Ni-NTA chromatography, cyanogen bromide cleavage, and reverse phase high performance liquid chromatography (RP-HPLC), isotopically-labeled APPTM was obtained in high yield and high purity. The reconstitution of APPTM into dodecylphosphocholine (DPC) micelle generated mono dispersed 2D 15N-1H HSQC spectra in high quality. We successfully established an efficient and reliable method for the expression, purification and reconstruction of APPTM, which may facilitate future investigation of APPTM and its complex in more native like membrane mimetics such as bicelle and nanodiscs.
Assuntos
Precursor de Proteína beta-Amiloide , Micelas , Humanos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Espectroscopia de Ressonância Magnética , Proteínas RecombinantesRESUMO
Peptides targeting disease-relevant protein-protein interactions are an attractive class of therapeutics covering the otherwise undruggable space between small molecules and therapeutic proteins. However, peptides generally suffer from poor metabolic stability and low membrane permeability. Hence, peptide cyclization has become a valuable approach to develop linear peptide motifs into metabolically stable and potentially cell-permeable cyclic leads. Furthermore, cyclization of side chains, also known as "stapling", can stabilize particular secondary peptide structures. Here, we demonstrate that a comprehensive examination of cyclization strategies in terms of position, chemistry, and length is a prerequisite for the selection of optimal cyclic peptide scaffolds. Our systematic approach identifies cyclic APP dodecamer peptides targeting the phosphotyrosine binding domain of Mint2 with substantially improved affinity. We show that especially all-hydrocarbon stapling provides improved metabolic stability, a significantly stabilized secondary structure and membrane permeability.
Assuntos
Precursor de Proteína beta-Amiloide , Peptídeos Cíclicos , Ciclização , Peptídeos Cíclicos/química , Estrutura Secundária de Proteína , Precursor de Proteína beta-Amiloide/química , Ligação Proteica , Fosfotirosina/químicaRESUMO
Amyloid precursor protein (APP) is a major contributor to the pathology of Alzheimer's and other neurodegenerative diseases through the accumulation of extracellular plaques. Here, we have studied changes in APP translation and aggregation of the APP intracellular domain when the Gαq/PLCß signaling system is activated by neurotransmitters. Using RT-PCR and a molecular beacon that follows APP mRNA in live cells, we find that Gαq activation sequesters APP mRNA similar to the stress granule response found in heat shock and hypo-osmotic shock thereby shutting down the production of APP. Following the intracellular domain of eGFP-APP, we find that Gαq stimulation increases aggregation as followed by number and brightness (N&B) analysis of single molecule fluorescence time series. Additionally, we show that APP aggregation is affected by changes in the levels of PLCß1 and its cytosolic binding partners. Our studies show the neurotransmitter activation of Gαq/PLCß reduces translation of APP and increases aggregation of its intracellular domain. These studies better establish a link between APP production and complexation and Gαq stimulation.
Assuntos
Precursor de Proteína beta-Amiloide , Transdução de Sinais , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , RNA Mensageiro , Neurotransmissores , Imagem Óptica , Peptídeos beta-AmiloidesRESUMO
The proteolytic cleavage of C99 by γ-secretase is the last step in the production of amyloid-ß (Aß) peptides. Previous studies have shown that membrane lipid composition, cholesterol concentration, and mutation in the transmembrane helix modified the structures and fluctuations of C99. In this study, we performed atomistic molecular dynamics simulations of the homodimer of the 55-residue congener of the C-terminal domain of the amyloid protein precursor, C99(1-55), in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine-cholesterol lipid bilayer and compared the conformational ensemble of wild-type (WT) sequence to those of the A2T and D23N variants. These mutations are particularly interesting as the protective Alzheimer's disease (AD) A2T mutation is known to decrease Aß production, whereas the early onset AD D23N mutation does not affect Aß production. We found noticeable differences in the structural ensembles of the three sequences. In particular, A2T varies from both WT and D23N by having long-range effects on the population of the extracellular juxtamembrane helix, the interface between the G29xxx-G33xxx-G37 motifs, and the fluctuations of the transmembrane helical topologies.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Fragmentos de Peptídeos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Colesterol , Humanos , Mutação , Fragmentos de Peptídeos/química , Multimerização ProteicaRESUMO
γ-Secretase (GS) is a transmembrane (TM) enzyme that plays important roles in the processing of approximately 90 substrates. The amyloid precursor protein (APP) is one of these substrates, and the peptides derived from their processing are related with the development of Alzheimer's disease. However, the mechanistic process involved in the GS substrate processing and regulation remains elusive. In this work, we employed extensive atomistic molecular dynamics simulations, reduction dimensionality, and network analysis to understand the dynamic behavior of GS in its apo form and bound to transmembrane fragments of APP-C99, APP-C83, and Notch. An evaluation of the global conformation of the enzyme revealed that GS and GS-C83 systems display extended and compact conformations. However, systems with long extracellular N-terminal substrates, such as APP-C99 and Notch, preferred compact conformations. Interestingly, our network analysis revealed that the NCT-lobule (residues 223-248) plays a crucial role in the communication and the dynamics between the extracellular and TM components of the enzyme, impacting the catalytic site. In our GS-C99 simulated system, the interaction paths of the substrate processing region encompass the ε-site and ζ-site, leading to more imprecise positioning of the catalytic residue Asp385. Conversely, our GS-C83 simulated system shows more stability at the ε-site. Our observations shed light on the important mechanics of the fascinating GS architecture and may contribute to propose new GS modulators able to impact on the Alzheimer's disease treatment.
Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/química , Domínio Catalítico , Humanos , Simulação de Dinâmica MolecularRESUMO
Amyloid precursor protein (APP) is cleaved and processed sequentially by γ-secretase yielding amyloid ß (Aß) peptides of different lengths. Longer Aß peptides are associated with the formation of neurotoxic plaques related to Alzheimer's disease. Based on the APP substrate-bound structure of γ-secretase, we investigated the enzyme-substrate interaction using molecular dynamics simulations and generated model structures that represent the sequentially cleaved intermediates during the processing reaction. The simulations indicated an internal docking site providing strong enzyme-substrate packing interaction. In the enzyme-substrate complex, it is located close to the region where the helical conformation of the substrate is interrupted and continues toward the active site in an extended conformation. The internal docking site consists of two non-polar pockets that are preferentially filled by large hydrophobic or aromatic substrate side chains to stabilize binding. Placement of smaller residues such as glycine can trigger a shift in the cleavage pattern during the simulations or results in destabilization of substrate binding. The reduced packing by smaller residues also influences the hydration of the active site and the formation of a catalytically active state. The simulations on processed substrate intermediates and a substrate G33I mutation offer an explanation of the experimentally observed relative increase of short Aß fragment production for this mutation. In addition, studies on a substrate K28A mutation indicate that the internal docking site opposes the tendency of substrate dissociation due to a hydrophobic mismatch at the membrane boundary caused by K28 during processing and substrate movement toward the enzyme active site. The proposed internal docking site could also be useful for the specific design of new γ-secretase modulators.
Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/química , Humanos , Simulação de Dinâmica Molecular , Especificidade por SubstratoRESUMO
Amyloid precursor protein (APP) is the core of the pathogenesis of Alzheimer's disease (AD). Existing studies have shown that the soluble secreted APP (sAPPα) fragment obtained from the hydrolysis of APP by α-secretase has a synaptic function. Thereinto, a nine-residue fragment (APP9mer) of the extension domain region of sAPPα can bind directly and selectively to the N-terminal sushi1 domain (SD1) of the γ-aminobutyric acid type B receptor subunit 1a (GABABR1a) protein, which can influence synaptic transmission and plasticity by changing the GABABR1a conformation. APP9mer is a highly flexible, disordered region, and as such it is difficult to experimentally determine the optimal APPmer-SD1 binding complex. In this study we constructed two types of APP9mer-SD1 complexes through molecular docking and molecular dynamics simulation, aiming to explore the recognition function and mechanism of the specific binding of APP9mer with SD1, from which the most probable APPmer-SD1 model conformation is predicted. All the data from the analyses of RMSD, RMSF, PCA, DCCM and MM/PBSA binding energy as well as comparison with the experimental dissociation constant Kd suggest that 2NC is the most likely conformation to restore the crystal structure of the experimental APP9mer-SD1 complex. Of note, the key recognition residues of APP9mer are D24, D25, D27, W29 and W30, which mainly act on the 9-45 residue domain of SD1 (consisting of two loops and three short ß-chains at the N-terminus of SD1). The mini-model with key residues identified establishes the molecular basis with deep insight into the interaction between APP and GABABR1a and provides a target for the development of therapeutic strategies for modulating GABABR1a-specific signaling in neurological and psychiatric disorders. More importantly, the study offers a theoretical solution for how to determine a biomolecular structure with a highly flexible, disordered fragment embedded within. The flexible fragment involved in a protein structure has to be deserted usually during the structural determination with experimental methods (e.g. X-ray crystallography, etc.).
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Receptores de GABA , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/ultraestrutura , Humanos , Simulação de Acoplamento Molecular , Receptores de GABA/química , Receptores de GABA/ultraestrutura , Sindactilia , Ácido gama-Aminobutírico/metabolismoRESUMO
γ-Secretase is a membrane protein complex that proteolyzes within the transmembrane domain of >100 substrates, including those derived from the amyloid precursor protein and the Notch family of cell surface receptors. The nine-transmembrane presenilin is the catalytic component of this aspartyl protease complex that carries out hydrolysis in the lipid bilayer. Advances in cryoelectron microscopy have led to the elucidation of the structure of the γ-secretase complex at atomic resolution. Recently, structures of the enzyme have been determined with bound APP- or Notch-derived substrates, providing insight into the nature of substrate recognition and processing. Molecular dynamics simulations of substrate-bound enzymes suggest dynamic mechanisms of intramembrane proteolysis. Structures of the enzyme bound to small-molecule inhibitors and modulators have also been solved, setting the stage for rational structure-based drug discovery targeting γ-secretase.
Assuntos
Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Receptores Notch/metabolismoRESUMO
Alzheimer's disease (AD) is caused by synaptic and neuronal loss in the brain. One of the characteristic hallmarks of AD is senile plaques containing amyloid ß-peptide (Aß). Aß is produced from amyloid precursor protein (APP) by sequential proteolytic cleavages by ß-secretase and γ-secretase, and the polymerization of Aß into amyloid plaques is thought to be a key pathogenic event in AD. Since γ-secretase mediates the final cleavage that liberates Aß, γ-secretase has been widely studied as a potential drug target for the treatment of AD. γ-Secretase is a transmembrane protein complex containing presenilin, nicastrin, Aph-1, and Pen-2, which are sufficient for γ-secretase activity. γ-Secretase cleaves >140 substrates, including APP and Notch. Previously, γ-secretase inhibitors (GSIs) were shown to cause side effects in clinical trials due to the inhibition of Notch signaling. Therefore, more specific regulation or modulation of γ-secretase is needed. In recent years, γ-secretase modulators (GSMs) have been developed. To modulate γ-secretase and to understand its complex biology, finding the binding sites of GSIs and GSMs on γ-secretase as well as identifying transiently binding γ-secretase modulatory proteins have been of great interest. In this review, decades of findings on γ-secretase in AD are discussed.