RESUMO
Alzheimer disease (AD) is the most common progressive neurodegenerative disorder. AD causes enormous personal and economic burden to society as currently only limited palliative therapeutic options are available. The pathological hallmarks of the disease are extracellular plaques, composed of fibrillar amyloid-ß (Aß), and neurofibrillary tangles inside neurons, composed of Tau protein. Until recently, the search for AD therapeutics was focussed more on the Aß peptide and its pathology, but the results were unsatisfying. As an alternative, Tau might be a promising therapeutic target as its pathology is closely correlated to clinical symptoms. In addition, pathological Tau aggregation occurs in a large group of diseases, called Tauopathies, and in most of them Aß aggregation does not play a role in disease pathogenesis. The formation of Tau aggregates is triggered by two hexapeptide motifs within Tau; PHF6* and PHF6. Both fragments are interesting targets for the development of Tau aggregation inhibitors (TAI). Peptides represent a unique class of pharmaceutical compounds and are reasonable alternatives to chemical substances or antibodies. They are attributed with high biological activity, valuable specificity and low toxicity, and often are developed as drug candidates to interrupt protein-protein interactions. The preparation of peptides is simple, controllable and the peptides can be easily modified. However, their application may also have disadvantages. Currently, a few peptide compounds acting as TAI are described in the literature, most of them developed by structure-based design or phage display. Here, we review the current state of research in this promising field of AD therapy development.
Assuntos
Doença de Alzheimer , Tauopatias , Humanos , Doença de Alzheimer/metabolismo , Proteínas tau/metabolismo , Tauopatias/metabolismo , Emaranhados Neurofibrilares/metabolismo , Peptídeos beta-Amiloides/metabolismoRESUMO
Alzheimer's disease and other Tauopathies are associated with neurofibrillary tangles composed of Tau protein, as well as toxic Tau oligomers. Therefore, inhibitors of pathological Tau aggregation are potentially useful candidates for future therapies targeting Tauopathies. Two hexapeptides within Tau, designated PHF6* (275-VQIINK-280) and PHF6 (306-VQIVYK-311), are known to promote Tau aggregation. Recently, the PHF6* segment has been described as the more potent driver of Tau aggregation. We therefore employed mirror-image phage display with a large peptide library to identify PHF6* fibril binding peptides consisting of D-enantiomeric amino acids. The suitability of D-enantiomeric peptides for inâ vivo applications, which are protease stable and less immunogenic than L-peptides, has already been demonstrated. The identified D-enantiomeric peptide MMD3 and its retro-inverso form, designated MMD3rev, inhibited inâ vitro fibrillization of the PHF6* peptide, the repeat domain of Tau as well as full-length Tau. Dynamic light scattering, pelleting assays and atomic force microscopy demonstrated that MMD3 prevents the formation of tau ß-sheet-rich fibrils by diverting Tau into large amorphous aggregates. NMR data suggest that the D-enantiomeric peptides bound to Tau monomers with rather low affinity, but ELISA (enzyme-linked immunosorbent assay) data demonstrated binding to PHF6* and full length Tau fibrils. In addition, molecular insight into the binding mode of MMD3 to PHF6* fibrils were gained by in silico modelling. The identified PHF6*-targeting peptides were able to penetrate cells. The study establishes PHF6* fibril binding peptides consisting of D-enantiomeric amino acids as potential molecules for therapeutic and diagnostic applications in AD research.
Assuntos
Peptídeos/farmacologia , Proteínas tau/antagonistas & inibidores , Humanos , Biblioteca de Peptídeos , Peptídeos/química , Agregados Proteicos/efeitos dos fármacos , Proteínas tau/metabolismoRESUMO
BACKGROUND: Amyloid fibrils such as Semen-Derived Enhancer of Viral Infection (SEVI) or amyloid-ß-peptide (Aß) enhance HIV-1 attachment and entry. Inhibitors destroying or converting those fibrils into non-amyloidogenic aggregates effectively reduce viral infectivity. Thus, they seem to be suitable as therapeutic drugs expanding the current HIV-intervening repertoire of antiretroviral compounds. FINDINGS: In this study, we demonstrate that the small D-amino acid peptide D3, which was investigated for therapeutic studies on Alzheimer's disease (AD), significantly reduces both SEVI and Aß fibril boosted infectivity of HIV-1. CONCLUSIONS: Since amyloids could play an important role in the progression of AIDS dementia complex (ADC), the treatment of HIV-1 infected individuals with D3, that inhibits Aß fibril formation and converts preformed Aß fibrils into non-amyloidogenic and non-fibrillar aggregates, may reduce the vulnerability of the central nervous system of HIV patients for HIV associated neurological disorders.
RESUMO
In neurodegenerative diseases like Alzheimer's disease (AD), endogenous proteins or peptides aggregate with themselves. These proteins may lose their function or aggregates and/or oligomers can obtain toxicity, causing injury or death to cells. Aggregation of two major proteins characterizes AD. Amyloid-ß peptide (Aß) is deposited in amyloid plaques within the extracellular space of the brain and Tau in so-called neurofibrillary tangles in neurons. Finding peptide ligands to halt protein aggregation is a promising therapeutical approach. Using mirror-image phage display with a commercially available, randomized 12-mer peptide library, we have selected D-amino acid peptides, which bind to the Tau protein and modulate its aggregation in vitro. Peptides can bind specifically and selectively to a target molecule, but natural L-amino acid peptides may have crucial disadvantages for in vivo applications, as they are sensitive to protease degradation and may elicit immune responses. One strategy to circumvent these disadvantages is the use of non-naturally occurring D-amino acid peptides as they exhibit increased protease resistance and generally do not activate the immune system. To perform mirror-image phage display, the target protein needs to be synthesized as D-amino acid version. If the target protein sequence is too long to be synthesized properly, smaller peptides derived from the full length protein can be used for the selection process. This also offers the possibility to influence the binding region of the selected D-peptides in the full-length target protein. Here we provide the protocols for mirror-image phage display selection on the PHF6* peptide of Tau, based on the commercially available Ph.D.™-12 Phage Display Peptide Library Kit, leading to D-peptides that also bind the full length Tau protein (Tau441), next to PHF6*. In addition, we provide protocols and data for the first characterization of those D-peptides that inhibit Tau aggregation in vitro. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Mirror image phage display selection against D-PHF6* fibrils Support Protocol 1: Single phage ELISA Basic Protocol 2: Sequencing and D-peptide generation Basic Protocol 3: Thioflavin-T (ThT) test to control inhibition of Tau aggregation Support Protocol 2: Purification of full-length Tau protein Basic Protocol 4: ELISA to demonstrate the binding of the generated D-peptides to PHF6* and full-length Tau fibrils.
Assuntos
Doença de Alzheimer , Bacteriófagos , Humanos , Proteínas tau/genética , Proteínas tau/química , Proteínas tau/metabolismo , Aminoácidos , Biblioteca de Peptídeos , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeo Hidrolases/metabolismo , Bacteriófagos/metabolismoRESUMO
The aggregation of the Aß plays a fundamental role in the pathology of AD. Recently, N-terminally modified Aß species, pE-Aß, have been described as major constituents of Aß deposits in the brains of AD patients. pE-Aß has an increased aggregation propensity and shows increased toxicity compared with Aß1-40 and Aß1-42. In the present work, high-resolution NMR spectroscopy was performed to study pE-Aß3-40 in aqueous TFE-containing solution. Two-dimensional TOCSY and NOESY experiments were performed. On the basis of NOE and chemical shift data, pE-Aß3-40 was shown to contain two helical regions formed by residues 14-22 and 30-36. This is similar as previously described for Aß1-40. However, the secondary chemical shift data indicate decreased helical propensity in pE-Aß3-40 when compared with Aß1-40 under exactly the same conditions. This is in agreement with the observation that pE-Aß3-40 shows a drastically increased tendency to form ß-sheet-rich structures under more physiologic conditions. Structural studies of pE-Aß are crucial for better understanding the structural basis of amyloid fibril formation in the brain during development of AD, especially because an increasing number of reports indicate a decisive role of pE-Aß for the pathogenesis of AD.
Assuntos
Peptídeos beta-Amiloides/química , Espectroscopia de Ressonância Magnética , Ácido Pirrolidonocarboxílico/química , Doença de Alzheimer/patologia , Humanos , Isoformas de Proteínas/químicaRESUMO
BACKGROUND: Alzheimer's disease (AD), the most common form of dementia, is a progressive neurodegenerative disorder that mainly affects older adults. One of the pathological hallmarks of AD is abnormally aggregated Tau protein that forms fibrillar deposits in the brain. In AD, Tau pathology correlates strongly with clinical symptoms, cognitive dysfunction, and neuronal death. METHODS: We aimed to develop novel therapeutic D-amino acid peptides as Tau fibrillization inhibitors. It has been previously demonstrated that D-amino acid peptides are protease stable and less immunogenic than L-peptides, and these characteristics may render them suitable for in vivo applications. Using a phage display procedure against wild type full-length Tau (TauFL), we selected a novel Tau binding L-peptide and synthesized its D-amino acid version ISAD1 and its retro inversed form, ISAD1rev, respectively. RESULTS: While ISAD1rev inhibited Tau aggregation only moderately, ISAD1 bound to Tau in the aggregation-prone PHF6 region and inhibited fibrillization of TauFL, disease-associated mutant full-length Tau (TauFLΔK, TauFL-A152T, TauFL-P301L), and pro-aggregant repeat domain Tau mutant (TauRDΔK). ISAD1 and ISAD1rev induced the formation of large high molecular weight TauFL and TauRDΔK oligomers that lack proper Thioflavin-positive ß-sheet conformation even at lower concentrations. In silico modeling of ISAD1 Tau interaction at the PHF6 site revealed a binding mode similar to those known for other PHF6 binding peptides. Cell culture experiments demonstrated that ISAD1 and its inverse form are taken up by N2a-TauRDΔK cells efficiently and prevent cytotoxicity of externally added Tau fibrils as well as of internally expressed TauRDΔK. CONCLUSIONS: ISAD1 and related peptides may be suitable for therapy development of AD by promoting off-pathway assembly of Tau, thus preventing its toxicity.
Assuntos
Doença de Alzheimer , Peptídeos , Proteínas tau , Idoso , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Aminoácidos/uso terapêutico , Células Cultivadas , Humanos , Peptídeos/uso terapêutico , Conformação Proteica em Folha beta , Proteínas tau/metabolismo , Proteínas tau/toxicidadeRESUMO
N-Terminally truncated and pyroglutamate (pGlu) modified amyloid beta (Abeta) peptides are major constituents of amyloid deposits in sporadic and inherited Alzheimer's disease (AD). Formation of pGlu at the N-terminus confers resistance against cleavage by most aminopeptidases, increases toxicity of the peptides, and may seed Abeta aggregate formation. Similarly, the deposited amyloid peptides ABri and ADan, which cause a very similar histopathology in familial British dementia (FBD) and familial Danish dementia (FDD), are N-terminally blocked by pGlu. Triggered by the coincidence of pGlu-modified amyloid peptides and similar pathology in AD, FBD, and FDD, we investigated the impact of N-terminal pGlu on biochemical and biophysical properties of Abeta, ABri, and ADan. N-Terminal pGlu increases the hydrophobicity and changes the pH-dependent solubility profile, rendering the pGlu-modified peptides less soluble in the basic pH range. The pGlu residue increases the aggregation propensity of all amyloid peptides as evidenced by ThT fluorescence assays and dynamic light scattering. The far-UV CD spectroscopic analysis points toward an enhanced beta-sheet structure of the pGlu-Abeta. Importantly, changes in fibril morphology are clearly caused by the N-terminal pGlu, resulting in the formation of short fibers, which are frequently arranged in bundles. The effect of pGlu on the morphology is virtually indistinguishable between ABri, ADan, and Abeta. The data provide evidence for a comparable influence of the pGlu modification on the aggregation process of structurally different amyloid peptides, thus likely contributing to the molecularly distinct neurodegenerative diseases AD, FBD, and FDD. The main driving force for the aggregation is apparently an increase in the hydrophobicity and thus an accelerated seed formation.
Assuntos
Amiloide/metabolismo , Peptídeos/metabolismo , Ácido Pirrolidonocarboxílico/metabolismo , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Amiloide/química , Amiloide/ultraestrutura , Dicroísmo Circular , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Dados de Sequência Molecular , Peptídeos/química , Solubilidade , Espectrofotometria UltravioletaRESUMO
Soluble amyloid beta(1-42) (A beta(1-42)) peptide has recently been assigned a key role in early Alzheimer's disease (AD) pathophysiology accounting for synaptic dysfunction before amyloid plaque formation and neurodegeneration can occur. Following sublethal A beta(1-42) administration, we observed an acute but transient reduction of the spike and burst rate of spontaneously active cortical networks cultured on microelectrode arrays. This simple experimental system appears suitable for future long-term pharmacological and genetic studies of A beta(1-42) signaling, thus providing a valuable new tool in AD research.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Córtex Cerebral/citologia , Rede Nervosa/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Potenciais de Ação/fisiologia , Peptídeos beta-Amiloides/administração & dosagem , Peptídeos beta-Amiloides/farmacologia , Animais , Técnicas de Cultura de Células , Eletrofisiologia , Microeletrodos , Rede Nervosa/fisiologia , Neurônios/fisiologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/farmacologia , RatosRESUMO
Prion diseases, Alzheimer's disease, and Parkinson's disease are age-related neurodegenerative diseases that are characterized by the formation of protein aggregates during the progress of the disease. Although it is still not known whether these aggregates are causative for, or symptoms of, the disease. Many studies show that aggregates or even oligomers of the according proteins are neurotoxic and thus may lead to neurodegeneration. To understand disease-associated or causative mechanisms in respect to protein aggregation, an ultrasensitive tool to quantify these disease-related aggregates is required. In this study we introduce a specificity-enhanced version of surface-FIDA as an approach to count even single aggregates in tissue homogenate and body liquids.
Assuntos
Doença de Alzheimer/diagnóstico , Bioensaio/métodos , Doenças Priônicas/diagnóstico , Animais , Bovinos , Estrutura Quaternária de Proteína , Sensibilidade e EspecificidadeRESUMO
Alzheimer's disease (AD) is a chronic neurodegenerative disorder and the most common cause of dementia. Aging is among the most significant risk factors. Today, AD can be diagnosed with certainty only post mortem, detecting insoluble beta-amyloid peptide (Abeta) aggregates in the patient's brain tissue. We have developed an ultrasensitive assay for early and non-invasive diagnosis of AD. This highly specific and sensitive assay uses fluorescence correlation spectroscopy (FCS) and is sensitive enough to detect even single aggregates in body fluids of AD patients. We investigate the correlation of aggregated Abeta concentrations in body fluids with clinical symptoms of AD.
Assuntos
Doença de Alzheimer/diagnóstico , Espectrometria de Fluorescência/métodos , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Peptídeos beta-Amiloides/síntese química , Peptídeos beta-Amiloides/química , Humanos , Estrutura Quaternária de Proteína , Sensibilidade e EspecificidadeRESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia. Today, AD can be diagnosed with certainty only post-mortem, by histopathologic staining of Abeta plaques and neurofibrillary tangles in brain tissue sections. We have developed an ultra-sensitive assay potentially suitable for early and non-invasive diagnosis of AD. This highly specific and sensitive assay uses fluorescence correlation spectroscopy (FCS) and is sensitive enough to detect even single aggregates in body fluids of AD patients. First results show a clear distinction between AD diseased people and non-demented controls by analysing cerebrospinal fluids (CSF) by confocal scanning of surface captured Abeta aggregates and subsequent two-dimensional fluorescence intensity distribution analysis.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/ultraestrutura , Líquido Cefalorraquidiano/citologia , Líquido Cefalorraquidiano/metabolismo , Microscopia de Fluorescência/métodos , Humanos , Aumento da Imagem/métodos , Microscopia Confocal/métodos , Tamanho da PartículaRESUMO
The amyloid cascade hypothesis assigns the amyloid-beta peptide (Abeta) a central role in the pathogenesis of Alzheimer's disease (AD). Although there are strong efforts to biophysically characterize formation of Abeta aggregates and fibrils, as well as their prevention, progress is still severly hampered by the availability of tens of milligrams of recombinant Abeta(1-42). Here, we describe a reliable and easy procedure to recombinantly express and purify Abeta(1-42), which is fully cytotoxic and able to form fibrils without any further refolding steps. The yield of the procedure is 5-8 mg of tag-less peptide per liter culture volume.
Assuntos
Peptídeos beta-Amiloides/isolamento & purificação , Fragmentos de Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Peptídeos beta-Amiloides/farmacologia , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Primers do DNA , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/farmacologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologiaRESUMO
Protein misfolding and aggregation are fundamental features of the majority of neurodegenerative diseases, like Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, and prion diseases. Proteinaceous deposits in the brain of the patient, e.g., amyloid plaques consisting of the amyloid-ß (Aß) peptide and tangles composed of tau protein, are the hallmarks of AD. Soluble oligomers of Aß and tau play a fundamental role in disease progression, and specific detection and quantification of the respective oligomeric proteins in cerebrospinal fluid may provide presymptomatically detectable biomarkers, paving the way for early diagnosis or even prognosis. Several studies on the development of techniques for the specific detection of Aß oligomers were published, but some of the existing tools do not yet seem to be satisfactory, and the study results are contradicting. The detection of oligomers is challenging due to their polymorphous and unstable nature, their low concentration, and the presence of competing proteins and Aß monomers in body fluids. Here, we present an overview of the current state of the development of methods for Aß oligomer specific detection and quantitation. The methods are divided in the three subgroups: (i) enzyme linked immunosorbent assays (ELISA), (ii) methods for single oligomer detection, and (iii) others, which are mainly biosensor based methods.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Técnicas Biossensoriais , Fragmentos de Peptídeos/líquido cefalorraquidiano , Ensaio de Imunoadsorção Enzimática , Humanos , Proteínas tau/análise , Proteínas tau/líquido cefalorraquidianoRESUMO
Alzheimer´s disease is the most prominent type of dementia and currently no causative treatment is available. According to recent studies, oligomeric species of the amyloid beta (Aß) peptide appear to be the most toxic Aß assemblies. Aß monomers, however, may be not toxic per se and may even have a neuroprotective role. Here we describe a competitive mirror image phage display procedure that allowed us to identify preferentially Aß1-42 monomer binding and thereby stabilizing peptides, which destabilize and thereby eliminate toxic oligomer species. One of the peptides, called Mosd1 (monomer specific d-peptide 1), was characterized in more detail. Mosd1 abolished oligomers from a mixture of Aß1-42 species, reduced Aß1-42 toxicity in cell culture, and restored the physiological phenotype in neuronal cells stably transfected with the gene coding for human amyloid precursor protein.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Técnicas de Visualização da Superfície Celular , Peptídeos/farmacologia , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas , Secretases da Proteína Precursora do Amiloide/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , HumanosRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0147470.].
RESUMO
A variety of neurodegenerative disorders, including Alzheimer disease (AD), are associated with neurofibrillary tangles composed of the tau protein, as well as toxic tau oligomers. Inhibitors of pathological tau aggregation, interrupting tau self-assembly, might be useful for the development of therapeutics. Employing mirror image phage display with a large peptide library (over 109 different peptides), we have identified tau fibril binding peptides consisting of d-enantiomeric amino acids. d-enantiomeric peptides are extremely protease stable and not or less immunogenic than l-peptides, and the suitability of d-peptides for in vivo applications have already been demonstrated. Phage display selections were performed using fibrils of the d-enantiomeric hexapeptide VQIVYK, representing residues 306 to 311 of the tau protein, as a target. VQIVYK has been demonstrated to be important for fibril formation of the full lengths protein and forms fibrils by itself. Here, we report on d-enantiomeric peptides, which bind to VQIVYK, tau isoforms like tau3RD (K19) as well as to full lengths tau fibrils, and modulate the aggregation of the respective tau form. The peptides are able to penetrate cells and might be interesting for therapeutic and diagnostic applications in AD research.
Assuntos
Oligopeptídeos/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Sítios de Ligação , Difusão Dinâmica da Luz , Fluoresceínas/química , Humanos , Simulação de Dinâmica Molecular , Oligopeptídeos/química , Oligopeptídeos/uso terapêutico , Biblioteca de Peptídeos , Agregados Proteicos , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Estereoisomerismo , Proteínas tau/química , Proteínas tau/genéticaRESUMO
The aggregation of amyloid-ß (Aß) is postulated to be the crucial event in Alzheimer's disease (AD). In particular, small neurotoxic Aß oligomers are considered to be responsible for the development and progression of AD. Therefore, elimination of thesis oligomers represents a potential causal therapy of AD. Starting from the well-characterized d-enantiomeric peptide D3, we identified D3 derivatives that bind monomeric Aß. The underlying hypothesis is that ligands bind monomeric Aß and stabilize these species within the various equilibria with Aß assemblies, leading ultimately to the elimination of Aß oligomers. One of the hereby identified d-peptides, DB3, and a head-to-tail tandem of DB3, DB3DB3, were studied in detail. Both peptides were found to: (i) inhibit the formation of Thioflavin T-positive fibrils; (ii) bind to Aß monomers with micromolar affinities; (iii) eliminate Aß oligomers; (iv) reduce Aß-induced cytotoxicity; and (v) disassemble preformed Aß aggregates. The beneficial effects of DB3 were improved by DB3DB3, which showed highly enhanced efficacy. Our approach yielded Aß monomer-stabilizing ligands that can be investigated as a suitable therapeutic strategy against AD.
Assuntos
Peptídeos beta-Amiloides/química , Oligopeptídeos/química , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , RatosRESUMO
Chiral compounds can be produced efficiently by using biocatalysts. However, wild-type enzymes often do not meet the requirements of a production process, making optimization by rational design or directed evolution necessary. Here, we studied the lipase-catalyzed hydrolysis of the model substrate 1-(2-naphthyl)ethyl acetate both theoretically and experimentally. We found that a computational equivalent of alanine scanning mutagenesis based on QM/MM methodology can be applied to identify amino acid positions important for the activity of the enzyme. The theoretical results are consistent with concomitant experimental work using complete saturation mutagenesis and high-throughput screening of the target biocatalyst, a lipase from Bacillus subtilis. Both QM/MM-based calculations and molecular biology experiments identify histidine 76 as a residue that strongly affects the catalytic activity. The experiments demonstrate its important influence on enantioselectivity.
Assuntos
Biologia Computacional/métodos , Evolução Molecular Direcionada/métodos , Biblioteca Gênica , Substituição de Aminoácidos/genética , Bacillus subtilis/química , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Hidrólise , Lipase/química , Lipase/genética , Modelos Químicos , Mutagênese , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
Recent evidence suggests Alzheimer-Disease (AD) to be driven by aggregated Aß. Capitalizing on the mechanism of molecular mimicry and applying several selection layers, we screened peptide libraries for moieties inducing antibodies selectively reacting with Aß-aggregates. The technology identified a pool of peptide candidates; two, AFFITOPES AD01 and AD02, were assessed as vaccination antigens and compared to Aß1-6, the targeted epitope. When conjugated to Keyhole Limpet Hemocyanin (KLH) and adjuvanted with aluminum, all three peptides induced Aß-targeting antibodies (Abs). In contrast to Aß1-6, AD01- or AD02-induced Abs were characterized by selectivity for aggregated forms of Aß and absence of reactivity with related molecules such as Amyloid Precursor Protein (APP)/ secreted APP-alpha (sAPPa). Administration of AFFITOPE-vaccines to APP-transgenic mice was found to reduce their cerebral amyloid burden, the associated neuropathological alterations and to improve their cognitive functions. Thus, the AFFITOME-technology delivers vaccines capable of inducing a distinct Ab response. Their features may be beneficial to AD-patients, a hypothesis currently tested within a phase-II-study.
Assuntos
Doença de Alzheimer/terapia , Vacinas contra Alzheimer/uso terapêutico , Peptídeos beta-Amiloides/imunologia , Anticorpos/imunologia , Doença de Alzheimer/imunologia , Vacinas contra Alzheimer/imunologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Biblioteca de PeptídeosRESUMO
One of the characteristic pathological hallmarks of Alzheimer's disease (AD) is neuritic plaques. The sequence of events leading to deposition of amyloid-ß (Aß) peptides in plaques is not clear. Here we investigate the effects of D3, an Aß oligomer directed D-enantiomeric peptide that was obtained from a mirror image phage display selection against monomeric or small oligomeric forms of Aß42, on Aß deposition in aged AßPP/PS1 double transgenic AD-model mice. Using Alzet minipumps, we infused the brains of these AD model mice for 8 weeks with FITC-labeled D3, and examined the subsequent changes in pathology and cognitive deficits. Initial cognitive deficits are similar comparing control and D3-FITC-treated mice, but the treated mice show a significant improvement on the last day of testing. Further, we show that there is a substantial reduction in the amount of amyloid deposits in the animals treated with D3-FITC, compared to the control mice. Finally, the amount of activated microglia and astrocytes surrounding Aß deposits is dramatically reduced in the D3-FITC-treated mice. Our findings demonstrate that treatments with the high affinity Aß42 oligomer binding D-enantiomeric peptide D3 significantly decrease Aß deposits and the associated inflammatory response, and improve cognition even when applied only at late stages and high age. Together, this suggests that the treatment reduces the level of Aß peptide in the brains of AßPP/PS1 mice, possibly by increasing Aß outflow from the brain. In conclusion, treatments with this D-peptide have great potential to be successful in AD patients.