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1.
Nat Commun ; 11(1): 4052, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792510

RESUMO

Turn-on fluorescence imaging is routinely studied; however, turn-on chemiluminescence has been rarely explored for in vivo imaging. Herein, we report the design and validation of chemiluminescence probe ADLumin-1 as a turn-on probe for amyloid beta (Aß) species. Two-photon imaging indicates that ADLumin-1 can efficiently cross the blood-brain barrier and provides excellent contrast for Aß plaques and cerebral amyloid angiopathy. In vivo brain imaging shows that the chemiluminescence signal of ADLumin-1 from 5-month-old transgenic 5xFAD mice is 1.80-fold higher than that from the age-matched wild-type mice. Moreover, we demonstrate that it is feasible to further dually-amplify signal via chemiluminescence resonance energy transfer (DAS-CRET) using two non-conjugated smart probes (ADLumin-1 and CRANAD-3) in solutions, brain homogenates, and in vivo whole brain imaging. Our results show that DAS-CRET can provide a 2.25-fold margin between 5-month-old 5xFAD mice and wild type mice. We believe that our strategy could be extended to other aggregating-prone proteins.


Assuntos
Peptídeos beta-Amiloides/química , Luminescência , Animais , Medições Luminescentes/métodos , Camundongos , Imagem Molecular/métodos , Imagem Óptica/métodos , Agregados Proteicos
2.
PLoS One ; 15(7): e0235543, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645028

RESUMO

Senile plaques frequently contain Aß-pE(3), a N-terminally truncated Aß species that is more closely linked to AD compared to other Aß species. Tau protein is highly phosphorylated at several residues in AD, and specifically phosphorylation at Ser202/Thr205 is known to be increased in AD. Several studies suggest that formation of plaques and tau phosphorylation might be linked to each other. To evaluate if Aß-pE(3) and ptau Ser202/Thr205 levels correlate in human and transgenic AD mouse models, we analyzed human cortical and hippocampal brain tissue of different Braak stages as well as murine brain tissue of two transgenic mouse models for levels of Aß-pE(3) and ptau Ser202/Thr205 and correlated the data. Our results show that Aß-pE(3) formation is increased at early Braak stages while ptau Ser202/Thr205 mostly increases at later stages. Further analyses revealed strongest correlations between the two pathologies in the temporal, frontal, cingulate, and occipital cortex, however correlation in the hippocampus was weaker. Evaluation of murine transgenic brain tissue demonstrated a slow but steady increase of Aß-pE(3) from 6 to 12 months of age in the cortex and hippocampus of APPSL mice, and a very early and strong Aß-pE(3) increase in 5xFAD mice. ptau Ser202/Thr205 levels increased at the age of 9 months in APPSL mice and at 6 months in 5xFAD mice. Our results show that Aß-pE(3) and ptau Ser202/Thr205 levels strongly correlate in human as well as murine tissues, suggesting that tau phosphorylation might be amplified by Aß-pE(3).


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Proteínas tau/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Fosforilação , Ácido Pirrolidonocarboxílico/química , Especificidade da Espécie , Proteínas tau/genética
3.
Nat Struct Mol Biol ; 27(7): 660-667, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32541895

RESUMO

Amyloid deposits consisting of fibrillar islet amyloid polypeptide (IAPP) in pancreatic islets are associated with beta-cell loss and have been implicated in type 2 diabetes (T2D). Here, we applied cryo-EM to reconstruct densities of three dominant IAPP fibril polymorphs, formed in vitro from synthetic human IAPP. An atomic model of the main polymorph, built from a density map of 4.2-Å resolution, reveals two S-shaped, intertwined protofilaments. The segment 21-NNFGAIL-27, essential for IAPP amyloidogenicity, forms the protofilament interface together with Tyr37 and the amidated C terminus. The S-fold resembles polymorphs of Alzheimer's disease (AD)-associated amyloid-ß (Aß) fibrils, which might account for the epidemiological link between T2D and AD and reports on IAPP-Aß cross-seeding in vivo. The results structurally link the early-onset T2D IAPP genetic polymorphism (encoding Ser20Gly) with the AD Arctic mutation (Glu22Gly) of Aß and support the design of inhibitors and imaging probes for IAPP fibrils.


Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Doença de Alzheimer/fisiopatologia , Substituição de Aminoácidos , Peptídeos beta-Amiloides/química , Microscopia Crioeletrônica , Diabetes Mellitus Tipo 2 , Humanos , Concentração de Íons de Hidrogênio , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica
4.
Proc Natl Acad Sci U S A ; 117(24): 13509-13518, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32493749

RESUMO

Protein misfolding and aggregation is the hallmark of numerous human disorders, including Alzheimer's disease. This process involves the formation of transient and heterogeneous soluble oligomers, some of which are highly cytotoxic. A major challenge for the development of effective diagnostic and therapeutic tools is thus the detection and quantification of these elusive oligomers. Here, to address this problem, we develop a two-step rational design method for the discovery of oligomer-specific antibodies. The first step consists of an "antigen scanning" phase in which an initial panel of antibodies is designed to bind different epitopes covering the entire sequence of a target protein. This procedure enables the determination through in vitro assays of the regions exposed in the oligomers but not in the fibrillar deposits. The second step involves an "epitope mining" phase, in which a second panel of antibodies is designed to specifically target the regions identified during the scanning step. We illustrate this method in the case of the amyloid ß (Aß) peptide, whose oligomers are associated with Alzheimer's disease. Our results show that this approach enables the accurate detection and quantification of Aß oligomers in vitro, and in Caenorhabditis elegans and mouse hippocampal tissues.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Anticorpos/imunologia , Agregados Proteicos , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Animais , Anticorpos/química , Anticorpos/metabolismo , Especificidade de Anticorpos , Caenorhabditis elegans , Modelos Animais de Doenças , Epitopos , Hipocampo/metabolismo , Camundongos , Ligação Proteica , Conformação Proteica , Anticorpos de Domínio Único
5.
Arch Biochem Biophys ; 690: 108446, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32593678

RESUMO

A simple NMR method to analyze the data obtained by NMR titration experiment of amyloid formation inhibitors against uniformly 15N-labeled amyloid-ß 1-42 peptide (Aß(1-42)) was described. By using solution nuclear magnetic resonance (NMR) measurement, the simplest method for monitoring the effects of Aß fibrilization inhibitors is the NMR chemical shift perturbation (CSP) experiment using 15N-labeled Aß(1-42). However, the flexible and dynamic nature of Aß(1-42) monomer may hamper the interpretation of CSP data. Here we introduced principal component analysis (PCA) for visualizing and analyzing NMR data of Aß(1-42) in the presence of amyloid inhibitors including high concentration osmolytes. We measured 1H-15N 2D spectra of Aß(1-42) at various temperatures as well as of Aß(1-42) with several inhibitors, and subjected all the data to PCA (PCA-HSQC). The PCA diagram succeeded in differentiating the various amyloid inhibitors, including epigallocatechin gallate (EGCg), rosmarinic acid (RA) and curcumin (CUR) from high concentration osmolytes. We hypothesized that the CSPs reflected the conformational equilibrium of intrinsically disordered Aß(1-42) induced by weak inhibitor binding rather than the specific molecular interactions.


Assuntos
Peptídeos beta-Amiloides/química , Fenóis/química , Análise de Componente Principal/métodos , Amiloide/metabolismo , Peptídeos beta-Amiloides/genética , Catequina/análogos & derivados , Catequina/química , Cinamatos/química , Curcumina/química , Depsídeos/química , Escherichia coli/genética , Humanos , Espectroscopia de Ressonância Magnética , Isótopos de Nitrogênio/química , Conformação Proteica , Temperatura , Termodinâmica
6.
Nat Commun ; 11(1): 3014, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32541820

RESUMO

Formation of amyloid-beta (Aß) oligomer pores in the membrane of neurons has been proposed to explain neurotoxicity in Alzheimer's disease (AD). Here, we present the three-dimensional structure of an Aß oligomer formed in a membrane mimicking environment, namely an Aß(1-42) tetramer, which comprises a six stranded ß-sheet core. The two faces of the ß-sheet core are hydrophobic and surrounded by the membrane-mimicking environment while the edges are hydrophilic and solvent-exposed. By increasing the concentration of Aß(1-42) in the sample, Aß(1-42) octamers are also formed, made by two Aß(1-42) tetramers facing each other forming a ß-sandwich structure. Notably, Aß(1-42) tetramers and octamers inserted into lipid bilayers as well-defined pores. To establish oligomer structure-membrane activity relationships, molecular dynamics simulations were carried out. These studies revealed a mechanism of membrane disruption in which water permeation occurred through lipid-stabilized pores mediated by the hydrophilic residues located on the core ß-sheets edges of the oligomers.


Assuntos
Peptídeos beta-Amiloides/química , Membrana Celular/química , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Conformação Proteica , Multimerização Proteica , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Membrana Celular/metabolismo , Condutividade Elétrica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/metabolismo , Síndromes Neurotóxicas/metabolismo , Fragmentos de Peptídeos/metabolismo , Água/metabolismo
7.
PLoS One ; 15(5): e0232266, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469918

RESUMO

Oligomeric amyloid ß (Aß) is currently considered the most neurotoxic form of the Aß peptide implicated in Alzheimer's disease (AD). The molecular structures of the oligomers have remained mostly unknown due to their transient nature. As a result, the molecular mechanisms of interactions between conformation-specific antibodies and their Aß oligomer (AßO) cognates are not well understood. A monoclonal conformation-specific antibody, m5E3, was raised against a structural epitope of Aß oligomers. m5E3 binds to AßOs with high affinity, but not to Aß monomers or fibrils. In this study, a computational model of the variable fragment (Fv) of the m5E3 antibody (Fv5E3) is introduced. We further employ docking and molecular dynamics simulations to determine the molecular details of the antibody-oligomer interactions, and to classify the AßOs as Fv5E3-positives and negatives, and to provide a rationale for the low affinity of Fv5E3 for fibrils. This information will help us to perform site-directed mutagenesis on the m5E3 antibody to improve its specificity and affinity toward oligomeric Aß species. We also provide evidence for the possible capability of the m5E3 antibody to disaggregate AßOs and to fragment protofilaments.


Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/imunologia , Anticorpos Monoclonais/imunologia , Multimerização Proteica , Sequência de Aminoácidos , Ligação Proteica , Estrutura Quaternária de Proteína
8.
Proc Natl Acad Sci U S A ; 117(21): 11265-11273, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32439711

RESUMO

The nucleation of Alzheimer-associated Aß peptide monomers can be catalyzed by preexisting Aß fibrils. This leads to autocatalytic amplification of aggregate mass and underlies self-replication and generation of toxic oligomers associated with several neurodegenerative diseases. However, the nature of the interactions between the monomeric species and the fibrils during this key process, and indeed the ultrastructural localization of the interaction sites have remained elusive. Here we used NMR and optical spectroscopy to identify conditions that enable the capture of transient species during the aggregation and secondary nucleation of the Aß42 peptide. Cryo-electron microscopy (cryo-EM) images show that new aggregates protrude from the entire length of the progenitor fibril. These protrusions are morphologically distinct from the well-ordered fibrils dominating at the end of the aggregation process. The data provide direct evidence that self-replication through secondary nucleation occurs along the sides of fibrils, which become heavily decorated under the current solution conditions (14 µM Aß42, 20 mM sodium phosphate, 200 µM EDTA, pH 6.8).


Assuntos
Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/ultraestrutura , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/ultraestrutura , Doença de Alzheimer/patologia , Amiloide/metabolismo , Amiloide/ultraestrutura , Peptídeos beta-Amiloides/química , Benzotiazóis/química , Benzotiazóis/metabolismo , Microscopia Crioeletrônica , Humanos , Concentração de Íons de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Fragmentos de Peptídeos/química , Imagem com Lapso de Tempo
9.
PLoS Comput Biol ; 16(5): e1007767, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365068

RESUMO

Many proteins have the potential to aggregate into amyloid fibrils, protein polymers associated with a wide range of human disorders such as Alzheimer's and Parkinson's disease. The thermodynamic stability of amyloid fibrils, in contrast to that of folded proteins, is not well understood: the balance between entropic and enthalpic terms, including the chain entropy and the hydrophobic effect, are poorly characterised. Using a combination of theory, in vitro experiments, simulations of a coarse-grained protein model and meta-data analysis, we delineate the enthalpic and entropic contributions that dominate amyloid fibril elongation. Our prediction of a characteristic temperature-dependent enthalpic signature is confirmed by the performed calorimetric experiments and a meta-analysis over published data. From these results we are able to define the necessary conditions to observe cold denaturation of amyloid fibrils. Overall, we show that amyloid fibril elongation is associated with a negative heat capacity, the magnitude of which correlates closely with the hydrophobic surface area that is buried upon fibril formation, highlighting the importance of hydrophobicity for fibril stability.


Assuntos
Amiloide/química , Amiloide/fisiologia , Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/fisiologia , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/fisiologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Teóricos , Simulação de Dinâmica Molecular , Desnaturação Proteica , Dobramento de Proteína , Temperatura , Termodinâmica
10.
DNA Cell Biol ; 39(6): 920-925, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32379486

RESUMO

A central event that underlies the etiology of Alzheimer's disease (AD) is the self-assembly of the amyloid-ß (Aß) peptide into aggregates termed amyloids. Increasing evidence implicates soluble prefibrillar Aß oligomers in the neurodegeneration and synaptic dysfunction in AD. Recently we introduced a new class of highly promising antagonists of Aß amyloidogenesis: designed cell-penetrating peptides (CPPs). These CPPs combine the attractive intrinsic properties of peptides (high target specificity and selectivity, biocompatibility, biodegradability, and ease and low cost of production) with potent therapeutic effects (inhibition of Aß oligomerization, fiber formation, and neurotoxicity) and highly efficient delivery (to target cells and subcellular organelles).


Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/toxicidade , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/farmacologia , Sistema Nervoso/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , Engenharia de Proteínas , Animais , Humanos
11.
Proc Natl Acad Sci U S A ; 117(22): 12087-12094, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414930

RESUMO

The spontaneous assembly of proteins into amyloid fibrils is a phenomenon central to many increasingly common and currently incurable human disorders, including Alzheimer's and Parkinson's diseases. Oligomeric species form transiently during this process and not only act as essential intermediates in the assembly of new filaments but also represent major pathogenic agents in these diseases. While amyloid fibrils possess a common, defining set of physicochemical features, oligomers, by contrast, appear much more diverse, and their commonalities and differences have hitherto remained largely unexplored. Here, we use the framework of chemical kinetics to investigate their dynamical properties. By fitting experimental data for several unrelated amyloidogenic systems to newly derived mechanistic models, we find that oligomers present with a remarkably wide range of kinetic and thermodynamic stabilities but that they possess two properties that are generic: they are overwhelmingly nonfibrillar, and they predominantly dissociate back to monomers rather than maturing into fibrillar species. These discoveries change our understanding of the relationship between amyloid oligomers and amyloid fibrils and have important implications for the nature of their cellular toxicity.


Assuntos
Amiloide/química , Proteínas Amiloidogênicas/química , Cinética , Doença de Alzheimer , Peptídeos beta-Amiloides/química , Amiloidose , Modelos Teóricos , Agregados Proteicos , Termodinâmica
12.
Proc Natl Acad Sci U S A ; 117(19): 10322-10328, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32345723

RESUMO

Atomistic description of protein fibril formation has been elusive due to the complexity and long time scales of the conformational search. Here, we develop a multiscale approach combining numerous atomistic simulations in explicit solvent to construct Markov State Models (MSMs) of fibril growth. The search for the in-register fully bound fibril state is modeled as a random walk on a rugged two-dimensional energy landscape defined by ß-sheet alignment and hydrogen-bonding states, whereas transitions involving states without hydrogen bonds are derived from kinetic clustering. The reversible association/dissociation of an incoming peptide and overall growth kinetics are then computed from MSM simulations. This approach is applied to derive a parameter-free, comprehensive description of fibril elongation of Aß16-22 and how it is modulated by phenylalanine-to-cyclohexylalanine (CHA) mutations. The trajectories show an aggregation mechanism in which the peptide spends most of its time trapped in misregistered ß-sheet states connected by weakly bound states twith short lifetimes. Our results recapitulate the experimental observation that mutants CHA19 and CHA1920 accelerate fibril elongation but have a relatively minor effect on the critical concentration for fibril growth. Importantly, the kinetic consequences of mutations arise from cumulative effects of perturbing the network of productive and nonproductive pathways of fibril growth. This is consistent with the expectation that nonfunctional states will not have evolved efficient folding pathways and, therefore, will require a random search of configuration space. This study highlights the importance of describing the complete energy landscape when studying the elongation mechanism and kinetics of protein fibrils.


Assuntos
Peptídeos beta-Amiloides/química , Amiloide/química , Mutação , Fragmentos de Peptídeos/química , Fenilalanina/análogos & derivados , Fenilalanina/genética , Peptídeos beta-Amiloides/genética , Simulação por Computador , Humanos , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Fragmentos de Peptídeos/genética , Estrutura Secundária de Proteína , Termodinâmica
13.
Gen Physiol Biophys ; 39(2): 135-144, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32329441

RESUMO

The structure and dynamics of membranes depend on many external and internal factors that in turn determine their biological functions. One of the widely accepted and studied characteristics of biomembranes is their fluidity. We research a simple system with variable fluidity tweakable via its composition. The addition of cholesterol is employed to increase the order of lipid chains, thus decreasing the membrane fluidity, while melatonin is shown to elevate the chain disorder, thus also the membrane fluidity. We utilize the densitometric measurements to show a shift of studied systems closer or further from the gel-to-fluid phase transition. The structural changes represented by changes to membrane thickness are evaluated from small angle neutron scattering. Finally, we look at the ability of the two additives to control the interactions between membrane and amyloid-beta peptides. Our results suggest that fluidizing effect of melatonin can promote an insertion of peptide within the membrane interior. Intriguingly, the latter structure relates possibly to an Alzheimer's disease preventing mechanism postulated in the case of melatonin.


Assuntos
Colesterol/química , Melatonina/química , Fluidez de Membrana , Peptídeos beta-Amiloides/química , Bicamadas Lipídicas/química , Conformação Molecular , Fosfatidilcolinas/química
14.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32211892

RESUMO

The accumulation of aberrant proteins leads to various neurodegenerative disorders. Mammalian cells contain several intracellular protein degradation systems, including autophagy and proteasomal systems, that selectively remove aberrant intracellular proteins. Although mammals contain not only intracellular but also extracellular proteins, the mechanism underlying the quality control of aberrant extracellular proteins is poorly understood. Here, using a novel quantitative fluorescence assay and genome-wide CRISPR screening, we identified the receptor-mediated degradation pathway by which misfolded extracellular proteins are selectively captured by the extracellular chaperone Clusterin and undergo endocytosis via the cell surface heparan sulfate (HS) receptor. Biochemical analyses revealed that positively charged residues on Clusterin electrostatically interact with negatively charged HS. Furthermore, the Clusterin-HS pathway facilitates the degradation of amyloid ß peptide and diverse leaked cytosolic proteins in extracellular space. Our results identify a novel protein quality control system for preserving extracellular proteostasis and highlight its role in preventing diseases associated with aberrant extracellular proteins.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Membrana Celular/metabolismo , Clusterina/metabolismo , Endocitose , Heparitina Sulfato/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Células A549 , Peptídeos beta-Amiloides/química , Clusterina/genética , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Proteínas Intrinsicamente Desordenadas/química , Lisossomos/metabolismo , Dobramento de Proteína , Proteólise , Proteostase , Propriedades de Superfície , Fatores de Tempo
15.
Chemistry ; 26(43): 9449-9453, 2020 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-32167218

RESUMO

HIV transactivator of transcription (Tat) protein could interact with amyloid ß (Aß) peptide which cause the growth of Aß plaques in the brain and result in Alzheimer's disease in HIV-infected patients. Herein, we employ high-resolution atomic force microscopy and quantitative nanomechanical mapping to investigate the effects of Tat protein in Aß peptide aggregation. Our results demonstrate that the Tat protein could bind to the Aß fibril surfaces and result in the formation of Tat-Aß multifibrillar structures. The resultant Tat-Aß multifibrillar aggregates represent an increase in stiffness compared with Aß fibrils due to the increase in ß-sheet formation. The identification and characterization of the Tat-Aß intermediate aggregates is important to understanding the interactions between Tat protein and Aß peptide, and the development of novel therapeutic strategy for Alzheimer's disease-like disorder in HIV infected individuals.


Assuntos
Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/química , Amiloide/química , Produtos do Gene tat/química , Microscopia de Força Atômica/métodos , Placa Amiloide/química , Peptídeos beta-Amiloides/análise , Produtos do Gene tat/metabolismo , Humanos , Placa Amiloide/metabolismo
16.
J Chem Phys ; 152(4): 045101, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32007046

RESUMO

The formation of amyloid fibrils from soluble peptide is a hallmark of many neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Characterization of the microscopic reaction processes that underlie these phenomena have yielded insights into the progression of such diseases and may inform rational approaches for the design of drugs to halt them. Experimental evidence suggests that most of these reaction processes are intrinsically catalytic in nature and may display enzymelike saturation effects under conditions typical of biological systems, yet a unified modeling framework accounting for these saturation effects is still lacking. In this paper, we therefore present a universal kinetic model for biofilament formation in which every fundamental process in the reaction network can be catalytic. The single closed-form expression derived is capable of describing with high accuracy a wide range of mechanisms of biofilament formation and providing the first integrated rate law of a system in which multiple reaction processes are saturated. Moreover, its unprecedented mathematical simplicity permits us to very clearly interpret the effects of increasing saturation on the overall kinetics. The effectiveness of the model is illustrated by fitting it to the data of in vitro Aß40 aggregation. Remarkably, we find that primary nucleation becomes saturated, demonstrating that it must be heterogeneous, occurring at interfaces and not in solution.


Assuntos
Amiloide/química , Modelos Químicos , Agregação Patológica de Proteínas , Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Catálise , Humanos , Cinética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo
17.
Int J Mol Sci ; 21(3)2020 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-32046252

RESUMO

The self-assembly of amyloid ß (Aß) proteins into oligomers is the major pathogenic event leading to Alzheimer's disease (AD). Typical in vitro experiments require high protein concentrations, whereas the physiological concentration of Aß is in the picomolar to low nanomolar range. This complicates the translation of results obtained in vitro to understanding the aggregation process in vivo. Here, we demonstrate that Aß42 self-assembles into aggregates on membrane bilayers at low nanomolar concentrations - a pathway in which the membrane plays the role of a catalyst. Additionally, physiological ionic conditions (150 mM NaCl) significantly enhance on-membrane aggregation, leading to the rapid formation of oligomers. The self-assembly process is reversible, so assembled aggregates can dissociate from the membrane surface into the bulk solution to further participate in the aggregation process. Molecular dynamics simulations demonstrate that the transient membrane-Aß interaction dramatically changes the protein conformation, facilitating the assembly of dimers. The results indicate peptide-membrane interaction is the critical step towards oligomer formation at physiologically low protein concentrations.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Doença de Alzheimer/genética , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica
18.
Chemistry ; 26(33): 7425-7432, 2020 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-32022335

RESUMO

Protein deposits are associated with many devastating diseases and fluorescent ligands able to visualize these pathological entities are essential. Here, we report the synthesis of thiophene-based donor-acceptor-donor heptameric ligands that can be utilized for spectral assignment of distinct amyloid-ß (Aß) aggregates, one of the pathological hallmarks in Alzheimer's disease. The ability of the ligands to selectively distinguish Aß deposits was abolished when the chemical composition of the ligands was altered. Our findings provide the structural and functional basis for the development of new fluorescent ligands that can distinguish between aggregated proteinaceous species consisting of the same peptide or protein. In addition, such ligands might aid in interpreting the potential role of polymorphic Aß deposits in the pathogenesis of Alzheimer's disease.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Encéfalo/patologia , Tiofenos/síntese química , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Humanos , Ligantes , Tiofenos/química
19.
J Mater Chem B ; 8(11): 2256-2268, 2020 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-32100783

RESUMO

Amyloid ß-peptide (Aß) aggregation induced by metal ions such as Cu2+ has been recognized as a crucial step in the pathogenesis of Alzheimer's disease (AD), so development of multifunctional agents that are able to inhibit Aß aggregation and modulate Cu2+-Aß species is considered as a promising strategy for fighting against AD. Our recent work proved that basification of human lysozyme (hLys) is in favor of enhancing the inhibition of Aß aggregation. Based on the finding, we have herein designed R-hLys, a conjugate of bifunctional alkaline decapeptide (RTHLVFFARK, RK10) coupled onto hLys via reaction with the carboxyl groups on hLys. The design created an even more basic protein than hLys, thus increasing the potency of hLys on inhibiting Aß fibrillation. Moreover, the RK10 conjugation onto hLys introduced a specific Cu2+-chelator and an additional peptide inhibitor (LVFFARK). Thus, a multifunctional modulator on Cu2+-mediated Aß aggregation and cytotoxicity was developed. The multifunctional effects of R-hLys were systematically investigated on Aß aggregation, Cu2+-mediated Aß aggregation, ROS production, and remodeling mature Aß species by comparison to its counterparts. The results revealed the following: (1) R-hLys possesses high potency on inhibiting Aß fibrillogenesis; R-hLys at 1/5 to 1/4 of hLys concentration works similarly to hLys. (2) R-hLys exhibits high performance on inhibiting Cu2+-induced Aß aggregation and cytotoxicity; it increased the cell viability from 48.9% to 86.1% at an equimolar concentration to Aß. (3) R-hLys arrests the production of reactive oxygen species catalyzed by Cu2+ or Cu2+-Aß42 species. (4) R-hLys remodels mature Aß42 fibrils and Cu2+-Aß42 aggregates into small amorphous aggregates of lower cytotoxicity; by co-incubation with R-hLys, the cytotoxicities of mature Aß42 fibrils and Cu2+-Aß42 aggregates are reduced from 31.4% to 14.2% and 48.1% to 10.4%, respectively. Taken together, R-hLys is a potent multifunctional modulator for inhibiting Aß/Cu2+-mediated-Aß aggregation, suppressing ROS production and remodeling mature Aß42/Cu2+-Aß42 species.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/química , Desenho de Fármacos , Muramidase/química , Peptídeos/química , Doença de Alzheimer/etiologia , Sequência de Aminoácidos , Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Cobre/farmacologia , Citotoxinas/farmacologia , Humanos , Agregados Proteicos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
20.
J Mol Biol ; 432(7): 2080-2098, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32061932

RESUMO

The self-assembly of the 42-residue amyloid-ß peptide, Aß42, into fibrillar aggregates is associated with neuronal dysfunction and toxicity in Alzheimer's disease (AD) patient brains, suggesting that small molecules acting on this process might interfere with pathogenesis. Here, we present experimental evidence that the small molecule sclerotiorin (SCL), a natural product belonging to the group of azaphilones, potently delays both seeded and nonseeded Aß42 polymerization in cell-free assays. Mechanistic biochemical studies revealed that the inhibitory effect of SCL on fibrillogenesis is caused by its ability to kinetically stabilize small Aß42 oligomers. These structures exhibit low ß-sheet content and do not possess seeding activity, indicating that SCL acts very early in the amyloid formation cascade before the assembly of seeding-competent, ß-sheet-rich fibrillar aggregates. Investigations with NMR WaterLOGSY experiments confirmed the association of Aß42 assemblies with SCL in solution. Furthermore, using ion mobility-mass spectrometry, we observed that SCL directly interacts with a small fraction of Aß42 monomers in the gas phase. In comparison to typical amyloid fibrils, small SCL-stabilized Aß42 assemblies are inefficiently taken up into mammalian cells and have low toxicity in cell-based assays. Overall, these mechanistic studies support a pathological role of stable, ß-sheet-rich Aß42 fibrils in AD, while structures with low ß-sheet content may be less relevant.


Assuntos
Peptídeos beta-Amiloides/química , Amiloide/antagonistas & inibidores , Benzopiranos/farmacologia , Proliferação de Células , Neuroblastoma/tratamento farmacológico , Fragmentos de Peptídeos/química , Multimerização Proteica/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Animais , Humanos , Camundongos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Células PC12 , Fragmentos de Peptídeos/metabolismo , Conformação Proteica em Folha beta , Ratos , Células Tumorais Cultivadas
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