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1.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208561

RESUMO

It has been proposed that a "common core" of pathologic pathways exists for the large family of amyloid-associated neurodegenerations, including Alzheimer's, Parkinson's, type II diabetes and Creutzfeldt-Jacob's Disease. Aggregates of the involved proteins, independently from their primary sequence, induced neuron membrane permeabilization able to trigger an abnormal Ca2+ influx leading to synaptotoxicity, resulting in reduced expression of synaptic proteins and impaired synaptic transmission. Emerging evidence is now focusing on low-molecular-weight prefibrillar oligomers (PFOs), which mimic bacterial pore-forming toxins that form well-ordered oligomeric membrane-spanning pores. At the same time, the neuron membrane composition and its chemical microenvironment seem to play a pivotal role. In fact, the brain of AD patients contains increased fractions of anionic lipids able to favor cationic influx. However, up to now the existence of a specific "common structure" of the toxic aggregate, and a "common mechanism" by which it induces neuronal damage, synaptotoxicity and impaired synaptic transmission, is still an open hypothesis. In this review, we gathered information concerning this hypothesis, focusing on the proteins linked to several amyloid diseases. We noted commonalities in their structure and membrane activity, and their ability to induce Ca2+ influx, neurotoxicity, synaptotoxicity and impaired synaptic transmission.


Assuntos
Amiloide/química , Amiloide/metabolismo , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/metabolismo , Multimerização Proteica , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Suscetibilidade a Doenças , Humanos , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Relação Estrutura-Atividade
2.
Nat Commun ; 12(1): 3962, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172723

RESUMO

Missense mutations in p53 are severely deleterious and occur in over 50% of all human cancers. The majority of these mutations are located in the inherently unstable DNA-binding domain (DBD), many of which destabilize the domain further and expose its aggregation-prone hydrophobic core, prompting self-assembly of mutant p53 into inactive cytosolic amyloid-like aggregates. Screening an oligopyridylamide library, previously shown to inhibit amyloid formation associated with Alzheimer's disease and type II diabetes, identified a tripyridylamide, ADH-6, that abrogates self-assembly of the aggregation-nucleating subdomain of mutant p53 DBD. Moreover, ADH-6 targets and dissociates mutant p53 aggregates in human cancer cells, which restores p53's transcriptional activity, leading to cell cycle arrest and apoptosis. Notably, ADH-6 treatment effectively shrinks xenografts harboring mutant p53, while exhibiting no toxicity to healthy tissue, thereby substantially prolonging survival. This study demonstrates the successful application of a bona fide small-molecule amyloid inhibitor as a potent anticancer agent.


Assuntos
Amiloide/antagonistas & inibidores , Antineoplásicos/farmacologia , Agregação Patológica de Proteínas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Amidas/química , Amidas/farmacologia , Amidas/uso terapêutico , Amiloide/química , Amiloide/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Camundongos , Mutação , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Agregação Patológica de Proteínas/tratamento farmacológico , Domínios Proteicos , Piridinas/química , Piridinas/farmacologia , Piridinas/uso terapêutico , Transcrição Genética/efeitos dos fármacos , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética
3.
Int J Mol Sci ; 22(9)2021 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-34063223

RESUMO

Proteolytic enzymes are known to be involved in the formation and degradation of various monomeric proteins, but the effect of proteases on the ordered protein aggregates, amyloid fibrils, which are considered to be extremely stable, remains poorly understood. In this work we study resistance to proteolytic degradation of lysozyme amyloid fibrils with two different types of morphology and beta-2-microglobulun amyloids. We showed that the proteolytic enzyme of the pancreas, trypsin, induced degradation of amyloid fibrils, and the mechanism of this process was qualitatively the same for all investigated amyloids. At the same time, we found a dependence of efficiency and rate of fibril degradation on the structure of the amyloid-forming protein as well as on the morphology and clustering of amyloid fibrils. It was assumed that the discovered relationship between fibrils structure and the efficiency of their degradation by trypsin can become the basis of a new express method for the analysis of amyloids polymorphism. Unexpectedly lower resistance of both types of lysozyme amyloids to trypsin exposure compared to the native monomeric protein (which is not susceptible to hydrolysis) was attributed to the higher availability of cleavage sites in studied fibrils. Another intriguing result of the work is that the cytotoxicity of amyloids treated with trypsin was not only failing to decline, but even increasing in the case of beta-2-microglobulin fibrils.


Assuntos
Amiloide/metabolismo , Tripsina/metabolismo , Amiloide/química , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Naftalenossulfonato de Anilina , Benzotiazóis , Corantes Fluorescentes , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Muramidase/metabolismo , Proteólise , Tripsina/química , Microglobulina beta-2/química , Microglobulina beta-2/metabolismo
4.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064883

RESUMO

Prion protein aggregation into amyloid fibrils is associated with the onset and progression of prion diseases-a group of neurodegenerative amyloidoses. The process of such aggregate formation is still not fully understood, especially regarding their polymorphism, an event where the same type of protein forms multiple, conformationally and morphologically distinct structures. Considering that such structural variations can greatly complicate the search for potential antiamyloid compounds, either by having specific propagation properties or stability, it is important to better understand this aggregation event. We have recently reported the ability of prion protein fibrils to obtain at least two distinct conformations under identical conditions, which raised the question if this occurrence is tied to only certain environmental conditions. In this work, we examined a large sample size of prion protein aggregation reactions under a range of temperatures and analyzed the resulting fibril dye-binding, secondary structure and morphological properties. We show that all temperature conditions lead to the formation of more than one fibril type and that this variability may depend on the state of the initial prion protein molecules.


Assuntos
Amiloide/química , Proteínas Priônicas/química , Multimerização Proteica , Temperatura , Conformação Proteica
5.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34066237

RESUMO

CsgA is an aggregating protein from bacterial biofilms, representing a class of functional amyloids. Its amyloid propensity is defined by five fragments (R1-R5) of the sequence, representing non-perfect repeats. Gate-keeper amino acid residues, specific to each fragment, define the fragment's propensity for self-aggregation and aggregating characteristics of the whole protein. We study the self-aggregation and secondary structures of the repeat fragments of Salmonella enterica and Escherichia coli and comparatively analyze their potential effects on these proteins in a bacterial biofilm. Using bioinformatics predictors, ATR-FTIR and FT-Raman spectroscopy techniques, circular dichroism, and transmission electron microscopy, we confirmed self-aggregation of R1, R3, R5 fragments, as previously reported for Escherichia coli, however, with different temporal characteristics for each species. We also observed aggregation propensities of R4 fragment of Salmonella enterica that is different than that of Escherichia coli. Our studies showed that amyloid structures of CsgA repeats are more easily formed and more durable in Salmonella enterica than those in Escherichia coli.


Assuntos
Amiloide/química , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Salmonella enterica/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Agregados Proteicos , Conformação Proteica , Salmonella enterica/genética , Salmonella enterica/crescimento & desenvolvimento , Homologia de Sequência
6.
J Med Chem ; 64(9): 6273-6299, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33939422

RESUMO

In immunoglobulin light-chain (LC) amyloidosis, transient unfolding or unfolding and proteolysis enable aggregation of LC proteins, causing potentially fatal organ damage. A drug that kinetically stabilizes LCs could suppress aggregation; however, LC sequences are variable and have no natural ligands, hindering drug development efforts. We previously identified high-throughput screening hits that bind to a site at the interface between the two variable domains of the LC homodimer. We hypothesized that extending the stabilizers beyond this initially characterized binding site would improve affinity. Here, using protease sensitivity assays, we identified stabilizers that can be divided into four substructures. Some stabilizers exhibit nanomolar EC50 values, a 3000-fold enhancement over the screening hits. Crystal structures reveal a key π-π stacking interaction with a conserved tyrosine residue that was not utilized by the screening hits. These data provide a foundation for developing LC stabilizers with improved binding selectivity and enhanced physicochemical properties.


Assuntos
Amiloide/química , Cumarínicos/química , Desenho de Fármacos , Cadeias Leves de Imunoglobulina/química , Cristalografia por Raios X , Ensaios de Triagem em Larga Escala , Cinética , Modelos Moleculares , Domínios Proteicos , Estabilidade Proteica/efeitos dos fármacos
7.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810433

RESUMO

Alzheimer's disease affects millions of lives worldwide. This terminal disease is characterized by the formation of amyloid aggregates, so-called amyloid oligomers. These oligomers are composed of ß-sheet structures, which are believed to be neurotoxic. However, the actual secondary structure that contributes most to neurotoxicity remains unknown. This lack of knowledge is due to the challenging nature of characterizing the secondary structure of amyloids in cells. To overcome this and investigate the molecular changes in proteins directly in cells, we used synchrotron-based infrared microspectroscopy, a label-free and non-destructive technique available for in situ molecular imaging, to detect structural changes in proteins and lipids. Specifically, we evaluated the formation of ß-sheet structures in different monogenic and bigenic cellular models of Alzheimer's disease that we generated for this study. We report on the possibility to discern different amyloid signatures directly in cells using infrared microspectroscopy and demonstrate that bigenic (amyloid-ß, α-synuclein) and (amyloid-ß, Tau) neuron-like cells display changes in ß-sheet load. Altogether, our findings support the notion that different molecular mechanisms of amyloid aggregation, as opposed to a common mechanism, are triggered by the specific cellular environment and, therefore, that various mechanisms lead to the development of Alzheimer's disease.


Assuntos
Doença de Alzheimer/metabolismo , Amiloide/química , Espectrofotometria Infravermelho/métodos , Doença de Alzheimer/diagnóstico por imagem , Peptídeos beta-Amiloides/metabolismo , Amiloidose/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Camundongos , Microscopia de Fluorescência , Neuroblastoma/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Síncrotrons , alfa-Sinucleína/química
8.
Biochem Biophys Res Commun ; 554: 145-150, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33798940

RESUMO

Recent structural investigation of amyloid filaments extracted from human patients demonstrated that the ex vivo filaments associated with different disease phenotypes adopt diverse molecular conformations, which are different from those of in vitro amyloid filaments. A very recent cryo-EM structural study also revealed that ex vivo α-synuclein filaments extracted from multiple system atrophy patients adopt distinct molecular structures from those of in vitro α-synuclein filaments, suggesting the presence of co-factors for α-synuclein aggregation in vivo. Here, we report structural characterizations of α-synuclein filaments formed in the presence of a potential co-factor, tau, using cryo-EM and solid-state NMR. Our cryo-EM structure of the tau-promoted α-synuclein filaments reveals some similarities to one of the previously reported polymorphs of in vitro α-synuclein filaments in the core region, while illustrating distinct conformations in the N- and C-terminal regions. The structural study highlights the conformational plasticity of α-synuclein filaments and the importance of the co-factors, requiring additional structural investigation of not only more ex vivo α-synuclein filaments, but also in vitro α-synuclein filaments formed in the presence of diverse co-factors. The comparative structural analyses will help better understand molecular basis of diverse structures of α-synuclein filaments and possible relevance of each structure to the disease phenotype.


Assuntos
Amiloide/química , Microscopia Crioeletrônica/métodos , Espectroscopia de Ressonância Magnética/métodos , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Amiloide/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Química Encefálica , Humanos , Microscopia Imunoeletrônica/métodos , Conformação Proteica , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
9.
ACS Appl Mater Interfaces ; 13(15): 18184-18193, 2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33826292

RESUMO

Tunable optical properties in nanomaterials enable a variety of applications in multidisciplinary areas. These properties are directly related to several different factors such as solvent conditions, synthesis methods, and most significantly, the oxidation states of metals participating in the absorption or emission properties. Lanthanide metals containing ABO3 perovskites are among such nanomaterials that can be tuned to a great extent by only modifying the charged states on the metals in the composition. We report a green synthesis method through sonication to synthesize ABO3 perovskites to incorporate Tb4+ into the perovskite composition at room temperature. The optical properties of the nanomaterial show emission in the entire ultraviolet-visible-near-infrared spectral regions through charge transfer between europium and terbium. The combination of cerium (C), molybdenum (M), europium (E), and terbium (T) results in a sheet-like CMET perovskite obeying hexagonal geometry. The nanomaterial is highly stable in an aqueous medium, showing finely suspended Tyndall effect due to particle size <300 nm. Owing to their wide range of emission behavior, surface charge, and aqueous stability, CMET perovskites were used to study the defibrillation of hen egg-white lysozyme (HEWL) as an amyloid model protein. The intrinsic property of the nanomaterial assists in the interaction of the fibrils with the perovskite and the emission range becomes the reporter of the defibrillation. Infrared spectroscopy shows the change in the material properties during the defibrillation. A preliminary test on the varying concentration of HEWL incubated with CMET perovskites shows linear behavior with R2 = 0.9841. The tunable emission characteristic and aqueous stability of the perovskite material make it suitable for future biological applications.


Assuntos
Amiloide/química , Óxidos/química , Óxidos/farmacologia , Temperatura , Animais , Cério/química , Európio/química , Muramidase/química , Tamanho da Partícula , Agregados Proteicos/efeitos dos fármacos , Térbio/química
10.
ACS Appl Mater Interfaces ; 13(15): 18089-18099, 2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33829756

RESUMO

Fibrillogenesis of amyloid ß-protein (Aß) is pathologically associated with Alzheimer's disease (AD), so modulating Aß aggregation is crucial for AD prevention and treatment. Herein, a zwitterionic polymer with short dimethyl side chains (pID) is synthesized and conjugated with a heptapeptide inhibitor (Ac-LVFFARK-NH2, LK7) to construct zwitterionic polymer-inhibitor conjugates for enhanced inhibition of Aß aggregation. However, it is unexpectedly found that the LK7@pID conjugates remarkably promote Aß fibrillization to form more fibrils than the free Aß system but effectively eliminate Aß-induced cytotoxicity. Such an unusual behavior of the LK7@pID conjugates is unraveled by extensive mechanistic studies. First, the hydrophobic environment within the assembled micelles of LK7@pID promotes the hydrophobic interaction between Aß molecules and LK7@pID, which triggers Aß aggregation at the very beginning, making fibrillization occur at an earlier stage. Second, in the aggregation process, the LK7@pID micelles disassemble by the intensive interactions with Aß, and LK7@pID participates in the fibrillization by being embedded in the Aß fibrils, leading to the formation of hybrid and heterogeneous fibrillar aggregates with a different structure than normal Aß fibrils. This unique Trojan horse-like feature of LK7@pID conjugates has not been observed for any other inhibitors reported previously and may shed light on the design of new modulators against ß-amyloid cytotoxicity.


Assuntos
Amiloide/química , Amiloide/toxicidade , Citotoxinas/química , Citotoxinas/toxicidade , Oligopeptídeos/química , Polímeros/química , Polímeros/farmacologia , Sequência de Aminoácidos , Desenho de Fármacos , Interações Hidrofóbicas e Hidrofílicas , Agregados Proteicos/efeitos dos fármacos
11.
Int J Mol Sci ; 22(9)2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33919421

RESUMO

Amyloid fibrils are supramolecular protein assemblies represented by a cross-ß structure and fibrous morphology, whose structural architecture has been previously investigated. While amyloid fibrils are basically a main-chain-dominated structure consisting of a backbone of hydrogen bonds, side-chain interactions also play an important role in determining their detailed structures and physicochemical properties. In amyloid fibrils comprising short peptide segments, a steric zipper where a pair of ß-sheets with side chains interdigitate tightly is found as a fundamental motif. In amyloid fibrils comprising longer polypeptides, each polypeptide chain folds into a planar structure composed of several ß-strands linked by turns or loops, and the steric zippers are formed locally to stabilize the structure. Multiple segments capable of forming steric zippers are contained within a single protein molecule in many cases, and polymorphism appears as a result of the diverse regions and counterparts of the steric zippers. Furthermore, the ß-solenoid structure, where the polypeptide chain folds in a solenoid shape with side chains packed inside, is recognized as another important amyloid motif. While side-chain interactions are primarily achieved by non-polar residues in disease-related amyloid fibrils, the participation of hydrophilic and charged residues is prominent in functional amyloids, which often leads to spatiotemporally controlled fibrillation, high reversibility, and the formation of labile amyloids with kinked backbone topology. Achieving precise control of the side-chain interactions within amyloid structures will open up a new horizon for designing useful amyloid-based nanomaterials.


Assuntos
Amiloide/química , Amiloide/metabolismo , Animais , Humanos , Modelos Moleculares , Conformação Proteica , Estabilidade Proteica
12.
Int J Mol Sci ; 22(9)2021 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-33922648

RESUMO

Transthyretin (TTR) is an essential transporter of a thyroid hormone and a holo-retinol binding protein, found abundantly in human plasma and cerebrospinal fluid. In addition, this protein is infamous for its amyloidogenic propensity, causing various amyloidoses in humans, such as senile systemic amyloidosis, familial amyloid polyneuropathy, and familial amyloid cardiomyopathy. It has been known for over two decades that decreased stability of the native tetrameric conformation of TTR is the main cause of these diseases. Yet, mechanistic details on the amyloidogenic transformation of TTR were not clear until recent multidisciplinary investigations on various structural states of TTR. In this review, we discuss recent advancements in the structural understanding of TTR misfolding and amyloidosis processes. Special emphasis has been laid on the observations of novel structural features in various amyloidogenic species of TTR. In addition, proteolysis-induced fragmentation of TTR, a recently proposed mechanism facilitating TTR amyloidosis, has been discussed in light of its structural consequences and relevance to acknowledge the amyloidogenicity of TTR.


Assuntos
Neuropatias Amiloides Familiares/patologia , Amiloide/química , Pré-Albumina/química , Dobramento de Proteína , Neuropatias Amiloides Familiares/metabolismo , Animais , Humanos
13.
Int J Mol Sci ; 22(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923609

RESUMO

Neurodegenerative disorders are a highly prevalent class of diseases, whose pathological mechanisms start before the appearance of any clear symptoms. This fact has prompted scientists to search for biomarkers that could aid early treatment. These currently incurable pathologies share the presence of aberrant aggregates called amyloids in the nervous system, which are composed of specific proteins. In this review, we discuss how these proteins, their conformations and modifications could be exploited as biomarkers for diagnostic purposes. We focus on proteins that are associated with the most prevalent neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, amyotrophic lateral sclerosis, and frontotemporal dementia. We also describe current challenges in detection, the most recent techniques with diagnostic potentials and possible future developments in diagnosis.


Assuntos
Amiloide/metabolismo , Doenças Neurodegenerativas/metabolismo , Agregação Patológica de Proteínas/metabolismo , Amiloide/química , Amiloide/genética , Animais , Biomarcadores/metabolismo , Humanos , Doenças Neurodegenerativas/patologia , Agregação Patológica de Proteínas/patologia
14.
Int J Mol Sci ; 22(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923726

RESUMO

Alzheimer's disease (AD) is a complex multifactorial disorder, mainly characterized by the progressive loss of memory and cognitive, motor, and functional capacity. The absence of effective therapies available for AD alongside the consecutive failures in the central nervous system (CNS) drug development has been motivating the search for new disease-modifying therapeutic strategies for this disease. To address this issue, the multitarget directed ligands (MTDLs) are emerging as a therapeutic alternative to target the multiple AD-related factors. Following this concept, herein we describe the design, synthesis, and biological evaluation of a family of chromeno[3,4-b]xanthones as well as their (E)-2-[2-(propargyloxy)styryl]chromone precursors, as first-in-class acetylcholinesterase (AChE) and ß-amyloid (Aß) aggregation dual-inhibitors. Compounds 4b and 10 emerged as well-balanced dual-target inhibitors, with IC50 values of 3.9 and 2.9 µM for AChE and inhibitory percentages of 70 and 66% for Aß aggregation, respectively. The molecular docking showed that most of the compounds bound to AChE through hydrogen bonds with residues of the catalytic triad and π-stacking interactions between the main scaffold and the aromatic residues present in the binding pocket. The interesting well-balanced activities of these compounds makes them interesting templates for the development of new multitarget compounds for AD.


Assuntos
Amiloide/efeitos dos fármacos , Inibidores da Colinesterase/síntese química , Fármacos Neuroprotetores/síntese química , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Amiloide/química , Amiloide/metabolismo , Sítios de Ligação , Inibidores da Colinesterase/farmacologia , Cromonas/química , Humanos , Fármacos Neuroprotetores/farmacologia , Ligação Proteica , Multimerização Proteica , Xantonas/química
15.
Int J Mol Sci ; 22(6)2021 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-33806726

RESUMO

A wide variety of neurodegenerative diseases are characterized by the accumulation of protein aggregates in intraneuronal or extraneuronal brain regions. In Alzheimer's disease (AD), the extracellular aggregates originate from amyloid-ß proteins, while the intracellular aggregates are formed from microtubule-binding tau proteins. The amyloid forming peptide sequences in the amyloid-ß peptides and tau proteins are responsible for aggregate formation. Experimental studies have until the date reported many of such amyloid forming peptide sequences in different proteins, however, there is still limited molecular level understanding about their tendency to form aggregates. In this study, we employed umbrella sampling simulations and subsequent electronic structure theory calculations in order to estimate the energy profiles for interconversion of the helix to ß-sheet like secondary structures of sequences from amyloid-ß protein (KLVFFA) and tau protein (QVEVKSEKLD and VQIVYKPVD). The study also included a poly-alanine sequence as a reference system. The calculated force-field based free energy profiles predicted a flat minimum for monomers of sequences from amyloid and tau proteins corresponding to an α-helix like secondary structure. For the parallel and anti-parallel dimer of KLVFFA, double well potentials were obtained with the minima corresponding to α-helix and ß-sheet like secondary structures. A similar double well-like potential has been found for dimeric forms for the sequences from tau fibril. Complementary semi-empirical and density functional theory calculations displayed similar trends, validating the force-field based free energy profiles obtained for these systems.


Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Amiloide/química , Teoria da Densidade Funcional , Fragmentos de Peptídeos/química , Proteínas tau/química , Sequência de Aminoácidos , Amiloide/metabolismo , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Conformação Proteica em alfa-Hélice , Relação Estrutura-Atividade , Proteínas tau/metabolismo
17.
Molecules ; 26(8)2021 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-33921801

RESUMO

Waste valorization represents one of the main social challenges when promoting a circular economy and environmental sustainability. Here, we evaluated the effect of the polyphenols extracted from apple peels, normally disposed of as waste, on the amyloid aggregation process of κ-casein from bovine milk, a well-used amyloidogenic model system. The effect of the apple peel extract on protein aggregation was examined using a thioflavin T fluorescence assay, Congo red binding assay, circular dichroism, light scattering, and atomic force microscopy. We found that the phenolic extract from the peel of apples of the cultivar "Fuji", cultivated in Sicily (Caltavuturo, Italy), inhibited κ-casein fibril formation in a dose-dependent way. In particular, we found that the extract significantly reduced the protein aggregation rate and inhibited the secondary structure reorganization that accompanies κ-casein amyloid formation. Protein-aggregated species resulting from the incubation of κ-casein in the presence of polyphenols under amyloid aggregation conditions were reduced in number and different in morphology.


Assuntos
Amiloide/química , Caseínas/química , Malus/química , Microscopia de Força Atômica
18.
Biochemistry ; 60(10): 756-764, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33645214

RESUMO

Misfolding and aggregation of transthyretin (TTR) are linked to amyloid disease. Amyloidosis occurs when the TTR homotetramer dissociates into aggregation-prone monomers that self-assemble into amyloid. In familial transthyretin amyloidosis, hereditary amino acid substitutions destabilize TTR and promote aggregation. In this work, we used 19F nuclear magnetic resonance (NMR) to determine the effect of mutations in the EF helix (Y78F, K80D, K80E, and A81T) and EF loop (G83R and I84S) on the aggregation kinetics and stability of the TTR tetramer and monomer. The EF region acts as a scaffold that stabilizes interactions in both the strong and weak dimer interfaces of the tetramer and is the site of a cluster of pathogenic mutations. K80D and K80E are non-natural mutants that destabilize the EF helix and yield an equilibrium mixture of tetramer and monomer at neutral pH, providing a unique opportunity to determine the thermodynamic parameters for tetramer assembly under nondenaturing conditions. Of the pathogenic mutants studied, only A81T formed appreciable monomer at neutral pH. Real-time 19F NMR measurements showed that the pathogenic Y78F mutation accelerates aggregation by destabilizing both the tetrameric and monomeric species. The pathogenic mutations A81T, G83R, and I84S destabilize the monomer and increase its aggregation rate by disrupting a Schellman helix C-capping motif. These studies provide new insights into the mechanism by which relatively subtle mutations that affect tetramer or monomer stability promote entry of TTR into the dissociation-aggregation pathway.


Assuntos
Amiloide/química , Pré-Albumina/química , Pré-Albumina/metabolismo , Termodinâmica , Sítios de Ligação , Humanos , Cinética , Modelos Moleculares , Mutação , Pré-Albumina/genética , Conformação Proteica
19.
Int J Biol Macromol ; 178: 424-433, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33662415

RESUMO

Amyloid proteins were recognized as the crucial cause of many senile diseases. In this study, the inhibitory effects of Sennoside A (SA) and Sennoside C (SC) on amyloid fibrillation were evaluated by the combination of biophysical approaches and molecular docking tool using human lysozyme (HL) as amyloid-forming model. The results of thioflavin-T (ThT), 8-anilino-1-naphthalenesulfonic acid (ANS) and congo red (CR) assays indicated that both SA and SC could inhibit the amyloid fibrillation of HL in a dose-dependent manner. The IC50 value of SA and SC on HL fibrillation was 200.09 µM and 186.20 µM, respectively. These findings were further verified by transmission electron microscopy (TEM) and atomic force microscopy (AFM), which showed that the addition of SA or SC could sharply reduce the amyloid fibrillation of HL. Additionally, the interactions of HL with SA and SC were investigated by steady-state fluorescence spectra and molecular docking studies. The results suggested that both SA and SC could bind to the binding pocket of HL and form a stable complex mainly via hydrogen bonds, van-der-Waals forces and hydrophobic interactions. In conclusion, our experiments revealed that both SA and SC can significantly inhibit amyloid fibrillation of HL.


Assuntos
Amiloide/química , Muramidase/química , Agregados Proteicos , Extrato de Senna/química , Senosídeos/química , Humanos
20.
ACS Appl Mater Interfaces ; 13(13): 14875-14884, 2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33759489

RESUMO

Grafting biomolecules on nanostructures' surfaces is an increasingly used strategy to target pathogenic cells, with both diagnostic and therapeutic applications. However, nanomaterials monofunctionalized by conjugating a single type of ligand find limited uses in pathologies/therapies that require two or more targets/receptors to be targeted and/or activated with a single molecular entity simultaneously. Therefore, multivalent nanomaterials for dual- or multitargeting are attracting significant interest. This study provides a proof of concept of such nanostructures. We have recently developed a modular methodology that allows obtaining amyloid-based materials decorated with active globular domains. Here, this approach is exploited to generate functional amyloid fibrils displaying antibody capture moieties. A high antibody binding affinity and capacity for the resulting nanofibrils, whose size can be manipulated to obtain homogeneous nanorods with high biocompatibility, are demonstrated. These nanorods are then used for specific antibody-mediated targeting of different cell types. Simultaneous conjugation of these nanorods with different antibodies allows obtaining a mimic of a bispecific antibody that redirects T lymphocytes to tumoral cells, holding high potential for immunotherapy. Overall, the work illustrates a modular and straightforward strategy to obtain preparative quantities of multivalent antibody-functionalized nanomaterials with multitargeting properties without the need for covalent modification.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Comunicação Celular/efeitos dos fármacos , Imunoconjugados/farmacologia , Nanotubos , Amiloide/química , Amiloide/farmacologia , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/química , Linhagem Celular Tumoral , Humanos , Imunoconjugados/química , Nanotubos/química , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Termodinâmica
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