RESUMO
Proteins from the S100 family perform numerous functions and may contribute to Alzheimer's disease (AD). Herein, we report the effects of S100A8/S100A9 heterooligomer calprotectin (CP) and the S100B homodimer on metal-free and metal-bound amyloid-ß (Aß; Aß40 and Aß42) aggregation in vitro. Studies performed with CP-Ser [S100A8(C42S)/S100A9(C3S) oligomer] indicate that the protein influences the aggregation profile for Aß40 in both the absence and presence of metal ions [i.e., Zn(ii) and Cu(ii)]. Moreover, the detection of Aß40-CP-Ser complexes by mass spectrometry suggests a direct interaction as a possible mechanism for the involvement of CP in Aß40 aggregation. Although the interaction of CP-Ser with Aß40 impacts Aß40 aggregation in vitro, the protein does not attenuate Aß-induced toxicity in SH-SY5Y cells. In contrast, S100B has a slight effect on the aggregation of Aß. Overall, this work supports a potential association of CP with Aß in the absence and presence of metal ions in AD.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Complexo Antígeno L1 Leucocitário/metabolismo , Metais/metabolismo , Agregados Proteicos , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Humanos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Células Tumorais CultivadasRESUMO
A catecholamine neurotransmitter, dopamine (DA), is suggested to be linked to the pathology of dementia; however, the involvement of DA and its structural analogues in the pathogenesis of Alzheimer's disease (AD), the most common form of dementia, composed of multiple pathogenic factors has not been clear. Herein, we report that DA and its rationally designed structural derivatives (1-6) based on DA's oxidative transformation are able to modulate multiple pathological elements found in AD [i.e., metal ions, metal-free amyloid-ß (Aß), metal-bound Aß (metal-Aß), and reactive oxygen species (ROS)], with demonstration of detailed molecular-level mechanisms. Our multidisciplinary studies validate that the protective effects of DA and its derivatives on Aß aggregation and Aß-mediated toxicity are induced by their oxidative transformation with concomitant ROS generation under aerobic conditions. In particular, DA and the derivatives (i.e., 3 and 4) show their noticeable anti-amyloidogenic ability toward metal-free Aß and/or metal-Aß, verified to occur via their oxidative transformation that facilitates Aß oxidation. Moreover, in primary pan-microglial marker (CD11b)-positive cells, the major producers of inflammatory mediators in the brain, DA and its derivatives significantly diminish inflammation and oxidative stress triggered by lipopolysaccharides and Aß through the reduced induction of inflammatory mediators as well as upregulated expression of heme oxygenase-1, the enzyme responsible for production of antioxidants. Collectively, we illuminate how DA and its derivatives could prevent multiple pathological features found in AD. The overall studies could advance our understanding regarding distinct roles of neurotransmitters in AD and identify key interactions for alleviation of AD pathology.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Dopamina/metabolismo , Fragmentos de Peptídeos/metabolismo , Agregação Patológica de Proteínas/metabolismo , Cobre/metabolismo , Dopamina/análogos & derivados , Humanos , Inflamação , Metais , Microglia/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Zinco/metabolismoRESUMO
Multiple pathogenic factors [e.g., amyloid-ß (Aß), metal ions, metal-bound Aß (metal-Aß), reactive oxygen species (ROS)] are found in the brain of patients with Alzheimer's disease (AD). In order to elucidate the roles of pathological elements in AD, chemical tools able to regulate their activities would be valuable. Due to the complicated link among multiple pathological factors, however, it has been challenging to invent such chemical tools. Herein, we report novel small molecules as chemical tools toward modulation of single or multiple target(s), designed via a rational structure-property-directed strategy. The chemical properties (e.g., oxidation potentials) of our molecules and their coverage of reactivities toward the pathological targets were successfully differentiated through a minor structural variation [i.e., replacement of one nitrogen (N) or sulfur (S) donor atom in the framework]. Among our compounds (1-3), 1 with the lowest oxidation potential is able to noticeably modify the aggregation of both metal-free Aß and metal-Aß, as well as scavenge free radicals. Compound 2 with the moderate oxidation potential significantly alters the aggregation of Cu(II)-Aß42. The hardly oxidizable compound, 3, relative to 1 and 2, indicates no noticeable interactions with all pathogenic factors, including metal-free Aß, metal-Aß, and free radicals. Overall, our studies demonstrate that the design of small molecules as chemical tools able to control distinct pathological components could be achieved via fine-tuning of structures and properties.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Radicais Livres/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Doença de Alzheimer/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Humanos , Metais/metabolismo , Oxirredução , Agregados Proteicos/fisiologia , Bibliotecas de Moléculas Pequenas/metabolismoRESUMO
The complexity of Alzheimer's disease (AD) stems from the inter-relation of multiple pathological factors upon initiation and progression of the disease. To identify the involvement of metal-bound amyloid-ß (metal-Aß) aggregation in AD pathology, among the pathogenic features found in the AD-affected brain, small molecules as chemical tools capable of controlling metal-Aß aggregation were developed. Herein, we report a new class of 2,2'-bipyridine (bpy) derivatives (1-4) rationally designed to be chemical modulators toward metal-Aß aggregation over metal-free Aß analogue. The bpy derivatives were constructed through a rational design strategy employing straightforward structural variations onto the backbone of a metal chelator, bpy: (i) incorporation of an Aß interacting moiety; (ii) introduction of a methyl group at different positions. The newly prepared bpy derivatives were observed to bind to metal ions [i.e., Cu(II) and Zn(II)] and interact with metal-Aß over metal-free Aß to varying degrees. Distinguishable from bpy, the bpy derivatives (1-3) were indicated to noticeably modulate the aggregation pathways of Cu(II)-Aß and Zn(II)-Aß over metal-free Aß. Overall, our studies of the bpy derivatives demonstrate that the alteration of metal binding properties as well as the installation of an Aß interacting capability onto a metal chelating framework, devised via the rational structure-based design, were able to achieve evident modulating reactivity against metal-Aß aggregation. Obviating the need for complicated structures, our design approach, presented in this work, could be appropriately utilized for inventing small molecules as chemical tools for studying desired metal-related targets in biological systems.
Assuntos
2,2'-Dipiridil/farmacologia , Peptídeos beta-Amiloides/antagonistas & inibidores , Cobre/farmacologia , Desenho de Fármacos , Zinco/farmacologia , 2,2'-Dipiridil/síntese química , 2,2'-Dipiridil/química , Peptídeos beta-Amiloides/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cobre/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Agregados Proteicos/efeitos dos fármacos , Relação Estrutura-Atividade , Zinco/químicaRESUMO
An amyloidogenic peptide, amyloid-ß (Aß), has been implicated as a contributor to the neurotoxicity of Alzheimer's disease (AD) that continues to present a major socioeconomic burden for our society. Recently, the use of metal complexes capable of cleaving peptides has arisen as an efficient tactic for amyloid management; unfortunately, little has been reported to pursue this strategy. Herein, we report a novel approach to validate the hydrolytic cleavage of divalent metal complexes toward two major isoforms of Aß (Aß40 and Aß42) and tune their proteolytic activity based on the choice of metal centers (M = Co, Ni, Cu, and Zn) which could be correlated to their anti-amyloidogenic properties. Such metal-dependent tunability was facilitated employing a tetra-N-methylated cyclam (TMC) ligand that imparts unique geometric and stereochemical control, which has not been available in previous systems. Co(II)(TMC) was identified to noticeably cleave Aß peptides and control their aggregation, reporting the first Co(II) complex for such reactivities to the best of our knowledge. Through detailed mechanistic investigations by biochemical, spectroscopic, mass spectrometric, and computational studies, the critical importance of the coordination environment and acidity of the aqua-bound complexes in promoting amide hydrolysis was verified. The biological applicability of Co(II)(TMC) was also illustrated via its potential blood-brain barrier permeability, relatively low cytotoxicity, regulatory capability against toxicity induced by both Aß40 and Aß42 in living cells, proteolytic activity with Aß peptides under biologically relevant conditions, and inertness toward cleavage of structured proteins. Overall, our approaches and findings on reactivities of divalent metal complexes toward Aß, along with the mechanistic insights, demonstrate the feasibility of utilizing such metal complexes for amyloid control.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Compostos Heterocíclicos/farmacologia , Metais Pesados/farmacologia , Compostos Organometálicos/farmacologia , Peptídeos beta-Amiloides/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Compostos Heterocíclicos/química , Humanos , Hidrólise , Metais Pesados/química , Estrutura Molecular , Compostos Organometálicos/químicaRESUMO
Alzheimer's disease (AD) is characterized by an imbalance between production and clearance of amyloid-ß (Aß) species. Aß peptides can transform structurally from monomers into ß-stranded fibrils via multiple oligomeric states. Among the various Aß species, structured oligomers are proposed to be more toxic than fibrils; however, the identification of Aß oligomers has been challenging due to their heterogeneous and metastable nature. Multiple techniques have recently helped us gain a better understanding of oligomers' assembly details and structural properties. Moreover, some progress on elucidating the mechanisms of oligomer-triggered toxicity has been made. Based on the collection of current findings, there is growing consensus that control of toxic Aß oligomers could be a valid approach to regulate Aß-associated toxicity, which could advance development of new diagnostics and therapeutics for amyloid-related diseases. In this review, we summarize the recent understanding of Aß oligomers' assembly, structural properties, and toxicity, along with inhibitors against Aß aggregation, including oligomerization.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , HumanosRESUMO
Aggregates of amyloidogenic peptides are involved in the pathogenesis of several degenerative disorders. Herein, an iridium(III) complex, Ir-1, is reported as a chemical tool for oxidizing amyloidogenic peptides upon photoactivation and subsequently modulating their aggregation pathways. Ir-1 was rationally designed based on multiple characteristics, including 1)â photoproperties leading to excitation by low-energy radiation; 2)â generation of reactive oxygen species responsible for peptide oxidation upon photoactivation under mild conditions; and 3)â relatively easy incorporation of a ligand on the IrIII center for specific interactions with amyloidogenic peptides. Biochemical and biophysical investigations illuminate that the oxidation of representative amyloidogenic peptides (i.e., amyloid-ß, α-synuclein, and human islet amyloid polypeptide) is promoted by light-activated Ir-1, which alters the conformations and aggregation pathways of the peptides. Additionally, their potential oxidation sites are identified as methionine, histidine, or tyrosine residues. Overall, our studies on Ir-1 demonstrate the feasibility of devising metal complexes as chemical tools suitable for elucidating the nature of amyloidogenic peptides at the molecular level, as well as controlling their aggregation.
Assuntos
Peptídeos beta-Amiloides/química , Complexos de Coordenação/química , Irídio/química , Sequência de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Luz , Oxirredução , Agregados Proteicos/efeitos da radiação , Espectrometria de Massas por Ionização por Electrospray , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMO
The absence of effective therapeutics against Alzheimer's disease (AD) is a result of the limited understanding of its multifaceted aetiology. Because of the lack of chemical tools to identify pathological factors, investigations into AD pathogenesis have also been insubstantial. Here we report chemical regulators that demonstrate distinct specificity towards targets linked to AD pathology, including metals, amyloid-ß (Aß), metal-Aß, reactive oxygen species, and free organic radicals. We obtained these chemical regulators through a rational structure-mechanism-based design strategy. We performed structural variations of small molecules for fine-tuning their electronic properties, such as ionization potentials and mechanistic pathways for reactivity towards different targets. We established in vitro and/or in vivo efficacies of the regulators for modulating their targets' reactivities, ameliorating toxicity, reducing amyloid pathology, and improving cognitive deficits. Our chemical tools show promise for deciphering AD pathogenesis and discovering effective drugs.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/antagonistas & inibidores , Amiloide/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Desenho de Fármacos , Radicais Livres/antagonistas & inibidores , Radicais Livres/metabolismo , Humanos , Metais/antagonistas & inibidores , Metais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estrutura Molecular , Agregados Proteicos/efeitos dos fármacos , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Protein inactivation by reactive oxygen species (ROS) such as singlet oxygen ((1)O2) and superoxide radical (O2(â¢-)) is considered to trigger cell death pathways associated with protein dysfunction; however, the detailed mechanisms and direct involvement in photodynamic therapy (PDT) have not been revealed. Herein, we report Ir(III) complexes designed for ROS generation through a rational strategy to investigate protein modifications by ROS. The Ir(III) complexes are effective as PDT agents at low concentrations with low-energy irradiation (≤ 1 J cm(-2)) because of the relatively high (1)O2 quantum yield (> 0.78), even with two-photon activation. Furthermore, two types of protein modifications (protein oxidation and photo-cross-linking) involved in PDT were characterized by mass spectrometry. These modifications were generated primarily in the endoplasmic reticulum and mitochondria, producing a significant effect for cancer cell death. Consequently, we present a plausible biologically applicable PDT modality that utilizes rationally designed photoactivatable Ir(III) complexes.
Assuntos
Retículo Endoplasmático/metabolismo , Irídio/química , Compostos Organometálicos/química , Compostos Organometálicos/metabolismo , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/metabolismo , Receptor 3 Toll-Like/metabolismo , Transporte Biológico , Células HEK293 , Células HeLa , Humanos , Oxigênio Singlete/metabolismoRESUMO
Amyloidosis is a clinical disorder implicated with the formation of toxic amyloid aggregates. Despite their pathological significance, it is challenging to define the structural characteristics of amyloid oligomers owing to their metastable nature. Herein, we report structural and mechanistic investigations of human islet amyloid polypeptide (hIAPP) oligomers, found in type II diabetes mellitus, in both the absence and presence of disease-relevant metal ions [i.e., Cu(ii) and Zn(ii)]. These metal ions show suppressive effects on hIAPP fibrillation and facilitate the generation of toxic oligomers. Using circular dichroism spectroscopy, transmission electron microscopy, gel electrophoresis, small-angle X-ray scattering, and ion mobility-mass spectrometry, we investigated the assembly mechanisms of hIAPP oligomers in the presence and absence of metal ions. Oligomerization of both metal-free hIAPP and metal-associated hIAPP monomers is initiated following a similar growth model. However, in the presence of Cu(ii), hIAPP monomers self-assemble into small globular aggregates (Rg â¼ 45 Å) with a random coil structure. This Cu(ii)-associated hIAPP oligomer shows an off-pathway aggregation, and is suggested to be an end product which is toxic to pancreatic ß-cells. On the other hand, metal-free hIAPP and Zn(ii)-associated hIAPP monomers generate relatively less toxic aggregates that eventually grow into fibrils. We suggest that the coordination of hIAPP to Cu(ii) and the relatively high stability (Ka, ca. 108 M-1) of hIAPP-Cu(ii) complexes result in the abnormal conformation and toxicity of hIAPP oligomers. Overall, through combining multiple biophysical methods, our studies suggest that molecular interactions between hIAPP and Cu(ii) induce a different pathway for hIAPP assembly. This work will advance our knowledge of the conformational basis, assembly mechanism, and toxicity of small soluble amyloid oligomers.
RESUMO
Amyloid fibrils are insoluble protein aggregates comprised of highly ordered ß-sheet structures and they are involved in the pathology of amyloidoses, such as Alzheimer's disease. A supramolecular strategy is presented for inhibiting amyloid fibrillation by using cucurbit[7]uril (CB[7]). CB[7] prevents the fibrillation of insulin and ß-amyloid by capturing phenylalanine (Phe) residues, which are crucial to the hydrophobic interactions formed during amyloid fibrillation. These results suggest that the Phe-specific binding of CB[7] can modulate the intermolecular interaction of amyloid proteins and prevent the transition from monomeric to multimeric states. CB[7] thus has potential for the development of a therapeutic strategy for amyloidosis.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Hidrocarbonetos Aromáticos com Pontes/química , Imidazóis/química , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Linhagem Celular Tumoral , Humanos , Dados de Sequência MolecularRESUMO
α-Synuclein (α-Syn) is an intrinsically disordered protein, whose fibrillar aggregates are associated with the pathogenesis of Parkinson's disease. α-Syn associates with lipid membranes and forms helical structures upon membrane binding. In this study, we explored the helix formation of α-Syn in solution containing trifluoroethanol using small-angle X-ray scattering and electrospray ionization ion mobility mass spectrometry. We then investigated the structural transitions of α-Syn to helical structures via association with large unilamellar vesicles as model lipid membrane systems. Hydrogen-deuterium exchange combined with electrospray ionization mass spectrometry was further utilized to understand the details of the regional interaction mechanisms of α-Syn with lipid vesicles based on the polarity of the lipid head groups. The characteristics of the helical structures were observed with α-Syn by adsorption onto the anionic phospholipid vesicles via electrostatic interactions between the N-terminal region of the protein and the anionic head groups of the lipids. α-Syn also associates with zwitterionic lipid vesicles and forms helical structures via hydrophobic interactions. These experimental observations provide an improved understanding of the distinct structural change mechanisms of α-Syn that originate from different regional interactions of the protein with lipid membranes and subsequently provide implications regarding diverse protein-membrane interactions related to their fibrillation kinetics.
Assuntos
Membrana Celular/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Membrana Celular/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismoRESUMO
The hydration of cucurbit[6]uril (CB[6]) in the gas phase is investigated using electrospray ionization traveling wave ion mobility mass spectrometry (ESI-TWIM-MS). Highly abundant dihydrated and tetrahydrated species of diprotonated CB[6] are found in the ESI-TWIM-MS spectrum. The hydration patterns of the CB[6] ion and the dissociation patterns of the hydrated CB[6] ion indicate that two water molecules are bound to each other, forming a water dimer in the CB[6] complex. Ion mobility studies combined with the structures calculated by density functional theory suggest that the proton-bound water dimer is present as a Zundel-like structure in the CB[6] portal, forming a hydrogen bond network with carbonyl groups of the CB[6]. When a large guest molecule is bound to a CB[6] portal, water molecules cannot bind to the portal. In addition, the strong binding energy of the water dimer blocks the portal, hindering the insertion of the long alkyl chain of the guest molecule into the CB[6] cavity. With small alkali metal cations, such as Li(+) and Na(+), a single water molecule interacts with the CB[6] portal, forming hydrogen bonds with the carbonyl groups of CB[6]. A highly stable Zundel-like structure of the proton-bound water dimer or a metal-bound water molecule at the CB[6] portal is suggested as an initial hydration process for CB[6], which is only dissolved in aqueous solution with acid or alkali metal ions.
RESUMO
An investigation of the host-guest chemistry of cucurbit[n]uril (CB[n], n = 6 and 7) with α,ω-alkyldiammonium guests (H2N(CH2)xNH2, x = 4, 6, 8, 10, and 12) both in solution and in the gas phase elucidates their intrinsic host-guest properties and the contribution of solvent water. Isothermal titration calorimetry and nuclear magnetic resonance measurements indicate that all alkyldiammonium cations have inclusion interactions with CB[n] except for the CB[7]-tetramethylenediamine complex in aqueous solution. The electrospray ionization of mixtures of CB[n] and the alkyldiammonium guests reflects their solution phase binding constants. Low-energy collision-induced dissociations indicate that, after the transfer of the CB[n]-alkyldiammonium complex to the gas phase, its stability is no longer correlated with the binding properties in solution. Gas phase structures obtained from density functional theory calculations, which support the results from the ion mobility measurements, and molecular dynamics simulated structures in water provide a detailed understanding of the solvated complexes. In the gas phase, the binding properties of complexation mostly depend on the ion-dipole interactions. However, the ion-dipole integrity is strongly affected by hydrogen bonding with water molecules in the aqueous condition. Upon the inclusion of water molecules, the intrinsic characteristics of the host-guest binding are dominated by entropic-driven thermodynamics.
RESUMO
We report mechanistic studies of structural changes of ubiquitin (Ub) by host-guest chemistry with cucurbit[6]uril (CB[6]) using electrospray ionization mass spectrometry (ESI-MS) combined with circular dichroism spectroscopy and molecular dynamics (MD) simulation. CB[6] binds selectively to lysine (Lys) residues of proteins. Low energy collision-induced dissociation (CID) of the protein-CB[6] complex reveals CB[6] binding sites. We previously reported (Anal. Chem. 2011, 83, 7916-7923) shifts in major charge states along with Ub-CB[6] complexes in the ESI-MS spectrum with addition of CB[6] to Ub from water. We also reported that CB[6] is present only at Lys(6) or Lys(11) in high charge state (+13) complex. In this study, we provide additional information to explain unique conformational change mechanisms of Ub by host-guest chemistry with CB[6] compared with solvent-driven conformational change of Ub. Additional CID study reveals that CB[6] is bound only to Lys(48) and Lys(63) in low charge state (+7) complex. MD simulation studies reveal that the high charge state complexes are attributed to the CB[6] bound to Lys(11). The complexation prohibits salt bridge formation between Lys(11) and Glu(34) and induces conformational change of Ub. This results in formation of high charge state complexes in the gas phase. Then, by utilizing stronger host-guest chemistry of CB[6] with pentamethylenediamine, refolding of Ub via detaching CB[6] from the protein is performed. Overall, this study gives an insight into the mechanism of denatured Ub ion formation via host-guest interactions with CB[6]. Furthermore, this provides a direction for designing function-controllable supramolecular system comprising proteins and synthetic host molecules.