RESUMO
The aggregation of amyloid-ß (Aß) on lipid bilayers has been implicated as a mechanism by which Aß exerts its toxicity in Alzheimer's disease (AD). Lipid bilayer thinning has been observed during both oxidative stress and protein aggregation in AD, but whether these pathological modifications of the bilayer correlate with Aß misfolding is unclear. Here, we studied peptide-lipid interactions in synthetic bilayers of the short-chain lipid dilauroyl phosphatidylcholine (DLPC) as a simplified model for diseased bilayers to determine their impact on Aß aggregate, protofibril, and fibril formation. Aß aggregation and fibril formation in membranes composed of dioleoyl phosphatidylcholine (DOPC) or 1- palmitoyl-2-oleoyl phosphatidylcholine mimicking normal bilayers served as controls. Differences in aggregate formation and stability were monitored by a combination of thioflavin-T fluorescence, circular dichroism, atomic force microscopy, transmission electron microscopy, and NMR. Despite the ability of all three lipid bilayers to catalyze aggregation, DLPC accelerates aggregation at much lower concentrations and prevents the fibrillation of Aß at low micromolar concentrations. DLPC stabilized globular, membrane-associated oligomers, which could disrupt the bilayer integrity. DLPC bilayers also remodeled preformed amyloid fibrils into a pseudo-unfolded, molten globule state, which resembled on-pathway, protofibrillar aggregates. Whereas the stabilized, membrane-associated oligomers were found to be nontoxic, the remodeled species displayed toxicity similar to that of conventionally prepared aggregates. These results provide mechanistic insights into the roles that pathologically thin bilayers may play in Aß aggregation on neuronal bilayers, and pathological lipid oxidation may contribute to Aß misfolding.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Bicamadas Lipídicas/metabolismo , Peptídeos beta-Amiloides/ultraestrutura , Humanos , Fosfatidilcolinas/metabolismo , Agregados Proteicos , Estrutura Secundária de ProteínaRESUMO
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
To elucidate the involvement of individual and inter-related pathological factors [i.e., amyloid-ß (Aß), metals, and oxidative stress] in the pathogenesis of Alzheimer's disease (AD), chemical tools have been developed. Characteristics required for such tool construction, however, have not been clearly identified; thus, the optimization of available tools or new design has been limited. Here, key structural properties and mechanisms that can determine tools' regulatory reactivities with multiple pathogenic features found in AD are reported. A series of small molecules was built up through rational structural selection and variations onto the framework of a tool useful for in vitro and in vivo metal-Aß investigation. Variations include: (i)â location and number of an Aß interacting moiety; (ii)â metal binding site; and (iii)â denticity and structural flexibility. Detailed biochemical, biophysical, and computational studies were able to provide a foundation of how to originate molecular formulas to devise chemical tools capable of controlling the reactivities of various pathological components through distinct mechanisms. Overall, this multidisciplinary investigation illustrates a structure-mechanism-based strategy of tool invention for such a complicated brain disease.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cloretos/química , Cobre/química , Humanos , Metais/química , Metais/metabolismo , Estresse Oxidativo , Ligação Proteica , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria , Compostos de Zinco/químicaRESUMO
The complex and multifaceted pathology of Alzheimer's disease (AD) continues to present a formidable challenge to the establishment of long-term treatment strategies. Multifunctional compounds able to modulate the reactivities of various pathological features, such as amyloid-ß (Aß) aggregation, metal ion dyshomeostasis, and oxidative stress, have emerged as a useful tactic. Recently, an incorporation approach to the rational design of multipurpose small molecules has been validated through the production of a multifunctional ligand (ML) as a potential chemical tool for AD. In order to further the development of more diverse and improved multifunctional reagents, essential pharmacophores must be identified. Herein, we report a series of aminoquinoline derivatives (AQ1-4, AQP1-4, and AQDA1-3) based on ML's framework, prepared to gain a structure-reactivity understanding of ML's multifunctionality in addition to tuning its metal binding affinity. Our structure-reactivity investigations have implicated the dimethylamino group as a key component for supplying the antiamyloidogenic characteristics of ML in both the absence and presence of metal ions. Two-dimensional NMR studies indicate that structural variations of ML could tune its interaction sites along the Aß sequence. In addition, mass spectrometric analyses suggest that the ability of our aminoquinoline derivatives to regulate metal-induced Aß aggregation may be influenced by their metal binding properties. Moreover, structural modifications to ML were also observed to noticeably change its metal binding affinities and metal-to-ligand stoichiometries that were shown to be linked to their antiamyloidogenic and antioxidant activities. Overall, our studies provide new insights into rational design strategies for multifunctional ligands directed at regulating metal ions, Aß, and oxidative stress in AD and could advance the development of improved next-generation multifunctional reagents.
Assuntos
Aminoquinolinas/química , Peptídeos beta-Amiloides/química , Antioxidantes/química , Dimetilaminas/química , Fragmentos de Peptídeos/química , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Aminoquinolinas/síntese química , Aminoquinolinas/toxicidade , Animais , Antioxidantes/síntese química , Antioxidantes/toxicidade , Linhagem Celular Tumoral , Cobre/química , Dimetilaminas/síntese química , Dimetilaminas/toxicidade , Humanos , Camundongos , Simulação de Acoplamento Molecular , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Multimerização Proteica , Espécies Reativas de Oxigênio/química , Relação Estrutura-Atividade , Zinco/químicaRESUMO
The aggregation of amyloid-ß (Aß) on neuronal membranes is implicated in both neuronal toxicity and the progression of Alzheimer's disease. Unfortunately, the heterogeneous environment that results from peptide aggregation in the presence of lipids makes the details of these pathways difficult to interrogate. In this study, we report an investigation of the membrane interaction of an Aß fragment (K16LVFFAEDVGSNK28, KK13), which maintains the amyloidogenic nature of the full-length peptide and is implicated in membrane-mediated folding, through a combination of NMR spectroscopy and molecular dynamics simulations. Despite KK13's ability to form amyloids in solution, the monomer remains unstructured in the presence of lipid bilayers, unlike its full-length parent peptide. Additionally, NMR and molecular dynamics simulation results support that the presence of GM1 ganglioside, a lipid which strongly promotes binding between Aß and lipid bilayers, promotes KK13 binding to but not folding on the membrane. Finally, we show that the peptide partitions between the membrane and aqueous solution based on the hydrophobicity of the N-terminal residues, regardless of lipid composition. These results support previous discoveries suggesting the importance of GM1 ganglioside in exacerbating membrane-driven aggregation while identifying the potential importance of C-terminal residues in membrane binding and folding, which has previously been unclear.
RESUMO
Chemical reagents targeting and controlling amyloidogenic peptides have received much attention for helping identify their roles in the pathogenesis of protein-misfolding disorders. Herein, we report a novel strategy for redirecting amyloidogenic peptides into nontoxic, off-pathway aggregates, which utilizes redox properties of a small molecule (DMPD, N,N-dimethyl-p-phenylenediamine) to trigger covalent adduct formation with the peptide. In addition, for the first time, biochemical, biophysical, and molecular dynamics simulation studies have been performed to demonstrate a mechanistic understanding for such an interaction between a small molecule (DMPD) and amyloid-ß (Aß) and its subsequent anti-amyloidogenic activity, which, upon its transformation, generates ligand-peptide adducts via primary amine-dependent intramolecular cross-linking correlated with structural compaction. Furthermore, in vivo efficacy of DMPD toward amyloid pathology and cognitive impairment was evaluated employing 5xFAD mice of Alzheimer's disease (AD). Such a small molecule (DMPD) is indicated to noticeably reduce the overall cerebral amyloid load of soluble Aß forms and amyloid deposits as well as significantly improve cognitive defects in the AD mouse model. Overall, our in vitro and in vivo studies of DMPD toward Aß with the first molecular-level mechanistic investigations present the feasibility of developing new, innovative approaches that employ redox-active compounds without the structural complexity as next-generation chemical tools for amyloid management.
Assuntos
Peptídeos beta-Amiloides/química , Amiloide/química , Linhagem Celular , Humanos , Técnicas In Vitro , Simulação de Dinâmica Molecular , OxirreduçãoRESUMO
Cholesterol and metal ions have been suggested to be associated with the onset and progression of Alzheimer's disease (AD). Moreover, recent findings have demonstrated a potential interconnection between these two factors. For example, (a) cholesterol has been shown to be misregulated in AD-afflicted brains, and the aberrant activity of proteins (particularly, apolipoprotein E (ApoE) and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGR)) has been linked to cholesterol-related AD exacerbation; (b) dyshomeostasis of metal ions associated with misfolded proteins (i.e., amyloid-ß (Aß) aggregates) found in the brains of AD patients is shown to promote oxidative stress leading to the malfunction of multiple proteins, including cytochrome c oxidase (CcO), and Cu/Zn superoxide dismutase (SOD1); (c) metal ion misregulation has also been observed to disrupt the activity of proteins (e.g., HMGR, low-density lipoproteins (LDL)), required for cholesterol production and regulation. Herein, we briefly discuss the potential involvement of cholesterol and metal ions in AD neuropathogenesis in both individual and interrelated manners.
Assuntos
Doença de Alzheimer/patologia , Colesterol/metabolismo , Metais/metabolismo , Doença de Alzheimer/metabolismo , Apolipoproteínas E/metabolismo , Encéfalo/metabolismo , Colesterol/química , Humanos , Hidroximetilglutaril-CoA-Redutases NADP-Dependentes/metabolismo , Metais/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
Reactions and interactions between glyoxal and salts in aqueous solution were studied. Glyoxal was found to react with ammonium to form imidazole, imidazole-2-carboxaldehyde, formic acid, N-glyoxal substituted imidazole, and minor products at very low concentrations. Overall reaction orders and rates for each major product were measured. Sulfate ions have a strong and specific interaction with glyoxal in aqueous solution, which shifts the hydration equilibria of glyoxal from the unhydrated carbonyl form to the hydrated form. This ion-specific effect contributes to the observed enhancement of the effective Henry's law coefficient for glyoxal in sulfate-containing solutions. The results of UV-vis absorption and NMR spectroscopy studies of solutions of glyoxal with ammonium, methylamine, and dimethylamine salts reveal that light absorbing compounds require the formation of nitrogen containing molecules. These findings have implications on the role of glyoxal in the atmosphere, both in models of the contribution of glyoxal to form secondary organic aerosol (SOA), the role of nitrogen containing species for aerosol optical properties and in predictions of the behavior of other carbonyls or dicarbonyls in the atmosphere.
Assuntos
Sulfato de Amônio/química , Glioxal/química , Água/química , Atmosfera/química , Eletrólitos , Cinética , Padrões de Referência , Soluções , Espectrofotometria Ultravioleta , Sulfatos/química , Fatores de TempoRESUMO
Apoptosis is regulated by BCL-2 family proteins. Anti-apoptotic members suppress cell death by deploying a surface groove to capture the critical BH3 α-helix of pro-apoptotic members. Cancer cells hijack this mechanism by overexpressing anti-apoptotic BCL-2 family proteins to enforce cellular immortality. We previously identified and harnessed a unique cysteine (C55) in the groove of anti-apoptotic BFL-1 to selectively neutralize its oncogenic activity using a covalent stapled-peptide inhibitor. Here, we find that disulfide bonding between a native cysteine pair at the groove (C55) and C-terminal α9 helix (C175) of BFL-1 operates as a redox switch to control the accessibility of the anti-apoptotic pocket. Reducing the C55-C175 disulfide triggers α9 release, which promotes mitochondrial translocation, groove exposure for BH3 interaction and inhibition of mitochondrial permeabilization by pro-apoptotic BAX. C55-C175 disulfide formation in an oxidative cellular environment abrogates the ability of BFL-1 to bind BH3 domains. Thus, we identify a mechanism of conformational control of BFL-1 by an intramolecular redox switch.
Assuntos
Apoptose , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Cisteína/química , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Células HEK293 , Humanos , Camundongos , Antígenos de Histocompatibilidade Menor/química , Mitocôndrias/metabolismo , Modelos Moleculares , Oxirredução , Conformação Proteica , Conformação Proteica em alfa-Hélice , Proteínas Proto-Oncogênicas c-bcl-2/químicaRESUMO
Proton magic-angle-spinning NMR used for real-time analysis of amyloid aggregation reveals that mechanical rotation of Aß1-40 monomers increases the rate of formation of aggregates, and that the increasing lag-time with peptide concentration suggests the formation of growth-incompetent species. EGCG's ability to shift off-pathway aggregation is also demonstrated.
RESUMO
Cancer cells overexpress a diversity of anti-apoptotic BCL-2 family proteins, such as BCL-2, MCL-1, and BFL-1/A1, to enforce cellular immortality. Thus, intensive drug development efforts have focused on targeting this class of oncogenic proteins to overcome treatment resistance. Whereas a selective BCL-2 inhibitor has been FDA approved and several small molecule inhibitors of MCL-1 have recently entered phase I clinical testing, BFL-1/A1 remains undrugged. Here, we developed a series of stapled peptide design principles to engineer a functionally selective and cell-permeable BFL-1/A1 inhibitor that is specifically cytotoxic to BFL-1/A1-dependent human cancer cells. Because cancers harbor a diversity of resistance mechanisms and typically require multi-agent treatment, we further investigated BFL-1/A1 co-dependencies by mining a genome-scale CRISPR-Cas9 screen. We identified ataxia-telangiectasia-mutated (ATM) kinase as a BFL-1/A1 co-dependency in acute myeloid leukemia (AML), which informed the validation of BFL-1/A1 and ATM inhibitor co-treatment as a synergistic approach to subverting apoptotic resistance in cancer.
Assuntos
Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Células HEK293 , Humanos , Masculino , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Membrane-assisted amyloid formation is implicated in human diseases, and many of the aggregating species accelerate amyloid formation and induce cell death. While structures of membrane-associated intermediates would provide tremendous insights into the pathology and aid in the design of compounds to potentially treat the diseases, it has not been feasible to overcome the challenges posed by the cell membrane. Here, we use NMR experimental constraints to solve the structure of a type-2 diabetes related human islet amyloid polypeptide intermediate stabilized in nanodiscs. ROSETTA and MD simulations resulted in a unique ß-strand structure distinct from the conventional amyloid ß-hairpin and revealed that the nucleating NFGAIL region remains flexible and accessible within this isolated intermediate, suggesting a mechanism by which membrane-associated aggregation may be propagated. The ability of nanodiscs to trap amyloid intermediates as demonstrated could become one of the most powerful approaches to dissect the complicated misfolding pathways of protein aggregation.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Agregados Proteicos , Multimerização Proteica , Humanos , Espectroscopia de Ressonância Magnética , Membranas/química , Dobramento de ProteínaRESUMO
Alzheimer's disease (AD) is a multifactorial disease that is characterized by the formation of intracellular neurofibrillary tangles and extracellular amyloid-ß (Aß) plaque deposits. Increased oxidative stress, metal ion dysregulation, and the formation of toxic Aß peptide oligomers are all considered to contribute to the etiology of AD. In this work we have developed a series of ligands that are multi-target-directed in order to address several disease properties. 2-(1-(3-Hydroxypropyl)-1H-1,2,3-triazol-4-yl)phenol (POH), 2-(1-(2-morpholinoethyl)-1H-1,2,3-triazol-4-yl)phenol (PMorph), and 2-(1-(2-thiomorpholinoethyl)-1H-1,2,3-triazol-4-yl)phenol (PTMorph) have been synthesized and screened for their antioxidant capacity, Cu-binding affinity, interaction with the Aß peptide and modulation of Aß peptide aggregation, and the ability to limit Aß1-42-induced neurotoxicity in human neuronal culture. The synthetic protocol and structural variance incorporated via click chemistry, highlights the influence of R-group modification on ligand-Aß interactions and neuroprotective effects. Overall, this study demonstrates that the phenol-triazole ligand scaffold can target multiple factors associated with AD, thus warranting further therapeutic development.
RESUMO
Chemical tools have been valuable for establishing a better understanding of the relationships between metal ion dyshomeostasis, the abnormal aggregation and accumulation of amyloid-ß (Aß), and oxidative stress in Alzheimer's disease (AD). Still, very little information is available to correlate the structures of chemical tools with specific reactivities used to uncover such relationships. Recently, slight structural variations to the framework of a chemical tool were found to drastically determine the tool's reactivities toward multiple pathological facets to various extents. Herein, we report our rational design and characterization of a structural series to illustrate the extent to which the reactivities of small molecules vary toward different targets as a result of minor structural modifications. These compounds were rationally and systematically modified based on consideration of properties, including ionization potentials and metal binding, to afford their desired reactivities with metal-free or metal-bound Aß, reactive oxygen species (ROS), and free organic radicals. Our results show that although small molecules are structurally similar, they can interact with multiple factors associated with AD pathogenesis and alleviate their reactivities to different degrees. Together, our studies demonstrate the rational structure-directed design that can be used to develop chemical tools capable of regulating individual or interrelated pathological features in AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Antioxidantes/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Antioxidantes/síntese química , Antioxidantes/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Estrutura Molecular , Estresse Oxidativo/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Aggregation at the neuronal cell membrane's lipid bilayer surface is implicated in amyloid-ß (Aß) toxicity associated with Alzheimer's disease; however, structural and mechanistic insights into the process remain scarce. We have identified a conserved binding mode of Aß40 on lipid bilayer surfaces with a conserved helix containing the self-recognition site (K16-E22).
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Amiloide/química , Amiloide/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Dobramento de Proteína , Amiloide/biossíntese , Sítios de Ligação , Modelos Moleculares , Conformação Proteica , Propriedades de SuperfícieRESUMO
Both amyloid-ß (Aß) and transition metal ions are shown to be involved in the pathogenesis of Alzheimer's disease (AD), though the importance of their interactions remains unclear. Multifunctional molecules, which can target metal-free and metal-bound Aß and modulate their reactivity (e.g., Aß aggregation), have been developed as chemical tools to investigate their function in AD pathology; however, these compounds generally lack specificity or have undesirable chemical and biological properties, reducing their functionality. We have evaluated whether multiple polyphenolic glycosides and their esterified derivatives can serve as specific, multifunctional probes to better understand AD. The ability of these compounds to interact with metal ions and metal-free/-associated Aß, and further control both metal-free and metal-induced Aß aggregation was investigated through gel electrophoresis with Western blotting, transmission electron microscopy, UV-Vis spectroscopy, fluorescence spectroscopy, and NMR spectroscopy. We also examined the cytotoxicity of the compounds and their ability to mitigate the toxicity induced by both metal-free and metal-bound Aß. Of the polyphenols investigated, the natural product (Verbascoside) and its esterified derivative (VPP) regulate the aggregation and cytotoxicity of metal-free and/or metal-associated Aß to different extents. Our studies indicate Verbascoside represents a promising structure for further multifunctional tool development against both metal-free Aß and metal-Aß.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Metais/metabolismo , Polifenóis/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Animais , Antioxidantes/química , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Glicosilação , Humanos , Ligantes , Metais/química , Camundongos , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Polifenóis/química , Polifenóis/farmacologia , Agregados Proteicos , Agregação Patológica de Proteínas/metabolismo , Ligação ProteicaRESUMO
Alzheimer's disease (AD) is a complex, multifactorial, neurodegenerative disease that poses tremendous difficulties in pinpointing its precise etiology. A toolkit, which specifically targets and modulates suggested key players, may elucidate their roles in disease onset and progression. We report high-resolution insights on the activity of a small molecule (L2-NO) which exhibits reactivity toward Cu(II)-amyloid-ß (Aß) over Zn(II)-Aß.