RESUMO
A hallmark of the pancreatic hormone amylin is its high propensity toward the formation of amyloid fibrils, which makes it a challenging drug design effort. The amylin analogue pramlintide is commercially available for diabetes treatment as an adjunct to insulin therapy but requires three daily injections due to its short half-life. We report here the development of the stable, lipidated long-acting amylin analogue cagrilintide (23) and some of the structure-activity efforts that led to the selection of this analogue for clinical development with obesity as an indication. Cagrilintide is currently in clinical trial and has induced significant weight loss when dosed alone or in combination with the GLP-1 analogue semaglutide.
Assuntos
Desenvolvimento de Medicamentos , Hipoglicemiantes/farmacologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , Hipoglicemiantes/síntese química , Hipoglicemiantes/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The aberrant misfolding and self-assembly of human islet amyloid polypeptide (hIAPP)-a hormone that is co-secreted with insulin from pancreatic ß-cells-into toxic oligomers, protofibrils and fibrils has been observed in type 2 diabetes mellitus (T2DM). The formation of these insoluble aggregates has been linked with the death and dysfunction of ß-cells. Therefore, hIAPP aggregation has been identified as a therapeutic target for T2DM management. Several natural products are now being investigated for their potential to inhibit hIAPP aggregation and/or disaggregate preformed aggregates. In this study, we attempt to identify the anti-amyloidogenic potential of Myricetin (MYR)- a polyphenolic flavanoid, commonly found in fruits (like Syzygium cumini). Our results from biophysical studies indicated that MYR supplementation inhibits hIAPP aggregation and disaggregates preformed fibrils into non-toxic species. This protection was accompanied by inhibition of oxidative stress, reduction in lipid peroxidation and the associated membrane damage and restoration of mitochondrial membrane potential in INS-1E cells. MYR supplementation also reversed the loss of functionality in hIAPP exposed pancreatic islets via restoration of glucose-stimulated insulin secretion. Molecular dynamics simulation studies suggested that MYR molecules interact with the hIAPP pentameric fibril model at the amyloidogenic core region and thus prevents aggregation and distort the fibrils.
Assuntos
Flavonoides/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/antagonistas & inibidores , Ilhotas Pancreáticas/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Flavonoides/química , Frutas/química , Humanos , Células Secretoras de Insulina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/metabolismo , Simulação de Dinâmica Molecular , Substâncias Protetoras/síntese química , Substâncias Protetoras/química , Agregados Proteicos/efeitos dos fármacos , Syzygium/químicaRESUMO
The hexapeptide hIAPP22-27 (NFGAIL) is known as a crucial amyloid core sequence of the human islet amyloid polypeptide (hIAPP) whose aggregates can be used to better understand the wild-type hIAPP's toxicity to ß-cell death. In amyloid research, the role of hydrophobic and aromatic-aromatic interactions as potential driving forces during the aggregation process is controversially discussed not only in case of NFGAIL, but also for amyloidogenic peptides in general. We have used halogenation of the aromatic residue as a strategy to modulate hydrophobic and aromatic-aromatic interactions and prepared a library of NFGAIL variants containing fluorinated and iodinated phenylalanine analogues. We used thioflavin T staining, transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) to study the impact of side-chain halogenation on NFGAIL amyloid formation kinetics. Our data revealed a synergy between aggregation behavior and hydrophobicity of the phenylalanine residue. This study introduces systematic fluorination as a toolbox to further investigate the nature of the amyloid self-assembly process.
Assuntos
Hidrocarbonetos Halogenados/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Fenilalanina/química , Teoria da Densidade Funcional , Halogenação , Humanos , Hidrocarbonetos Halogenados/síntese química , Interações Hidrofóbicas e Hidrofílicas , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Cinética , Estrutura Molecular , Tamanho da Partícula , Agregados ProteicosRESUMO
Islet amyloid polypeptide (IAPP), a 37 residue polypeptide, is the main protein component of islet amyloid deposits produced in the pancreas in Type 2 diabetes. Human IAPP contains five serine residues at positions 19, 20, 28, 29, and 34. Models of the IAPP amyloid fibril indicate a structure composed of two closely aligned columns of IAPP monomers with each monomer contributing to two intermolecular ß-strands. Ser 19 and Ser 20 are in the partially ordered ß-turn region, which links the two strands, whereas Ser 28, Ser 29, and Ser 34 are in the core region of the amyloid fibril. Ser 29 is involved in contacts between the two columns of monomers and is the part of the steric zipper interface. We undertook a study of individual serine substitutions with the hydrophobic isostere 2-aminobutyric acid (2-Abu) to examine the site-specific role of serine side chains in IAPP amyloid formation. All five variants formed amyloid. The Ser 19 to 2-Abu mutant accelerates amyloid formation by a factor of 3 to 4, while the Ser 29 to 2-Abu mutation modestly slows the rate of amyloid formation. 2-Abu replacements at the other sites had even smaller effects. The data demonstrate that the cross-column interactions made by residue 29 are not essential for amyloid formation and also show that cross-strand networks of hydrogen-bonded Ser side chains, so called Ser-ladders, are not required for IAPP amyloid formation. The effect of the Ser 19 to 2-Abu mutant suggests that residues in this region are important for amyloid formation by IAPP.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Serina/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/isolamento & purificação , Cinética , Modelos Moleculares , Solventes/química , Propriedades de SuperfícieRESUMO
Misfolding and abnormal assembly of proteins cause many intractable diseases. The modulation of the assembly process of these proteins could contribute to understanding and controlling amyloid protein aggregation. Previous works focused mainly on the inhibition of the assembly process. To broaden the interaction modality of modulators with proteins for developing new modulators, in this work, we designed and synthesized two reactive poly ( p-phenylene vinylene) polymers, respectively, functionalized with N-hydroxysuccinimide ester (PPV-NHS) and pentafluorophenol ester (PPV-PFP), which exhibited the prevention or co-assembly effect on the aggregation process of islet amyloid polypeptide (IAPP). Cell assays demonstrated that both of the two polymers could effectively eliminate the cytotoxicity of IAPP. Moreover, PPV-NHS also could irreversibly disrupt preformed IAPP fibrils. We envision that PPV-NHS and PPV-PFP might offer a new design method for the modulation of protein assembly.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polímeros/química , Agregação Patológica de Proteínas/tratamento farmacológico , Sequência de Aminoácidos/genética , Ésteres/síntese química , Ésteres/química , Ésteres/farmacologia , Fluorbenzenos/síntese química , Fluorbenzenos/química , Fluorbenzenos/farmacologia , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Fenóis/síntese química , Fenóis/química , Fenóis/farmacologia , Polímeros/síntese química , Polímeros/farmacologia , Agregação Patológica de Proteínas/genética , Succinimidas/síntese química , Succinimidas/química , Succinimidas/farmacologiaRESUMO
Transiently populated oligomers formed en route to amyloid fibrils may constitute the most toxic aggregates associated with many amyloid-associated diseases. Most nucleation theories used to describe amyloid aggregation predict low oligomer concentrations and do not take into account free energy costs that may be associated with structural rearrangements between the oligomer and fiber states. We have used isotope labeling and two-dimensional infrared spectroscopy to spectrally resolve an oligomeric intermediate during the aggregation of the human islet amyloid protein (hIAPP or amylin), the protein associated with type II diabetes. A structural rearrangement includes the F23G24A25I26L27 region of hIAPP, which starts from a random coil structure, evolves into ordered ß-sheet oligomers containing at least 5 strands, and then partially disorders in the fibril structure. The supercritical concentration is measured to be between 150 and 250 µM, which is the thermodynamic parameter that sets the free energy of the oligomers. A 3-state kinetic model fits the experimental data, but only if it includes a concentration independent free energy barrier >3 kcal/mol that represents the free energy cost of refolding the oligomeric intermediate into the structure of the amyloid fibril; i.e., "oligomer activation" is required. The barrier creates a transition state in the free energy landscape that slows fibril formation and creates a stable population of oligomers during the lag phase, even at concentrations below the supercritical concentration. Largely missing in current kinetic models is a link between structure and kinetics. Our experiments and modeling provide evidence that protein structural rearrangements during aggregation impact the populations and kinetics of toxic oligomeric species.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Termodinâmica , Diabetes Mellitus Tipo 2 , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Cinética , Redobramento de Proteína , Fatores de TempoRESUMO
The interaction of the intrinsically disordered polypeptide islet amyloid polypeptide (IAPP), which is associated with type 2 diabetes (T2D), with the Alzheimer's disease amyloid-ß (Aß) peptide modulates their self-assembly into amyloid fibrils and may link the pathogeneses of these two cell-degenerative diseases. However, the molecular determinants of this interaction remain elusive. Using a systematic alanine scan approach, fluorescence spectroscopy, and other biophysical methods, including heterocomplex pulldown assays, far-UV CD spectroscopy, the thioflavin T binding assay, transmission EM, and molecular dynamics simulations, here we identified single aromatic/hydrophobic residues within the amyloid core IAPP region as hot spots or key residues of its cross-interaction with Aß40(42) peptide. Importantly, we also find that none of these residues in isolation plays a key role in IAPP self-assembly, whereas simultaneous substitution of four aromatic/hydrophobic residues with Ala dramatically impairs both IAPP self-assembly and hetero-assembly with Aß40(42). Furthermore, our experiments yielded several novel IAPP analogs, whose sequences are highly similar to that of IAPP but have distinct amyloid self- or cross-interaction potentials. The identified similarities and major differences controlling IAPP cross-peptide interaction with Aß40(42) versus its amyloid self-assembly offer a molecular basis for understanding the underlying mechanisms. We propose that these insights will aid in designing intervention strategies and novel IAPP analogs for the management of type 2 diabetes, Alzheimer's disease, or other diseases related to IAPP dysfunction or cross-amyloid interactions.
Assuntos
Aminoácidos/metabolismo , Peptídeos beta-Amiloides/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Modelos Moleculares , Fragmentos de Peptídeos/metabolismo , Agregação Patológica de Proteínas/metabolismo , Substituição de Aminoácidos , Aminoácidos/química , Aminoácidos Aromáticos , Peptídeos beta-Amiloides/síntese química , Peptídeos beta-Amiloides/química , Dicroísmo Circular , Humanos , Interações Hidrofóbicas e Hidrofílicas , Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Cinética , Metilação , Microscopia Eletrônica de Transmissão , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Estabilidade Proteica , Estrutura Secundária de Proteína , Técnicas de Síntese em Fase Sólida , Solubilidade , Espectrometria de FluorescênciaRESUMO
Aggregation of the islet amyloid polypeptide (IAPP) to form fibrils and oligomers is important in the progression of type 2 diabetes. This article describes X-ray crystallographic and solution-state NMR studies of peptides derived from residues 11-17 of IAPP that assemble to form tetramers. Incorporation of residues 11-17 of IAPP (RLANFLV) into a macrocyclic ß-sheet peptide results in a monomeric peptide that does not self-assemble to form oligomers. Mutation of Arg11 to the uncharged isostere citrulline gives peptide homologues that assemble to form tetramers in both the crystal state and in aqueous solution. The tetramers consist of hydrogen-bonded dimers that sandwich together through hydrophobic interactions. The tetramers share several features with structures reported for IAPP fibrils and demonstrate the importance of hydrogen bonding and hydrophobic interactions in the oligomerization of IAPP-derived peptides.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação ProteicaRESUMO
The aggregation of human islet amyloid polypeptide (hIAPP) has been closely associated with the pathogeny of type 2 diabetes mellitus (T2DM) and destruction of pancreatic islet ß-cells. Several amyloidogenic domains within the hIAPP sequence capable of self-association have been identified. Among them is the 8-20 region of hIAPP, which has formed ß-sheet fibrils despite being contained within an α-helical region of full-length hIAPP. To further understand the propensity of this region for self-assembly, two peptide fragments were compared, one consisting of the residues 8-20 (WT8-20) and a mutant fragment with a His18Pro substitution (H18P8-20). The conformational distribution and aggregation propensity of these peptides was determined using a combination of ion mobility mass spectrometry and atomic force microscopy. Our results reveal that the two peptide fragments have vastly differing assembly pathways. WT8-20 produces a wide range of oligomers up to decamer whereas the H18P8-20 mutant produces only low order oligomers. This study confirms the propensity of the 8-20 region to aggregate from its native α-helical structure into amyloid ß-sheet oligomers and highlights the significance of the charged His18 in the aggregation process.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Fragmentos de Peptídeos/química , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Espectrometria de Massas , Microscopia de Força Atômica , Fragmentos de Peptídeos/genéticaRESUMO
Versions of a previously discovered ß-hairpin peptide inhibitor of IAPP aggregation that are stabilized in that conformation, or even forced to remain in the hairpin conformation by a backbone cyclization constraint, display superior activity as inhibitors. The cyclized hairpin, cyclo-WW2, displays inhibitory activity at substoichiometric concentrations relative to this amyloidogenic peptide. The hairpin-binding hypothesis stands confirmed.
Assuntos
Insulina/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Peptídeos/química , Agregação Patológica de Proteínas/genética , Benzotiazóis , Dicroísmo Circular , Humanos , Insulina/genética , Polipeptídeo Amiloide das Ilhotas Pancreáticas/antagonistas & inibidores , Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Peptídeos/antagonistas & inibidores , Peptídeos/síntese química , Ligação Proteica/genética , Conformação Proteica/efeitos dos fármacos , Tiazóis/química , Tiazóis/metabolismoRESUMO
Pramlintide (Symlin®), a synthetic analogue of the neuroendocrine hormone amylin, is devoid of the tendency to form cytotoxic amyloid fibrils and is currently used in patients with type I and type II diabetes mellitus as an adjunctive therapy with insulin or insulin analogues. As part of an on-going search for a pramlintide analogue with improved pharmacokinetic properties, we herein report the synthesis of mono- and di-glycosylated analogues of pramlintide and their activity at the AMY1(a) receptor. Introduction of N-glycosylated amino acids into the pramlintide sequence afforded the native N-linked glycomimetics whilst use of Cu(i)-catalysed azide-alkyne 1,3-dipolar cycloaddition (click) chemistry delivered 1,2,3-triazole linked glycomimetics. AMY1(a) receptor activity was retained by incorporation of single or multiple GlcNAc moieties at positions 21 and 35 of native pramlintide. Importantly, no difference in AMY1(a) activity was observed between native N-linked glycomimetics and 1,2,3-triazole linked glycomimetics demonstrating that the click variants can act as surrogates for the native N-glycosides in a biological setting.
Assuntos
Materiais Biomiméticos/síntese química , Materiais Biomiméticos/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/metabolismo , Sequência de Aminoácidos , Materiais Biomiméticos/química , Química Click , Glicosilação , Polipeptídeo Amiloide das Ilhotas Pancreáticas/químicaRESUMO
Transition dipoles are an underutilized quantity for probing molecular structures. The transition dipole strengths in an extended system like a protein are modulated by the couplings and thus probe the structures. Here we measure the absolute transition dipole strengths of human and rat amylin in their solution, aggregated, membrane, and micelleular bound forms, using a combination of 1D and 2D infrared spectroscopy. We find that the vibrational modes of amyloid fibers made of human amylin can extend across as many as 12 amino acids, reflecting very ordered ß-sheets in the most carefully prepared samples. Rat amylin has FTIR spectra that are nearly identical in solution, micelles, and membranes. We show that the transition dipoles of rat amylin are much larger when bound to micelles and membranes than when in solution, consistent with rat amylin adopting an α-helical structure. We interpret the transition dipole strengths as experimental measurements of the inverse participation ratio often calculated in theoretical studies. The structure of aggregating and membrane-bound proteins can be difficult to identify with existing techniques, especially during kinetics. These results demonstrate how absolute transition dipoles measured via our 1D/2D spectroscopy method can provide important structural information.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Animais , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Tamanho da Partícula , Estrutura Secundária de Proteína , Ratos , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
By adding a mid-infrared pulse shaper to a sum-frequency generation (SFG) spectrometer, we have built a 2D SFG spectrometer capable of measuring spectra analogous to 2D IR spectra but with monolayer sensitivity and SFG selection rules. In this paper, we describe the experimental apparatus and provide an introduction to 2D SFG spectroscopy to help the reader interpret 2D SFG spectra. The main aim of this manuscript is to report 2D SFG spectra of the amyloid forming peptide FGAIL. FGAIL is a critical segment of the human islet amyloid polypeptide (hIAPP or amylin) that aggregates in people with type 2 diabetes. FGAIL is catalyzed into amyloid fibers by many types of surfaces. Here, we study the structure of FGAIL upon deposition onto a gold surface covered with a self-assembled monolayer of methyl-4-mercaptobenzoate (MMB) that produces an ester coating. FGAIL deposited on bare gold does not form ordered layers. The measured 2D SFG spectrum is consistent with amyloid fiber formation, exhibiting both the parallel (a+) and perpendicular (a-) symmetry modes associated with amyloid ß-sheets. Cross peaks are observed between the ester stretches of the coating and the FGAIL peptides. Simulations are presented for two possible structures of FGAIL amyloid ß-sheets that illustrate the sensitivity of the 2D SFG spectra to structure and orientation. These results provide some of the first molecular insights into surface catalyzed amyloid fiber structure.
Assuntos
Amiloide/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Espectrofotometria Infravermelho/instrumentação , Adsorção , Benzoatos/química , Ouro/química , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Agregados Proteicos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espectrofotometria Infravermelho/métodosRESUMO
Pramlintide (Symlin®), a synthetic analogue of the naturally occurring pancreatic hormone amylin, is currently used with insulin in adjunctive therapy for type 1 and type 2 diabetes mellitus. Herein we report a systematic study into the effect that N-glycosylation of pramlintide has on activation of amylin receptors. A highly efficient convergent synthetic route, involving a combination of solid phase peptide synthesis and enzymatic glycosylation, delivered a library of N-glycosylated variants of pramlintide bearing either GlcNAc, the core N-glycan pentasaccharide [Man3(GlcNAc)2] or a complex biantennary glycan [(NeuAcGalGlcNAcMan)2Man(GlcNAc)2] at each of its six asparagine residues. The majority of glycosylated versions of pramlintide were potent receptor agonists, suggesting that N-glycosylation may be used as a tool to optimise the pharmacokinetic properties of pramlintide and so deliver improved therapeutic agents for the treatment of diabetes and obesity.
Assuntos
Agonistas dos Receptores da Amilina/química , Agonistas dos Receptores da Amilina/farmacologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Receptores de Polipeptídeo Amiloide de Ilhotas Pancreáticas/metabolismo , Agonistas dos Receptores da Amilina/síntese química , Relação Dose-Resposta a Droga , Glicosilação , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The O-acyl isopeptide method was developed for the efficient preparation of difficult sequence-containing peptide. Furthermore, development of the O-acyl isodipeptide unit for Fmoc chemistry simplified its synthetic procedure by solid-phase peptide synthesis. Here, we report a novel isodipeptide unit for Boc chemistry, and the unit was successfully applied to the synthesis of amyloid ß peptide. Combination of Boc chemistry and the isodipeptide unit would be an effective method for the synthesis of many difficult peptides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
Assuntos
Peptídeos beta-Amiloides/síntese química , Peptídeos beta-Amiloides/isolamento & purificação , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/isolamento & purificação , Técnicas de Síntese em Fase Sólida/métodos , Peptídeos beta-Amiloides/química , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/isolamento & purificação , Fragmentos de Peptídeos/química , Estrutura Secundária de ProteínaRESUMO
The molecular mechanisms by which different proteins assemble into highly ordered fibrillar deposits and cause disease remain topics of debate. Human amylin (also known as islet amyloid polypeptide/hIAPP) is found in vivo as amyloid deposits in the pancreatic islets of sufferers of type II diabetes mellitus, and its self-aggregation is thought to be a pathogenic factor in disease and to contribute to the failure of islet transplants. Here, electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) has been used to monitor oligomer formation from IAPP. The detection, identification and characterization of oligomers from both human and rat amylin (rIAPP) are described. Oligomers up to and including hexamers have been detected for both peptides. From ESI-IMS-MS derived collision cross sections (CCS), these species are shown to be elongated in conformation. Collision-induced dissociation (CID-MS/MS) revealed differences in the gas-phase stability of the oligomers formed from hIAPP and rIAPP, which may contribute to their differences in amyloid propensity. Using ESI-IMS-MS, the mode of inhibition of amyloid formation from hIAPP using small molecules or co-incubation with rIAPP was also investigated. We show that the polyphenolic compounds epigallocatechin gallate (EGCG) and silibinin bind to specific conformers within a dynamic ensemble of hIAPP monomers, altering the progress of oligomerization and fibril assembly. Hetero-oligomer formation also occurs with rIAPP but leads only to inefficient inhibition. The results indicate that although different small molecules can be effective inhibitors of hIAPP self-assembly, their modes of action are distinct and can be distinguished using ESI-IMS-MS.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/antagonistas & inibidores , Polipeptídeo Amiloide das Ilhotas Pancreáticas/análise , Silimarina/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Ligantes , Ratos , Silibina , Silimarina/química , Bibliotecas de Moléculas Pequenas/química , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-AtividadeRESUMO
We modified amylin chemically by conjugating methoxyl polyethyleneglycol succinimidyl carbonate (mPEGSC) of varying molecular weights (1 kDa, 2 kDa and 5 kDa). The reaction occurred within a few minutes, resulting in at least four distinct PEGylated products. The reaction products were separated by reversed-phase chromatography and identified by mass-spectrometry. The monoPEGylated and diPEGylated amylin products were generated rapidly through conjugation to the two amino groups of the N-terminal lysine residue. Both PEGylated amylin products bound to the receptor activity-modifying protein 1 (RAMP1). Pharmacological evaluation by subcutaneous administration in mice of monoPEGylated and diPEGylated amylin obtained with mPEG-SC 5 kDa revealed that both compounds modulated glycemia for longer times than unmodified amylin. Collectively, these data demonstrate the potential of bioconjugation with mPEG for the design of amylin therapeutics with sustained action.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polietilenoglicóis/síntese química , Animais , Células COS , Chlorocebus aethiops , Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Proteínas de Membrana/química , Camundongos , Polietilenoglicóis/química , Ligação Proteica , Proteína 1 Modificadora da Atividade de Receptores/química , Proteína 1 Modificadora da Atividade de Receptores/metabolismoRESUMO
Bisphenol A (BPA) is a chemical compound widely used in manufacturing plastic products. Recent epidemiological studies suggest BPA exposure is positively associated with the incidence of type 2 diabetes mellitus (T2DM), however the mechanisms underlying this link remain unclear. Human islet amyloid polypeptide (hIAPP) is a hormone synthesized and secreted by the pancreatic ß-cells. Misfolding of hIAPP into toxic oligomers and mature fibrils can disrupt cell membrane and lead to ß-cell death, which is regarded as one of the causative factors of T2DM. To test whether there are any connections between BPA exposure and hIAPP misfolding, we investigated the effects of BPA on hIAPP aggregation using thioflavin-T based fluorescence, transmission electronic microscopy, circular dichroism, dynamic light scattering, size-exclusion chromatography, fluorescence-dye leakage assay in an artificial micelle system and the generation of reactive oxygen species in INS-1 cells. We demonstrated that BPA not only dose-dependently promotes the aggregation of hIAPP and enhances the membrane disruption effects of hIAPP, but also promotes the extent of hIAPP aggregation related oxidative stress. Taken together, our results suggest that BPA exposure increased T2DM risk may involve the exacerbated toxic aggregation of hIAPP.
Assuntos
Poluentes Ocupacionais do Ar/toxicidade , Compostos Benzidrílicos/toxicidade , Células Secretoras de Insulina/efeitos dos fármacos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/análise , Fenóis/toxicidade , Sequência de Aminoácidos , Animais , Benzotiazóis , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Dicroísmo Circular , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Relação Dose-Resposta a Droga , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/ultraestrutura , Microscopia Eletrônica de Transmissão , Anotação de Sequência Molecular , Estresse Oxidativo , Dobramento de Proteína/efeitos dos fármacos , Estrutura Secundária de Proteína , Ratos , TiazóisRESUMO
Age-related diseases, like Alzheimer's disease and type 2 diabetes mellitus, are characterized by protein misfolding and the subsequent pathological deposition of fibrillized protein, also called amyloid. Several classes of amyloid-inhibitors have recently been tested, traditionally under bulk conditions. However, it has become apparent that amyloid fibrils and oligomers assemble and exert their cytotoxic effect at cellular membranes, rather than in bulk solution. Knowledge is therefore required of inhibitor activity specifically at the phospholipid membrane interface. Here we show, using surface-specific sum-frequency generation (SFG) spectroscopy and atomic force microscopy (AFM), that the commonly used (-)-epigallocatechin gallate (EGCG) is a much less efficient amyloid inhibitor at a phospholipid interface than in bulk solution. Moreover, EGCG is not able to disaggregate existing amyloid fibrils at a phospholipid interface, in contrast to its behavior in bulk. Our results show that interfaces significantly affect the efficiency of inhibition by EGCG inhibitors and should therefore be considered during the design and testing of amyloid inhibitors.
Assuntos
Catequina/análogos & derivados , Polipeptídeo Amiloide das Ilhotas Pancreáticas/antagonistas & inibidores , Fosfolipídeos/química , Polifenóis/farmacologia , Catequina/química , Catequina/farmacologia , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Microscopia de Força Atômica , Modelos Moleculares , Estrutura Molecular , Tamanho da Partícula , Polifenóis/química , Soluções , Análise Espectral , Propriedades de SuperfícieRESUMO
The pathologic self-assembly of proteins is associated with typically late-onset disorders such as Alzheimer's disease, Parkinson's disease, and type 2 diabetes. Important mechanistic details of the self-assembly are unknown, but there is increasing evidence supporting the role of transient α-helices in the early events. Islet amyloid polypeptide (IAPP) is a 37-residue polypeptide that self-assembles into aggregates that are toxic to the insulin-producing ß cells. To elucidate early events in the self-assembly of IAPP, we used limited proteolysis to identify an exposed and flexible region in IAPP monomer. This region includes position 20 where a serine-to-glycine substitution (S20G) is associated with enhanced formation of amyloid fibrils and early onset type 2 diabetes. To perform detailed biophysical studies of the exposed and flexible region, we synthesized three peptides including IAPP(11-25)WT (wild type), IAPP(11-25)S20G, and IAPP(11-25)S20P. Solution-state NMR shows that all three peptides transiently populate the α-helical conformational space, but the S20P peptide, which does not self-assemble, transiently samples a broken helix. Under similar sample conditions, the WT and S20G peptides populate the α-helical intermediate state and ß-sheet end state, respectively, of fibril formation. Our results suggest a mechanism for self-assembly that includes the stabilization of transient α-helices through the formation of NMR-invisible helical intermediates followed by an α-helix to ß-sheet conformational rearrangement. Furthermore, our results suggest that reducing intermolecular helix-helix contacts as in the S20P peptide is an attractive strategy for the design of blockers of peptide self-assembly.