Islet Amyloid Research Articles

Understanding the structural dynamics of human islet amyloid polypeptide: Advancements in and applications of ion-mobility mass spectrometry

Human islet amyloid polypeptide (hIAPP) forms amyloid deposits that contribute to β-cell death in pancreatic islets and are considered a hallmark of Type II diabetes Mellitus (T2DM). Evidence suggests that the early oligomers of hIAPP formed during the aggregation process are the primary pathological agent in islet amyloid induced β-cell death. The self-assembly mechanism of hIAPP, however, remains elusive, largely due to limitations in conventional biophysical techniques for probing the distribution or capturing detailed structures of the early, structurally dynamic oligomers. The advent of Ion-mobility Mass Spectrometry (IM-MS) has enabled the characterisation of hIAPP early oligomers in the gas phase, paving the way towards a deeper understanding of the oligomerisation mechanism and the correlation of structural information with the cytotoxicity of the oligomers. The sensitivity and the rapid structural characterisation provided by IM-MS also show promise in screening hIAPP inhibitors, categorising their modes of inhibition through “spectral fingerprints”. This review delves into the application of IM-MS to the dissection of the complex steps of hIAPP oligomerisation, examining the inhibitory influence of metal ions, and exploring the characterisation of hetero-oligomerisation with different hIAPP variants. We highlight the potential of IM-MS as a tool for the high-throughput screening of hIAPP inhibitors, and for providing insights into their modes of action. Finally, we discuss advances afforded by recent advancements in tandem IM-MS and the combination of gas phase spectroscopy with IM-MS, which promise to deliver a more sensitive and higher-resolution structural portrait of hIAPP oligomers. Such information may help facilitate a new era of targeted therapeutic strategies for islet amyloidosis in T2DM.

TSPO in pancreatic beta cells and its possible involvement in type 2 diabetes

Amyloidosis forms a large family of pathologies associated with amyloid deposit generated by the formation of amyloid fibrils or plaques. The amyloidogenic proteins and peptides involved in these processes are targeted against almost all organs. In brain they are associated with neurodegenerative disease, and the Translocator Protein (TSPO), overexpressed in these inflammatory conditions, is one of the target for the diagnostic. Moreover, TSPO ligands have been described as promising therapeutic drugs for neurodegenerative diseases. Type 2 diabetes, another amyloidosis, is due to a beta cell mass decrease that has been linked to hIAPP (human islet amyloid polypeptide) fibril formation, leading to the reduction of insulin production. In the present study, in a first approach, we link overexpression of TSPO and inflammation in potentially prediabetic patients. In a second approach, we observed that TSPO deficient rats have higher level of insulin secretion in basal conditions and more IAPP fibrils formation compared with wild type animals. In a third approach, we show that diabetogenic conditions also increase TSPO overexpression and IAPP fibril formation in rat beta pancreatic cell line (INS-1E). These data open the way for further studies in the field of type 2 diabetes treatment or prevention.

Pathological evaluation of the pathogenesis of diabetes mellitus and diabetic peripheral neuropathy.

Currently, there are more than 10 million patients with diabetes mellitus in Japan. Therefore, the need to explore the pathogenesis of diabetes and the complications leading to its cure is becoming increasingly urgent. Pathological examination of pancreatic tissues from patients with type 2 diabetes reveals a decrease in the volume of beta cells because of a combination of various stresses. In human type 2 diabetes, islet amyloid deposition is a unique pathological change characterized by proinflammatory macrophage (M1) infiltration into the islets. The pathological changes in the pancreas with islet amyloid were different according to clinical factors, which suggests that type 2 diabetes can be further subclassified based on islet pathology. On the other hand, diabetic peripheral neuropathy is the most frequent diabetic complication. In early diabetic peripheral neuropathy, M1 infiltration in the sciatic nerve evokes oxidative stress or attenuates retrograde axonal transport, as clearly demonstrated by in vitro live imaging. Furthermore, islet parasympathetic nerve density and beta cell volume were inversely correlated in type 2 diabetic Goto-Kakizaki rats, suggesting that diabetic peripheral neuropathy itself may contribute to the decrease in beta cell volume. These findings suggest that the pathogenesis of diabetes mellitus and diabetic peripheral neuropathy may be interrelated.

Screening Carbon Nano Materials for Preventing Amyloid Protein Aggregation by Adopting a Facile Method.

The soluble-to-toxic transformation of intrinsically disordered amyloidogenic proteins such as amyloid beta (Aβ), α-synuclein, mutant Huntingtin Protein (mHTT) and islet amyloid polypeptide (IAPP) among others are associated with disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and Type 2 Diabetes (T2D), respectively. The dissolution of mature fibrils and toxic amyloidogenic intermediates, including oligomers, continues to be the pinnacle in the treatment of neurodegenerative disorders. Yet, methods to effectively and quantitatively report on the interconversion between amyloid monomers, oligomers and mature fibrils fall short. Here we describe a simplified method that implements the use of gel electrophoresis to address the transformation between soluble monomeric amyloid proteins and mature amyloid fibrils. The technique implements an optimized but well-known, simple, inexpensive, and quantitative assessment previously used to assess the oligomerization of amyloid monomers and subsequent amyloid fibrils. This method facilitates the screening of small molecules that disintegrate oligomers and fibrils into monomers, dimers, and trimers and/or retain amyloid proteins in their monomeric forms. Most importantly, our optimized method diminishes existing barriers associated with existing (alternative) techniques to evaluate fibril formation and intervention.

ROF-2 as an Aggregation-Induced Emission (AIE) Probe for Multi-Target Amyloid Detection and Screening of Amyloid Inhibitors.

Misfolding and aggregation of amyloid peptides into β-structure-rich fibrils represent pivotal pathological features in various neurodegenerative diseases, including Alzheimer's disease (AD), type II diabetes (T2D), and medullary thyroid carcinoma (MTC). The development of effective amyloid detectors and inhibitors for probing and preventing amyloid aggregation is crucial for diagnosing and treating debilitating diseases, yet it poses significant challenges. Here, an aggregation-induced emission (AIE) molecule of ROF2 with multifaceted functionalities as an amyloid probe and a screening tool for amyloid inhibitors using different biophysical, cellular, and worm assays, are reported. As an amyloid probe, ROF2 outperformed ThT, demonstrating its superior sensing capability in monitoring, detecting, and distinguishing amyloid aggregates of different sequences (Amyloid-β, human islet amyloid polypeptide, or human calcitonin) and sizes (monomers, oligomers, or fibrils). More importantly, the utilization of ROF2 as a screening molecule to identify and repurpose cardiovascular drugs as amyloid inhibitors is introduced. These drugs exhibit potent amyloid inhibition properties, effectively preventing amyloid aggregation and reducing amyloid-induced cytotoxicity both in cells and nematode. The findings present a novel strategy to discovery AIE-based amyloid probes and to be used to repurpose amyloid inhibitors, expanding diagnostic and therapeutic options for neurodegenerative diseases while addressing vascular congestion and amyloid aggregation risks.

Structural Insights into Seeding Mechanisms of hIAPP Fibril Formation.

The deposition of islet amyloid polypeptide (hIAPP) fibrils is a hallmark of β-cell death in type II diabetes. In this study, we employ state-of-the-art MAS solid-state spectroscopy to investigate the previously elusive N-terminal region of hIAPP fibrils, uncovering both rigidity and heterogeneity. Comparative analysis between wild-type hIAPP and a disulfide-deficient variant (hIAPPC2S,C7S) unveils shared fibril core structures yet strikingly distinct dynamics in the N-terminus. Specifically, the variant fibrils exhibit extended β-strand conformations, facilitating surface nucleation. Moreover, our findings illuminate the pivotal roles of specific residues in modulating secondary nucleation rates. These results deepen our understanding of hIAPP fibril assembly and provide critical insights into the molecular mechanisms underpinning type II diabetes, holding promise for future therapeutic strategies.

Phytochemical and pharmacological properties of the genus Tamarix: a comprehensive review.

The genus Tamarix in the Tamaricaceae family consists of more than 100 species of halophyte plants worldwide that are mainly used to improve saline-alkali land and for coastal windbreaks, sand fixation, and afforestation in arid areas. A considerable number of species in this genus are also used as traditional medicines to treat various human diseases, especially in Asian and African countries. This review presents a comprehensive summary of 655 naturally occurring compounds derived from the genus Tamarix, categorized into flavonoids (18.0%), phenols (13.9%), tannins (9.3%), terpenoids (10.5%), essential oils (31.0%), and others (17.3%). The investigation revealed that the crude extracts and phytochemicals of this genus exhibited significant therapeutic potential, including anti-inflammatory, anti-Alzheimer, anticancer, antidiabetic, antibacterial, and antifungal activities. Six species of Tamarix have anticancer effects by causing cancer cell death, inducing autophagy, and stopping cell division. Seven species from the same genus have the potential for treating diabetes by inhibiting α-glycosidase activity, suppressing human islet amyloid polypeptide, regulating blood glucose levels, and modulating autophagy or inflammation. The focus on antibacterial and antidiabetic effects is due to the presence of volatile oil and flavonoid components. Extensive research has been conducted on the biological activity of 30 constituents, including 15 flavonoids, 5 phenols, 3 terpenoids, 1 tannin, and 6 others. Therefore, future research should thoroughly study the mechanisms of action of these and similar compounds. This is the most comprehensive review of the phytochemistry and pharmacological properties of Tamarix species, with a critical assessment of the current state of knowledge.

Catalysis driven by an amyloid–substrate complex

Amine modification through nucleophilic attack of the amine functionality is a very common chemical transformation. Under biorelevant conditions using acidic-to-neutral pH buffer, however, the nucleophilic reaction of alkyl amines (pKa ≈ 10) is not facile due to the generation of ammonium ions lacking nucleophilicity. Here, we disclose a unique molecular transformation system, catalysis driven by amyloid-substrate complex (CASL), that promotes amine modifications in acidic buffer. Ammonium ions attached to molecules with amyloid-binding capability were activated through deprotonation due to the close proximity to the amyloid catalyst formed by Ac-Asn-Phe-Gly-Ala-Ile-Leu-NH2 (NL6), derived from islet amyloid polypeptide (IAPP). Under the CASL conditions, alkyl amines underwent various modifications, i.e., acylation, arylation, cyclization, and alkylation, in acidic buffer. Crystallographic analysis and chemical modification studies of the amyloid catalysts suggested that the carbonyl oxygen of the Phe-Gly amide bond of NL6 plays a key role in activating the substrate amine by forming a hydrogen bond. Using CASL, selective conversion of substrates possessing equivalently reactive amine functionalities was achieved in catalytic reactions using amyloids. CASL provides a unique method for applying nucleophilic conversion reactions of amines in diverse fields of chemistry and biology.

Structural evolution of the conformational ensembles in peptide fibrillar aggregates

Exploring the folding structures and intermolecular recognitions of proteins in their assemblies is essential for revealing the mechanisms of amyloid fibrillar aggregation. However, the molecular-level description of protein structural changes with aggregation progression is challenging due to the heterogeneity of assembled structures. Here we report the use of scanning tunneling microscopy (STM), which is competent in analyzing the highly heterogeneous structures with submolecular resolution, to probe the structures of human islet amyloid polypeptide (hIAPP) assemblies at the different time points of hIAPP fibrillation. The conformational substate ensembles of the β-strands formed by hIAPP as well as the interpeptide interactions are found to change with time in the growth and plateau phases. Detailed analyses of the distribution probability of interpeptide interactions at the different time points manifest that the enthalpic contribution to the structural conversions of β-sheet fibrillation becomes more significant. Our single-molecular level studies highlight the dynamic nature of protein structures within a β-sheet-assembled fibril.

Efficient Vascular and Neural Engraftment of Stem Cell-Derived Islets.

Pluripotent stem cell-derived islets (SC-islets) now emerge as a new source for beta-cell replacement therapy. While the function of human islet transplants is hampered by excessive cell death post-transplantation, contributing factors include inflammatory reactions, insufficient revascularization and islet amyloid formation, there is a gap in knowledge on the engraftment process of the SC-islets. In this experimental study, we investigated the engraftment capability of SC-islets at three months post-transplantation, and observed that the cell apoptosis rates were lower, but the vascular density was similar in SC-islets to that of human islets. While the human islet transplant vascular structures were a mixture of remnant donor endothelium and ingrowing blood vessels, the SC-islets contained ingrowing blood vessels only. The oxygenation of the SC-islet grafts was twice as high as in the corresponding grafts of human islets, suggesting better vascular functionality. Similar to the blood vessel ingrowth, also the reinnervation of the SC-islets was four- to five-fold higher than the human islets. Both SC-islets and the human islets contained amyloid at one and three months post-transplantation. We conclude that the vascular and neural engraftment of SC-islets is superior to human islets, but that grafts of both origins develop amyloid with potential long-term consequences.

Composition and Conformation of Hetero- versus Homo-Fluorinated Triazolamers Influence their Activity on Islet Amyloid Polypeptide Aggregation.

Novel fluorinated foldamers based on aminomethyl-1,4-triazolyl-difluoroacetic acid (1,4-Tz-CF2) units were synthesized and their conformational behaviour was studied by NMR and molecular dynamics. Their activity on the aggregation of the human islet amyloid polypeptide (hIAPP) amyloid protein was evaluated by fluorescence spectroscopy and mass spectrometry. The fluorine labelling of these foldamers allowed the analysis of their interaction with the target protein. We demonstrated that the preferred extended conformation of homotriazolamers of 1,4-Tz-CF2 unit increases the aggregation of hIAPP, while the hairpin-like conformation of more flexible heterotriazolamers containing two 1,4-Tz-CF2 units mixed with natural amino acids from the hIAPP sequence reduces it, and more efficiently than the parent natural peptide. The longer heterotriazolamers having three 1,4-Tz-CF2 units adopting more folded hairpin-like and ladder-like structures similar to short multi-stranded β-sheets have no effect. This work demonstrates that a good balance between the structuring and flexibility of these foldamers is necessary to allow efficient interaction with the target protein.

The Co-oligomers of Aβ42 and Human Islet Amyloid Polypeptide Exacerbate Neurotoxicity and Alzheimer-like Pathology at Cellular Level

The Aβ hypothesis has long been central to Alzheimer's disease (AD) theory, with a recent surge in attention following drug approvals targeting Aβ plaque clearance. Aβ42 oligomers (AβO) are key neurotoxins. While β-amyloid (Aβ) buildup is a hallmark of AD, postmortem brain analyses have unveiled human islet amyloid polypeptide (hIAPP) deposition in AD patients, suggesting a potential role in Alzheimer's pathology. This study investigates the neurotoxic effects of co-aggregates of Aβ42 and hIAPP, specifically focusing on their impact on cell survival, apoptosis, and AD-like pathology. We analyzed and compared the impact of AβO and Aβ42-hIAPP on cell survival in SH-SY5Y cells, apoptosis and inducing AD-like pathology in glutamatergic neurons. Aβ42-hIAPP co-oligomers exhibited significantly greater toxicity, causing 2.3–3.5 times higher cell death compared to AβO alone. Furthermore, apoptosis rates were significantly exacerbated in glutamatergic neurons when exposed to Aβ42-hIAPP co-oligomers. The study also revealed that Aβ42-hIAPP co-oligomers induced typical AD-like pathology in glutamatergic neurons, including the presence of Aβ deposits (detected by 6E10 and 4G8 immunofluorescence) and alterations in tau protein (changes in total tau HT7, phosphorylated tau AT8, AT180). Notably, Aβ42-hIAPP co-oligomers induced a more severe AD pathology compared to AβO alone. These findings provide compelling evidence for the heightened toxicity of Aβ42-hIAPP co-oligomers on neurons and their role in exacerbating AD pathology. The study contributes novel insights into the pathogenesis of Alzheimer's disease, highlighting the potential involvement of hIAPP in AD pathology. Together, these findings offer novel insights into AD pathogenesis and routes for constructing animal models.

Experimental and computational investigation of the effect of Hsc70 structural variants on inhibiting amylin aggregation

The misfolding and aggregation of human islet amyloid polypeptide (hIAPP), also known as amylin, have been implicated in the pathogenesis of type 2 diabetes (T2D). Heat shock proteins, specifically, heat shock cognate 70 (Hsc70), are molecular chaperones that protect against hIAPP misfolding and inhibits its aggregation. Nevertheless, there is an incomplete understanding of the mechanistic interactions between Hsc70 domains and hIAPP, thus limiting their potential therapeutic role in diabetes. This study investigates the inhibitory capacities of different Hsc70 variants, aiming to identify the structural determinants that strike a balance between efficacy and cytotoxicity. Our experimental findings demonstrate that the ATPase activity of Hsc70 is not a pivotal factor for inhibiting hIAPP misfolding. We underscore the significance of the C-terminal substrate-binding domain of Hsc70 in inhibiting hIAPP aggregation, emphasizing that the removal of the lid subdomain diminishes the inhibitory effect of Hsc70. Additionally, we employed atomistic discrete molecular dynamics simulations to gain deeper insights into the interaction between Hsc70 variants and hIAPP. Integrating both experimental and computational findings, we propose a mechanism by which Hsc70's interaction with hIAPP monomers disrupts protein-protein connections, primarily by shielding the β-sheet edges of the Hsc70-β-sandwich. The distinctive conformational dynamics of the alpha helices of Hsc70 potentially enhance hIAPP binding by obstructing the exposed edges of the β-sandwich, particularly at the β5-β8 region along the alpha helix interface. This, in turn, inhibits fibril growth, and similar results were observed following hIAPP dimerization. Overall, this study elucidates the structural intricacies of Hsc70 crucial for impeding hIAPP aggregation, improving our understanding of the potential anti-aggregative properties of molecular chaperones in diabetes treatment.

Delineating the impact of N21D mutation on the conformational preferences and structural transitions in human islet amyloid polypeptide

The amyloid deposits of human islet amyloid polypeptide (hIAPP) in the pancreatic β-cells are linked to a chronic metabolic disease known as type 2 diabetes (T2D). Recent studies reported that N21D mutation in hIAPP inhibited the formation of fibrillar aggregates, however, the underlying molecular mechanism of fibril inhibition in the N21D mutant remains largely unclear. Thus, molecular dynamics (MD) simulations have been employed to illuminate the effect of N21D mutation on the conformational dynamics and structural transformations in hIAPP at the atomistic level. Notably, the structural stability of WT-hIAPP (wild type-hIAPP) was enhanced upon N21D mutation. Interestingly, the sampling of helical conformation in hIAPP was significantly increased from 12.60 to 19.80% with a concomitant decrease in β-sheet from 7.40 to 1.20% upon N21D mutation that corroborates with the circular dichroism (CD). The dihedral principal component analysis (dPCA) depicted a notable reduction in the conformational phase space of [N21D]hIAPP as compared to WT-hIAPP, which highlights the prevention of conformational transitions and enhanced structural stability. Remarkably, a complete loss in the β-sheet content was noted in the conformations extracted from the free energy landscape of [N21D]hIAPP as compared to 24–25% sampling of β-sheets in minimum energy conformations of WT-hIAPP consistent with the transmission electron microscopy (TEM) and thioflavin T (ThT) fluorescence assay. The present study provided key insights into the N21D mutation-induced conformational changes in hIAPP and highlighted that Asn21 plays a key role in hIAPP aggregation. The insights from this work will be beneficial in the design of drug candidates by selectively targeting Asn21 of hIAPP which could interfere with its aggregation and associated cytotoxicity.

Natural compound plumbagin based inhibition of hIAPP revealed by Markov state models based on MD data along with experimental validations.

Human islet amyloid polypeptide (amylin or hIAPP) is a 37 residue hormone co-secreted with insulin from β cells of the pancreas. In patients suffering from type-2 diabetes, amylin self-assembles into amyloid fibrils, ultimately leading to the death of the pancreatic cells. However, a research gap exists in preventing and treating such amyloidosis. Plumbagin, a natural compound, has previously been demonstrated to have inhibitory potential against insulin amyloidosis. Our investigation unveils collapsible regions within hIAPP that, upon collapse, facilitates hydrophobic and pi-pi interactions, ultimately leading to aggregation. Intriguingly plumbagin exhibits the ability to bind these specific collapsible regions, thereby impeding the aforementioned interactions that would otherwise drive hIAPP aggregation. We have used atomistic molecular dynamics approach to determine secondary structural changes. MSM shows metastable states forming native like hIAPP structure in presence of PGN. Our in silico results concur with in vitro results. The ThT assay revealed a striking 50% decrease in fluorescence intensity at a 1:1 ratio of hIAPP to Plumbagin. This finding suggests a significant inhibition of amyloid fibril formation by plumbagin, as ThT fluorescence directly correlates with the presence of these fibrils. Further TEM images revealed disappearance of hIAPP fibrils in plumbagin pre-treated hIAPP samples. Also, we have shown that plumbagin disrupts the intermolecular hydrogen bonding in hIAPP fibrils leading to an increase in the average beta strand spacing, thereby causing disaggregation of pre-formed fibrils demonstrating overall disruption of the aggregation machinery of hIAPP. Our work is the first to report a detailed atomistic simulation of 22 μs for hIAPP. Overall, our studies put plumbagin as a potential candidate for both preventive and therapeutic candidate for hIAPP amyloidosis.

Identification of Electrostatic Hotspots at the Binding Interface of Amylin and InsulinDegrading Enzyme: A Structural and Biophysical Investigation

Amylin, also known as islet amyloid polypeptide (IAPP), is a metabolic homeostasis-related hormone that is produced and released by the β cells of the pancreas, the same cells that produce insulin. Insulin-degrading enzyme (IDE) is a protease enzyme that plays an essential role in the breakdown and degradation of various peptides, including insulin and amylin. Direct binding & interaction between amylin and IDE is inextricably linked to the degradation of amylin, and research and development effort in this area is crucial to understand the potential therapeutic implications of disrupting the IDE-amylin interaction in the context of conditions where metabolic homeostasis needs to be regulated, such as diabetes and obesity. Here, this article incorporates currently available experimental complex structure of amylin and IDE, and delves deep into the interstructural biophysics underlying the binding interface of the two interacting partners. With a set of comprehensive structural biophysical analysis, this article identified an intriguing region of high electrostatic potential indicative of strong binding sites between the first N-terminal lysine (Lys1, K1) residue of amylin and Glu341 (E341) of IDE. This unique electrostatic hotspot presented herein paves the way for the rational design of drug-like small molecules that can selectively disrupt this interaction, offering a targeted therapeutic strategy for improved metabolic homeostasis, particularly for patients with diabetes and obesity.

Probing the Molecular Structure and Dynamics of Membrane-Bound Proteins during Misfolding Processes by Sum-Frequency Generation Vibrational Spectroscopy.

Protein misfolding and amyloid formation are implicated in the protein dysfunction, but the underlying mechanism remains to be clarified due to the lack of effective tools for detecting the transient intermediates. Sum frequency generation vibrational spectroscopy (SFG-VS) has emerged as a powerful tool for identifying the structure and dynamics of proteins at the interfaces. In this review, we summarize recent SFG-VS studies on the structure and dynamics of membrane-bound proteins during misfolding processes. This paper first introduces the methods for determining the secondary structure of interfacial proteins: combining chiral and achiral spectra of amide A and amide I bands and combining amide I, amide II, and amide III spectral features. To demonstrate the ability of SFG-VS in investigating the interfacial protein misfolding and amyloid formation, studies on the interactions between different peptides/proteins (islet amyloid polypeptide, amyloid β, prion protein, fused in sarcoma protein, hen egg-white lysozyme, fusing fusion peptide, class I hydrophobin SC3 and class II hydrophobin HFBI) and surfaces such as lipid membranes are discussed. These molecular-level studies revealed that SFG-VS can provide a unique understanding of the mechanism of interfacial protein misfolding and amyloid formation in real time, in situ and without any exogenous labeling.

Flavones in pomelo peel resist fibril formation of human islet amyloid polypeptide

ObjectiveExploring the formation and aggregation of human islet amyloid polypeptide (hIAPP) (amylin) fibers is significant for promoting the prevention and treatment of type II diabetes mellitus (T2DM). Flavones in pomelo peel have visible biological activity in the anti-diabetes aspect. The present study aimed to investigate the effects of five flavones [naringin (NRG), narirutin (NRR), nobiletin (NOB), sinensetin (SIN), and neohesperidin (NHP)] in pomelo peel on peptide aggregation and explore its possible mechanisms. The cell viability of flavones against peptide aggregation was also evaluated. MethodsThe thioflavin T (ThT) assay and transmission electron microscopy (TEM) were used for evaluating the inhibition and disaggregation of flavones on peptide aggregation. The interaction mechanism was analyzed by endogenous fluorescence, molecular dynamics (MD) simulations, ultraviolet spectroscopy (UV) and isothermal titration calorimetry (ITC) experiments. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and immune assays were performed to characterize the cell viability of flavones against peptide aggregation. ResultsThe five flavones showed a decrease in fluorescence intensity, fiber number and size under incubation with different molar ratios of hIAPP. The compounds can bind to the aromatic tyrosine (Tyr) residueTyr 37, resulting in the intrinsic fluorescence quenching of the peptides. Five flavones can form hydrogen bonds with hIAPP, which is likely to be based on their phenolic hydroxyl structure. They showed strong binding affinity with peptides. The reaction system of NRG and NRR observed an exothermic reaction, and the others were endothermic reactions. The absorption peaks of the compounds with hIAPP changed and showed hypochromic effects, indicating that there may be π-π stacking interaction. Flavones noticeably increased the cell viability in the presence of amyloid peptides and reduced the absorption intensity induced by peptide oligomers. ConclusionFive flavones in pomelo peel have inhibitory and depolymerization effects on amyloid fibrils, and can significantly protect cells from the toxic effect of hIAPP and reduce the production of toxic oligomers.

Improving cryo-EM grids for amyloid fibrils using interface-active solutions and spectator proteins

Preparation of cryoelectron microscopy (cryo-EM) grids for imaging of amyloid fibrils is notoriously challenging. The human islet amyloid polypeptide (hIAPP) serves as a notable example, as the majority of reported structures have relied on the use of nonphysiological pH buffers, N-terminal tags, and seeding. This highlights the need for more efficient, reproducible methodologies that can elucidate amyloid fibril structures formed under diverse conditions. In this work, we demonstrate that the distribution of fibrils on cryo-EM grids is predominantly determined by the solution composition, which is critical for the stability of thin vitreous ice films. We discover that, among physiological pH buffers, HEPES uniquely enhances the distribution of fibrils on cryo-EM grids and improves the stability of ice layers. This improvement is attributed to direct interactions between HEPES molecules and hIAPP, effectively minimizing the tendency of hIAPP to form dense clusters in solutions and preventing ice nucleation. Furthermore, we provide additional support for the idea that denatured protein monolayers forming at the interface are also capable of eliciting a surfactant-like effect, leading to improved particle coverage. This phenomenon is illustrated by the addition of nonamyloidogenic rat IAPP (rIAPP) to a solution of preaggregated hIAPP just before the freezing process. The resultant grids, supplemented with this “spectator protein”, exhibit notably enhanced coverage and improved ice quality. Unlike conventional surfactants, rIAPP is additionally capable of disentangling the dense clusters formed by hIAPP. By applying the proposed strategies, we have resolved the structure of the dominant hIAPP polymorph, formed in vitro at pH 7.4, to a final resolution of 4 Å. The advances in grid preparation presented in this work hold significant promise for enabling structural determination of amyloid proteins which are particularly resistant to conventional grid preparation techniques.

Prion protein-derived cell-penetrating peptide inhibits type II diabetes associated islet amyloid polypeptide aggregation and cytotoxicity

Prion protein-derived cell-penetrating peptide inhibits type II diabetes associated islet amyloid polypeptide aggregation and cytotoxicity