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 ß-sheet edges of the Hsc70-ß-sandwich. The distinctive conformational dynamics of the alpha helices of Hsc70 potentially enhance hIAPP binding by obstructing the exposed edges of the ß-sandwich, particularly at the ß5-ß8 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.

Calcitonin receptor, calcitonin gene-related peptide and amylin distribution in C1/2 dorsal root ganglia.

BACKGROUND: The upper cervical dorsal root ganglia (DRG) are important for the transmission of sensory information associated with the back of the head and neck, contributing to head pain. Calcitonin receptor (CTR)-based receptors, such as the amylin 1 (AMY1) receptor, and ligands, calcitonin gene-related peptide (CGRP) and amylin, have been linked to migraine and pain. However, the contribution of this system to nociception involving the cervical DRG is unclear. Therefore, this study aimed to determine the relative distribution of the CTR, CGRP, and amylin in upper cervical DRG. METHODS: CTR, CGRP, and amylin immunofluorescence was examined relative to neural markers in C1/2 DRG from male and female mice, rats, and human cases. Immunofluorescence was supported by RNA-fluorescence in situ hybridization examining amylin mRNA distribution in rat DRG. RESULTS: Amylin immunofluorescence was observed in neuronal soma and fibres. Amylin mRNA (Iapp) was also detected. Amylin and CGRP co-expression was observed in 19% (mouse), 17% (rat), and 36% (human) of DRG neurons in distinct vesicle-like neuronal puncta from one another. CTR immunoreactivity was present in DRG neurons, and both peptides produced receptor signalling in primary DRG cell cultures. CTR-positive neurons frequently co-expressed amylin and/or CGRP (66% rat; 84% human), with some sex differences. CONCLUSIONS: Amylin and CGRP could both be local peptide agonists for CTR-based receptors in upper cervical DRG, potentially acting through autocrine and/or paracrine signalling mechanisms to modulate neuron function. Amylin and its receptors could represent novel pain targets.

Development of a Novel Assay for Direct Assessment of Selective Amylin Receptor Activation Reveals Novel Differences in Behavior of Selective and Nonselective Peptide Agonists.

Dual amylin and calcitonin receptor agonists (DACRAs) show promise as efficacious therapeutics for treatment of metabolic disease, including obesity. However, differences in efficacy in vivo have been observed for individual DACRAs, indicating that detailed understanding of the pharmacology of these agents across target receptors is required for rational drug development. To date, such understanding has been hampered by lack of direct, subtype-selective, functional assays for the amylin receptors (AMYRs). Here, we describe the generation of receptor-specific assays for recruitment of Venus-tagged Gs protein through fusion of luciferase to either the human calcitonin receptor (CTR), human receptor activity-modifying protein (RAMP)-1, RAMP1 (AMY1R), human RAMP2 (AMY2R), or human RAMP3 (AMY3R). These assays revealed a complex pattern of receptor activation by calcitonin, amylin, or DACRA peptides that was distinct at each receptor subtype. Of particular note, although both of the CT-based DACRAs, sCT and AM1784, displayed relatively similar behaviors at CTR and AMY1R, they generated distinct responses at AMY2R and AMY3R. These data aid the rationalization of in vivo differences in response to DACRA peptides in rodent models of obesity. Direct assessment of the pharmacology of novel DACRAs at AMYR subtypes is likely to be important for development of optimized therapeutics for treatment of metabolic diseases. SIGNIFICANCE STATEMENT: Amylin receptors (AMYRs) are important obesity targets. Here we describe a novel assay that allows selective functional assessment of individual amylin receptor subtypes that provides unique insight into the pharmacology of potential therapeutic ligands. Direct assessment of the pharmacology of novel agonists at AMYR subtypes is likely to be important for development of optimized therapeutics for treatment of metabolic diseases.

The processing intermediate of human amylin, pro-amylin(1-48), has in vivo and in vitro bioactivity.

Amylin is released by pancreatic beta-cells in response to a meal and its major soluble mature form (37 amino acid-peptide) produces its biological effects by activating amylin receptors. Amylin is derived from larger propeptides that are processed within the synthesizing beta-cell. There are suggestions that a partially processed form, pro-amylin(1-48) is also secreted. We tested the hypothesis that pro-amylin(1-48) has biological activity and that human pro-amylin(1-48) may also form toxic pre-amyloid species. Amyloid formation, the ability to cross-seed and in vitro toxicity were similar between human pro-amylin(1-48) and amylin. Human pro-amylin(1-48) was active at amylin-responsive receptors, though its potency was reduced at rat, but not human amylin receptors. Pro-amylin(1-48) was able to promote anorexia by activating neurons of the area postrema, amylin's primary site of action, indicating that amylin can tolerate significant additions at the N-terminus without losing bioactivity. Our studies help to shed light on the possible roles of pro-amylin(1-48) which may be relevant for the development of future amylin-based drugs.

Liraglutide versus pramlintide in protecting against cognitive function impairment through affecting PI3K/AKT/GSK-3ß/TTBK1 pathway and decreasing Tau hyperphosphorylation in high-fat diet- streptozocin rat model.

The American Diabetes Association guidelines (2021) confirmed the importance of raising public awareness of diabetes-induced cognitive impairment, highlighting the links between poor glycemic control and cognitive impairment. The characteristic brain lesions of cognitive dysfunction are neurofibrillary tangles (NFT) and senile plaques formed of amyloid-ß deposition, glycogen synthase kinase 3 beta (GSK3ß), and highly homologous kinase tau tubulin kinase 1 (TTBK1) can phosphorylate Tau proteins at different sites, overexpression of these enzymes produces extensive phosphorylation of Tau proteins making them insoluble and enhance NFT formation, which impairs cognitive functions. The current study aimed to investigate the potential contribution of liraglutide and pramlintide in the prevention of diabetes-induced cognitive dysfunction and their effect on the PI3K/AKT/GSK-3ß/TTBK1 pathway in type 2 diabetic (T2D) rat model. T2D was induced by administration of a high-fat diet for 10 weeks, then injection of a single dose of streptozotocin (STZ); treatment was started with either pramlintide (200 µg/kg/day sc) or liraglutide (0.6 mg/kg/day sc) for 6 weeks in addition to the HFD. At the end of the study, cognitive functions were assessed by novel object recognition and T-maze tests. Then, rats were sacrificed for biochemical and histological assessment of the hippocampal tissue. Both pramlintide and liraglutide treatment revealed equally adequate control of diabetes, prevented the decline in memory function, and increased PI3K/AKT expression while decreasing GSK-3ß/TTBK1 expression; however, liraglutide significantly decreased the number of Tau positive cells better than pramlintide did. This study confirmed that pramlintide and liraglutide are promising antidiabetic medications that could prevent associated cognitive disorders in different mechanisms.

Amylin in Alcohol Addiction: A Potential New Treatment Target or an Adjuvant to Other Treatments?

Amylin is a neuroendocrine hormone with a potential role in addictive disorders, including alcohol use disorder (AUD). In addition to reducing appetitive behavior, amylin has been shown to affect alcohol-related behaviors in rodents. Delineating the biobehavioral correlates of amylin in relation to alcohol seeking and consumption has the potential of identifying new treatment targets for AUD, yet additional translational and human research is needed.

On the importance of being amidated: Analysis of the role of the conserved C-terminal amide of amylin in amyloid formation and cytotoxicity.

The polypeptide hormone Amylin (also known as islet amyloid polypeptide) plays a role in regulation of glucose metabolism, but forms pancreatic islet amyloid deposits in type 2 diabetes. The process of islet amyloid formation contributes to ß-cell dysfunction and the development of the disease. Amylin is produced as a pro-from and undergoes processing prior to secretion. The mature hormone contains an amidated C-terminus. Analysis of an alignment of vertebrate amylin sequences reveals that the processing signal for amidation is strictly conserved. Furthermore, the enzyme responsible for C-terminal amidation is found in all of these organisms. Comparison of the physiologically relevant amidated form to a variant with a free C-terminus (Amylin-COO-) shows that replacement of the C-terminal amide with a carboxylate slows, but does not prevent amyloid formation. Pre-fibrillar species produced by both variants are toxic to cultured ß-cells, although hAmylin-COO- is moderately less so. Amyloid fibrils produced by either peptide are not toxic. Prior work (ACS Pharmacol. Translational. Sci. 1, 132-49 (2018)) shows that Amylin- COO- exhibits a 58-fold reduction in activation of the Amylin1 receptor and 20-fold reduction in activation of the Amylin3 receptor. Thus, hAmylin-COO- exhibits significant toxicity, but significantly reduced activity and offers a reagent for studies which aim to decouple hAmylin's toxic effects from its activity. The different behaviours of free and C-terminal amidated Amylin should be considered when designing systems to produce the polypeptide recombinantly.

Robust Chemical Synthesis of "Difficult Peptides" via 2-Hydroxyphenol-pseudoproline (ψ2-hydroxyphenolpro) Modifications.

The challenging preparation of "difficult peptides" has always hindered the development of peptide-active pharmaceutical ingredients. Pseudoproline (ψpro) building blocks have been proven effective and powerful tools for the synthesis of "difficult peptides". In this paper, we efficiently prepared a set of novel 2-(oxazolidin-2-yl)phenol compounds as proline surrogates (2-hydroxyphenol-pseudoprolines, ψ2-hydroxyphenolpro) and applied it in the synthesis of many well-known "difficult peptides", including human thymosin α1, amylin, and ß-amyloid (1-42) (Aß42).

Multiscale Modeling of Macromolecular Interactions between Tau-Amylin Oligomers and Asymmetric Lipid Nanodomains That Link Alzheimer's and Diabetic Diseases.

The molecular events of protein misfolding and self-aggregation of tau and amylin are associated with the progression of Alzheimer's and diabetes, respectively. Recent studies suggest that tau and amylin can form hetero-tau-amylin oligomers. Those hetero-oligomers are more neurotoxic than homo-tau oligomers. So far, the detailed interactions between the hetero-oligomers and the neuronal membrane are unknown. Using multiscale MD simulations, the lipid binding and protein folding behaviors of hetero-oligomers on asymmetric lipid nanodomains or raft membranes were examined. Our raft membranes contain phase-separated phosphatidylcholine (PC), cholesterol, and anionic phosphatidylserine (PS) or ganglioside (GM1) in one leaflet of the lipid bilayer. The hetero-oligomers bound more strongly to the PS and GM1 than other lipids via the hydrophobic and hydrophilic interactions, respectively, in the raft membranes. The hetero-tetramer disrupted the acyl chain orders of both PC and PS in the PS-containing raft membrane, but only the GM1 in the GM1-containing raft membrane as effectively as the homo-tau-tetramer. We discovered that the alpha-helical content in the heterodimer was greater than the sum of alpha-helical contents from isolated tau and amylin monomers on both raft membranes, indicative of a synergetic effect of tau-amylin interactions in surface-induced protein folding. Our results provide new molecular insights into understanding the cross-talk between Alzheimer's and diabetes.

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.

The Inhibition Effect of Epigallocatechin-3-Gallate on the Co-Aggregation of Amyloid-ß and Human Islet Amyloid Polypeptide Revealed by Replica Exchange Molecular Dynamics Simulations.

Alzheimer's disease and Type 2 diabetes are two epidemiologically linked diseases which are closely associated with the misfolding and aggregation of amyloid proteins amyloid-ß (Aß) and human islet amyloid polypeptide (hIAPP), respectively. The co-aggregation of the two amyloid proteins is regarded as the fundamental molecular mechanism underlying their pathological association. The green tea extract epigallocatechin-3-gallate (EGCG) has been extensively demonstrated to inhibit the amyloid aggregation of Aß and hIAPP proteins. However, its potential role in amyloid co-aggregation has not been thoroughly investigated. In this study, we employed the enhanced-sampling replica exchange molecular dynamics simulation (REMD) method to investigate the effect of EGCG on the co-aggregation of Aß and hIAPP. We found that EGCG molecules substantially diminish the ß-sheet structures within the amyloid core regions of Aß and hIAPP in their co-aggregates. Through hydrogen-bond, π-π and cation-π interactions targeting polar and aromatic residues of Aß and hIAPP, EGCG effectively attenuates both inter-chain and intra-chain interactions within the co-aggregates. All these findings indicated that EGCG can effectively inhibit the co-aggregation of Aß and hIAPP. Our study expands the potential applications of EGCG as an anti-amyloidosis agent and provides therapeutic options for the pathological association of amyloid misfolding disorders.

Amylin, Another Important Neuroendocrine Hormone for the Treatment of Diabesity.

Diabetes mellitus is a devastating chronic metabolic disease. Since the majority of type 2 diabetes mellitus patients are overweight or obese, a novel term-diabesity-has emerged. The gut-brain axis plays a critical function in maintaining glucose and energy homeostasis and involves a variety of peptides. Amylin is a neuroendocrine anorexigenic polypeptide hormone, which is co-secreted with insulin from ß-cells of the pancreas in response to food consumption. Aside from its effect on glucose homeostasis, amylin inhibits homeostatic and hedonic feeding, induces satiety, and decreases body weight. In this narrative review, we summarized the current evidence and ongoing studies on the mechanism of action, clinical pharmacology, and applications of amylin and its analogs, pramlintide and cagrilintide, in the field of diabetology, endocrinology, and metabolism disorders, such as obesity.

Structural diversity in the membrane-bound hIAPP dimer correlated with distinct membrane disruption mechanisms.

Amyloid deposits of the human islet amyloid polypeptide (hIAPP) have been identified in 90% of patients with type II diabetes. Cellular membranes accelerate the hIAPP fibrillation, and the integrity of membranes is also disrupted at the same time, leading to the apoptosis of ß cells in pancreas. The molecular mechanism of hIAPP-induced membrane disruption, especially during the initial membrane disruption stage, has not been well understood yet. Herein, we carried out extensive all-atom molecular dynamics simulations investigating the hIAPP dimerization process in the anionic POPG membrane, to provide the detailed molecular mechanisms during the initial hIAPP aggregation stage in the membrane environment. Compared to the hIAPP monomer on the membrane, we observed not only an increase of α-helical structures, but also a substantial increase of ß-sheet structures upon spontaneous dimerization. Moreover, the random coiled and α-helical dimer structures insert deep into the membrane interior with a few inter-chain contacts at the C-terminal region, while the ß-sheet-rich structures reside on the membrane surface accompanied by strong inter-chain hydrophobic interactions. The coexistence of α and ß structures constitutes a diverse structural ensemble of the membrane-bound hIAPP dimer. From α-helical to ß-sheet structures, the degree of membrane disruption decreases gradually, and thus the membrane damage induced by random coiled and α-helical structures precedes that induced by ß-sheet structures. We speculate that insertion of random coiled and α-helical structures contributes to the initial stage of membrane damage, while ß-sheet structures on the membrane surface are more involved in the later stage of fibril-induced membrane disruption.

Islet amyloid polypeptide tagged with green fluorescent protein localises to mitochondria and forms filamentous aggregates in Caenorhabditis elegans.

Type 2 diabetes (T2D) is the most common form of diabetes and represents a growing health concern. A characteristic feature of T2D is the aggregation of islet amyloid polypeptide (IAPP), which is thought to be associated with the death of pancreatic ß-cells. Inhibiting IAPP aggregation is a promising therapeutic avenue to treat T2D, but the mechanisms of aggregation and toxicity are not yet fully understood. Caenorhabditis elegans is a well-characterised multicellular model organism that has been extensively used to study protein aggregation diseases. In this study, we aimed to develop a simple in vivo model to investigate IAPP aggregation and toxicity based on expression in the C. elegans body wall muscle cells. We show that IAPP tagged with green fluorescent protein (GFP) localises to mitochondria not only in muscle cells but also when expressed in the intestine, in line with previous observations in mouse and human pancreatic ß-cells. The IAPP-GFP fusion protein forms solid aggregates, which have a filamentous appearance as seen by electron microscopy. However, the animals expressing IAPP-GFP in the body wall muscle cells do not display a strong motility phenotype, suggesting that the IAPP-GFP aggregates are not considerably toxic. Nevertheless, the mitochondrial localisation and aggregate formation may be useful read-outs to screen for IAPP-solubilizing compounds as a therapeutic strategy for T2D.

Non-fibril form but not fibril form of human islet amyloid polypeptide 8-20 changes brain functions in mice.

Whether fibril formation increases or decreases cytotoxicity remains unclear. Aggregation of human islet amyloid polypeptide (hIAPP), a pivotal regulator of glucose homeostasis, impairs the function and viability of pancreatic ß cells. Evidence suggests that low-order oligomers of hIAPP are more toxic to ß cells than fibril. However, it remains unclear whether non-fibril form of hIAPP specifically alters brain functions. This study produced fibril and non-fibril forms from a single hIAPP 8-20 peptide. The non-fibril form-injected mice showed changes in spontaneous motor activities, preference for location in the open field and social behavior. In contrast, the fibril-injected mice showed no changes in these behavioral tests. In line with the behavioral changes, the non-fibril form led to impaired neurite outgrowth of cultured neuron-like cells and the loss of neurons in the mouse hippocampus. These findings suggest that non-fibril form but not fibril form of hIAPP changes brain functions.

RIPK3 promotes islet amyloid-induced ß-cell loss and glucose intolerance in a humanized mouse model of type 2 diabetes.

OBJECTIVE: Aggregation of human islet amyloid polypeptide (hIAPP), a ß-cell secretory product, leads to islet amyloid deposition, islet inflammation and ß-cell loss in type 2 diabetes (T2D), but the mechanisms that underlie this process are incompletely understood. Receptor interacting protein kinase 3 (RIPK3) is a pro-death signaling molecule that has recently been implicated in amyloid-associated brain pathology and ß-cell cytotoxicity. Here, we evaluated the role of RIPK3 in amyloid-induced ß-cell loss using a humanized mouse model of T2D that expresses hIAPP and is prone to islet amyloid formation. METHODS: We quantified amyloid deposition, cell death and caspase 3/7 activity in islets isolated from WT, Ripk3-/-, hIAPP and hIAPP; Ripk3-/- mice in real time, and evaluated hIAPP-stimulated inflammation in WT and Ripk3-/- bone marrow derived macrophages (BMDMs) in vitro. We also characterized the role of RIPK3 in glucose stimulated insulin secretion (GSIS) in vitro and in vivo. Finally, we examined the role of RIPK3 in high fat diet (HFD)-induced islet amyloid deposition, ß-cell loss and glucose homeostasis in vivo. RESULTS: We found that amyloid-prone hIAPP mouse islets exhibited increased cell death and caspase 3/7 activity compared to amyloid-free WT islets in vitro, and this was associated with increased RIPK3 expression. hIAPP; Ripk3-/- islets were protected from amyloid-induced cell death compared to hIAPP islets in vitro, although amyloid deposition and caspase 3/7 activity were not different between genotypes. We observed that macrophages are a source of Ripk3 expression in isolated islets, and that Ripk3-/- BMDMs were protected from hIAPP-stimulated inflammatory gene expression (Tnf, Il1b, Nos2). Following 52 weeks of HFD feeding, islet amyloid-prone hIAPP mice exhibited impaired glucose tolerance and decreased ß-cell area compared to WT mice in vivo, whereas hIAPP; Ripk3-/- mice were protected from these impairments. CONCLUSIONS: In conclusion, loss of RIPK3 protects from amyloid-induced inflammation and islet cell death in vitro and amyloid-induced ß-cell loss and glucose intolerance in vivo. We propose that therapies targeting RIPK3 may reduce islet inflammation and ß-cell loss and improve glucose homeostasis in the pathogenesis of T2D.

Investigation of the Effect of Pyrogallol on the Formation of Amylin Amyloid Fibrils as a Strategy for the Treatment of Type 2 Diabetic Patients: A Theoretical and Experimental Study.

In more than 50 to 90% of type 2 diabetic patients, under the influence of various factors, the production of islet amyloid polypeptide or amylin in pancreatic beta cells increases. Spontaneous accumulation of amylin peptide in the form of insoluble amyloid fibrils and soluble oligomers is one of the main causes of beta cell death in diabetic patients. The objective of the present study was to evaluate the effect of pyrogallol, as a phenolic compound, on inhibiting the formation of amylin protein amyloid fibrils. In this study, different techniques such as the thioflavin T (ThT) and 1-Anilino-8-naphthalene sulfonate (ANS) fluorescence intensity and the circular dichroism (CD) spectrum, will be used to investigate the effects of this compound on inhibiting the formation of amyloid fibrils. To investigate the interaction sites of pyrogallol with amylin, docking studies were performed. Our results that pyrogallol in a dose-dependent manner (0.5:1, 1:1, and 5:1, Pyr to Amylin) inhibits the amylin amyloid fibrils formation. Docking analysis revealed that pyrogallol forms hydrogen bonds with valine 17 and asparagine 21. In addition, this compound forms 2 more hydrogen bonds with asparagine 22. This compound also forms hydrophobic bonds with histidine 18. Considering this data and the direct relationship between oxidative stress and the formation of amylin amyloid accumulations in diabetes, the use of compounds with both antioxidant and anti-amyloid properties can be considered an important therapeutic strategy for type 2 diabetes.

What can AlphaFold do for antimicrobial amyloids?

Amyloids, protein, and peptide assemblies in various organisms are crucial in physiological and pathological processes. Their intricate structures, however, present significant challenges, limiting our understanding of their functions, regulatory mechanisms, and potential applications in biomedicine and technology. This study evaluated the AlphaFold2 ColabFold method's structure predictions for antimicrobial amyloids, using eight antimicrobial peptides (AMPs), including those with experimentally determined structures and AMPs known for their distinct amyloidogenic morphological features. Additionally, two well-known human amyloids, amyloid-ß and islet amyloid polypeptide, were included in the analysis due to their disease relevance, short sequences, and antimicrobial properties. Amyloids typically exhibit tightly mated ß-strand sheets forming a cross-ß configuration. However, certain amphipathic α-helical subunits can also form amyloid fibrils adopting a cross-α structure. Some AMPs in the study exhibited a combination of cross-α and cross-ß amyloid fibrils, adding complexity to structure prediction. The results showed that the AlphaFold2 ColabFold models favored α-helical structures in the tested amyloids, successfully predicting the presence of α-helical mated sheets and a hydrophobic core resembling the cross-α configuration. This implies that the AI-based algorithms prefer assemblies of the monomeric state, which was frequently predicted as helical, or capture an α-helical membrane-active form of toxic peptides, which is triggered upon interaction with lipid membranes.

Human islet amyloid polypeptide-induced ß-cell cytotoxicity is linked to formation of α-sheet structure.

Type 2 diabetes (T2D) results from insulin secretory dysfunction arising in part from the loss of pancreatic islet ß-cells. Several factors contribute to ß-cell loss, including islet amyloid formation, which is observed in over 90% of individuals with T2D. The amyloid is comprised of human islet amyloid polypeptide (hIAPP). Here we provide evidence that early in aggregation, hIAPP forms toxic oligomers prior to formation of amyloid fibrils. The toxic oligomers contain α-sheet secondary structure, a nonstandard secondary structure associated with toxic oligomers in other amyloid diseases. De novo, synthetic α-sheet compounds designed to be nontoxic and complementary to the α-sheet structure in the toxic oligomers inhibit hIAPP aggregation and neutralize oligomer-mediated cytotoxicity in cell-based assays. In vivo administration of an α-sheet design to mice for 4 weeks revealed no evidence of toxicity nor did it elicit an immune response. Furthermore, the α-sheet designs reduced endogenous islet amyloid formation and mitigation of amyloid-associated ß-cell loss in cultured islets isolated from an hIAPP transgenic mouse model of islet amyloidosis. Characterization of the involvement of α-sheet in early aggregation of hIAPP and oligomer toxicity contributes to elucidation of the molecular mechanisms underlying amyloid-associated ß-cell loss.

Structural insight into selectivity of amylin and calcitonin receptor agonists.

Amylin receptors (AMYRs), heterodimers of the calcitonin receptor (CTR) and one of three receptor activity-modifying proteins, are promising obesity targets. A hallmark of AMYR activation by Amy is the formation of a 'bypass' secondary structural motif (residues S19-P25). This study explored potential tuning of peptide selectivity through modification to residues 19-22, resulting in a selective AMYR agonist, San385, as well as nonselective dual amylin and calcitonin receptor agonists (DACRAs), with San45 being an exemplar. We determined the structure and dynamics of San385-bound AMY3R, and San45 bound to AMY3R or CTR. San45, via its conjugated lipid at position 21, was anchored at the edge of the receptor bundle, enabling a stable, alternative binding mode when bound to the CTR, in addition to the bypass mode of binding to AMY3R. Targeted lipid modification may provide a single intervention strategy for design of long-acting, nonselective, Amy-based DACRAs with potential anti-obesity effects.