A Hot‐Segment‐Based Approach for the Design of Cross‐Amyloid Interaction Surface Mimics as Inhibitors of Amyloid Self‐Assembly

The design of inhibitors of protein–protein interactions mediating amyloid self‐assembly is a major challenge mainly due to the dynamic nature of the involved structures and interfaces. Interactions of amyloidogenic polypeptides with other proteins are important modulators of self‐assembly. Here we present a hot‐segment‐linking approach to design a series of mimics of the IAPP cross‐amyloid interaction surface with Aβ (ISMs) as nanomolar inhibitors of amyloidogenesis and cytotoxicity of Aβ, IAPP, or both polypeptides. The nature of the linker determines ISM structure and inhibitory function including both potency and target selectivity. Importantly, ISMs effectively suppress both self‐ and cross‐seeded IAPP self‐assembly. Our results provide a novel class of highly potent peptide leads for targeting protein aggregation in Alzheimer’s disease, type 2 diabetes, or both diseases and a chemical approach to inhibit amyloid self‐assembly and pathogenic interactions of other proteins as well.     

Mechanistic studies of peptide self-assembly: transient α-helices to stable β-sheets

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 β 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 β-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 β-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.     

Amyloidogenicity of recombinant human pro-islet amyloid polypeptide (ProIAPP)

BACKGROUND: Pancreatic amyloid has been associated with type II diabetes. The major constituent of pancreatic amyloid is the 37-residue peptide islet amyloid polypeptide (IAPP). IAPP is expressed as a 67-residue pro-peptide called ProIAPP which is processed to IAPP following stimulation. While the molecular events underlying IAPP amyloid formation in vitro have been studied, little is known about the role of ProIAPP in the formation of pancreatic amyloid. This has been due in part to the limited availability of purified ProIAPP for conformational and biochemical studies. RESULTS: We present a method for efficient recombinant expression and purification of ProIAPP and a processing site mutant, mutProIAPP, as thioredoxin (Trx) fusion proteins. Conformation and amyloidogenicity of cleaved ProIAPP and mutProIAPP and the fusion proteins were assessed by circular dichroism, electron microscopy and Congo red staining. We find that ProIAPP and mutProIAPP exhibit strong self-association potentials and are capable of forming amyloid. However, the conformational transitions of ProIAPP and mutProIAPP during aging and amyloidogenesis are distinct from the random coil-to-beta-sheet transition of IAPP. Both proteins are found to be less amyloidogenic than IAPP and besides fibrils a number of non-fibrillar but ordered aggregates form during aging of ProIAPP. ProIAPP aggregates are cytotoxic on pancreatic cells but less cytotoxic than IAPP while mutProIAPP aggregates essentially lack cytotoxicity. The Trx fusion proteins are neither amyloidogenic nor cytotoxic. CONCLUSIONS: Our studies suggest that ProIAPP has typical properties of an amyloidogenic polypeptide but also indicate that the pro-region suppresses the amyloidogenic and cytotoxic potentials of IAPP.     

The role of copper(II) and zinc(II) in the degradation of human and murine IAPP by insulin‐degrading enzyme

Amylin or islet amyloid polypeptide (IAPP) is a 37‐residue peptide hormone secreted from the pancreatic islets into the blood circulation and is cleared by peptidases in the kidney. IAPP aggregates are strongly associated with β‐cell degeneration in type 2 diabetes, as demonstrated by the fact that more than 95% of patients exhibit IAPP amyloid upon autopsy. Recently, it has been reported that metal ions such as copper(II) and zinc(II) are implicated in the aggregation of IAPP as well as able to modulate the proteolytic activity of IAPP degrading enzymes. For this reason, in this work, the role of the latter metal ions in the degradation of IAPP by insulin‐degrading enzyme (IDE) has been investigated by a chromatographic and mass spectrometric combined method. The latter experimental approach allowed not only to assess the overall metal ion inhibition of the human and murine IAPP degradation by IDE but also to have information on copper‐ and zinc‐induced changes in IAPP aggregation. In addition, IDE cleavage site preferences in the presence of metal ions are rationalized as metal ion‐induced changes in substrate accessibility. Copyright © 2014 John Wiley & Sons, Ltd.     

Structural Insight into IAPP‐Derived Amyloid Inhibitors and Their Mechanism of Action

Designed peptides derived from the islet amyloid polypeptide (IAPP) cross‐amyloid interaction surface with Aβ (termed interaction surface mimics or ISMs) have been shown to be highly potent inhibitors of Aβ amyloid self‐assembly. However, the molecular mechanism of their function is not well understood. Using solution‐state and solid‐state NMR spectroscopy in combination with ensemble‐averaged dynamics simulations and other biophysical methods including TEM, fluorescence spectroscopy and microscopy, and DLS, we characterize ISM structural preferences and interactions. We find that the ISM peptide R3‐GI is highly dynamic, can adopt a β‐like structure, and oligomerizes into colloid‐like assemblies in a process that is reminiscent of liquid–liquid phase separation (LLPS). Our results suggest that such assemblies yield multivalent surfaces for interactions with Aβ40. Sequestration of substrates into these colloid‐like structures provides a mechanistic basis for ISM function and the design of novel potent anti‐amyloid molecules.     

The Human Host‐Defense Peptide Cathelicidin LL‐37 is a Nanomolar Inhibitor of Amyloid Self‐Assembly of Islet Amyloid Polypeptide (IAPP)

Amyloid self‐assembly of islet amyloid polypeptide (IAPP) is linked to pancreatic inflammation, β‐cell degeneration, and the pathogenesis of type 2 diabetes (T2D). The multifunctional host‐defence peptides (HDPs) cathelicidins play crucial roles in inflammation. Here, we show that the antimicrobial and immunomodulatory polypeptide human cathelicidin LL‐37 binds IAPP with nanomolar affinity and effectively suppresses its amyloid self‐assembly and related pancreatic β‐cell damage in vitro. In addition, we identify key LL‐37 segments that mediate its interaction with IAPP. Our results suggest a possible protective role for LL‐37 in T2D pathogenesis and offer a molecular basis for the design of LL‐37‐derived peptides that combine antimicrobial, immunomodulatory, and T2D‐related anti‐amyloid functions as promising candidates for multifunctional drugs.     

Cucurbit[7]uril Inhibits Islet Amyloid Polypeptide Aggregation by Targeting N Terminus Hot Segments and Attenuates Cytotoxicity

Two “hot segments” within an islet amyloid polypeptide are responsible for its self-assembly, which in turn is linked to the decline of β-cells in type 2 diabetes (T2D). A readily available water-soluble, macrocyclic host, cucurbit[7]uril (CB[7]), effectively inhibits islet amyloid polypeptide (IAPP) aggregation through ion–dipole and hydrophobic interactions with different residues of the monomeric peptide in its random-coil conformation. A HSQC NMR study shows that CB[7] likely modulates IAPP self-assembly by interacting with and masking major residues present in the “hot segments” at the N terminus. CB[7] also prevents the formation of toxic oligomers and inhibits seed-catalyzed fibril proliferation. Importantly, CB[7] recovers rat insulinoma cells (RIN-m) from IAPP-assembly associated cytotoxicity.     

Semisynthetic and Enzyme‐Mediated Conjugate Preparations Illuminate the Ubiquitination‐Dependent Aggregation of Tau Protein

In the brain of individuals with Alzheimer's disease, the regulatory protein ubiquitin is found conjugated to different lysine residues of tau protein assembled into pathological paired helical filaments. To shed light on the hitherto unexplored ubiquitination‐linked conformational transitions of tau, the availability of in vitro ubiquitin conjugation methods is of primary importance. In our work, we focused on the four‐repeat domain of tau and assembled an enzymatic machinery formed by UBE1, Ubc13, and CHIP enzymes. The enzymatic reaction resulted in monoubiquitination at multiple sites, reminiscent of the ubiquitination pattern observed in vivo. We further exploited chemoselective disulfide coupling reactions to construct three tau regioisomers with site‐specific monoubiquitination. Protein aggregation experiments revealed that the multiple enzyme‐derived products were unable to convert into amyloid fibrils, while the semisynthetic conjugates exhibited diverse capability to form filaments. This study contributes novel insight into the effects of a key post‐translational modification on aberrant protein self‐assembly.     

Intramolecular Charge Interactions as a Tool to Control the Coiled‐Coil‐to‐Amyloid Transformation

Under the influence of a changed environment, amyloid‐forming proteins partially unfold and assemble into insoluble β‐sheet rich fibrils. Molecular‐level characterization of these assembly processes has been proven to be very challenging, and for this reason several simplified model systems have been developed over recent years. Herein, we present a series of three de novo designed model peptides that adopt different conformations and aggregate morphologies depending on concentration, pH value, and ionic strength. The design strictly follows the characteristic heptad repeat of the α‐helical coiled‐coil structural motif. In all peptides, three valine residues, known to prefer the β‐sheet conformation, have been incorporated at the solvent‐exposed b, c, and f positions to make the system prone to amyloid formation. Additionally, pH‐controllable intramolecular electrostatic repulsions between equally charged lysine (peptide A) or glutamate (peptide B) residues were introduced along one side of the helical cylinder. The conformational behavior was monitored by circular dichroism spectroscopic analysis and thioflavin T fluorescence, and the resulting aggregates were further characterized by transmission electron microscopy. Whereas uninterrupted α‐helical aggregates are found at neutral pH, Coulomb repulsions between lysine residues in peptide A destabilize the helical conformation at acidic pH values and trigger an assembly into amyloid‐like fibrils. Peptide B features a glutamate‐based switch functionality and exhibits opposite pH‐dependent folding behavior. In this case, α‐helical aggregates are found under acidic conditions, whereas amyloids are formed at neutral pH. To further validate the pH switch concept, peptide C was designed by including serine residues, thus resulting in an equal distribution of charged residues. Surprisingly, amyloid formation is observed at all pH values investigated for peptide C. The results of further investigations into the effect of different salts, however, strongly support the crucial role of intramolecular charge repulsions in the model system presented herein.     

Inhibition of amyloid aggregation by formation of helical assemblies

The formation of amyloid aggregates is responsible for a wide range of diseases, including Alzheimer's and Parkinson's disease. Although the amyloid-forming proteins have different structures and sequences, all undergo a conformational change to form amyloid aggregates that have a characteristic cross-β-structure. The mechanistic details of this process are poorly understood, but different strategies for the development of inhibitors of amyloid formation have been proposed. In most cases, chemically diverse compounds bind to an elongated form of the protein in a β-strand conformation and thereby exert their therapeutic effect. However, this approach could favor the formation of prefibrillar oligomeric species, which are thought to be toxic. Herein, we report an alternative approach in which a helical coiled-coil-based inhibitor peptide has been designed to engage a coiled-coil-based amyloid-forming model peptide in a stable coiled-coil arrangement, thereby preventing rearrangement into a β-sheet conformation and the subsequent formation of amyloid-like fibrils. Moreover, we show that the helix-forming peptide is able to disassemble mature amyloid-like fibrils.     

Cyclic Peptides as Inhibitors of Amyloid Fibrillation

Many neurodegenerative diseases, like Parkinson’s, Alzheimer’s, or Huntington’s disease, occur as a result of amyloid protein fibril formation and cell death induced by this process. Cyclic peptides (CPs) and their derivatives form a new class of powerful inhibitors that prevent amyloid fibrillation and decrease the cytotoxicity of aggregates. The strategies for designing CPs are described, with respect to their amino acid sequence and/or conformational similarity to amyloid fibrils. The implications of CPs for the study and possible treatment of amyloid‐related diseases are discussed.     

Modulating Aβ33–42 Peptide Assembly by Graphene Oxide

Graphene oxide (GO) is utilized as the modulator to tune the formation and development of amyloid fibrils (Aβ_33–42). Atomic force microscopy temporal evolution measurements reveal that the initial binding between the peptide monomer and the large available surface of the GO sheets can redirect the assembly pathway of amyloid beta. The results support the possibility to develop graphene‐based materials to inhibit amyloidosis.     

Confined Water in Amyloid‐Competent Oligomers of the Prion Protein

Conformational switching of the prion protein into the abnormal form involves the formation of (obligatory) molten‐oligomers that mature into ordered amyloid fibrils. The role of water in directing the course of amyloid formation remains poorly understood. Here, we show that the mobility of the water molecules within the on‐pathway oligomers is highly retarded. The water relaxation time within the oligomers was estimated to be ≈1 ns which is about three orders of magnitude slower than the bulk water and resembles the characteristics of (trapped) nano‐confined water. We propose that the coalescence of these obligatory oligomers containing trapped water is entropically favored because of the release of ordered water molecules in the bulk milieu and results in the sequestration of favorable inter‐chain amyloid contacts via nucleated conformational conversion. The dynamic role of water in protein aggregation will have much broader implications in a variety of protein misfolding diseases.     

Multifaceted Influences of Melanin‐Like Particles on Amyloid‐beta Aggregation

The properties of eumelanin‐like particles (EMPs) and pheomelanin‐like particles (PMPs) in regulating the process of amyloid formation of amyloid‐beta 42 (Aβ42) were examined. EMPs and PMPs are effective both in interfering with amyloid aggregation of Aβ42 and in remodeling matured Αβ42 fibers. The results suggest that some (but not all) molecular species consisting of melanin‐like particles (MPs) are responsible for their inhibiting property toward amyloid formation, and the influence is likely manifested by long‐range interactions. Incubating preformed Aβ42 fibers with catechols or MPs leads to the formation of mesh‐like, interconnected Aβ42 fibers encapsulated with melanin‐like material. MPs are kinetically more effective than catechol monomers in this process, and a detailed investigation reveals that 4,5‐dihydroxyindole, a major intermediate in the formation of melanin‐like species, and its derivatives are mainly responsible for remodeling amyloid fibers.     

Low pH acts as inhibitor of membrane damage induced by human islet amyloid polypeptide

Human islet amyloid polypeptide (IAPP) is the major component of the amyloid deposits found in the pancreatic islets of patients with type 2 diabetes mellitus. After synthesis, IAPP is stored in the β-cell granules of the pancreas at a pH of approximately 5.5 and released into the extracellular compartment at a pH of 7.4. To gain insight into the possible consequences of pH differences for properties and membrane interaction of IAPP, we here compared the aggregational and conformational behavior of IAPP as well as IAPP-membrane interactions at pH 5.5 and pH 7.4. Our data reveal that a low pH decreases the rate of fibril formation both in solution and in the presence of membranes. We observed by CD spectroscopy that these differences in kinetics are directly linked to changes in the conformational behavior of the peptide. Mechanistically, the processes that occur at pH 5.5 and pH 7.4 appear to be similar. At both pH values, we found that the kinetic profile of IAPP fibril growth matches the kinetic profile of IAPP-induced membrane damage, and that both are characterized by a lag phase and a sigmoidal transition. Furthermore, monolayer studies as well as solid-state NMR experiments indicate that the differences in kinetics and conformational behavior as function of pH are not due to a different mode of membrane insertion. Our study suggests that a low pH prevents aggregation and membrane damage of IAPP in the secretory granules, most likely by affecting the ionization properties of the peptide.     

Atomic force microscopic imaging of seeded fibril formation and fibril branching by the Alzheimer's disease amyloid-β protein

Background: Amyloid plaques composed of the fibrillar form of the amyloid-β protein (Aβ) are the defining neuropathological feature of Alzheimer's disease (AD). A detailed understanding of the time course of amyloid formation could define steps in disease progression and provide targets for therapeutic intervention. Amyloid fibrils, indistinguishable from those derived from an AD brain, can be produced in vitro using a seeded polymerization mechanism. In its simplest form, this mechanism involves a cooperative transition from monomeric Aβ to the amyloid fibril without the buildup of intermediates. Recently, however, a transient species, the Aβ amyloid protofibril, has been identified. Here, we report studies of Aβ amyloid protofibril and its seeded transition into amyloid fibrils using atomic force microscopy.Results: Seeding of the protofibril-to-fibril transition was observed. Preformed fibrils, but not protofibrils, effectively seeded this transition. The assembly state of Aβ influenced the rate of seeded growth, indicating that protofibrils are fibril assembly precursors. The handedness of the helical surface morphology of fibrils depended on the chirality of Aβ. Finally, branched and partially wound fibrils were observed.Conclusions: The temporal evolution of morphologies suggests that the protofibril-to-fibril transition is nucleation-dependent and that protofibril winding is involved in that transition. Fibril unwinding and branching may be essential for the post-nucleation growth process. The protofibrillar assembly intermediate is a potential target for AD therapeutics aimed at inhibiting amyloid formation and AD diagnostics aimed at detecting presymptomatic disease.     

Co‐existence of Two Different α‐Synuclein Oligomers with Different Core Structures Determined by Hydrogen/Deuterium Exchange Mass Spectrometry

Neurodegenerative disorders are characterized by the formation of protein oligomers and amyloid fibrils, which in the case of Parkinson’s disease involves the protein α‐synuclein (αSN). Cytotoxicity is mainly associated with the oligomeric species, but we still know little about their assembly and structure. Hydrogen/deuterium exchange (HDX) monitored by mass spectrometry is used to analyze oligomers formed by wild‐type (wt) αSN and also three familial αSN mutants (A30P, E46K, and A53T). All four variants show co‐existence of two different oligomers. The backbone amides of oligomer type I are protected from exchange with D₂O until they dissociate into monomeric αSN by EX1 exchange kinetics. Fewer residues are protected against exchange in oligomer type II, but this type does not revert to αSN monomers. Both oligomers are protected in the core sequence Y39–A89. Based on incubation studies, oligomer type I appears to form straight fibrils, while oligomer type II forms amorphous clusters that do not directly contribute to the fibrillation process.     

Cold Denaturation of α‐Synuclein Amyloid Fibrils

Although amyloid fibrils are associated with numerous pathologies, their conformational stability remains largely unclear. Herein, we probe the thermal stability of various amyloid fibrils. α‐Synuclein fibrils cold‐denatured to monomers at 0–20 °C and heat‐denatured at 60–110 °C. Meanwhile, the fibrils of β2‐microglobulin, Alzheimer’s Aβ1‐40/Aβ1‐42 peptides, and insulin exhibited only heat denaturation, although they showed a decrease in stability at low temperature. A comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in fibril cores contributed to the cold denaturation of α‐synuclein fibrils. We propose that although cold‐denaturation is common to both native proteins and misfolded fibrillar states, the main‐chain dominated amyloid structures may explain amyloid‐specific cold denaturation arising from the unfavorable burial of charged side‐chains in fibril cores.     

Size Effect of Graphene Oxide on Modulating Amyloid Peptide Assembly

Protein misfolding and abnormal assembly could lead to aggregates such as oligomer, proto‐fibril, mature fibril, and senior amyloid plaques, which are associated with the pathogenesis of many amyloid diseases. These irreversible amyloid aggregates typically form in vivo and researchers have been endeavoring to find new modulators to invert the aggregation propensity in vitro, which could increase understanding in the mechanism of the aggregation of amyloid protein and pave the way to potential clinical treatment. Graphene oxide (GO) was shown to be a good modulator, which could strongly control the amyloidosis of Aβ (33–42). In particular, quartz crystal microbalance (QCM), circular dichroism (CD) spectroscopy, and atomic force microscopy (AFM) measurements revealed the size‐dependent manner of GO on modulating the assembly of amyloid peptides, which could be a possible way to regulate the self‐assembled nanostructure of amyloid peptide in a predictable manner.     

Electrostatic Complementarity Drives Amyloid/Nucleic Acid Co‐Assembly

Proteinaceous plaques associated with neurodegenerative diseases contain many biopolymers including the polyanions glycosaminoglycans and nucleic acids. Polyanion‐induced amyloid fibrillation has been implicated in disease etiology, but structural models for amyloid/nucleic acid co‐assemblies remain limited. Here we constrain nucleic acid/peptide interactions with model peptides that exploit electrostatic complementarity and define a novel amyloid/nucleic acid co‐assembly. The structure provides a model for nucleic acid/amyloid co‐assembly as well as insight into the energetic determinants involved in templating amyloid assembly.