Lydia M. Young

Screening and classifying small-molecule inhibitors of amyloid formation using ion mobility spectrometry–mass spectrometry

The search for therapeutic agents that bind specifically to precursor protein conformations and inhibit amyloid assembly is an important challenge. Identifying such inhibitors is difficult because many protein precursors of aggregation are partially folded or intrinsically disordered, which rules out structure-based design. Furthermore, inhibitors can act by a variety of mechanisms, including specific or nonspecific binding, as well as colloidal inhibition. Here we report a high-throughput method based on ion mobility spectrometry–mass spectrometry (IMS–MS) that is capable of rapidly detecting small molecules that bind to amyloid precursors, identifying the interacting protein species and defining the mode of inhibition. Using this method we have classified a variety of small molecules that are potential inhibitors of human islet amyloid polypeptide (hIAPP) aggregation or amyloid-beta 1-40 aggregation as specific, nonspecific, colloidal or non-interacting. We also demonstrate the ability of IMS–MS to screen for inhibitory small molecules in a 96-well plate format and use this to discover a new inhibitor of hIAPP amyloid assembly. A method for rapidly screening small-molecule inhibitors of amyloid assembly has been developed. This method uses electrospray ionization–ion mobility spectrometry–mass spectrometry to detect and identify the type of inhibition. A screen of this nature could help in the discovery of therapeutics for numerous diseases associated with aberrant protein aggregation.

Ion Mobility Spectrometry–Mass Spectrometry Defines the Oligomeric Intermediates in Amylin Amyloid Formation and the Mode of Action of Inhibitors

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.

Mutational analysis of the ability of resveratrol to inhibit amyloid formation by islet amyloid polypeptide: Critical evaluation of the importance of aromatic-inhibitor and histidine-inhibitor interactions

The process of amyloid formation by the normally soluble hormone islet amyloid polypeptide (IAPP) contributes to β-cell death in type 2 diabetes and in islet transplants. There are no clinically approved inhibitors of islet amyloidosis, and the mode of action of existing inhibitors is not well-understood. Resveratrol, a natural polyphenol, has been reported to inhibit amyloid formation by IAPP and by the Alzheimer’s disease Aβ peptide. The mechanism of action of this compound is not known, nor is its mode of interaction with IAPP. In this study, we use a series of IAPP variants to examine possible interactions between resveratrol and IAPP. Fluorescence assays, transmission electron microscopy, and mass spectrometry demonstrate that resveratrol is much less effective as an inhibitor of IAPP amyloid formation than the polyphenol (−)-epigallocatechin 3-gallate (EGCG) and, unlike EGCG, does not significantly disaggregate preformed IAPP amyloid fibrils. Resveratrol is also shown to interfere with thioflavin-T assays. His-18 mutants, a truncation mutant, mutants of each of the aromatic residues, and mutants of Arg-11 of IAPP were examined. Mutation of His to Gln or Leu weakens the ability of resveratrol to inhibit amyloid formation by IAPP, as do mutations of Arg-11, Phe-15, or Tyr-37 to Leu, and truncation to form the variant Ac 8−37-IAPP, which removes the first seven residues to eliminate Lys-1 and the N-terminal amino group. In contrast, replacement of Phe-23 with Leu has a smaller effect. The data highlight Phe-15, His-18, and Tyr-37 as being important for IAPP–resveratrol interactions and are consistent with a potential role of the N-terminus and Arg-11 in polypeptide–resveratrol interactions.

An in vivo platform for identifying inhibitors of protein aggregation

Protein aggregation underlies an array of human diseases, yet only one small molecule therapeutic has been successfully developed to date. Here, we introduce an in vivo system, based on a β-lactamase tripartite fusion construct, capable of identifying aggregation-prone sequences in the periplasm of Escherichia coli and inhibitors that prevent their aberrant self-assembly. We demonstrate the power of the system using a range of proteins, from small unstructured peptides (islet amyloid polypeptide and amyloid β) to larger, folded immunoglobulin domains. Configured in a 48-well format, the split β-lactamase sensor readily differentiates between aggregation-prone and soluble sequences. Performing the assay in the presence of 109 compounds enabled a rank ordering of inhibition and revealed a new inhibitor of IAPP aggregation. This platform can be applied to both amyloidogenic and other aggregation-prone systems, independent of sequence or size, and can identify small molecules or other factors able to ameliorate or inhibit protein aggregation.

ESI-IMS-MS: A method for rapid analysis of protein aggregation and its inhibition by small molecules.

Graphical abstract

De novo design of a biologically active amyloid

Aggregation by design Amyloid aggregation is driven by short sequences within proteins that self-assemble into characteristic amyloid structures. About 30 human proteins are implicated in amyloid-associated diseases, but many more contain short sequences that are potentially amyloidogenic. Gallardo et al. designed a peptide based on an amyloidogenic sequence in the vascular endothelial growth factor receptor VEGFR2. The peptide induced VEGFR2 to form aggregates with features characteristic of amyloids. Amyloids were toxic only in cells that required VEGFR2 activity, suggesting that the toxicity was due to loss of function of VEGFR2, rather than to inherent toxicity of the aggregates. The peptide inhibited VEGFR2-dependent tumor growth in a mouse tumor model. Science, this issue p. 10.1126/science.aah4949 A designed peptide drives a protein that does not usually aggregate to form amyloids. INTRODUCTION It has been shown that most proteins possess amyloidogenic segments. However, only about 30 human proteins are known to be involved in amyloid-associated pathologies, and it is still not clear what determines amyloid toxicity in these diseases. We investigated whether an endogenously expressed protein that contains sequences with known amyloidogenic segments, but is not known to aggregate either under normal or pathological conditions, can be induced to do so by seeding it with a peptide comprising the protein’s own amyloidogenic fragment. We chose to target the protein vascular endothelial growth factor receptor 2 (VEGFR2) because it has well-characterized biological function and so could provide a model system with which to investigate the relationship between protein loss of function and amyloid toxicity in different cellular contexts. RATIONALE The capacity of the amyloid conformation of disease proteins to catalyze their own amyloid conversion demonstrates the sequence specificity of amyloid assembly. Because the core of amyloids consists of short amyloidogenic sequence fragments, we hypothesized that a short amyloidogenic protein sequence of VEGFR2, a protein normally not associated with protein aggregation, should be able to interact with and specifically induce the aggregation of VEGFR2, resulting in its functional knockdown. We used TANGO, an algorithm that predicts aggregation-prone sequences, to identify potential amyloidogenic fragments in VEGFR2. We synthesized these fragments as a tandem repeat in a peptide framework in which each unit is flanked by charged residues and coupled by a short peptide linker. The thinking behind this design was that the tandem repeats would promote the formation of diffusable soluble oligomeric aggregates, whereas the charged residues would kinetically stabilize these oligomers and reduce the rate of insoluble fibril formation. RESULTS By screening for loss of function, we identified one peptide, termed “vascin,” that was highly potent at inhibiting VEGFR2. This sequence was derived from the translocation signaling sequence of VEGFR2. We found that vascin is an amyloidogenic peptide that readily forms small β-structured oligomers, ranging from dimers to nonamers, that slowly convert to amyloid fibrils. When added to cell culture medium, these oligomers are efficiently absorbed by the cell, where they interact with and promote the aggregation and partial degradation of nascent VEGFR2. Vascin aggregation does not induce the aggregation of known disease amyloids. Neither do vascin oligomers affect the function of the related EGF receptor or the surface translocation of other receptors. We found vascin only to be toxic to cells that are dependent on VEGFR2 function, suggesting that toxicity is due to loss of VEGFR2 function and not to vascin aggregation or vascin-induced VEGFR2 aggregation. Consistent with this, we found that vascin is active in vivo and could reduce tumor growth in a VEGFR2-sensitive subcutaneous B16 melanoma syngenic tumor model in mice but is not intrinsically toxic to other tissues. CONCLUSION We found that a short amyloidogenic protein fragment can induce the aggregation of a protein normally not associated with amyloidosis in a manner that recapitulates key biophysical and biochemical characteristics of natural amyloids. In addition, we found that amyloid toxicity is observed only in cells that both express VEGFR2 and are dependent on VEGFR2 activity for survival. Thus, rather than being generic, amyloid toxicity here appears to be both protein-specific and conditional on a requirement for VEGFR2 protein function. A synthetic amyloid peptide induces aggregation. We designed vascin, a synthetic amyloid peptide based on an amyloidogenic fragment of the signal peptide of VEGFR2. Vascin forms prefibrillar oligomers that penetrate mammalian cells and interacts with the nascent VEGFR2 protein, resulting in its aggregation and functional knockdown. [Composition includes parts of an image from iStock.com/luismmolina.] Most human proteins possess amyloidogenic segments, but only about 30 are associated with amyloid-associated pathologies, and it remains unclear what determines amyloid toxicity. We designed vascin, a synthetic amyloid peptide, based on an amyloidogenic fragment of vascular endothelial growth factor receptor 2 (VEGFR2), a protein that is not associated to amyloidosis. Vascin recapitulates key biophysical and biochemical characteristics of natural amyloids, penetrates cells, and seeds the aggregation of VEGFR2 through direct interaction. We found that amyloid toxicity is observed only in cells that both express VEGFR2 and are dependent on VEGFR2 activity for survival. Thus, amyloid toxicity here appears to be both protein-specific and conditional—determined by VEGFR2 loss of function in a biological context in which target protein function is essential.

Small molecule probes of protein aggregation

Understanding the mechanisms of amyloid formation and toxicity remain major challenges. Although substantial progress has been made in the development of methods able to identify the species formed during self-assembly and to describe the kinetic mechanisms of aggregation, the structure(s) of non-native species, including potentially toxic oligomers, remain elusive. Moreover, how fibrils contribute to disease remains unclear. Here we review recent advances in the development of small molecules and other reagents that are helping to define the mechanisms of protein aggregation in molecular detail. Such probes form a powerful platform with which to better define the mechanisms of structural conversion into amyloid fibrils and may provide the much-needed stepping stone for future development of successful therapeutic agents.

Monitoring oligomer formation from self-aggregating amylin peptides using ESI-IMS-MS

Amyloid diseases are a serious cause for concern world-wide. To understand the mechanism of formation of the fibrillar structures associated with such disorders, it is necessary to study the progression from soluble protein or peptide monomer through an array of oligomers to the final, insoluble, fibrils. The protein IAPP is found in vivo in the form of insoluble amyloid deposits in the pancreatic islets of diabetes type II sufferers. Here, we have studied the in vitro self-aggregation of three fibril-forming peptides from the amyloidogenic core of IAPP. Using electrospray ionization—mass spectrometry coupled with ion mobility spectrometry, the mass and cross-sectional area of each oligomer present in the heterogeneous assembly mixtures can be determined individually in a single, rapid experiment over time. For the three peptides studied, oligomers ≤20-mer were characterized. Conversely, no oligomers higher than a dimer were detected for a non-assembling peptide control. The rate in which the cross-sectional area of the oligomers increases with increasing number of peptide sub-units indicates that assembly for the amyloid-forming peptides proceeds in a linear fashion until an oligomer of a certain size is attained. After this, a step increase in cross-sectional area occurs for the next higher-order oligomer. This behaviour can be explained by molecular modelling of singly, doubly, triply and quadruply stacked β-stranded structures. Using one peptide as an example, the cross-sectional areas of the lower order oligomers (dimer to pentamer) were found to be consistent with a single β-sheet model, whereas the higher order oligomers were consistent with double-stranded (hexamer to decamer oligomers), triply-stranded (11-mers to 15-mers) and quadruply-stranded (16-mers to 20-mers) β-sheet models.

Aggregating sequences that occur in many proteins constitute weak spots of bacterial proteostasis

Aggregation is a sequence-specific process, nucleated by short aggregation-prone regions (APRs) that can be exploited to induce aggregation of proteins containing the same APR. Here, we find that most APRs are unique within a proteome, but that a small minority of APRs occur in many proteins. When aggregation is nucleated in bacteria by such frequently occurring APRs, it leads to massive and lethal inclusion body formation containing a large number of proteins. Buildup of bacterial resistance against these peptides is slow. In addition, the approach is effective against drug-resistant clinical isolates of Escherichiacoli and Acinetobacterbaumannii, reducing bacterial load in a murine bladder infection model. Our results indicate that redundant APRs are weak points of bacterial protein homeostasis and that targeting these may be an attractive antibacterial strategy.Aggregation is sequence-specific and nucleated by short aggregating protein segments (APR). Here authors use a multidisciplinary approach to show that in E.coli some frequently occurring APRs lead to protein aggregation and ultimately bacterial cell death, which could serve as antibacterial strategy.

A peptide-display protein scaffold to facilitate single molecule force studies of aggregation-prone peptides: Developing a Peptide Display System for Force Spectroscopy

Protein aggregation is linked with the onset of several neurodegenerative disorders, including Parkinsons disease (PD), which is associated with the aggregation of α‐synuclein (αSyn). The structural mechanistic details of protein aggregation, including the nature of the earliest protein–protein interactions, remain elusive. In this study, we have used single molecule force spectroscopy (SMFS) to probe the first dimerization events of the central aggregation‐prone region of αSyn (residues 71–82) that may initiate aggregation. This region has been shown to be necessary for the aggregation of full length αSyn and is capable of forming amyloid fibrils in isolation. We demonstrate that the interaction of αSyn71‐82 peptides can be studied using SMFS when inserted into a loop of protein L, a mechanically strong and soluble scaffold protein that acts as a display system for SMFS studies. The corresponding fragment of the homolog protein γ‐synuclein (γSyn), which has a lower aggregation propensity, has also been studied here. The results from SMFS, together with native mass spectrometry and aggregation assays, demonstrate that the dimerization propensity of γSyn71‐82 is lower than that of αSyn71‐82, but that a mixed αSyn71‐82: γSyn71‐82 dimer forms with a similar propensity to the αSyn71‐82 homodimer, slowing amyloid formation. This work demonstrates the utility of a novel display method for SMFS studies of aggregation‐prone peptides, which would otherwise be difficult to study.