Maria Andreasen

Coexistence of ribbon and helical fibrils originating from hIAPP(20-29) revealed by quantitative nanomechanical atomic force microscopy.

Uncontrolled misfolding of proteins leading to the formation of amyloid deposits is associated with more than 40 types of diseases, such as neurodegenerative diseases and type-2 diabetes. These irreversible amyloid fibrils typically assemble in distinct stages. Transitions among the various intermediate stages are the subject of many studies but are not yet fully elucidated. Here, we combine high-resolution atomic force microscopy and quantitative nanomechanical mapping to determine the self-assembled structures of the decapeptide hIAPP20–29, which is considered to be the fibrillating core fragment of the human islet amyloid polypeptide (hIAPP) involved in type-2 diabetes. We successfully follow the evolution of hIAPP20–29 nanostructures over time, calculate the average thickening speed of small ribbon-like structures, and provide evidence of the coexistence of ribbon and helical fibrils, highlighting a key step within the self-assembly model. In addition, the mutations of individual side chains of wide-type hIAPP20–29 shift this balance and destabilize the helical fibrils sufficiently relative to the twisted ribbons to lead to their complete elimination. We combine atomic force microscopy structures, mechanical properties, and solid-state NMR structural information to build a molecular model containing β sheets in cross-β motifs as the basis of self-assembled amyloids.

Human Phenotypically Distinct TGFBI Corneal Dystrophies Are Linked to the Stability of the Fourth FAS1 Domain of TGFBIp

Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. The protein consists of an N-terminal Cys-rich EMI domain and four consecutive fasciclin 1 (FAS1) domains. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substitutions in the first FAS1 (FAS1-1) domain (R124H, R124L, and R124C) did not alter the stability. However, substitutions within the fourth FAS1 (FAS1-4) domain (A546T, R555Q, and R555W) affected the overall stability of intact TGFBIp revealing the following stability ranking R555W>WT>R555Q>A546T. Significantly, the stability ranking of the isolated FAS1-4 domains mirrored the behavior of the intact protein. In addition, it was linked to the aggregation propensity as the least stable mutant (A546T) forms amyloid fibrils while the more stable variants generate non-amyloid amorphous deposits in vivo. Significantly, the data suggested that both an increase and a decrease in the stability of FAS1-4 may unleash a disease mechanism. In contrast, amino acid substitutions in FAS1-1 did not affect the stability of the intact TGFBIp suggesting that molecular the mechanism of disease differs depending on the FAS1 domain carrying the mutation.

Interactions between misfolded protein oligomers and membranes: A central topic in neurodegenerative diseases?

The deposition of amyloid material has been associated with many different diseases. Although these diseases are very diverse the amyloid material share many common features such as cross-β-sheet structure of the backbone of the proteins deposited. Another common feature of the aggregation process for a wide variety of proteins is the presence of prefibrillar oligomers. These oligomers are linked to the cytotoxicity occurring during the aggregation of proteins. These prefibrillar oligomers interact extensively with lipid membranes and in some cases leads to destabilization of lipid membranes. This interaction is however highly dependent on the nature of both the oligomer and the lipids. Anionic lipids are often required for interaction with the lipid membrane while increased exposure of hydrophobic patches from highly dynamic protein oligomers are structural determinants of cytotoxicity of the oligomers. To explore the oligomer lipid interaction in detail the interaction between oligomers of α-synuclein and the 4th fasciclin-1 domain of TGFBIp with lipid membranes will be examined here. For both proteins the dynamic species are the ones causing membrane destabilization and the membrane interaction is primarily seen when the lipid membranes contain anionic lipids. Hence the dynamic nature of oligomers with exposed hydrophobic patches alongside the presence of anionic lipids could be essential for the cytotoxicity observed for prefibrillar oligomers in general. This article is part of a Special Issue entitled: Lipid-protein interactions.

Electrostatically-guided inhibition of Curli amyloid nucleation by the CsgC-like family of chaperones

Polypeptide aggregation into amyloid is linked with several debilitating human diseases. Despite the inherent risk of aggregation-induced cytotoxicity, bacteria control the export of amyloid-prone subunits and assemble adhesive amyloid fibres during biofilm formation. An Escherichia protein, CsgC potently inhibits amyloid formation of curli amyloid proteins. Here we unlock its mechanism of action, and show that CsgC strongly inhibits primary nucleation via electrostatically-guided molecular encounters, which expands the conformational distribution of disordered curli subunits. This delays the formation of higher order intermediates and maintains amyloidogenic subunits in a secretion-competent form. New structural insight also reveal that CsgC is part of diverse family of bacterial amyloid inhibitors. Curli assembly is therefore not only arrested in the periplasm, but the preservation of conformational flexibility also enables efficient secretion to the cell surface. Understanding how bacteria safely handle amyloidogenic polypeptides contribute towards efforts to control aggregation in disease-causing amyloids and amyloid-based biotechnological applications.

The importance of being capped: Terminal capping of an amyloidogenic peptide affects fibrillation propensity and fibril morphology.

The formation of aggregated fibrillar β-sheet structures has been proposed to be a generic feature of proteins. Aggregation propensity is highly sequence dependent, and often only part of the protein is incorporated into the fibril core. Therefore, shorter peptide fragments corresponding to the fibril core are attractive fibrillation models. The use of peptide models introduces new termini into the fibrils, yet little attention has been paid to the role these termini may play in fibrillation. Here, we report that terminal modifications of a 10-residue peptide fragment of human islet amyloid polypeptide strongly affect fibrillation kinetics and the resulting fibril morphology. Capping of the N-terminus abolishes fibrillation, while C-terminal capping results in fibrils with a twisted morphology. Peptides with either both termini free or both termini capped form flat fibrils. Molecular dynamics simulations reveal that the N-terminal acetyl cap folds up and interacts with the peptides hydrophobic side chains, while the uncapped N-terminus in the C-terminally capped version results in twisting of the fibrils due to charge repulsion from the free N-termini. Our results highlight the role of terminal interactions in fibrillation of small peptides and provide molecular insight into the consequences of C-terminal modifications frequently found in peptide hormones in vivo.

Polymorphic Fibrillation of the Destabilized Fourth Fasciclin-1 Domain Mutant A546T of the Transforming Growth Factor-β-induced Protein (TGFBIp) Occurs through Multiple Pathways with Different Oligomeric Intermediates

Background: Corneal dystrophies are linked to aggregation of mutants of transforming growth factor β-induced Protein (TGFBIp) leading to blindness. Results: Depending on concentration, the fourth fasciclin-1 domain carrying the A546T substitution follows different fibrillation pathways involving different oligomeric intermediates. Conclusion: Aggregate species forming under different conditions have different biochemical properties. Significance: Understanding the molecular events causing aggregation is crucial for development of possible drugs. Mutations in the transforming growth factor β-induced protein (TGFBIp) are linked to the development of corneal dystrophies in which abnormal protein deposition in the cornea leads to a loss of corneal transparency and ultimately blindness. Different mutations give rise to phenotypically distinct corneal dystrophies. Most mutations are located in the fourth fasciclin-1 domain (FAS1–4). The amino acid substitution A546T in the FAS1–4 domain is linked to the development of lattice corneal dystrophy with amyloid deposits in the superficial and deep stroma, classifying it as an amyloid disease. Here we provide a detailed description of the fibrillation of the isolated FAS1–4 domain carrying the A546T substitution. The A546T substitution leads to a significant destabilization of FAS1–4 and induces a partially folded structure with increased surface exposure of hydrophobic patches. The mutation also leads to two distinct fibril morphologies. Long straight fibrils composed of pure β-sheet structure are formed at lower concentrations, whereas short and curly fibrils containing a mixture of α-helical and β-sheet structures are formed at higher concentrations. The formation of short and curly fibrils is preceded by the formation of a small number of oligomeric species with high membrane permeabilization potential and rapid fibril formation. The long straight fibrils are formed more slowly and through progressively bigger oligomers that lose their membrane permeabilization potential as fibrillation proceeds beyond the lag phase. These different fibril classes and associated biochemical differences may lead to different clinical symptoms associated with the mutation.

Modulation of fibrillation of hIAPP core fragments by chemical modification of the peptide backbone.

The well-ordered cross β-strand structure found in amyloid aggregates is stabilized by many different side chain interactions, including hydrophobic interactions, electrostatic charge and the intrinsic propensity to form β-sheet structures. In addition to the side chains, backbone interactions are important because of the regular hydrogen-bonding pattern. β-Sheet breaking peptide analogs, such as those formed by N-methylation, interfere with the repetitive hydrogen bonding pattern of peptide strands. Here we test backbone contributions to fibril stability using analogs of the 6-10 residue fibril core of human islet amyloid polypeptide, a 37 amino acid peptide involved in the pathogenesis of type II diabetes. The Phe-Gly peptide bond has been replaced by a hydroxyethylene or a ketomethylene group and the nitrogen-atom has been methylated. In addition, we have prepared peptoids where the side chain is transferred to the nitrogen atom. The backbone turns out to be extremely sensitive to substitution, since only the minimally perturbed ketomethylene analog (where only one of the -NH- groups has been replaced by -CH(2)-) can elongate wildtype fibrils but cannot fibrillate on its own. The resulting fibrils displayed differences in both secondary structure and overall morphology. No analog could inhibit the fibrillation of the parent peptide, suggesting an inability to bind to existing fibril surfaces. In contrast, side chain mutations that left the backbone intact but increased backbone flexibility or removed stabilizing side-chain interactions had very small effect on fibrillation kinetics. We conclude that fibrillation is very sensitive to even small modifications of the peptide backbone.

Preventing peptide and protein misbehavior.

Proteins provide remarkably diverse functionality in living systems by interacting and self-assembling together with other proteins into complexes, which represent key components of the nano-scale machinery of life (1). Such self-assembly processes generate intricate structures of considerable complexity that can perform a wide variety of functional roles, which range from generating motion to controlling transport across membranes. Where assembly processes are involved, there is, however, an inherent danger of misassembly, which can occur when a protein binds an incorrect partner or interacts with the appropriate partner in an incorrect manner. These types of aberrant behavior can lead to the formation of dysfunctional and even toxic molecular species. A particularly striking example of such misbehavior is the formation of amyloid fibrils (2), highly ordered elongated structures initially identified through their association with neurodegenerative disorders, such as Alzheimer’s and Parkinson’s diseases (3, 4). This kind of pathological protein self-assembly is now known to occur in the context of more than 30 disorders, and the associated neurodegenerative diseases have been estimated to affect about 45 million people worldwide, a number that is expected to triple in the next 35 y as a consequence of the general increase in life expectancy in modern societies and the fact that aging is one of the key risk factors for this class of protein misfolding diseases (5). Hence, there is an urgent need for progresses in diagnosis and drug development against this class of disorders (6). A fundamental prerequisite to achieve these goals is a detailed understanding of the molecular mechanisms underlying the aberrant assembly of peptides and proteins, and how these mechanisms can be perturbed by the presence of other compounds. A crucial advance in this direction is presented in the PNAS report by Abelein et al. (7), which reveals the mechanism underlying the inhibition activity of the metal ion zinc against the aggregation of the amyloid β (Aβ) peptide, a peptide associated with Alzheimer’s disease.

Scaffolded multimers of hIAPP20–29 peptide fragments fibrillate faster and lead to different fibrils compared to the free hIAPP20–29 peptide fragment

Applying fibril-forming peptides in nanomaterial design is still challenged by the difficulties in understanding and controlling how fibrils form. The present work investigates the influence of motional restriction on peptide fibrillation. We use cyclotriphosphazene and cyclodextrin as templates to make conjugates of the fibril-forming core of human islet amyloid polypeptide. Attachment of the peptide to the templates resulted in multimers containing six peptide fragments at different positions. ThT fluorescence, CD and FTIR spectroscopy, and AFM and TEM imaging reveal that in both conjugates the peptide retained its fibrillating properties and formed fibrils. However, the conjugate fibrils formed more rapidly than the free peptide and were long and thin, as opposed to the thick and twisted morphology of the intact peptide. Thus the motional restrictions introduced by the scaffold modulate the structure of the fibrils but do not impede the actual fibrillation process.

The Molecular Basis For TGFBIp-Related Corneal Dystrophies

Abstract Several forms of the familial protein aggregation disease corneal dystrophy (CD) have been linked to mutations in transforming growth factor β-induced protein (TGFBIp). More than 30 point mutations in TGFBIp lead to CD, but the mutations induce many different aggregates in the cornea, ranging from granular to lattice and rod-like deposits. Biophysical methods have begun to help us elucidate how and why these mutations lead to polymorphic aggregates. Most CD-inducing mutations are found in the fourth fasciclin-1 domain of TGFBIp, and this domain also controls the stability of the entire TGFBIp molecule. Some mutations decrease TGFBIp stability, others increase it, and there is as yet no simple link between phenotype and stability. The mutations also affect surface electrostatics, proteolytic cleavage susceptibility, oligomerization propensities and interactions with other macromolecules. We highlight ways in which these changes can affect corneal aggregation. Future investigations will hopefully provide us with a clearer view of the links between biophysical properties and clinical manifestations.