The inverse scattering problem is based on the scattering theory in physics, where measured data such as radiation from an object is used to determine the unique structure of the object in question. This approach has been widely successful in fields ranging from geophysics and medical imaging, to quantum field theory. In 1996 Henrik Flyvbjerg suggested that a similar approach could be used to study a reaction far from equilibrium of the self-assembly of a nucleation dependent biopolymer and, under certain conditions, uniquely determine the kinetics of the assembly. Here we use this approach to elucidate the unique structure of human islet amyloid polypeptide, also known as amylin, in-vitro. We use a systematic phenomenological analysis of the amount of monomer in fibril, of amylin, for various initial concentrations from an unstructured monomer pool. Using the assumption that nucleation is the rate-limiting step in fibril formation, we invoke mass action to develop our model. We find that the fibrillogenesis of amylin is well described by a nucleation dependent polymerization event that is characteristic of the sigmoidal shape of the reaction profile generated by our data. Furthermore, we find a second nucleation event is needed to accurately match model predictions to the observed data for the kinetic profiles of fibril formation, and the experimental length distributions of mature fibrils from in-vitro assays. This analysis allows for the theoretical determination of each step of assembly in the nucleation process. Specifically, we find the number of steps to nucleation, the size of each oligomer formed in the nucleation process, the nucleus size, and the elongation kinetics of fibrils. The secondary nucleation process is found to be a fibril dependent surface mediated nucleation event and is similar in reaction order to the primary nucleation step. Model predictions are found to be congruent with experimental assay results of oligomer populations and monomer concentration. We demonstrate that, a persistent oligomer formation is a natural and necessary consequence of nucleated fibril formation, given certain qualitative features of the kinetic profile of fibril formation. Furthermore, the modeling assumptions about monomer and fibril mass are in agreement with experiment.

The inverse scattering problem is based on the scattering theory in physics, where measured data such as radiation from an object is used to determine the unique structure of the object in question. This approach has been widely successful in fields ranging from geophysics and medical imaging, to quantum field theory. In 1996 Henrik Flyvbjerg suggested that a similar approach could be used to study a reaction far from equilibrium of the self-assembly of a nucleation dependent biopolymer and, under certain conditions, uniquely determine the kinetics of the assembly. Here we use this approach to elucidate the unique structure of human islet amyloid polypeptide, also known as amylin, in-vitro. We use a systematic phenomenological analysis of the amount of monomer in fibril, of amylin, for various initial concentrations from an unstructured monomer pool. Using the assumption that nucleation is the rate-limiting step in fibril formation, we invoke mass action to develop our model. We find that the fibrillogenesis of amylin is well described by a nucleation dependent polymerization event that is characteristic of the sigmoidal shape of the reaction profile generated by our data. Furthermore, we find a second nucleation event is needed to accurately match model predictions to the observed data for the kinetic profiles of fibril formation, and the experimental length distributions of mature fibrils from in-vitro assays. This analysis allows for the theoretical determination of each step of assembly in the nucleation process. Specifically, we find the number of steps to nucleation, the size of each oligomer formed in the nucleation process, the nucleus size, and the elongation kinetics of fibrils. The secondary nucleation process is found to be a fibril dependent surface mediated nucleation event and is similar in reaction order to the primary nucleation step. Model predictions are found to be congruent with experimental assay results of oligomer populations and monomer concentration. We demonstrate that, a persistent oligomer formation is a natural and necessary consequence of nucleated fibril formation, given certain qualitative features of the kinetic profile of fibril formation. Furthermore, the modeling assumptions about monomer and fibril mass are in agreement with experiment.

The inverse scattering problem is based on the scattering theory in physics, where measured data such as radiation from an object is used to determine the unique structure of the object in question. This approach has been widely successful in fields ranging from geophysics and medical imaging, to quantum field theory. In 1996 Henrik Flyvbjerg suggested that a similar approach could be used to study a reaction far from equilibrium of the self-assembly of a nucleation dependent biopolymer and, under certain conditions, uniquely determine the kinetics of the assembly. Here we use this approach to elucidate the unique structure of human islet amyloid polypeptide, also known as amylin, in-vitro. We use a systematic phenomenological analysis of the amount of monomer in fibril, of amylin, for various initial concentrations from an unstructured monomer pool. Using the assumption that nucleation is the rate-limiting step in fibril formation, we invoke mass action to develop our model. We find that the fibrillogenesis of amylin is well described by a nucleation dependent polymerization event that is characteristic of the sigmoidal shape of the reaction profile generated by our data. Furthermore, we find a second nucleation event is needed to accurately match model predictions to the observed data for the kinetic profiles of fibril formation, and the experimental length distributions of mature fibrils from in-vitro assays. This analysis allows for the theoretical determination of each step of assembly in the nucleation process. Specifically, we find the number of steps to nucleation, the size of each oligomer formed in the nucleation process, the nucleus size, and the elongation kinetics of fibrils. The secondary nucleation process is found to be a fibril dependent surface mediated nucleation event and is similar in reaction order to the primary nucleation step. Model predictions are found to be congruent with experimental assay results of oligomer populations and monomer concentration. We demonstrate that, a persistent oligomer formation is a natural and necessary consequence of nucleated fibril formation, given certain qualitative features of the kinetic profile of fibril formation. Furthermore, the modeling assumptions about monomer and fibril mass are in agreement with experiment. / Science, Faculty of / Mathematics, Department of / Graduate

abstract: Human islet amyloid polypeptide (hIAPP), also known as amylin, is a 37-residue intrinsically disordered hormone involved in glucose regulation and gastric emptying. The aggregation of hIAPP into amyloid fibrils is believed to play a causal role in type 2 diabetes. To date, not much is known about the monomeric state of hIAPP or how it undergoes an irreversible transformation from disordered peptide to insoluble aggregate. IAPP contains a highly conserved disulfide bond that restricts hIAPP(1-8) into a short ring-like structure: N_loop. Removal or chemical reduction of N_loop not only prevents cell response upon binding to the CGRP receptor, but also alters the mass per length distribution of hIAPP fibers and the kinetics of fibril formation. The mechanism by which N_loop affects hIAPP aggregation is not yet understood, but is important for rationalizing kinetics and developing potential inhibitors. By measuring end-to-end contact formation rates, Vaiana et al. showed that N_loop induces collapsed states in IAPP monomers, implying attractive interactions between N_loop and other regions of the disordered polypeptide chain . We show that in addition to being involved in intra-protein interactions, the N_loop is involved in inter-protein interactions, which lead to the formation of extremely long and stable β-turn fibers. These non-amyloid fibers are present in the 10 μM concentration range, under the same solution conditions in which hIAPP forms amyloid fibers. We discuss the effect of peptide cyclization on both intra- and inter-protein interactions, and its possible implications for aggregation. Our findings indicate a potential role of N_loop-N_loop interactions in hIAPP aggregation, which has not previously been explored. Though our findings suggest that N_loop plays an important role in the pathway of amyloid formation, other naturally occurring IAPP variants that contain this structural feature are incapable of forming amyloids. For example, hIAPP readily forms amyloid fibrils in vitro, whereas the rat variant (rIAPP), differing by six amino acids, does not. In addition to being highly soluble, rIAPP is an effective inhibitor of hIAPP fibril formation . Both of these properties have been attributed to rIAPP's three proline residues: A25P, S28P and S29P. Single proline mutants of hIAPP have also been shown to kinetically inhibit hIAPP fibril formation. Because of their intrinsic dihedral angle preferences, prolines are expected to affect conformational ensembles of intrinsically disordered proteins. The specific effect of proline substitutions on IAPP structure and dynamics has not yet been explored, as the detection of such properties is experimentally challenging due to the low molecular weight, fast reconfiguration times, and very low solubility of IAPP peptides. High-resolution techniques able to measure tertiary contact formations are needed to address this issue. We employ a nanosecond laser spectroscopy technique to measure end-to-end contact formation rates in IAPP mutants. We explore the proline substitutions in IAPP and quantify their effects in terms of intrinsic chain stiffness. We find that the three proline mutations found in rIAPP increase chain stiffness. Interestingly, we also find that residue R18 plays an important role in rIAPP's unique chain stiffness and, together with the proline residues, is a determinant for its non-amyloidogenic properties. We discuss the implications of our findings on the role of prolines in IDPs. / Dissertation/Thesis / Ph.D. Physics 2013

Islet amyloid polypeptide (IAPP) is a 37-residue amyloidogenic hormone implicated in the progression of Type II Diabetes (T2D). T2D affects an estimated 422 million people yearly and is a co-morbidity with numerous diseases. IAPP forms toxic oligomers and amyloid fibrils that reduce pancreatic β-cell mass and exacerbate the T2D disease state. Toxic oligomer formation is attributed, in part, to the formation of inter-peptide β-strands comprised of residues 23-27 (FGAIL). Flavonoids, a class of polyphenolic natural products, have been found experimentally to inhibit IAPP aggregate formation. Many of these known IAPP aggregation attenuating small flavonoids differ structurally only slightly; the influence of functional group placement on inhibiting the aggregation of the IAPP(20-29) has yet to be explored. To probe the role of small-molecule structural features that impede IAPP aggregation, molecular dynamics (MD) simulations were performed on a model fragment of IAPP(20-29) in the presence of morin, quercetin, dihydroquercetin, epicatechin, and myricetin. Contacts between Phe23 residues are critical to oligomer formation, and small-molecule contacts with Phe23 are a key predictor of β-strand reduction. Structural properties influencing the ability of compounds to disrupt Phe23-Phe23 contacts include carbonyl and hydroxyl group placement. These structural features influence aromaticity and hydrophobicity, principally affecting ability to disrupt IAPP(20-29) oligomer formation. This work provides key information on design considerations for T2D therapeutics. / Master of Science in Life Sciences / Type II Diabetes (T2D) affects an estimated 422 million people worldwide, with the World Health Organization (WHO) reporting that approximately 1.5 million deaths were directly caused by T2D in 2019. The progression of T2D has been attributed to a protein, called islet amyloid polypeptide (IAPP, or amylin) that is co-secreted with insulin after individuals eat or consumes calories. IAPP has been discovered to form toxic aggregates or clumps of protein material that worsen the disease state and cause a loss of mass of pancreatic cells. There is a large market for therapeutics of T2D and more small molecule drugs are needed to slow progression and severity of T2D. Flavonoids, a class of natural molecules, have been found to inhibit the processes by which IAPP promotes T2D disease progression by stopping the aggregation of IAPP. The structures of these flavonoid compounds differ slightly but show difference in ability to slow IAPP aggregation. By understanding how those differences confer more or less protection against T2D and inhibit IAPP aggregation, we can design more potent and specific drugs to target IAPP. To probe the role of molecular structure in preventing IAPP aggregation, molecular dynamics (MD) simulations — a powerful computational technique — were performed on a model fragment of IAPP in the presence of molecules morin, quercetin, dihydroquercetin, epicatechin, and myricetin. MD simulations provide extremely detailed information about potential drug interactions with a given target, serving as an important tool in the development of new drugs. This work has identified key features and predictors of effective IAPP drugs, providing a framework for the further development of therapeutics against T2D and similar diseases.

De nombreuses protéines associées aux maladies neurodégénératives humaines sont intrinsèquement désordonnées. Ce sont des protéines qui sont dépourvues de structure tertiaire ou secondaire stable dans des conditions physiologiques. Plus précisément, les protéines intrinsèquement désordonnées (IDPs) subissent diverses changements conformationnels entre la pelote aléatoire, des conformations hélicoïdales et des structures en feuillet-β, ces deux dernières étant généralement impliquées dans la reconnaissance protéine-protéine. Parmi une vingtaine de peptides amyloïdogènes connus liés aux maladies dégénératives humaines, notre étude porte sur deux protéines désordonnées: le peptide Amyloïde-β (Aβ) associé à la maladie d'Alzheimer et l'Islet Amyloid Polypeptide (IAPP) impliqué dans le diabète de type II. Aβ possède deux alloformes courants de 40 et 42 résidus, tandis que IAPP est une hormone peptidique de 37 résidus. Les agrégats de Aβ sont toxiques pour les cellules du cerveau, tandis que la fibrillisation de IAPP affecte les cellules-β du pancréas. Le mécanisme d'agrégation de ces deux peptides reste encore mal connu, mais il a été proposé qu’en solution, ces peptides visitent différentes conformations, l'une d'entre elles étant riche en feuillets-β. Cela conduirait à l’oligomérisation de ces peptides, par le biais d’interactions feuillet-β / feuillet-β et, éventuellement, à la formation de fibrilles. Le but de notre étude est de mieux caractériser la dynamique conformationnelle de ces deux peptides, dans leur forme monomérique et oligomérique. Comprendre les premières étapes de leur agrégation est crucial pour le développement de nouvelles molécules thérapeutiques efficaces contre ces protéines amyloïdes. / Many proteins associated with human neurodegenerative diseases are intrinsically disordered. They are proteins which lack stable tertiary or secondary structure under physiological conditions. More specifically, intrinsically disordered proteins (IDPs) undergo various structural conversions between random coil, helical conformations and β-strand structures, these two latter being generally involved in protein-protein recognition. Among about twenty known amyloidogenic peptides related to human degenerative diseases, we focus our study on two disordered proteins: the Amyloid-β peptide (Aβ) associated to the Alzheimer’s disease and the Islet Amyloid Polypeptide (IAPP) involved in type II diabetes. Aβ has two common alloforms of 40 and 42 residues in length, meanwhile IAPP is a 37-residues peptide hormone. Aggregates of Aβ are toxic to the brain cells, meanwhile IAPP fibrillization affects the pancreatic β-cells. The aggregation mechanism of these two peptides is not known in detail, but it was proposed that in solution, these peptides visit various conformations, one of them being rich in β-strands. This would lead to peptide oligomerization, through β-strand / β-strand interactions and eventually to the fibril formation. The aim of our study is to provide insights into the conformational dynamics of these two peptides in monomeric and oligomeric forms. Understanding the early steps of their aggregation is crucial for the development of new effective therapeutic molecules against these amyloid proteins.De nombreuses protéines associées aux maladies neurodégénératives humaines sont intrinsèquement désordonnées. Ce sont des protéines qui sont dépourvues de structure tertiaire ou secondaire stable dans des conditions physiologiques. Plus précisément, les protéines intrinsèquement désordonnées (IDPs) subissent diverses changements conformationnels entre la pelote aléatoire, des conformations hélicoïdales et des structures en feuillet-β, ces deux dernières étant généralement impliquées dans la reconnaissance protéine-protéine. Parmi une vingtaine de peptides amyloïdogènes connus liés aux maladies dégénératives humaines, notre étude porte sur deux protéines désordonnées: le peptide Amyloïde-β (Aβ) associé à la maladie d'Alzheimer et l'Islet Amyloid Polypeptide (IAPP) impliqué dans le diabète de type II. Aβ possède deux alloformes courants de 40 et 42 résidus, tandis que IAPP est une hormone peptidique de 37 résidus. Les agrégats de Aβ sont toxiques pour les cellules du cerveau, tandis que la fibrillisation de IAPP affecte les cellules-β du pancréas. Le mécanisme d'agrégation de ces deux peptides reste encore mal connu, mais il a été proposé qu’en solution, ces peptides visitent différentes conformations, l'une d'entre elles étant riche en feuillets-β. Cela conduirait à l’oligomérisation de ces peptides, par le biais d’interactions feuillet-β / feuillet-β et, éventuellement, à la formation de fibrilles. Le but de notre étude est de mieux caractériser la dynamique conformationnelle de ces deux peptides, dans leur forme monomérique et oligomérique. Comprendre les premières étapes de leur agrégation est crucial pour le développement de nouvelles molécules thérapeutiques efficaces contre ces protéines amyloïdes.

Transplantation of pancreatic islets is a potential treatment of type 1 diabetes that aims to restore normal blood glucose control. By encapsulating the islets in alginate, they can be protected from rejection. The aim of this thesis was to study the biology of encapsulated islets and to use the technique of microencapsulation to study the effect of transplantation in a system that is separated from direct contact with the vascular system and the host tissue at the transplantation site. Encapsulated islets can effectively reverse hyperglycaemia after transplantation into the peritoneal cavity of diabetic mice. A period of culture before encapsulation and transplantation did not affect their insulin release or curative capability. Pre-culture with exendin-4 improved insulin secretion, but not to the extent that the long term outcome in our transplantation model was improved. Despite being able to reach and retain normoglycaemia, microencapsulated islets transplanted intraperitoneally decreased in size. More specifically the number of beta cells in each individual islet was decreased. However, in contrast to previous studies using non-encapsulated islets, the alpha cell number was maintained, and thus the capsule seems to protect these peripherally located and otherwise exposed cells. As the capsule also prevents revascularisation of the islets, the model was used to study the importance of vascular supply for islet amyloid formation. Islet amyloid is a possible reason for the long-term failure of transplanted islets. It is likely that their low vascular density causes a disturbed local clearance of IAPP and insulin that starts the aggregation of IAPP. Indeed, encapsulated islets had an accelerated amyloid formation compared to normal islets, and might serve as a model for further studies of this process. In conclusion, although revascularisation is not a prerequisite for islet graft function, it plays an important role for islet transplantation outcome.

The misfolding and aggregation of the beta cell hormone islet amyloid polypeptide (IAPP) into amyloid fibrils is the main pathological finding in islets of Langerhans in type 2 diabetes. Pathological assemblies of IAPP are cytotoxic and believed to contribute to the loss of insulin-producing beta cells. Changes in the microenvironment that could trigger the aggregation of IAPP are largely unknown. So is the possibility that islet amyloid can spread within or between tissues. The present thesis have explored the roles of glycosaminoglycan heparan sulfate (HS) and the novel anti-amyloid chaperone Bri2 BRICHOS domain in the assembly of IAPP amyloid and cytotoxic IAPP aggregates. Furthermore, cross-seeding as a molecular interaction between the observed connection of type 2 diabetes and Alzheimer disease has been examined. The N-terminal region of IAPP was required for binding to HS structures and induction of binding promoted amyloid formation. Interference in the HS-IAPP interaction by heparanase degradation of HS or by introducing short, soluble HS-structure fragments reduced amyloid deposition in cultured islets. Cytotoxicity induced by extracellular, aggregating IAPP was mediated via interactions with cell-surface HS. This suggests that HS plays an important role in islet amyloid deposition and associated toxicity. BRICHOS domain containing protein Bri2 was highly expressed in human beta cells and colocalized with IAPP intracellularly and in islet amyloid deposits. The BRICHOS domain effectively attenuated both IAPP amyloid formation and IAPP-induced cytotoxicity. These results propose Bri2 BRICHOS as a novel chaperone preventing IAPP aggregation in beta cells. The intravenous injection of IAPP, proIAPP or amyloid-β (Aβ) fibrils enhanced islet amyloidosis in transgenic human IAPP mice, demonstrating that both homologous- and heterologous seeding of islet amyloid can occur in vivo. IAPP colocalized with Aβ in brain amyloid from AD patients, and AD patients diagnosed with T2D displayed increased proportions of neuritic plaques, the more pathogenic plaque subtype. In conclusion, both IAPP amyloid formation and the cytotoxic effects of IAPP is dependent on interactions with HS whereas interactions with Bri2 BRICHOS is protective. Cross-seeding between Aβ and IAPP can occur in vivo and the two peptides colocalize in brain amyloid in AD patients.

Type II diabetes mellitus (T2D) and Alzheimer's disease (AD) share aetiology and have a high incidence of co-morbidity. Evidence suggests that both diseases are caused by the pathogenic aggregation of an intrinsically disordered native amyloid peptide. Furthermore, T2D and AD share risk factors such as age, obesity and vascular health. Recent studies demonstrate that amylin, an amyloidogenic pancreatic hormone deposited in the pancreas in T2D, is also deposited in the brain in AD. We hypothesised that amylin directly contributes to AD through deposition in the brain and activation of pathogenic signalling cascades. We provide evidence to validate that amylin is deposited in the brain parenchyma and vasculature. Furthermore, we present data demonstrating amylin (IAPP) expression in the brain is significantly elevated in AD; and that amylin treatment increases amyloid-beta (AB) secretion in neuronal culture. Soluble oligomeric species of AB cause AD by initiation of downstream signalling cascades that dysregulate kinase activity, promote tau phosphorylation and result in neuronal death. One such pathway involves AB oligomer activation of the Src-family kinase Fyn, through binding to the cellular prion protein (PrPC) receptor complex. We provide evidence that amylin activates Fyn in neuroblastoma and stem cell derived neurons, this activation is possibly mediated through PrPC. Together the data presented in this thesis demonstrate multiple modes of action whereby amylin may directly propagate or indirectly exacerbate AD-associated processes. Amylin aggregation, deposition, up-regulation and signalling should be considered one of several links between T2D and AD. The pathogenic actions of AB and amylin are mediated by oligomer species. Therefore therapeutics which prevent oligomerisation or oligomer action may be valuable in AD and T2D. One such class of therapeutic are flavonoids. Our collaborators have recently demonstrated the flavonoids rutin and quercetin reduce amylin aggregation and extend lifespan in diabetic animal models. As a result of this we investigated the anti-amyloidogenic and anti-oligomeric properties of the flavonoid quercetin against AB. Quercetin treatment prevented AB oligomerisation, cell binding of pre-formed AB oligomers and also reduced APP processing in cell models. These data suggest quercetin is a multimodal therapeutic with potential utility in AD and T2D and should be explored for further drug development.

Aggregation of proteins into amyloid deposits is a common feature among dozens of diseases. Two such diseases that feature amyloid deposits are Alzheimer's disease (AD) and type 2 diabetes (T2D). AD toxicity has been associated with the aggregation and accumulation of the amyloid β-peptide (Aβ); Aβ exerts its toxic effects through interactions with neuronal cell membranes. A characteristic feature of T2D is the deposition of the islet amyloid polypeptide (IAPP) in the pancreatic islets of Langerhans. It is currently unknown if IAPP aggregation is a cause or consequence of T2D, but it does lead to β-cell dysfunction and death, exacerbating the effects of diabetes. Characterizing the fundamental interactions between both Aβ and IAPP with lipid membranes and in solution will give greater insight into mechanisms of toxicity exhibited by amyloid proteins. In this work, molecular dynamics (MD) simulations were used to study the secondary, tertiary, and quatnary structure of Aβ and IAPP, in addition to peptide-membrane interactions and membrane perturbation as independently caused by both peptides. Studies were conducted to address the following questions: (1) what influence do solution conditions and oxidation state have on monomeric Aβ] (2) how and in what way does monomeric Aβ interact with model lipid membranes and what role does sequence play on these peptide-membrane interactions; (3) can MD simulations be utilized to understand Aβ tetramer formation, rearrangement, and tetramer-membrane interactions; (4) how does IAP interact with model membranes and how does that vary from non-toxic (rat) IAPP peptide-membrane interactions. These studies led to conclusions that showed variance in lipid affinity and degree of perturbation as based on peptide sequence, in addition to insight into the type of perturbation caused to membranes by these amyloid peptides. Understanding the differences in peptide-membrane interactions of amyloidogenic and non-amyloidogenic (rat) peptides gave insight into the overall mechanism of amyloidogenicity, leading to the detection of specific amino acids essential in peptide-membrane perturbation. These residues can then be targeted for novel therapeutic design to attenuate the perturbation and potential cell death as caused by these peptides. / Ph. D.