Expression, purification and characterisation of large quantities of recombinant human IAPP for mechanistic studies.

Malfunction and amyloid formation of the Islet Amyloid Polypeptide (IAPP) are factors contributing to Type 2 diabetes. Unravelling the mechanism of IAPP aggregate formation may forward our understanding of this process and its effect on pancreatic ß-islet cell. Such mechanistic studies require access to sequence homogeneous and highly pure IAPP. Here we present a new facile protocol for the production of pure recombinant human IAPP at relatively high yield. The protocol uses a His-tagged version of the Npro mutant EDDIE, which drives expression to inclusion bodies, from which the peptide is purified using sonication, refolding and auto-cleavage, removal of EDDIE using Ni-NTA chromatography and reverse-phase HPLC. The purified material is used at multiple concentrations in aggregation kinetics measurements monitored by thioflavin-T fluorescence. Global analysis of the data implies a double nucleation aggregation mechanism including both primary and secondary nucleation.

Differential effects of serine side chain interactions in amyloid formation by islet amyloid polypeptide.

Islet amyloid polypeptide (IAPP), a 37 residue polypeptide, is the main protein component of islet amyloid deposits produced in the pancreas in Type 2 diabetes. Human IAPP contains five serine residues at positions 19, 20, 28, 29, and 34. Models of the IAPP amyloid fibril indicate a structure composed of two closely aligned columns of IAPP monomers with each monomer contributing to two intermolecular ß-strands. Ser 19 and Ser 20 are in the partially ordered ß-turn region, which links the two strands, whereas Ser 28, Ser 29, and Ser 34 are in the core region of the amyloid fibril. Ser 29 is involved in contacts between the two columns of monomers and is the part of the steric zipper interface. We undertook a study of individual serine substitutions with the hydrophobic isostere 2-aminobutyric acid (2-Abu) to examine the site-specific role of serine side chains in IAPP amyloid formation. All five variants formed amyloid. The Ser 19 to 2-Abu mutant accelerates amyloid formation by a factor of 3 to 4, while the Ser 29 to 2-Abu mutation modestly slows the rate of amyloid formation. 2-Abu replacements at the other sites had even smaller effects. The data demonstrate that the cross-column interactions made by residue 29 are not essential for amyloid formation and also show that cross-strand networks of hydrogen-bonded Ser side chains, so called Ser-ladders, are not required for IAPP amyloid formation. The effect of the Ser 19 to 2-Abu mutant suggests that residues in this region are important for amyloid formation by IAPP.

Unpacking the aggregation-oligomerization-fibrillization process of naturally-occurring hIAPP amyloid oligomers isolated directly from sera of children with obesity or diabetes mellitus.

The formation of amyloid oligomers and fibrils of the human islet amyloid polypeptide (hIAPP) has been linked with ß- cell failure and death which causes the onset, progression, and comorbidities of diabetes. We begin to unpack the aggregation-oligomerization-fibrillization process of these oligomers taken from sera of pediatric patients. The naturally occurring or real hIAPP (not synthetic) amyloid oligomers (RIAO) were successfully isolated, we demonstrated the presence of homo (dodecamers, hexamers, and trimers) and hetero-RIAO, as well as several biophysical characterizations which allow us to learn from the real phenomenon taking place. We found that the aggregation/oligomerization process is active in the sera and showed that it happens very fast. The RIAO can form fibers and react with anti-hIAPP and anti-amyloid oligomers antibodies. Our results opens the epistemic horizon and reveal real differences between the four groups (Controls vs obesity, T1DM or T2DM) accelerating the process of understanding and discovering novel and more efficient prevention, diagnostic, transmission and therapeutic pathways.

Study of forced degradation behavior of pramlintide acetate by HPLC and LC-MS.

Pramlintide acetate (Symlin®), a synthetic analogue of the human hormone amylin. It was approved in March 2005 as a subcutaneous injection for the adjunctive treatment of patients who have type 1 or 2 diabetes mellitus. The objective of current investigation was to study the degradation behavior of pramlintide acetate under different ICH recommended stress conditions by HPLC and LC-MS. Pramlintide acetate was subjected to stress conditions of hydrolysis (acidic or alkaline), oxidation, photolysis and thermal decomposition. Extensive degradation products were observed under the hydrolysis, oxidation or thermal stress conditions, while minimal degradation was found in the photolytic conditions. Successful separation of drug from the degradation products was achieved by the validated chromatography (RP-HPLC and SCX-HPLC) methods. Subsequent to isolation, the molecular weight of each component was determined by LC-MS. The LC-MS m/z values and fragmentation patterns of 4 impurities matched with the predicted degradation products of pramlintide acetate.

In Vitro Studies of Membrane Permeability Induced by Amyloidogenic Polypeptides Using Large Unilamellar Vesicles.

The process of amyloid formation is cytotoxic and contributes to a wide range of human diseases, but the mechanisms of amyloid-induced cytotoxicity are not well understood. It has been proposed that amyloidogenic peptides exert their toxic effects by damaging membranes. Membrane disruption is clearly not the only mechanism of toxicity, but the literature suggests that loss of membrane integrity may be a contributing factor. In this chapter we describe the measurement of in vitro membrane leakage induced by amyloidogenic proteins via the use of model vesicles. We use islet amyloid polypeptide (IAPP, amylin) as an example, but the methods are general.

Structural basis for the inhibition of truncated islet amyloid polypeptide aggregation by Cu(II): insights into the bioinorganic chemistry of type II diabetes.

Type 2 diabetes (T2D) is one of the most common chronic diseases, affecting over 300 million people worldwide. One of the hallmarks of T2D is the presence of amyloid deposits of human islet amyloid polypeptide (IAPP) in the islets of Langerhans of pancreatic ß-cells. Recent reports indicate that Cu(II) can inhibit the aggregation of human IAPP, although the mechanism for this inhibitory effect is not clear. In this study, different spectroscopic techniques and model fragments of IAPP were employed to shed light on the structural basis for the interaction of Cu(II) with human IAPP. Our results show that Cu(II) anchors to His18 and the subsequent amide groups toward the C-terminal, forming a complex with an equatorial coordination mode 3N1O at physiological pH. Cu(II) binding to truncated IAPP at the His18 region is the key event for its inhibitory effect in amyloid aggregation. Electron paramagnetic resonance studies indicate that the monomeric Cu(II)-IAPP(15-22) complex differs significantly from Cu(II) bound to mature IAPP(15-22) fibers, suggesting that copper binding to monomeric IAPP(15-22) competes with the conformation changes needed to form ß-sheet structures, thus delaying fibril formation. A general mechanism is proposed for the inhibitory effect of copper and other imidazole-binding metal ions in IAPP amyloid formation, providing further insights into the bioinorganic chemistry of T2D.

Cloning, expression and purification of the human Islet Amyloid Polypeptide (hIAPP) from Escherichia coli.

Type II diabetes is characterized by deposition of the hormone human Islet Amyloid Polypeptide (hIAPP). Formation of hIAPP amyloid fibrils and aggregates is considered to be responsible for pancreatic ß-cell losses. Therefore, insight into the structure of hIAPP in the solid-state and in solution is of fundamental importance in order to better understand the action of small molecules, which can potentially dissolve protein aggregates and modulate cell toxicity. So far, no procedure has been described that allows to obtain the native human IAPP peptide at high yields. We present here a cloning, expression and purification protocol that permits the production of 2.5 and 3mg of native peptide per liter of minimal and LB medium, respectively. In the construct, hIAPP is fused to a chitin binding domain (CBD). The CBD is subsequently cleaved off making use of intein splicing reaction which yield amidation of the C-terminus. The N-terminus contains a solubilization domain which is cleaved by V8 protease, avoiding additional residues at the N-terminus. The correct formation of the disulfide bond is achieved by oxidation with H2O2.

Dewetting transition assisted clearance of (NFGAILS) amyloid fibrils from cell membranes by graphene.

Clearance of partially ordered oligomers and monomers deposited on cell membrane surfaces is believed to be an effective route to alleviate many potential protein conformational diseases (PCDs). With large-scale all-atom molecular dynamics simulations, here we show that graphene nanosheets can easily and quickly win a competitive adsorption of human islet amyloid polypeptides (hIAPP22-28) NFGAILS and associated fibrils against cell membrane, due to graphene's unique two-dimensional, highly hydrophobic surface with its all-sp(2) hybrid structure. A nanoscale dewetting transition was observed at the interfacial region between the fibril (originally deposited on the membrane) and the graphene nanosheet, which significantly assisted the adsorption of fibrils onto graphene from the membrane. The π-π stacking interaction between Phe23 and graphene played a crucial role, providing the driving force for the adsorption at the graphene surface. This study renders new insight towards the importance of water during the interactions between amyloid peptides, the phospholipidic membrane, and graphene, which might shed some light on future developments of graphene-based nanomedicine for preventing/curing PCDs like type II diabetes mellitus.

O-acyl isopeptide method: development of an O-acyl isodipeptide unit for Boc SPPS and its application to the synthesis of Aß1-42 isopeptide.

The O-acyl isopeptide method was developed for the efficient preparation of difficult sequence-containing peptide. Furthermore, development of the O-acyl isodipeptide unit for Fmoc chemistry simplified its synthetic procedure by solid-phase peptide synthesis. Here, we report a novel isodipeptide unit for Boc chemistry, and the unit was successfully applied to the synthesis of amyloid ß peptide. Combination of Boc chemistry and the isodipeptide unit would be an effective method for the synthesis of many difficult peptides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Preparation of uniformly (13)C,(15)N-labeled recombinant human amylin for solid-state NMR investigation.

A number of diseases are caused by the formation of amyloid fibrils. Detailed understanding of structural features of amyloid fibers is of great importance for our understanding of disease progression and design of agents for diagnostics or potential prevention of protein aggregation. In lack of 3D crystal ordering, solid-state NMR forms the most suited method to determine the structures of the fibrils with atomic resolution. To exploit this potential, large amounts of isotopic-labeled protein need to be obtained through recombinant protein expression. However, expression and purification of amyloidogenic proteins in large amounts remains challenging due to their aggregation potential, toxicity for cells and difficult purification. In this work, we report a method for the production of large amounts of uniformly labeled (13)C,(15)N-human amylin, being one of the most amyloidogenic peptides known. This method utilizes inclusion bodies-directed expression and cheap chemical cleavage with cyanogen bromide in order to minimize the cost of the procedure compared to the use of less efficient proteolytic enzymes. We demonstrate the formation of amylin fibrils in vitro characterized using biophysical methods and electron microscopy, show toxicity towards human cells, and demonstrate that produced material may form the basis for structure determination using solid-state NMR.

Application of photochemical cross-linking to the study of oligomerization of amyloidogenic proteins.

Assembly of amyloidogenic proteins into toxic oligomers and fibrils is an important pathogenic feature of over 30 amyloid-related diseases. Understanding the structures and mechanisms involved in the assembly process is necessary for rational approaches geared at inhibiting formation of these toxic species. Here, we review the application of photo-induced cross-linking of unmodified proteins (PICUP) to two disease-related amyloidogenic proteins (1) islet amyloid polypeptide (IAPP), whose toxic oligomers are thought to cause the demise of pancreatic ß-cells in type-2 diabetes mellitus and (2) α-synuclein, which aggregates into toxic oligomers and precipitates in Lewy bodies in Parkinson's disease. PICUP is an effective method allowing chemical "freezing" of dynamically changing oligomers and subsequent study of the oligomer size distribution that existed before cross-linking. The method has provided insights into the factors controlling early oligomerization, which could not be obtained by other means. We discuss sample preparation, experimental details, optimization of parameters, and troubleshooting.