The formation of amyloid fibrils from proteins in the lysozyme family.

Amyloid fibrils are highly ordered protein assemblies known to contribute to the pathology of a variety of genetic and aging-associated diseases. More recently, these fibrils have been shown to be useful as structural scaffolds in both natural biological systems and nanotechnology applications. The intense interest in amyloid fibrils has led to the investigation of well-characterized proteins, such as hen egg white lysozyme (HEWL), as model systems to examine structural and mechanistic principles that may be generally applicable to all amyloid fibrils. The purpose of this review is to critically examine the fibril-formation literature of proteins in the lysozyme family with respect to the known structure and folding properties of these proteins. The goal is to identify similarities and differences within the family, examine general misfolding / aggregation principles, and identify key areas of importance for future work on the fibril formation of these proteins.

The component polypeptide chains of bovine insulin nucleate or inhibit aggregation of the parent protein in a conformation-dependent manner.

Amyloid fibrils are typically rigid, unbranched structures with diameters of approximately 10 nm and lengths up to several micrometres, and are associated with more than 20 diseases including Alzheimer's disease and type II diabetes. Insulin is a small, predominantly alpha-helical protein consisting of 51 residues in two disulfide-linked polypeptide chains that readily assembles into amyloid fibrils under conditions of low pH and elevated temperature. We demonstrate here that both the A-chain and the B-chain of insulin are capable of forming amyloid fibrils in isolation under similar conditions, with fibrillar morphologies that differ from those composed of intact insulin. Both the A-chain and B-chain fibrils were found to be able to cross-seed the fibrillization of the parent protein, although these reactions were substantially less efficient than self-seeding with fibrils composed of full-length insulin. In both cases, the cross-seeded fibrils were morphologically distinct from the seeding material, but shared common characteristics with typical insulin fibrils, including a very similar helical repeat. The broader distribution of heights of the cross-seeded fibrils compared to typical insulin fibrils, however, indicates that their underlying protofilament hierarchy may be subtly different. In addition, and remarkably in view of this seeding behavior, the soluble forms of the A-chain and B-chain peptides were found to be capable of inhibiting insulin fibril formation. Studies using mass spectrometry suggest that this behavior might be attributable to complex formation between insulin and the A-chain and B-chain peptides. The finding that the same chemical form of a polypeptide chain in different physical states can either stimulate or inhibit the conversion of a protein into amyloid fibrils sheds new light on the mechanisms underlying fibril formation, fibril strain propagation and amyloid disease initiation and progression.

Insulin amyloid at injection sites of patients with diabetes.

The formation of insulin amyloid can dramatically impact glycemic control in patients with diabetes, making it an important therapeutic consideration. In addition, the cost associated with the excess insulin required by patients with amyloid is estimated to be $3K per patient per year, which adds to the growing financial burden of this disease. Insulin amyloid has been observed with every mode of therapeutic insulin administration (infusion, injection and inhalation), and the number of reported cases has increased significantly since 2002. The new cases represent a much broader demographic, and include many patients who have used exclusively human insulin and human insulin analogs. The reason for the increase in case reports is unknown, but this review explores the possibility that changes in patient care, improved differential diagnosis and/or changes in insulin type and insulin delivery systems may be important factors. The goal of this review is to raise key questions that will inspire proactive measures to prevent, identify and treat insulin amyloid. Furthermore, this comprehensive examination of insulin amyloid can provide insight into important considerations for other injectable drugs that are prone to form amyloid deposits.

Pancreatic beta-cell granule peptides form heteromolecular complexes which inhibit islet amyloid polypeptide fibril formation.

Islet amyloid polypeptide (IAPP), or 'amylin', is co-stored with insulin in secretory granules of pancreatic islet beta-cells. In Type 2 diabetes, IAPP converts into a beta-sheet conformation and oligomerizes to form amyloid fibrils and islet deposits. Granule components, including insulin, inhibit spontaneous IAPP fibril formation in vitro. To determine the mechanism of this inhibition, molecular interactions of insulin with human IAPP (hIAPP), rat IAPP (rIAPP) and other peptides were examined using surface plasmon resonance (BIAcore), CD and transmission electron microscopy (EM). hIAPP and rIAPP complexed with insulin, and this reaction was concentration-dependent. rIAPP and insulin, but not pro-insulin, bound to hIAPP. Insulin with a truncated B-chain, to prevent dimerization, also bound hIAPP. In the presence of insulin, hIAPP did not spontaneously develop beta-sheet secondary structure or form fibrils. Insulin interacted with pre-formed IAPP fibrils in a regular repeating pattern, as demonstrated by immunoEM, suggesting that the binding sites for insulin remain exposed in hIAPP fibrils. Since rIAPP and hIAPP form complexes with insulin (and each other), this could explain the lack of amyloid fibrils in transgenic mice expressing hIAPP. It is likely that IAPP fibrillogenesis is inhibited in secretory granules (where the hIAPP concentration is in the millimolar range) by heteromolecular complex formation with insulin. Alterations in the proportions of insulin and IAPP in granules could disrupt the stability of the peptide. The increase in the proportion of unprocessed pro-insulin produced in Type 2 diabetes could be a major factor in destabilization of hIAPP and induction of fibril formation.

Chemical modification of insulin in amyloid fibrils.

We have investigated the chemical modification of insulin under conditions that promote the conversion of the soluble protein into amyloid fibrils. The modifications that are incorporated into the fibrils include deamidation of Asn A21, Asn B3, and Gln B4. In order to prepare fibrils with minimal deamidation of these residues, the kinetics of aggregation were accelerated by seeding with aliquots of a solution containing preformed fibrils. The resulting fibrils were then reincubated to determine the extent to which chemical modification occurs in the fibril itself. The deamidation of Asn A21 in particular could be followed in detail. Deamidation of this residue in the fibrillar form of insulin was found to occur in only 52 +/- 5% of molecules. This result indicates that there are at least two different packing environments of insulin molecules in the fibrils and suggests that the characterization of chemical modifications may be a useful probe of the environment of polypeptide chains within amyloid fibrils.

Low levels of asparagine deamidation can have a dramatic effect on aggregation of amyloidogenic peptides: implications for the study of amyloid formation.

The polypeptide hormone amylin forms amyloid deposits in Type 2 diabetes mellitus and a 10-residue fragment of amylin (amylin(20-29)) is commonly used as a model system to study this process. Studies of amylin(20-29) and several variant peptides revealed that low levels of deamidation can have a significant effect on the secondary structure and aggregation behavior of these molecules. Results obtained with a variant of amylin(20-29), which has the primary sequence SNNFPAILSS, are highlighted. This peptide is particularly interesting from a technical standpoint. In the absence of impurities the peptide does not spontaneously aggregate and is not amyloidogenic. This peptide can spontaneously deamidate, and the presence of less than 5% of deamidation impurities leads to the formation of aggregates that have the hallmarks of amyloid. In addition, small amounts of deamidated material can induce amyloid formation by the purified peptide. These results have fundamental implications for the definition of an amyloidogenic sequence and for the standards of purity of peptides and proteins used for studies of amyloid formation.

Techniques to study amyloid fibril formation in vitro.

Amyloid fibrils are ordered aggregates of peptides or proteins that are fibrillar in structure and contribute to the complications of many diseases (e.g., type 2 diabetes mellitus, Alzheimer's disease, and primary systemic amyloidosis). These fibrils can also be prepared in vitro and there are three criteria that define a protein aggregate as an amyloid fibril: green birefringence upon staining with Congo Red, fibrillar morphology, and beta-sheet secondary structure. The purpose of this review is to describe the techniques used to study amyloid fibril formation in vitro, address common errors in the collection and interpretation of data, and open a discussion for a critical review of the criteria currently used to classify a protein aggregate as an amyloid fibril.

In vitro characterization of lactoferrin aggregation and amyloid formation.

Lactoferrin has previously been identified in amyloid deposits in the cornea, seminal vesicles, and brain. We report in this paper a highly amyloidogenic region of lactoferrin (sequence of NAGDVAFV). This region was initially identified by sequence comparison with medin, a 5.5 kDa amyloidogenic fragment derived from lactadherin. Subsequent characterization revealed that this peptide forms amyloid fibrils at pH 7.4 when incubated at 37 degrees C. Furthermore, although full-length lactoferrin does not by itself form amyloid fibrils, the protein does bind to the peptide fibrils as revealed by an increase in thioflavin T fluorescence and the presence of enlarged fibrils by transmission electron microscopy and polarized light microscopy. The binding of lactoferrin is a selective interaction with the NAGDVAFV fibrils. Lactoferrin does not bind to insulin or lysozyme fibrils, and the NAGDVAFV fibrils do not bind to soluble insulin or lysozyme. The lactoferrin appears to coat the peptide fibril surface to form mixed peptide/protein fibrils, but again there is no evidence for the formation of lactoferrin-only fibrils. This interaction, therefore, seems to involve selective binding rather than conventional seeding of fibril formation. We suggest that such a process could be generally important in the formation of amyloid fibrils in vivo since the identification of both full-length protein and protein fragments is common in ex vivo amyloid deposits.