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UBC Theses and Dissertations

Domains in islet amyloid polypeptide important in fibril formation and toxicity Park, Kirily


Islet amyloid deposits are a characteristic pathologic lesion o f the pancreas in type 2 diabetes and are composed primarily of the islet beta cell peptide islet amyloid polypeptide (IAPP or amylin). Islet amyloid fibrils are toxic to insulin-producing beta cells. Heparan sulfate proteoglycans (HSPGs) are also a component of islet amyloid in vivo and accelerate amyloid fibril formation in vitro. Although the cause of amyloid formation in pancreatic islets is unknown, HSPGs have been proposed to play an initiating role. Impaired processing of proIAPP, the IAPP precursor, has also been implicated in the mechanism of islet amyloid formation. The N - and C-terminal cleavage sites at which proIAPP is processed by prohormone convertases contain series of basic amino acid residues which we hypothesized may interact with heparan sulfate proteoglycans. We found that a monomeric fragment of proIAPP (residues 1-30) bound both heparin and heparan sulfate affinity columns. Substitution of alanine residues for two basic residues in the N-terminal cleavage site abolished heparin and heparan sulfate binding activity. These data suggest that monomeric N-terminal human proIAPP contains a heparin-binding domain that is lost during normal processing of proIAPP. Although islet amyloid fibrils bind to heparin and heparan sulfate, it is unknown which residues in IAPP may be responsible for this interaction or whether nonfibrillar (monomeric) human IAPP also binds to heparin. Three basic residues in human IAPP (Lys1 , A r g 1 1 , and His1 8 ) could potentially contribute to this binding by interacting with negatively charged sulfate groups in heparin. We found heparin significantly enhanced fibril formation by IAPP 1-37 and 11-37, but not IAPP 12-37, suggesting that A r g 1 1 may be important in the ability of heparin to enhance IAPP fibril formation. Fibrils generated from IAPP 20-29 (which contains no basic residues) did not bind heparin, supporting the possibility that specific basic residues within IAPP may be important in the formation of heparin binding domains within amyloid fibrils. These findings suggest that basic amino acids may play important roles in IAPP and amyloid fibril interaction with heparin/heparan sulfate and may contribute to islet amyloid formation. A point mutation (serine to glycine at position 20) in IAPP has been identified in a subpopulation of Japanese patients with a severe, early onset form of type 2 diabetes. We find S20G IAPP, when applied to INS-1 beta cells in culture, is cytotoxic at lower concentrations than wild-type IAPP. Using electron microscopy and thioflavin T assays, we also found that the S20G mutant fibrils form much more rapidly. Increased fibrillogenicity of S20G IAPP could cause the increased beta cell toxicity of this peptide and may contribute to early beta cell death and onset of diabetes in patients carrying this mutation. In conclusion, we speculate that i f N-terminally extended proIAPP is secreted in excessive amounts from the beta cell in type 2 diabetes, this peptide may be retained by heparan sulfate proteoglycans in the extracellular matrix and form a nidus for subsequent islet amyloid formation. The possible importance of the three basic residues in mature IAPP in amyloid fibril formation suggests potential therapeutic targets in preventing islet amyloid formation and beta cell death in type 2 diabetes. Finally, IAPP toxicity is increased under conditions that hasten the onset of fibrillogenesis, such as a substitution of glycine for serine at position 20 of the molecule. Domains in IAPP and proIAPP involved in H S PG binding, fibrillogenesis, and toxicity may make novel therapeutic targets for inhibition o f islet amyloid formation and the progressive loss of beta cells in type 2 diabetes.

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