Andisheh Abedini

The flavanol (-)-epigallocatechin 3-gallate inhibits amyloid formation by islet amyloid polypeptide, disaggregates amyloid fibrils, and protects cultured cells against IAPP-induced toxicity.

Islet amyloid polypeptide (IAPP, amylin) is the major protein component of the islet amyloid deposits associated with type 2 diabetes. The polypeptide lacks a well-defined structure in its monomeric state but readily assembles to form amyloid. Amyloid fibrils formed from IAPP, intermediates generated in the assembly of IAPP amyloid, or both are toxic to β-cells, suggesting that islet amyloid formation may contribute to the pathology of type 2 diabetes. There are relatively few reported inhibitors of amyloid formation by IAPP. Here we show that the tea-derived flavanol, (-)-epigallocatechin 3-gallate [(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3-yl 3,4,5-trihydroxybenzoate] (EGCG), is an effective inhibitor of in vitro IAPP amyloid formation and disaggregates preformed amyloid fibrils derived from IAPP. The compound is thus one of a very small set of molecules which have been shown to disaggregate IAPP amyloid fibrils. Fluorescence-detected thioflavin-T binding assays and transmission electron microscopy confirm that the compound inhibits unseeded amyloid fibril formation as well as disaggregates IAPP amyloid. Seeding studies show that the complex formed by IAPP and EGCG does not seed amyloid formation by IAPP. In this regard, the behavior of IAPP is similar to the reported interactions of Aβ and α-synuclein with EGCG. Alamar blue assays and light microscopy indicate that the compound protects cultured rat INS-1 cells against IAPP-induced toxicity. Thus, EGCG offers an interesting lead structure for further development of inhibitors of IAPP amyloid formation and compounds that disaggregate IAPP amyloid.

A role for helical intermediates in amyloid formation by natively unfolded polypeptides

Amyloid formation and aberrant protein aggregation have been implicated in more than 15 different human diseases and an even wider range of proteins form amyloid in vitro. From a structural perspective the proteins which form amyloid can be divided into two classes: those which adopt a compact globular fold and must presumably at least partially unfold to form amyloid and those which are unstructured in their monomeric state. Important examples of the latter include the Abeta peptide of Alzheimers disease, atrial natriuretic factor, calcitonin, pro-calcitonin, islet amyloid polypeptide (IAPP, amylin), alpha-synuclein and the medin polypeptide. The kinetics of amyloid assembly are complex and typically involve a lag phase during which little or no fibril material is formed, followed by a rapid growth stage leading to the beta-sheet-rich amyloid structure. Increasing evidence suggests that some natively unfolded polypeptides populate a helical intermediate during the lag phase. We propose a model in which early oligomerization is linked to helix formation and is promoted by helix-helix association. Recent work has highlighted the potential importance of polypeptide membrane interactions in amyloid formation and helical intermediates appear to play an important role here as well. Characterization of helical intermediates is experimentally challenging but new spectroscopic techniques are emerging which hold considerable promise and even have the potential to provide residue specific information.

A critical assessment of the role of helical intermediates in amyloid formation by natively unfolded proteins and polypeptides

Amyloidogenic proteins and polypeptides can be divided into two structural classes, namely those which are flexible and are intrinsically disordered in their unaggregated state and those which form a compact globular structure with a well-defined tertiary fold in their normally soluble state. This review article is focused on amyloid formation by natively disordered polypeptides. Important examples of this class include islet amyloid polypeptide (IAPP, amylin), pro-IAPP processing intermediates, alpha-synuclein, the Abeta peptide, atrial natriuretic factor, calcitonin, pro-calcitonin, the medin polypeptide, as well as a range of de novo designed peptides. Amyloid formation is a complex process consisting of a lag phase during which no detectable fibril material is formed, followed by a rapid growth phase that leads to amyloid fibrils. A critical analysis of the literature suggests that a subset of intrinsically disordered polypeptides populate a helical intermediate during the lag phase. In this scenario, early formation of multimeric species is promoted by helix-helix association involving one region of the polypeptide chain which leads to a high effective concentration of an amyloidogenic sequence located in a different region of the chain. Helical intermediates appear to be particularly important in membrane-catalyzed amyloid formation and have been implicated in glycosaminoglycan mediated amyloid formation as well. There is suggestive evidence that targeting helix-helix interactions can be a viable strategy to inhibit amyloid formation. The characterization of transient helical intermediates is challenging, but new methods are being developed that offer the prospect of providing residue-specific information in real time.

Islet amyloid: From fundamental biophysics to mechanisms of cytotoxicity

Pancreatic islet amyloid is a characteristic feature of type 2 diabetes. The major protein component of islet amyloid is the polypeptide hormone known as islet amyloid polypeptide (IAPP, or amylin). IAPP is stored with insulin in the β‐cell secretory granules and is released in response to the stimuli that lead to insulin secretion. IAPP is normally soluble and is natively unfolded in its monomeric state, but forms islet amyloid in type 2 diabetes. Islet amyloid is not the cause of type 2 diabetes, but it leads to β‐cell dysfunction and cell death, and contributes to the failure of islet cell transplantation. The mechanism of IAPP amyloid formation is not understood and the mechanisms of cytotoxicity are not fully defined.

Islet amyloid deposition limits the viability of human islet grafts but not porcine islet grafts

Islet transplantation is a promising treatment for diabetes but long-term success is limited by progressive graft loss. Aggregates of the beta cell peptide islet amyloid polypeptide (IAPP) promote beta cell apoptosis and rapid amyloid formation occurs in transplanted islets. Porcine islets are an attractive alternative islet source as they demonstrate long-term graft survival. We compared the capacity of transplanted human and porcine islets to form amyloid as an explanation for differences in graft survival. Human islets were transplanted into streptozotocin-diabetic immune-deficient mice. Amyloid deposition was detectable at 4 weeks posttransplantation and was associated with islet graft failure. More extensive amyloid deposition was observed after 8 weeks. By contrast, no amyloid was detected in transplanted neonatal or adult porcine islets that had maintained normoglycemia for up to 195 days. To determine whether differences in IAPP sequence between humans and pigs could explain differences in amyloid formation and transplant viability, we sequenced porcine IAPP. Porcine IAPP differs from the human sequence at 10 positions and includes substitutions predicted to reduce its amyloidogenicity. Synthetic porcine IAPP was considerably less amyloidogenic than human IAPP as determined by transmission electron microscopy, circular dichroism, and thioflavin T binding. Viability assays indicated that porcine IAPP is significantly less toxic to INS-1 beta cells than human IAPP. Our findings demonstrate that species differences in IAPP sequence can explain the lack of amyloid formation and improved survival of transplanted porcine islets. These data highlight the potential of porcine islet transplantation as a therapeutic approach for human diabetes.

Mechanisms of islet amyloidosis toxicity in type 2 diabetes

Amyloid formation by the neuropancreatic hormone, islet amyloid polypeptide (IAPP or amylin), one of the most amyloidogenic sequences known, leads to islet amyloidosis in type 2 diabetes and to islet transplant failure. Under normal conditions, IAPP plays a role in the maintenance of energy homeostasis by regulating several metabolic parameters, such as satiety, blood glucose levels, adiposity and body weight. The mechanisms of IAPP amyloid formation, the nature of IAPP toxic species and the cellular pathways that lead to pancreatic β‐cell toxicity are not well characterized. Several mechanisms of toxicity, including receptor and non‐receptor‐mediated events, have been proposed. Analogs of IAPP have been approved for the treatment of diabetes and are under investigation for the treatment of obesity.

Islet amyloid polypeptide toxicity and membrane interactions

Significance Amyloid formation is a hallmark of a range of human diseases. The polypeptide hormone amylin forms amyloid deposits in type 2 diabetes, and amyloid formation is thought to be a contributor to the decline in β-cell mass in the disease, however the basis of amylin-induced cytotoxicity is not fully understood. Amylin-induced membrane disruption has attracted considerable interest, and the interactions of amylin with model membranes have been characterized, but the relationship between studies with model membranes and toxicity is not understood. In this article the relationship between cell toxicity and the ability to disrupt model membranes is examined. There is no direct correlation between the two processes. Islet amyloid polypeptide (IAPP) is responsible for amyloid formation in type 2 diabetes and contributes to the failure of islet cell transplants, however the mechanisms of IAPP-induced cytotoxicity are not known. Interactions with model anionic membranes are known to catalyze IAPP amyloid formation in vitro. Human IAPP damages anionic membranes, promoting vesicle leakage, but the features that control IAPP–membrane interactions and the connection with cellular toxicity are not clear. Kinetic studies with wild-type IAPP and IAPP mutants demonstrate that membrane leakage is induced by prefibrillar IAPP species and continues over the course of amyloid formation, correlating additional membrane disruption with fibril growth. Analyses of a set of designed mutants reveal that membrane leakage does not require the formation of β-sheet or α-helical structures. A His-18 to Arg substitution enhances leakage, whereas replacement of all of the aromatic residues via a triple leucine mutant has no effect. Biophysical measurements in conjunction with cytotoxicity studies show that nonamyloidogenic rat IAPP is as effective as human IAPP at disrupting standard anionic model membranes under conditions where rat IAPP does not induce cellular toxicity. Similar results are obtained with more complex model membranes, including ternary systems that contain cholesterol and are capable of forming lipid rafts. A designed point mutant, I26P-IAPP; a designed double mutant, G24P, I26P-IAPP; a double N-methylated variant; and pramlintide, a US Food and Drug Administration–approved IAPP variant all induce membrane leakage, but are not cytotoxic, showing that there is no one-to-one relationship between disruption of model membranes and induction of cellular toxicity.

Rational design of potent domain antibody inhibitors of amyloid fibril assembly

Antibodies hold significant potential for inhibiting toxic protein aggregation associated with conformational disorders such as Alzheimer’s and Huntington’s diseases. However, near-stoichiometric antibody concentrations are typically required to completely inhibit protein aggregation. We posited that the molecular interactions mediating amyloid fibril formation could be harnessed to generate antibodies with potent antiaggregation. Here we report that grafting small amyloidogenic peptides (6–10 residues) into the complementarity-determining regions of a single-domain (VH) antibody yields potent domain antibody inhibitors of amyloid formation. Grafted AMyloid-Motif AntiBODIES (gammabodies) presenting hydrophobic peptides from Aβ (Alzheimer’s disease), α-Synuclein (Parkinsons disease), and islet amyloid polypeptide (type 2 diabetes) inhibit fibril assembly of each corresponding polypeptide at low substoichiometric concentrations (1:10 gammabody:monomer molar ratio). In contrast, sequence- and conformation-specific antibodies that were obtained via immunization are unable to prevent fibrillization at the same substoichiometric concentrations. Gammabodies prevent amyloid formation by converting monomers and/or fibrillar intermediates into small complexes that are unstructured and benign. We expect that our antibody design approach—which eliminates the need for immunization or screening to identify sequence-specific domain antibody inhibitors—can be readily extended to generate potent aggregation inhibitors of other amyloidogenic polypeptides linked to human disease.

Aggregation of islet amyloid polypeptide: from physical chemistry to cell biology

Amyloid formation in the pancreas by islet amyloid polypeptide (IAPP) leads to β-cell death and dysfunction, contributing to islet transplant failure and to type-2 diabetes. IAPP is stored in the β-cell insulin secretory granules and cosecreted with insulin in response to β-cell secretagogues. IAPP is believed to play a role in the control of food intake, in controlling gastric emptying and in glucose homeostasis. The polypeptide is natively unfolded in its monomeric state, but is one of the most amyloidogenic sequences known. The mechanisms of IAPP amyloid formation in vivo and in vitro are not understood; the mechanisms of IAPP induced cell death are unclear; and the nature of the toxic species is not completely defined. Recent work is shedding light on these important issues.

The ability of rodent islet amyloid polypeptide to inhibit amyloid formation by human islet amyloid polypeptide has important implications for the mechanism of amyloid formation and the design of inhibitors.

Islet amyloid polypeptide (IAPP) is a 37-residue polypeptide hormone that is responsible for islet amyloid formation in type II diabetes. Human IAPP is extremely amyloidogenic, while rat IAPP and mouse IAPP do not form amyloid in vitro or in vivo. Rat IAPP and mouse IAPP have identical primary sequences, but differ from the human polypeptide at six positions, five of which are localized between residues 20 and 29. The ability of rat IAPP to inhibit amyloid formation by human IAPP was tested, and the rat peptide was found to be an effective inhibitor. Thioflavin-T fluorescence-monitored kinetic experiments, transmission electron microscopy, and circular dichroism showed that rat IAPP lengthened the lag phase for amyloid formation by human IAPP, slowed the growth rate, reduced the amount of amyloid fibrils produced in a dose-dependent manner, and altered the morphology of the fibrils. The inhibition of human IAPP amyloid formation by rat IAPP can be rationalized by a model that postulates formation of an early helical intermediate during amyloid formation where the helical region is localized to the N-terminal region of IAPP. The model predicts that proline mutations in the putative helical region should lead to ineffective inhibitors as should mutations that alter the peptide-peptide interaction interface. We confirmed this by testing the ability of A13P and F15D point mutants of rat IAPP to inhibit amyloid formation by human IAPP. Both these mutants were noticeably less effective inhibitors than wild-type rat IAPP. The implications for inhibitor design are discussed.