The hormone islet amyloid polypeptide (IAPP, or amylin) plays a role in glucose homeostasis but aggregates to form islet amyloid in type-2 diabetes. Islet amyloid formation contributes to β-cell dysfunction and death in the disease and to the failure of islet transplants. Recent work suggests a role for IAPP aggregation in cardiovascular complications of type-2 diabetes and hints at a possible role in type-1 diabetes. The mechanisms of IAPP amyloid formation in vivo or in vitro are not understood and the mechanisms of IAPP induced β-cell death are not fully defined. Activation of the inflammasome, defects in autophagy, ER stress, generation of reactive oxygen species, membrane disruption, and receptor mediated mechanisms have all been proposed to play a role. Open questions in the field include the relative importance of the various mechanisms of β-cell death, the relevance of reductionist biophysical studies to the situation in vivo, the molecular mechanism of amyloid formation in vitro and in vivo, the factors which trigger amyloid formation in type-2 diabetes, the potential role of IAPP in type-1 diabetes, the development of clinically relevant inhibitors of islet amyloidosis toxicity, and the design of soluble, bioactive variants of IAPP for use as adjuncts to insulin therapy.
Islet amyloid polypeptide (IAPP, Amylin) is responsible for amyloid formation in type 2 diabetes and in islet cell transplants. The only known natural mutation found in mature human IAPP is a Ser-20 to Gly mis-sense mutation, found with small frequency in Chinese and Japanese populations. The mutation appears to be associated with increased risk of early onset type 2 diabetes. Early measurements in the presence of organic co-solvents showed that S20G-IAPP formed amyloid more quickly than the wild type. We confirm that the mutant accelerates amyloid formation under a range of conditions including in the absence of co-solvents. Ser-20 adopts a normal backbone geometry and the side chain makes no steric clashes in models of IAPP amyloid fibers, suggesting that the increase rate of amyloid formation by the mutant does not result from the relief of steric incompatibility in the fiber state. Transmission electronic microscopy, circular dichroism and seeding studies were used to probe the structure of the resulting fibers. The S20G-IAPP peptide is toxic to cultured rat INS-1 β-cells. The sensitivity of amyloid formation to the identity of residue-20 was exploited to design a variant which is much slower to aggregate and which inhibits amyloid formation by wild type IAPP. A S20K mutant forms amyloid with an 18-fold longer lag phase. Thioflavin-T binding assays, together with experiments using a p-cyanophenylalanine variant of human IAPP, show that the designed S20K mutant inhibits amyloid formation by human IAPP. The experiments illustrate how p-cyanophenylalanine can be exploited to monitor amyloid formation even in the presence of other amyloidogenic proteins.
Ion mobility coupled to mass spectrometry (IM-MS) is widely used to study protein dynamics and structure in the gas phase. Increasing the energy with which the protein ions are introduced to the IM cell can induce them to unfold, providing information on the comparative energetics of unfolding between different proteoforms. Recently, a high-resolution cyclic IM-mass spectrometer (cIM-MS) was introduced, allowing multiple, consecutive tandem IM experiments (IM n ) to be carried out. We describe a tandem IM technique for defining detailed protein unfolding pathways and the dynamics of disordered proteins. The method involves multiple rounds of IM separation and collision activation (CA): IM-CA-IM and CA-IM-CA-IM. Here, we explore its application to studies of a model protein, cytochrome C, and dimeric human islet amyloid polypeptide (hIAPP), a cytotoxic and amyloidogenic peptide involved in type II diabetes. In agreement with prior work using single stage IM-MS, several unfolding events are observed for cytochrome C. IM n -MS experiments also show evidence of interconversion between compact and extended structures. IM n -MS data for hIAPP shows interconversion prior to dissociation, suggesting that the certain conformations have low energy barriers between them and transition between compact and extended forms.
Islet amyloid formation contributes to β-cell death and dysfunction in type-2 diabetes and to the failure of islet transplants. Amylin (islet amyloid polypeptide, IAPP), a normally soluble 37 residue polypeptide hormone produced in the pancreatic β-cells, is responsible for amyloid formation in type-2 diabetes and is deficient in type-1 diabetes. Amylin normally plays an adaptive role in metabolism, and the development of nontoxic, non-amyloidogenic, bioactive variants of human amylin are of interest for use as adjuncts to insulin therapy. Naturally occurring non-amyloidogenic variants are of interest for xenobiotic transplantation and because they can provide clues toward understanding the amyloidogenicity of human amylin. The sequence of amylin is well-conserved among species, but sequence differences strongly correlate with in vitro amyloidogenicity and with islet amyloid formation in vivo. Bovine amylin differs from the human peptide at 10 positions and is one of the most divergent among known amylin sequences. We show that bovine amylin oligomerizes but is not toxic to cultured β-cells and that it is considerably less amyloidogenic than the human polypeptide and is only a low-potency agonist at human amylin-responsive receptors. The bovine sequence contains several nonconservative substitutions relative to human amylin, including His to Pro, Ser to Pro, and Asn to Lys replacements. The effect of these substitutions is analyzed in the context of wild-type human amylin; the results provide insight into their role in receptor activation, the mode of assembly of human amylin, and the design of soluble amylin analogues.
Antibodies with conformational specificity are important for detecting and interfering with polypeptide aggregation linked to several human disorders. We are developing a motif-grafting approach for designing lead antibody candidates specific for amyloid-forming polypeptides such as the Alzheimer peptide (A). This approach involves grafting amyloidogenic peptide segments into the complementarity-determining regions (CDRs) of single-domain (V H ) antibodies. Here we have investigated the impact of polar mutations inserted at the edges of a large hydrophobic A42 peptide segment (A residues 17-42) in CDR3 on the solubility and conformational specificity of the corresponding V H domains. We find that V H expression and solubility are strongly enhanced by introducing multiple negatively charged or asparagine residues at the edges of CDR3, whereas other polar mutations are less effective (glutamine and serine) or ineffective (threonine, lysine, and arginine). Moreover, A V H domains with negatively charged CDR3 mutations show significant preference for recognizing A fibrils relative to A monomers, whereas the same V H domains with other polar CDR3 mutations recognize both A conformers. We observe similar behavior for a V H domain grafted with a large hydrophobic peptide from islet amyloid polypeptide (residues 8 -37) that contains negatively charged mutations at the edges of CDR3. These findings highlight the sensitivity of antibody binding and solubility to residues at the edges of CDRs, and provide guidelines for designing other grafted antibody fragments with hydrophobic binding loops.The misfolding and assembly of peptides and proteins into prefibrillar oligomers and amyloid fibrils is linked to several neurodegenerative diseases (1-4). To understand the contributions of polypeptide aggregation to such disorders, it is important to characterize the biochemical properties of aggregates. However, pre-amyloid and amyloid aggregates are difficult to characterize using many traditional biochemical methods that are routinely used for soluble proteins. High-resolution structural analysis of such aggregates is particularly challenging and must be performed using specialized methods such as solid state NMR (5-13) or x-ray crystallography of small peptide fragments (14 -16). These powerful structural methods generally lack the time resolution to probe pre-amyloid intermediates and oligomers unless they can be kinetically trapped.Given these challenges, antibodies with conformational specificity for prefibrillar oligomers and amyloid fibrils have proven valuable for biochemical characterization (17-28). An obvious strength of such antibodies is their ability to detect specific types of aggregates formed both in vivo and in vitro. The ability of conformational antibodies to bridge in vivo and in vitro studies is important for understanding the biochemical mechanisms that contribute to protein misfolding disorders.Several approaches have been used to generate conformational antibodies. The most widely used one is immuni...
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