All three charge-loaded peptides fibrillize poorly on their own and inhibit fibril elongation of WT-amylin at physiological ionic strength. Here, we describe the development and detailed characterization of three novel assays for amylin degradation, two based on a fluoresceinated and biotinylated form of rodent amylin (fluorescein-rodent amylin-biotin, FrAB), which can be used for any amylin protease, and another based on an internally quenched fluorogenic substrate (FRET-based amylin, FRAM), which is more specific for IDE. β-cell dysfunction and the decline in β-cell mass are attributed to several factors, including glucolipotoxicity, inflammation, accumulation of cholesterol, and islet amyloid formation [117–122]. The biologically active sequences all contain a disulfide bridge between Cys-2 and Cys-7 and have an amidated C-terminus. Soluble insulin is one of the most potent inhibitors of IAPP aggregation and may play a role in modulating intragranule aggregation; however insulin is in a semicrystalline state in the granule [71–75]. The differences between the various studies might be related to the level of production of hIAPP [4, 114–116]. Young, “Inhibition of insulin secretion,”, G. J. S. Cooper, B. Leighton, G. D. Dimitriadis et al., “Amylin found in amyloid deposits in human type-2 diabetes-mellitus may be a hormone that regulates glycogen-metabolism in skeletal-muscle,”, D. L. Morris and L. Rui, “Recent advances in understanding leptin signaling and leptin resistance,”, J. D. Roth, H. Hughes, E. Kendall, A. D. Baron, and C. M. Anderson, “Antiobesity effects of the, T. A. Lutz, “The interaction of amylin with other hormones in the control of eating,”, J. Islet amyloid polypeptide (IAPP, amylin) is secreted from pancreatic islet β-cells and converted to amyloid deposits in type 2 diabetes. Insulin with a truncated B-chain, to prevent dimerization, also bound hIAPP. , pp. The literature on IAPP mutations has been critically reviewed in 2013 and, in the interest of space, we refer the interested reader to that work for a more detailed discussion [12]. A range of mechanisms have been proposed to account for the toxic effects of amyloidosis, but the exact causes of cell death are still not completely defined. Type 2 diabetes mellitus (T2DM) has been clearlylinked to oxidative stress and amylin amyloidosis in pancreatic β-cells. Not all mammals form islet amyloid; notably mice and rats do not [19, 21]. Non-insulin-dependent (type II) diabetes mellitus (NIDDM) is characterized by dysfunction and depletion of these β-cells and also, in more than 90% of patients, amyloid plaques containing fibrillar IAPP. pancreas. The model shares several of the general features of the NMR and fragment based models in that each IAPP molecule bends into an approximate U-shaped structure and contains two β-strands; in the case of the EPR model, these are made up of residues 12 to 19 and of 31 to 36 with residues 7 to 10 in a transition region. Given the analogous cellular stress environments shared by both T2DM and AD, the objective of this study is to explore T2DM pathogenesis from the viewpoint of amyloidogenic evolvability. (a) Primary sequence of human IAPP. (a) Primary sequence of human IAPP. Commonly used model systems contain a much higher percentage of anionic lipids than that found in the β-cell membrane [110] and usually lack cholesterol and gangliosides. (c) The sequence of the mature 37-residue human IAPP. A further complication arises from the fact that many studies have made use of a truncated fragment of IAPP which lacks the first seven residues (IAPP8–37). The amidated C-terminus is produced after further processing by CPE/PAM. Open questions in the eld include the relative im, reductionist biophysical studies to the si, the factors which trigger amyloid formation in type- diabetes, the poten, Hyaline lesions in the pancreas were rst described more than, e deposits were originally assumed to be composed of, residue polypeptide neuropancreatic hormone, now known, metabolism and glucose homeostasis, helping to control, IAPP has been found in all mammals studied to date, mature hormone (Figure ) [, ]. Arg-1 protects against WT-amylin cytotoxicity towards a MIN6 mouse model of pancreatic í µí»½-cells, and Arg-2 protects at higher concentrations, whereas Mem-T has no effect. The dibasic Lys-Arg pair at the C-terminus is removed by carboxypeptidase and the Gly acts as the nitrogen donor for amidation of the C-terminal Tyr by the peptidyl amidating monooxygenase complex (PAM). -cell death are not fully dened. ere are no clinically a, inhibitors of IAPP toxicity and very few, if any. IAPP amyloid fibrils were visible by electron microscopy in lysosomes of pancreatic macrophages in man, monkeys and human IAPP transgenic mice. The S20G mutation has been described as pathological related, and G33R mutation may have a deleterious effect. e si, are still not understood nor are the factors which trigger, islet amyloidosis in type- diabetes (TD). In particular, the rate amyloid formation is significantly accelerated with increasing salt and the effects depend on the choice of anion. action on islet amyloid polypeptide ber formation, of membrane-bound islet amyloid polypeptide with soluble and, the transport and ligand specicity of the calcitonin- recept, action with the calcitonin receptor gene p. control body weight and energy expenditure, and the - fragment of islet amyloid polypeptide on insulin, found in amyloid deposits in human type- diabetes-melli, may be a hormone that regulates glycogen-metabolism in, [] D. L. Morris and L. Rui, “Recent advances in understandin. Consistent with this hypothesis, experimental, [, ]. M, substitution of residues , , and i, corresponding amino acids of hIAPP led to a weakly amy-, loidogenic polypeptide even though it still contained the . e model shar, several of the general features of the NMR and fragment, key dierence between the EPR based model and the others, A with respect to each other in the EPR mo, the staggered relationship leads to a le-handed twist. Rehana Akter, Ping Cao, Harris Noor, Zachary Ridgway, Ling-Hsien Tu, Hui Wang, Amy G. Wong, Xiaoxue Zhang, Andisheh Abedini, Ann Marie Schmidt, Daniel P. Raleigh, "Islet Amyloid Polypeptide: Structure, Function, and Pathophysiology", Journal of Diabetes Research, vol. The human islet amyloid polypeptide (hIAPP) is an intrinsically disordered protein that can self-assemble into fibrillar aggregates that play a key role in the pathogenesis of the type II diabetes mellitus. This mutation, which is found at low levels in certain Asian populations, has been proposed to lead to a slightly higher risk of diabetes, although the statistical significance has been questioned [4, 49–52]. The human plasma-protein transthyretin (TTR), a well-known amyloid-inhibiting protein, is interestingly also expressed within the IAPP producing β-cells. molecule, but there are dierences in the C-terminal half. A. Williamson and A. D. Miranker, “Direct de, and cooperative assembly of membrane-bound, insulin on brillogenesis of islet amyloid polypeptide. Unfortunately, Pramlintide is not soluble at the appropriate pH. e biologically active sequences all con, be signicantly less amyloidogenic than huma, TPIESHQVEKR KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY, -residue signal sequence is shown in italics; the N- and C-terminal, in this issue for coverage of other topics a, are described in other articles in this issue and have been, been published in recent years which provide addition, information on various aspects of the biology and biophysics, disease, but there is still much that is unclear. A similar approach could be used to probe the role of the different Thr residues via Val substitutions. Similar to AD-related APs, protofibrillar amylin might confer resistance against the multiple stressors in β-cells and be transmitted to offspring to deliver stress information, in the absence of which, type 1 DM (T1DM) in offspring might develop. Replacement of His-18 with either a Gln or Leu significantly accelerated amyloid formation. There are no clinically approved inhibitors of IAPP toxicity and very few, if any, effective “drug-like” inhibitors of IAPP amyloid formation have been reported in the literature. Islet amyloid deposition is also a key factor contributing to the failure of islet cell transplants. The effects of crowding agents and osmolytes on amyloid formation by IAPP are reviewed in detail in this volume by Gao and Winter [169]. Inhibiting IAPP secretion reduced amyloid formation, but increasing secretion increased toxicity and amyloid formation [115]. Finally, we underscore an urgent need for developing effective therapeutic strategies aimed at hindering hIAPP-phospholipid interactions. We suggest that future studies should include more physiologically-relevant and in-cell studies to allow a more accurate model of in vivo interactions. We do not discuss efforts at inhibitor design as they are described in other articles in this issue and have been reviewed elsewhere [12]. e, studied in detail and seems ripe for further investigation. , vol. Amylin plays a role in glycemic regulation by slowing gastric emptying and promoting satiety, thereby preventing post-prandial spikes in blood glucose levels. IAPP is most similar to CGRP. Residues color-coded red are found in the first β-strand in the fibril; those colored blue in the second β-strand and the ones located in the partially ordered loop that connects the two stands are colored green. However, despite the important role of chaperones on protein folding, less is known about chaperonal modulation of protein aggregation and fibrillation considering different classes of chaperones. , the molecular mechanism of amyloid formation Thus, the 20–29 sequence cannot be the only factor governing amyloid formation, but there is no doubt that it is important and single proline substitutions within the 20–29 segment have been shown to significantly reduce the amyloidogenicity of hIAPP as have double N-methyl modifications in this region [32–34]. However, a subsequent study revealed that mutants which were designed to disrupt the putative His-Tyr interaction actually accelerated amyloid formation, indicating that the interaction is not essential for amyloid formation [60]. In addition, there is experimental evidence that membrane gangliosides and cholesterol play a role in the uptake and clearance of hIAPP [111, 148]. Nontoxic variants of hIAPP with considerably improved solubility at pH 7.4 have been developed by rationally redesigning the sequence to incorporate strategic proline residues together with additional charges (Figure 4) [176]. Interaction of exogenous or endogenous hIAPP aggregates with Fas, also known as the death receptor, leads to caspase-3 activation, while deletion of Fas protects β-cells from hIAPP induced toxicity and inhibition of caspase-3 in vivo protects β-cells from hIAPP induced β-cell apoptosis [15, 134]. Indeed, it is now clear that the effects of many crowding agents, and by implication the cellular environment, cannot be explained solely on the basis of excluded volume. Two structures were proposed based on the solid state NMR data, both of which are consistent with the experimental restraints. This segment is well ordered in the model and both Ser-28 and Ser-29 are involved in critical contacts (Figure 3), rationalizing why the three Pro residues in rat IAPP impact amyloid formation. ese observations led to the initial, by studies with ten residue peptides derived from, of the rat/mouse sequence to the sequence of hIAPP, between residues –. We find that the pharmacological chaperone O4, the chemical chaperone proline as well as the protein chaperone serum amyloid P component (SAP) are inhibitors of the type 2 diabetes mellitus-related aggregation process of islet amyloid polypeptide (IAPP). D'Alessio, M. W. Schwartz et al., “Evidence of cosecretion of islet amyloid polypeptide and insulin by, M. Stridsberg, S. Sandler, and E. Wilander, “Cosecretion of islet amyloid polypeptide (IAPP) and insulin from isolated rat pancreatic islets following stimulation or inhibition of beta-cell function,”, P. Cao, P. Marek, H. Noor et al., “Islet amyloid: from fundamental biophysics to mechanisms of cytotoxicity,”, P. Cao, A. Abedini, and D. P. Raleigh, “Aggregation of islet amyloid polypeptide: from physical chemistry to cell biology,”, M. S. Fernández, “Human IAPP amyloidogenic properties and pancreatic, A. Abedini and A. M. Schmidt, “Mechanisms of islet amyloidosis toxicity in type 2 diabetes,”, S. Despa, S. Sharma, T. R. Harris et al., “Cardioprotection by controlling hyperamylinemia in a ‘humanized’ diabetic rat model,”, S. Zhang, H. Liu, C. L. Chuang et al., “The pathogenic mechanism of diabetes varies with the degree of overexpression and oligomerization of human amylin in the pancreatic islet, J. F. Paulsson, J. Ludvigsson, A. Carlsson et al., “High plasma levels of islet amyloid polypeptide in young with new-onset of type 1 diabetes mellitus,”, P. Westermark, U. Engstrom, K. H. Johnson, G. T. Westermark, and C. Betsholtz, “Islet amyloid polypeptide: pinpointing amino acid residues linked to amyloid fibril formation,”, T. T. Ashburn and P. T. Lansbury Jr., “Interspecies sequence variations affect the kinetics and thermodynamics of amyloid formation: peptide models of pancreatic amyloid,”, C. Betsholtz, L. Christmansson, U. Engstrom et al., “Sequence divergence in a specific region of islet amyloid polypeptide (IAPP) explains differences in islet amyloid formation between species,”, M. R. Nilsson and D. P. Raleigh, “Analysis of amylin cleavage products provides new insights into the amyloidogenic region of human amylin,”, L. A. Scrocchi, K. Ha, Y. Chen, L. Wu, F. Wang, and P. E. Fraser, “Identification of minimal peptide sequences in the (8–20) domain of human islet amyloid polypeptide involved in fibrillogenesis,”, S. Gilead and E. Gazit, “The role of the 14–20 domain of the islet amyloid polypeptide in amyloid formation,”, Y. Mazor, S. Gilead, I. Benhar, and E. Gazit, “Identification and characterization of a novel molecular-recognition and self-assembly domain within the islet amyloid polypeptide,”, J. J. W. Wiltzius, S. A. Sievers, M. R. Sawaya, and D. Eisenberg, “Atomic structures of IAPP (amylin) fusions suggest a mechanism for fibrillation and the role of insulin in the process,”, S. Gilead, H. Wolfenson, and E. Gazit, “Molecular mapping of the recognition interface between the islet amyloid polypeptide and insulin,”, E. Andreetto, L. M. Yan, M. Tatarek-Nossol, A. Velkova, and A. Kapurniotu, “Identification of hot regions of the A beta-IAPP interaction interface as high-affinity binding sites in both cross- and self-association,”, A. Abedini and D. P. Raleigh, “Destabilization of human IAPP amyloid fibrils by proline mutations outside of the putative amyloidogenic domain: is there a critical amyloidogenic domain in human IAPP?”, B. W. Koo, J. which inhibit islet amyloid polypeptide bril formation. increased the net charge on the polypeptide. The in-register arrangement implies that there can be significant electrostatic interactions in amyloids. An interesting fea, modication work is that the eects of the modications o, the normal activity of hIAPP were found to depend on the, site modied and thus provide indirect information about, A fourth approach has made use of N-methylatio, include identifying the initiation site(s) of am, mouse models that highly overexpress hIAPP and h, that warrants further eort. showing the arrangement of the two stacks as a ribbon diagram. Pro-hIAPP is cleaved by the prohormone convertases PC(1/3) and PC2 at two conserved dibasic sites, indicated by arrows. These considerations mean that considerable caution needs to be employed when comparing data generated in different laboratories. Such evolvability-associated processes might be affected by parental diabetic conditions, including T1DM and T2DM. Secretion of an incompletely processed proIAPP intermediate that includes the N-terminal prosequence, denoted here by Npro-hIAPP, has been reported to be increased in T2D and to be incorporated into islet amyloid [4, 62, 63]. An exper, imental support for the key role of the phen, domain in the N-terminal cleavage site of p. convertase PC leads to amyloid formation and cell death, diabetes: from molecular misfolding to islet pa, and insulin on brillogenesis of islet am. The effect of these variants can be evaluated by means of computational tools, such as molecular dynamics simulations. and membrane potential. The data are consistent with an N-terminal β-strand made up of residues 8 through 18 and a C-terminal strand comprised of residues 26 to 37 [37]. in vitro Studies on intact hIAPP also indicate that the 20–29 segment is not the sole amyloidogenic determinant. The N-terminal extension actually makes hIAPP less amyloidogenic and more soluble but enhances interactions with GAGs. Utilizing graphical description it is explained how a smart targeted nano-delivery system could promote β-cell growth and development by inducing the Wnt signaling pathway (inhibiting Gsk3β), inhibiting inflammasome (inhibiting NLRP3), and activating autophagic target points (protecting Atg3/Atg7 complex from oxidative stress) thereby might ameliorate the severity of T2D. Our circular dichroism and liquid state NMR data reveal that 10–30% of cholesterol slightly affects the aggregational and conformational behaviour of hIAPP. A fourth approach has made use of N-methylation and builds upon the development of N-methylated hIAPP analogs as potent inhibitors of wild type aggregation and toxicity [34]. -strands in all of the models of hIAPP am, -sheet structure. A number of review articles have been published in recent years which provide additional information on various aspects of the biology and biophysics of IAPP [4–6, 12–15]. In the fragment model, Ser-28 and Ser-29 are involved in a steric zipper and make extensive hydrogen bonding interactions [26]. Isosteric replacement of other residues in hIAPP requires nongenetically coded amino acids. The Asn14Leu and Asn21Leu mutants did not form amyloid on the experimental timescale of these studies. hIAPP adopts a helix-kink-helix structure on model membranes with the helices located between residues 5 to 17 and 20 to 27. Type 2 diabetes (T2D) is a common protein misfolding disease (PMD), and its pathogenesis is considered to be tightly associated with the aggregation of the disease-causative hIAPP (or amylin). It is important to emphasize that both structures are models based on, and consistent with, separate sets of experimental data which are sufficient to constrain the models but not to completely define a precise, three-dimensional, high resolution structure. Two complimentary sets of reagents were employed: one that inhibited IAPP secretion, but maintained the level of production of IAPP and a second that increased IAPP secretion but did not increase the amount of IAPP produced. Significant overproduction of the polypeptide could play a role. It is also worth noting that exogenously added IAPP has been reported to induce different toxic effects on closely related cell types, arguing that nonspecific membrane disruption cannot be the only mechanism of toxicity [150]. Several recent reviews provide a more in-depth view of the physiological role of hIAPP [4, 5, 88]. All three assays yield highly quantitative data and are resistant to DMSO, and the FRAM and FP-based FrAB assay are ideally suited to HTS applications. Upregulation of autophagy is a common protective response to the accumulation of toxic amyloidogenic aggregates in degenerative disease. Unfortunately some papers do not provide all of the details required to repeat measurements. Burial of a charged Arg side chain will be energetically unfavorable and the structure in which it is exposed seems more likely. IAPP receptors are generated from coexpression of the calcitonin (CT) receptor with receptor activity-modifying proteins (RAMPs) [76–79]. (Figure ). High resolution models of the IAPP amyloid fibril have been developed based upon solid state NMR studies, and on X-ray diffraction studies of microcrystals of small peptide fragments of hIAPP which form steric zippers. Introduction. However, it is not known whether cholesterol enhances membrane-interaction or membrane-insertion of hIAPP. II diabetes. e dierences between, the various studies might be related to the level of p, of hIAPP [, –]. For example, substitution of Ser with 2-aminobuytric acid represents an isosteric replacement and would allow the role of the OH group to be probed. Recent work also highlights a role for hIAPP aggregation and hyperamylinemia in cardiovascular complications of diabetes [16, 127]. Surprisingly, however, uncleaved substrate 1 could not be precipitated by avidin-agarose, apparently because the biotin moiety was inaccessible ( Figure 1b). In this study, we investigated the effect of cholesterol incorporated in zwitterionic and anionic membranes. In addition, is asymmetric with the anionic lipids localized on the inner, leaet. The importance of electrostatic interactions in hIAPP amyloid is also reflected in the dependence of the kinetics of hIAPP amyloid formation on ionic strength and on the type of salt. IAPP has been proposed to help regulate blood glucose levels by inhibiting insulin secretion [83, 84]. The mechanism(s) of IAPP amyloid formation in vivo and in vitro are still not understood nor are the factors which trigger islet amyloidosis in type-2 diabetes (T2D). In hIAPP, Arg-11 and His-18 are in the structured core of the fibril, or immediately adjacent to it, arguing that they will make net unfavorable electrostatic contributions to the stability of the fibril. The N- and C-terminal flanking regions of proIAPP are cleaved by the prohormone convertases PC2 and PC1/3 [7]. Pramlintide was designed based on comparison of the sequences of rat and human IAPP and is simply human IAPP with the three Pro substitutions found in the rat polypeptide. in the granule is noticeably lower than that of insulin, Soluble insulin is one of the most potent inhibitors of IAPP, aggregation; however insulin is in a semicrystalline state in, be on the order of to picomolar and to rise to to , be much higher and this is the more relevant num, mediated, but are still not fully understood. 6. If nothing else, the truncation removes the charge on the side chain of Lys-1 and, depending upon whether or not the N-terminus is acetylated, the charge on the N-terminus as well. Additionally, several targeting molecules associated with autophagic and epigenetic factors are also highlighted, which can be exploited in future diabetic research. (a) The primary sequence of the 89-residue human PreProIAPP. hIAPP plays a role in maintaining glucose homeostasis, in controlling gastric emptying, and in the suppression of glucagon release [4–6]. The cellular environment determines the structure and function of proteins. Quantitative mutational studies of amyloid fibril stability and of the kinetics of amyloid formation are much more challenging than studies with soluble, monomeric, globular proteins. Channel formation is dependent upon lipid membrane composition, ionic strength, A. Hebda, and A. D. Miranker, “Conserved and cooperative assembly of membrane-bound, R. P. R. Nanga, J. R. Brender, J. Xu, K. Hartman, V. Subramanian, and A. Ramamoorthy, “Three-dimensional structure and orientation of rat islet amyloid polypeptide protein in a membrane environment by solution NMR spectroscopy,”, R. P. R. Nanga, J. R. Brender, J. Xu, G. Veglia, and A. Ramamoorthy, “Structures of rat and human islet amyloid polypeptide IAPP, R. P. R. Nanga, J. R. Brender, S. Vivekanandan, and A. Ramamoorthy, “Structure and membrane orientation of IAPP in its natively amidated form at physiological pH in a membrane environment,”, J. R. Brender, K. Hartman, K. R. Reid, R. T. Kennedy, and A. Ramamoorthy, “A single mutation in the nonamyloidogenic region of islet amyloid polypeptide greatly reduces toxicity,”, P. J. Marek, V. Patsalo, D. F. Green, and D. P. Raleigh, “Ionic strength effects on amyloid formation by amylin are a complicated interplay among Debye screening, ion selectivity, and Hofmeister effects,”, Y. Li, W. Xu, Y. Mu, and J. In contrast to the mouse studies, a recent investigation used a cultured transgenic islet model to show that secretion of IAPP is an important factor in β-cell toxicity and islet amyloid formation. In rats, the circulating concentration of IAPP is reported to be on the order of 3 to 5 picomolar and to rise to 15 to 20 picomolar with elevation of blood glucose [4, 5]. IAPP is coexpressed and cosecreted with insulin by pancreatic β-cells (21– 23). Islet amyloid formation contributes to β-cell dysfunction and death in the disease and to the failure of islet transplants. Islet amyloid polypeptide: structure, function, and pathophysiology Hamilton, "Synthetic [alpha]-helix mimetics as agonists and antagonists of islet amyloid polypeptide aggregation," Angewandte Chemie--International Edition, vol.
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