Amylin Proprotein Processing Generates Progressively More Amyloidogenic Peptides that Initially Sample the Helical State

Abstract

Human amylin, or islet amyloid polypeptide, is a peptide co-secreted with insulin by the beta cells of the pancreatic islets of Langerhans. The 37-residue, C-terminally amidated human amylin peptide derives from a proprotein that undergoes disulfide bond formation in the endoplasmic reticulum and is then subjected to four enzymatic processing events in the immature secretory granule. Human amylin forms both intracellular and extracellular amyloid deposits in the pancreas of most Type II diabetic subjects, likely reflecting compromised secretory cell function. In addition, amylin processing intermediates, postulated to initiate intracellular amyloidogenesis, have been reported as components of intracellular amyloid in beta cells. We investigated the amyloidogenicity of amylin and its processing intermediates in vitro. Chaotrope-denatured amylin and amylin processing intermediates were subjected to size exclusion chromatography, affording high concentrations of monomeric peptides. NMR studies reveal that human amylin samples helical conformations. Under conditions mimicking the immature secretory granule (37°C, pH 6), amylin forms amyloid aggregates more rapidly than its processing intermediates, and more rapidly than its reduced counterparts. Our studies also show that the amyloidogenicity of amylin and its processing intermediates is negatively correlated with net charge and charge at the C-terminus. Although our conditions may not precisely reflect the environment of amyloidogenesis in vivo, the lower amyloidogenicity of the processing intermediates relative to amylin suggests their presence in intracellular amyloid deposits in the increasingly stressed beta cells of diabetic subjects may be a consequence of general defects in protein homeostasis control known to occur in diabetes.In type II diabetes, an increasing demand for insulin places a substantial stress on the protein secretion system of the beta cells of the islets of Langerhans, which results in cellular dysfunction and, eventually, beta cell death. As the population of beta cells continues to diminish, the stress on the remaining cells increases as they struggle to produce the insulin † We thank the NIH (DK46335 and AG18917), The Skaggs Institute of Chemical Biology, and the Lita Annenberg Hazen Foundation for financial support. *Corresponding author: jkelly@scripps.edu, phone: +1-858-784-9880, fax: +1-858-784-9610. SUPPORTING INFORMATION AVAILABLERepresentative analytical ultracentrifugation data; NMR spectra, NMR resonance assignments and a table of resonances; as well as representative amyloidogenesis time courses are included. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 September 16. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript necessary to mount a proper host homeostatic response. This feedback loop is thought to be responsible for the progressive nature of type II diabetes (1).Cro...

Molecular Tweezers Inhibit Islet Amyloid Polypeptide Assembly and Toxicity by a New Mechanism

“…However, this state likely does not reflect the physiologic conformation of IAPP monomers, which have been reported to have a higher α-helical content. 9,37 Following 1 h of incubation, the minimum at 201 nm decreased in intensity, a local maximum developed at 208 nm, and a minimum developed at 220 nm, suggesting formation of a mixture containing both α-helix and β-sheet structures. 38 With continued incubation, the maximum at 208 nm increased in intensity and shifted slightly toward shorter wavelengths.…”

α-helix to β-hairpin transition of human amylin monomer

“…Subsequent CD studies demonstrated that the hIAPP monomer adopts primarily a random coil structure. 16,[33][34][35][36][37] Recent NMR studies on human amylin, 38 rat amylin and pramlintide peptides, which have sequences similar to that of amylin, suggest that an α-helical structure is adopted near the N-terminus. 39,40 By interpreting the results of ion mobility mass spectroscopy experiments with a molecular implicit-solvent model, Dupuis et al 29 showed that the hIAPP monomer adopts an α-helical conformation between residues 9-17 on the terminus end, and a short β-hairpin between residues 24-28 and 31-35 on the C-terminus.…”

Structure and Thermodynamics of Amylin Dimer Studied by Hamiltonian-Temperature Replica Exchange Molecular Dynamics Simulations

“…Brender et al, 7 for example, showed that membrane disruption and amyloid formation are two separate processes that happen in different regions of hIAPP. Liposome leakage experiments on full-length hIAPP and hIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] fragments at low concentration confirmed that the pathological membrane-disrupting activity comes from the IAPP N-terminal region as both sequences induce almost identical ABSTRACT: The loss of the insulin-producing β-cells in the pancreatic islets of Langerhans, responsible for type-II diabetes, is associated with islet amyloid deposits. The main component of these deposits is the amyloid fibrils formed by the 37-residue human islet amyloid polypeptide (hIAPP also known as amylin).…”

“…However, the peptide is exposed to the water, whereas hIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] is buried within the micelle. Interestingly, protonation of His18 of hIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] drives the peptide to the surface of the membrane and decreases its toxicity. 29,30 To better understand the role of the disulfide bridge and the amino acid sequence on the early stage of aggregation in solution, we study the full-length hIAPP dimer, with and without the disulfide bridge, and compare its conformational properties with that of the monomer, already studied by us, 20 as well as with rIAPP in both monomeric and dimeric forms.…”