Journal of Molecular Biology
Phospholipid Catalysis of Diabetic Amyloid Assembly
Introduction
Amyloid formation is a critical component of the pathology of many degenerative diseases, including Alzheimer's and type II diabetes (NIDDM).1., 2. For each disease, a unique protein undergoes changes in conformation resulting in polymerization to a fibrous structure. Amyloid fibers are characterized by a cross-β structure in which β strands are organized orthogonal to the long axis of an unbranched fiber.3 Biochemically, these fibers are characterized by extreme apparent resistance to chemical and proteolytic degradation. As a result, in vivo deposits of amyloid are not readily cleared by cellular processes. Fiber formation is destructive either through macroscopic obstruction, e.g. in light chain amyloidosis,4 or by leading to cellular dysfunction and death, e.g. in NIDDM.1., 5.
NIDDM is a disease that afflicts more than 150 million individuals worldwide. The pathology of NIDDM includes the presence of amyloid deposits in the islets of Langerhans. The main component of these deposits, called islet amyloid polypeptide (IAPP) or amylin, is a 37-residue hormone (Figure 1) that is normally stored with insulin in vesicles and secreted by islet β-cells. Islet amyloidosis in patients with NIDDM is associated with a reduced mass of insulin-producing β-cells.6 In addition, oligomeric intermediates of fiber formation have been shown to be cytotoxic to cultured β-cells.7., 8. Fiber formation is therefore an important factor in the development of β-cell failure. Since IAPP isolated from amyloid fibers in diabetic patients is wild-type, the obvious candidates for agents that might affect fiber formation are the other granule components. The most abundant protein in the granule is insulin, which is secreted in ∼100-fold molar excess over IAPP. In vitro, however, substoichiometric amounts of insulin readily inhibit IAPP fiber formation.9 Therefore, fiber formation in vivo must involve an accelerant and/or isolation of IAPP from interaction with this inhibitor.
Several mechanisms for induction of β-cell death in NIDDM have been proposed.10 One possibility is that IAPP forms cation-permeable pores, which disrupt membranes and lead to cell death.11., 12. Indeed, preamyloid conformations of IAPP are able to induce membrane destabilization and apoptosis in healthy cultured islet cells.7 An alternative hypothesis is that elevated glucose and lipid levels associated with early NIDDM overstimulate metabolic and signaling pathways, resulting in β-cell failure and apoptosis.13., 14. These two hypotheses need not be mutually exclusive. It is reasonable to suggest that alterations in metabolism accompanying NIDDM onset provide the stimulus for IAPP fiber formation. Immunostaining studies of IAPP in healthy human β-cells show localization of the protein in a narrow region of the granule between a central core of insulin and the lipid bilayer.15 Furthermore, in transgenic mice which express human IAPP, fiber formation requires high fat intake or genetic alteration of lipid metabolism.16., 17. It is therefore plausible that alterations in lipid metabolism accompanying obesity and NIDDM provide for changes of the bilayer environment which lead to pathological interactions with IAPP.
We have previously characterized the fiber formation pathway of IAPP upon its in vitro dilution into physiological buffer from a stock solution in organic solvent.9., 18., 19. IAPP fibrillogenesis kinetics generally follow a sigmoidal profile; an initial lag phase of relatively slow fiber nucleation precedes a rapid elongation phase during which the remainder of the soluble protein is converted into fiber. One central result from our studies is that the rate of fiber formation is exceptionally sensitive to the residual presence of the cosolvent hexafluoroisopropanol (HFIP). For example, at 4% HFIP, fiber formation occurs rapidly following a lag phase of a few minutes. In contrast, at 2% HFIP, the midpoint of fiber formation is reached after several hours.19 Fluorinated alcohols have both hydrophobic and polar moieties and are therefore capable of providing a variety of interactions that can affect conformation and assembly processes.19., 20. A similar variety of interactions can be provided in vivo by phospholipid membranes. Phospholipids can stabilize polar and electrostatic interactions through their head groups and hydrophobic interactions in the transmembrane region.21., 22. Here, we use an established lipid binding assay and kinetic studies coupled with electron microscopy to elucidate the mechanism of IAPP fibrillogenesis in the presence of synthetic and tissue-derived lipid membranes.
Section snippets
Results
In this study, we investigate the mechanism of IAPP fiber formation under conditions approximating those found in the NIDDM β-cell. First, we establish an in vitro system amenable to kinetic studies in the presence of lipid bilayers. We then assess the effects of patient-derived and synthetic lipid bilayers on IAPP fibrillogenesis. Our analyses focus on the kinetic consequences of varying lipid headgroup and protein:lipid ratio, explicit measurement of protein–lipid binding, and the
Discussion
IAPP is a peptide hormone, packaged in proximity to the lipid bilayer of a secretory granule. Its conversion to amyloid is central to the progression of NIDDM. The following in vitro observations offer insight into the mechanism of lipid-mediated fibrillogenesis: (i) IAPP fiber formation is dramatically accelerated by negatively charged phospholipid bilayers, including those derived from diabetic pancreas. (ii) Lipid binding by IAPP is rapid compared to the timescale of fiber formation. (iii)
Materials
Synthetic lipids were obtained from Avanti (Alabaster, AL) dissolved in chloroform; DMSO was from J.T. Baker or Sigma; Thioflavin T (ThT) was from Acros; heparin and salmon sperm DNA were from Sigma. Human IAPP was synthesized using standard TBOC methods and purified by the W.M. Keck facility (New Haven, CT). TAMRA-IAPP, rIAPP, IAPP8-37, and IAPPK1E were synthesized using Fmoc methods and cyclized and purified in house.
Isolation of tissue phospholipids and liposome preparation
Pancreatic tissue was obtained post-mortem and flash-frozen from a
Acknowledgements
The authors thank Drs V. Unger and D. Chester for assistance with EM and helpful discussions, Dr D. Engelman for helpful discussion, Dr F. Sigworth for use of fluorimeter, and Drs S. Jaswal and C. Atreya for critical reading of this manuscript. This work was supported by a grant from the National Institutes of Health (DK54899). J.D.K. is supported by a National Science Foundation Graduate Research Fellowship.
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