Skip to main content

Molecular biophysical study of tilted peptide-lipid interactions in lipid bilayers and their application in membrane fusion

Final Activity Report Summary - TILTED PEPTIDES (Molecular biophysical study of tilted peptide-lipid interactions in lipid bilayers and their application in membrane fusion.)

Amyloid formation has been implicated in a wide range of human diseases including Alzheimer disease, Parkinson disease and type 2 diabetes mellitus (DM2). DM2 is histopathologically characterised by the presence of fibrillar amyloid deposits in the pancreatic islets of Langerhans. Amyloid cytotoxicity is thought to be an early mechanism involved in death of insulin-producing islet ß-cells in DM2. The main component of islet amyloid, and the actual fibril-forming molecule, is a 37 amino acid peptide called human islet amyloid polypeptide (hIAPP) or amylin, which is produced along with insulin in the pancreatic islet b-cells. The normal physiological role of soluble hIAPP is not entirely clear, but it is believed to play a role in gastric emptying, suppression of food intake and glucose homeostasis.

Increasing evidence suggests the importance of IAPP misfolding and amyloid fibril formation in the pathogenesis of DM2. Fibril formation is a multi-step process in which the formation of smaller oligomeric protein structures precedes the formation of mature fibrils. There is still debate as to whether oligomers, mature fibrils or something else are responsible for toxicity.

The purpose of this research was to improve understanding of the interaction between IAPP and the membrane and to study the mechanism of toxicity. In particular, we addressed the following issues:

1. not only the mechanism of action of amyloidogenic proteins remains unknown, but the nature of the cytotoxic species is still a topic of discussion. The prevailing view is that membrane damage and concomitant ß-cell death are caused by cytotoxic hIAPP oligomers. In addition to this hypothesis, recent reports suggest that the fibrillar form of amyloidogenic proteins can also be cytotoxic. Therefore, the first question we wanted to answer was the identification of cytotoxicity causes.
2. hIAPP is synthesized in ß-cells as a preprohormone and processing occurs in several steps. It was suggested that in type 2 diabetes processing is defective and the intracellular aggregation of the processing intermediates may represent the initial step of the formation of amyloid. We investigated this possibility by analysing the aggregation, structure and membrane interaction of mature hIAPP and its precursors in vitro.
3. a previous study showed that it was the N-terminal part of hIAPP (hIAPP1-19), and not the amyloidogenic region (hIAPP20-29), which played a major role in the insertion of the peptide into the membrane. We decided to fully examine the aggregation in the presence of membranes, the membrane damage, the structure and the insertion of the peptide hIAPP1-19 in comparison with the full-length hIAPP.
4. hIAPP is stored in a relatively low pH environment in the pancreatic secretory granules prior to its release to the extracellular environment, i.e. in a higer pH environnment. We would like to know whether the pH could influence the rate of fibril formation and membrane leakage.

To investigate the mechanism of hIAPP and the lipid-peptide interaction we used a multidisciplinary approach employing different techniques as for example dichroism circular (CD), monolayer techniques, microscopy, solid-state nuclear magnetic resonance (NMR) and fluorescence spectroscopy. Moreover, in order to mimic the ß-cells membranes, vesicles with zwitterionic and negatively charged phospholipid were used.