The impact of Cellular Environment on IAPP Fibril Polymorphism - Cryo-EM Structural Analysis

Sammanfattning

Diabetes type 2 Mellitus (T2DM) is a worldwide disease that millions suffer from. Characteristic for the disease is the misfolding and aggregation of the hormone hIAPP into insoluble fibrils that disturb and lead to death of pancreatic beta cell function in patients. Structural research of these fibrils is of great importance to discover new therapeutic targets and develop therapies for TD2M. Research is mostly done in vitro with synthetic peptide or with patient derived fibrils. This study focuses on uncovering different aspects that complement the ongoing research. The study aimed to by cryo electron microscopy (Cryo-EM) uncover the structure of hIAPP fibrils that had been extracted from a pancreatic beta cell-line overexpressing hIAPP (INS1-E hiAPP CLON3). This was done by culturing of INS1-E hiAPP CLON3 cells that were later induced with glucose to start overexpression of hIAPP. The cells were harvested and fibrils extracted by lysis with added N-lauroylsarcosine sodium salt (sarkosyl) and ultracentrifugation. Lastly the fibrils were imaged both by Atomic force microscopy (AFM) and by Cryo-EM, resulting in images of fibrillar structures as well as unknown round structures with membranes/vesicles. Another aim of this study was to form fibrils from synthetic hIAPP in INS1-E cell-conditioned medium and analyze with Cryo-EM. Mimicking an environment of native beta cells, to resemble the conditions for fibril formation in T2DM patients and as well as research the secretome of INS1-E cells. Thioflavin T (ThT) assays were performed to compare fibrillization kinetics between hIAPP in cell-conditioned medium and HEPES, from which it could be determined that the cell-secreted particles in the medium slowed down fibrillization and gave fewer fibrils. Addition of protease inhibitor cocktail to the medium led to more rapid and higher amounts of fibrillization than without. Cryo-EM showed fibrillar structures after addition of protease inhibitor cocktail to the medium and washing with sarkosyl. Indicating that proteases from the medium may be degrading fibrils or early intermediates.