Nano-scale imaging and dynamics of amylin-membrane interactions and its implication in type II diabetes mellitus.
Amylin, a 37-amino acid peptide hormone produced and secreted by pancreatic beta-cells, is the principal constituent of amyloid deposits in Type II Diabetes Mellitus (TTDM). Although much progress has been made in the understanding of amylin aggregation, molecular determinants that contribute to amylin aggregation in the pancreas and TTDM remain largely unknown. In order to better understand amylin aggregation and how membranes contribute to this process, visualization of amylin aggregation and deposition on membrane surface is of utmost importance. Here, we describe a new atomic force microscopy (AFM) approach to visualize amylin aggregation and to asses amylin-surface interactions. Using AFM in contact or tapping mode in fluid, amylin phase transitions on different supports were studied in real time and with high spatial nanometer-resolution. On mica, a two-stage sequential conversion of amylin from soluble monomer to small oligomers and further to mature amyloid fibrils was revealed by the AFM. This amylin conversion was accompanied by peptide conformational transition from random coil to beta-sheets assessed by CD spectroscopy. In contrast to mica, amylin formed amorphous amyloid deposits on planar lipid membranes consistent with pathological findings in diabetic subjects. Anionic lipid phosphatidylserine (PS) and membrane cholesterol had opposing effect on the kinetics and the extent of amylin aggregation. PS stimulated amylin aggregation, whereas cholesterol reversed the effect of PS. In addition, cholesterol sequestered amylin aggregates into membrane microdomains that in turn decreased amyloid deposition across the membranes. Hence, this reconstituted AFM approach offers new molecular insights to the etiology of diabetes that could be extended to investigate amylin aggregation in living islet cells at a subcellular resolution.
Cho
Won-Jin
WJ
Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Jena
Bhanu P
BP
Jeremic
Aleksandar M
AM
D000538:Aluminum Silicates; D000682:Amyloid / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D052160:Benzothiazoles; D001681:Biological Assay; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002784:Cholesterol / Q000378:metabolism; D002942:Circular Dichroism; D003924:Diabetes Mellitus, Type 2 / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D058228:Islet Amyloid Polypeptide; D008081:Liposomes / Q000378:metabolism; D018625:Microscopy, Atomic Force; D008954:Models, Biological; D036103:Nanotechnology / Q000379:methods*; D044367:Phase Transition; D010743:Phospholipids / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D051381:Rats; D012996:Solutions; D013499:Surface Properties; D013844:Thiazoles / Q000378:metabolism
D000538:Aluminum Silicates; D000682:Amyloid; D052160:Benzothiazoles; D058228:Islet Amyloid Polypeptide; D008081:Liposomes; D010743:Phospholipids; D012996:Solutions; D013844:Thiazoles; C009462:thioflavin T; D002784:Cholesterol; C011934:mica