molecular mechanism of insulin and amylin release in the -cell and etiology of type ii diabetes...
TRANSCRIPT
Molecular Mechanism of Insulin and Amylin Release Molecular Mechanism of Insulin and Amylin Release
in the in the -cell and Etiology of Type II -cell and Etiology of Type II Diabetes MellitusDiabetes Mellitus
Aleksandar Jeremic
Department of Biological SciencesThe George Washington University
Phone: (202) 994-7899
Email: [email protected]
http://www.gwu.edu/%7Eclade/faculty/jeremic/
Overall Goal: To dissect the “nanomechaniscs” of the insulin and
amylin release in the -cell and etiology of TTDM
insulinamylin
2D / 3D AFM image of amylin fibril growth
Aim 1. Unraveling machinery regulating insulin and amylin release in -cells: A. Role of intracellular water channels and heterotrimeric G-proteins
B. Role of GTP-ase dynamin and actin
Aim 2. Molecular mechanism of amylin –derived islet amyloidosis:
A. Role of chaperones
B. Role of metal (Zn)-peptide complexes
Research Aims
Aim 1. Nanoscale Imaging of Hormone Secretion and Membrane Dynamics in the -cell
Approach:-Combined AFM and Confocal imaging
Secretory vesicle
NPY-RFP (cargo)Phogrin-GFP (membrane)
NPY
Phogrin
Role of intracellular water channels and heterotrimeric G-proteins in
regulation of insulin release
Approach:-AFM, Confocal Microscopy, ELISA-Blockers (Hg; Ab)-AQPs (RNA interference)
Role of dynamin and actin in the fusion pore dynamics and vesicle
recycling in the -cell
Approach:-AFM, Confocal, ELISA- RNA interference- Inhibitors (cytoch. D)
-cellinsulin
amylin
amyloid fibrils
Aim 2. Molecular mechanism of islet amyloidosis
18 23 25 29
hAmylin: KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY
rAmylin: KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY
Approach:-Dynamics of amyloid fibril assembly (time-lapse AFM)-Dysfunction of insulin release (TIRF and ELISA)-Cell integrity (Ca2+ imaging, Caspases activation)-Peptide conformation transitions (CD and Raman Spectroscopy)-Protein-Lipid interactions (AFM-Force Imaging; microcalorimetry)
(MIN-6)
Role of chaperones in amylin processing and fibrilogenesis
pre-pro-amylin amylin
PC2
7B2 Chaperone
Approach:- RNA interference- Over expression of its dominant negative form
Role of metal-peptide complexes in islet amyloid fibril formation
Zn
insulin amylin
Approach:- AFM, CD, ITC- Single aa substitution: 1. His18 (Zn-peptide complex) 2. Phe23 (aromatic interactions) 3. Pro25-29 (secondary structure)