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: jerema@gwu.edu

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)