1. Molecular Mechanism of Insulin and Amylin Release in the  -cell and Etiology of Type II Diabetes Mellitus Aleksandar Jeremic Department of Biological Sciences The George Washington University Phone: (202) 994-7899 Email: jerema@gwu.edujerema@gwu.edu http://www.gwu.edu/%7Eclade/faculty/jeremic /

2. Overall Goal: To dissect the “nanomechaniscs” of the insulin and amylin release in the  -cell and etiology of TTDM insulin amylin 2D / 3D AFM image of amylin fibril growth

3. 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

4. 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

5. 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)

6.  -cell insulin amylin amyloid fibrils Aim 2. Molecular mechanism of islet amyloidosis 18 23 25 29 hAmylin: KCNTATCATQRLANFLV H SSNN F G AI L SS TNVGSNTY rAmylin: KCNTATCATQRLANFLV R SSNN L G PV L PP TNVGSNTY Approach: -Dynamics of amyloid fibril assembly (time-lapse AFM) -Dysfunction of insulin release (TIRF and ELISA) -Cell integrity (Ca 2+ imaging, Caspases activation) -Peptide conformation transitions (CD and Raman Spectroscopy) -Protein-Lipid interactions (AFM-Force Imaging; microcalorimetry) (MIN-6)

7. Role of chaperones in amylin processing and fibrilogenesis pre-pro-amylinamylin PC2 7B2 Chaperone Approach: - RNA interference - Over expression of its dominant negative form Role of metal-peptide complexes in islet amyloid fibril formation Zn insulinamylin Approach: - AFM, CD, ITC - Single aa substitution: 1. His 18 (Zn-peptide complex) 2. Phe 23 (aromatic interactions) 3. Pro 25-29 (secondary structure)