Presentation Title

Identifying Amyloidogenic IAPP Protein Sequences from Diabetic and Nondiabetic Organisms

Faculty Mentor

David Moffet

Start Date

18-11-2017 9:30 AM

End Date

18-11-2017 9:45 AM

Location

9-273

Session

Bio Sciences 3

Type of Presentation

Oral Talk

Subject Area

biological_agricultural_sciences

Abstract

Roughly 1.4 million individuals in the United States are diagnosed with type-II diabetes each year. As the disease progresses, patients lose pancreatic β cells (the cells that produce insulin) with up to a 45% loss of pancreatic mass. Although the cause of the disease is unknown, it is believed that islet amyloid polypeptide (IAPP) is one of the agents responsible for the death of β cells. IAPP accumulates in the pancreas, where it aggregates into a variety of toxic forms that are harmful to β cells. In this experiment, we synthesized animal IAPP genes and attached them to green fluorescent protein, EGFP. Upon expression in E. coli, the fluorescence, or lack thereof, serves as an assay to determine amyloidogenicity. Additionally, we viewed IAPP aggregation through Atomic Force Microscopy (AFM) to identify fiber formation of various animal IAPP sequences. Finally, we imported our sequences into TANGO computational software to indicate regions of amyloidogenicity in the IAPP gene. By comparing IAPP amyloidogenicity with an organism’s propensity to develop type 2 diabetes, we hope to establish a direct causality between IAPP aggregation and type 2 diabetes within the animal kingdom.

Summary of research results to be presented

We have produced high-resolution atomic force microscopy images for thirteen unique animal sequences. Four of these sequences, cat, chicken, horse, and seal, clearly demonstrate significant amyloid formation. In order to confirm our AFM findings, we have turned to a variety of supporting techniques. We analyzed our sequences computationally with the Tango algorithm, designed to predict aggregating regions with polypeptide chains. The results of which largely coincide with our AFM data. Furthermore, we have employed a unique assay of combining IAPP sequences of interest with EGFP in a recombinant plasmid, which is expressible in E. coli. Fluorescence is then correlated to amyloid formation inversely, as EGFP is unable to fold and thus unable to fluoresce when bound to sequences that quickly aggregate. We have performed this assay for human, guinea pig, monkey, orangutan, and degu and found these results to be consistent with the existing literature. Finally, circular dichroism spectroscopy is currently being performed to determine B-sheet structure of sequences that have demonstrated amyloidogenicity in other assays. These CD studies are ongoing, but promising, and will completed by the time of SCCUR 2017.

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Nov 18th, 9:30 AM Nov 18th, 9:45 AM

Identifying Amyloidogenic IAPP Protein Sequences from Diabetic and Nondiabetic Organisms

9-273

Roughly 1.4 million individuals in the United States are diagnosed with type-II diabetes each year. As the disease progresses, patients lose pancreatic β cells (the cells that produce insulin) with up to a 45% loss of pancreatic mass. Although the cause of the disease is unknown, it is believed that islet amyloid polypeptide (IAPP) is one of the agents responsible for the death of β cells. IAPP accumulates in the pancreas, where it aggregates into a variety of toxic forms that are harmful to β cells. In this experiment, we synthesized animal IAPP genes and attached them to green fluorescent protein, EGFP. Upon expression in E. coli, the fluorescence, or lack thereof, serves as an assay to determine amyloidogenicity. Additionally, we viewed IAPP aggregation through Atomic Force Microscopy (AFM) to identify fiber formation of various animal IAPP sequences. Finally, we imported our sequences into TANGO computational software to indicate regions of amyloidogenicity in the IAPP gene. By comparing IAPP amyloidogenicity with an organism’s propensity to develop type 2 diabetes, we hope to establish a direct causality between IAPP aggregation and type 2 diabetes within the animal kingdom.