FG-Nup Aggregation with Specific Crowding Agents

Reskin, Sophie

Undergraduate Honors Thesis

FG-Nup Aggregation with Specific Crowding Agents 公开 Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/sb3978678

Abstract

A eukaryotic cell's genetic material is stored in the nucleus, surrounded and protected by the nuclear envelope. Spanning the nuclear envelope are nuclear pore complexes (NPC). The NPC's mechanism to regulate which macromolecules may pass between the nucleus and cytoplasm and which macromolecules are blocked remains undetermined. It is known that the NPC is filled with nucleoporins that have repeating phenylalanine and glycine residues (FG-nups). These FG-nups are intrinsically disordered proteins that act as the filter to determine what moves between the nucleus and cytoplasm. These proteins readily aggregate within living cells and their cellular state is unclear. Moreover, FG Nup aggregation resembles that of many other, primarily pathological, protein aggregates, and can be used as a model system to determine what features promote the aggregation state of this broad class of proteins. It is important to research the NPC and its selective filter because it is essential to understanding how the body maintains normal physiology by microcellular control. Alzheimer’s disease and other amyloid pathologies are associated with the NPC’s and its role in protein production. A fibrillation assay has been established to investigate amyloid formation of a specific protein domain taken from the NPC, FG124. In vitro molecular crowding is often used to simulate cellular conditions. Here we show that crowding agents differently affect amyloid formation. A 96-well plate and plate reader was used to record thioflavin fluorescence, which is an established readout of amyloid formation, to investigate how specific conditions affect FG124 over time. The basic goal is to obverse how fast it aggregates, how much it aggregates and any other notable differences between specific agents that modulate aggregation. These properties were quantitated by using MATLAB's curve fitting algorithms. Based on our results, we hypothesized that each specific crowder will have an individualized interaction with proteins due to the unique molecular structure. However, to prove this speculation, future experiments are required to test FG124 interactions with different concentration of crowders and other aromatic polymers.

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