Date of this Version
Truong, T., Bilyeu, C. (2018). Self-Assembling Nanoluciferase Fragments as Probes for Alpha-Synuclein Aggregation (Unpublished undergraduate thesis). University of Nebraska-Lincoln, Lincoln, Nebraska.
Alpha-Synuclein (ASYN) is a protein that is abundant in the human brain and is found mainly at the tips of nerve cells (neurons) in specialized structures called presynaptic terminals. Recent efforts have shown that ASYN is the major constituent of Lewy bodies, protein clumps that are pathological hallmarks of Parkinson’s disease. Given this clear role of protein aggregation in diseases such as Alzheimer’s, Type II Diabetes, Huntington’s, and Parkinson’s, there is a critical need for assays capable of quantifying ASYN aggregation in living systems. To address this need, we have employed our recently discovered self-assembling Nanoluciferase (Nluc) fragments to generate a sensitive luminescence-based assay for ASYN aggregation in living cells. Our design entailed the fusion of ASYN to the N-terminal fragment of Nluc, allowing for analysis of ASYN aggregation in cells by monitoring luminescence signal output as a result of assembly with the complementary C-terminal Nluc fragment. We leveraged this system to interrogate the influence of mutations as well as small molecules on ASYN aggregation. The advantages of this proposed system include low background and compatibility with living systems. In the long term, this assay provides the basis for analysis of the effect of mutations on ASYN aggregation as well as a platform for the identification of inhibitors of ASYN aggregation.