Date of Award

2020

Document Type

Thesis

Degree Name

M. S.

Department

Chemistry and Biochemistry

First Advisor

Erich G. Chapman

Second Advisor

Martin Margittai

Third Advisor

Scott Horowitz

Keywords

Aggregation, Dimer, Electrolytes, TDP-43

Abstract

Trans-active response (TAR) DNA-binding protein 43 (TDP-43) is essential for RNA processing but can also form toxic cytoplasmic inclusions in neurons of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RNA-binding has been shown to have the potential to decrease or inhibit the aggregation of TDP-43, indicating that supplying RNA therapeutics may be a solution to treat these neurodegenerative disorders. However, the mechanism of aggregation, transitioning from TDP-43’s native dimeric state to small oligomers to toxic aggregates, is still relatively unknown. This needs to be established before determining how to target and disrupt this aggregation. Using GFP-TDP-43, we first sought to further classify the native state of TDP-43 by characterizing its dimer interfaces by structure-guided mutagenesis. Combined, our results indicate that TDP-43 can form head-to-tail dimerization between two N-terminal domains (NTD) and head-to-head dimerization between two RNA-recognition motifs 2 (RRM2). When both interfaces are disrupted, we isolate a tentatively assigned monomeric species. The second aim of our study was to determine the propensity for aggregation of the same family of TDP-43 mutants. Using a denaturing purification method and low ionic strength buffers, we obtain a metastable intermediate of GFP-TDP-43 that undergoes inducible aggregation when electrolytes are added back to solution. By monitoring protein aggregation using turbidity assays and dynamic light scattering (DLS) measurements we observe that the addition of smaller divalent cations induces faster aggregation and produces larger aggregates than corresponding amounts of larger monovalent cations. These data support a model in which different electrolytes alter the zeta potential of TDP-43. Finally, we show that aggregation can be mitigated by the addition of DNA oligomers with some degree of sequence specificity. Excess TG12 DNA significantly reduces the rate of aggregation and size of aggregated species of GFP-TDP-43 whereas excess AC12 DNA has no significant difference in aggregation from GFP-TDP-43 without DNA. This result supports the hypothesis that UG-rich RNA should be considered for development of a RNA therapeutic drug to help patients with ALS.

Publication Statement

Copyright is held by the author. User is responsible for all copyright compliance.

Provenance

Received from ProQuest

Rights holder

Elizabeth Ehrhardt

File size

71 p.

File format

application/pdf

Language

en

Discipline

Biochemistry

Included in

Biochemistry Commons

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