Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Biochemistry

Supervisor

Dr. Murray Junop

Abstract

ALS is a progressive and fatal neurodegenerative disorder whose pathologic hallmark is the presence of neuronal cytoplasmic inclusions (NCIs). In approximately 97% of ALS cases, NCIs are found to be TDP-43+. Rho-guanine nucleotide exchange factor (RGNEF) has recently been implicated in ALS pathophysiology through its co-localization and coimmunoprecipitation with TDP-43+. RGNEF has also been shown to harbour cytoprotective effects in the N-terminal region and is responsible for the regulation of low molecular weight-neurofilament (NFL), intimately involved neural structure, through its predicted RNA-binding domain (RBD). This study looked to purify constructs of RGNEF through nickel immobilized metal affinity chromatography (Ni-IMAC) and validate the previously documented functions of RGNEF ex-vivo. N-terminal constructs of RGNEF tended to self-associate into higher order structure and did not display a direct interaction with TDP-43 through SPR. pMJ5922, an RBD construct, was successfully purified and did demonstrate RNA-binding ex-vivo, validating maintenance of functionality following purification.

Summary for Lay Audience

Amyotrophic Lateral Sclerosis (ALS) is a progressive and fatal neurodegenerative disorder for which the cause is often unknown. Progressive neurodegeneration is in part caused by a combination of maladaptive protein and RNA processing. Typically, proteins involved in these defective systems are RNA-binding proteins (RBPs). One such protein is Tar DNA-binding protein-43 (TDP-43), whose involvement in ALS pathologic mechanisms leads to its aggregation and formation of neuronal cytoplasmic inclusions (NCIs). TDP-43 has been shown to co-localize with a secondary protein, Rho-guanine nucleotide exchange factor (RGNEF), in ALS-affected cells. Expression of RGNEF within cells typically helps with cell survival and the regulation of important structural protein RNA. Association of RGNEF into the NCIs may decrease these functions. In this study, segments of RGNEF were purified and tested to determine biophysical characteristics and if function could be maintained outside of the cell. Tests showed that with a small segment of RGNEF, TDP-43 did not interact directly. This segment of the protein also tended to self-associate into larger structures. A second segment of RGNEF did display binding to RNA, validating this function outside of a cellular environment.

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