A Characterization of RGNEF Biophysical Properties and Interactome

Brooke E. Wile, The University of Western Ontario

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.