Abstract
We have investigated the behavior of second RNA-recognition motif (RRM2) of neuropathological protein TDP43 under the effect of oxidative stress as modeled in vitro. Toward this end we have used the specially adapted version of H/D exchange experiment, NMR relaxation and diffusion measurements, dynamic light scattering, controlled proteolysis, gel electrophoresis, site-directed mutagenesis and microsecond MD simulations. Under oxidizing conditions RRM2 forms disulfide-bonded dimers that experience unfolding and then assemble into aggregate particles (APs). These particles are strongly disordered, highly inhomogeneous and susceptible to proteolysis; some of them withstand the dithiothreitol treatment. They can recruit/release monomeric RRM2 through thiol-disulfide exchange reactions. By using a combination of dynamic light scattering and NMR diffusion data we were able to approximate the size distribution function for the APs. The key to the observed aggregation behavior is the diminished ability of disulfide-bonded RRM2 dimers to refold and their increased propensity to misfold, which makes them vulnerable to large thermal fluctuations. The emerging picture provides detailed insight on how oxidative stress can contribute to neurodegenerative disease, with unfolding, aggregation, and proteolytic cleavage as different facets of the process.
Highlights
The system of redox signaling and regulation in the cell is vast and complicated[1]
Oxidative stress leads to formation of disulfide-linked aggregates of superoxide dismutase SOD1 which are associated with amyotrophic lateral sclerosis[6, 7]
Our results suggest that (i) aggregate particles (APs) formation does not depend on the presence of disulfide-linked network, rather it is sufficient to have two protein units connected by a single disulfide bond and (ii) the limiting step in the formation of APs is oxidative dimerization of RRM2
Summary
The system of redox signaling and regulation in the cell is vast and complicated[1]. One important element of this system is antioxidant defense. The same mechanism is relevant for inclusion bodies formed by neuropathological protein TDP-43 which are associated with amyotrophic lateral sclerosis, as well as frontotemporal lobar degeneration[8]. It appears that such disulfide-linked proteinaceous deposits are formed when the cell is nearing its death and redox homeostasis is severely impaired. When exposed to oxidizing environment, RRM2 forms disulfide-bonded dimers (db-RRM2) These dimers tend to self-associate and assemble into aggregate particles (AP), i.e. moderately sized soluble bodies consisting of partially cross-linked disordered peptide chains. Upon reduction by dithiothreitol the protein is largely returned into monomeric state, but a sizeable fraction remains aggregated[16], with the smaller APs no longer dependent on disulfide linkages
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