The anti-oxidant enzyme Cu,Zn superoxide dismutase (SOD1) is associated with amyotrophic lateral sclerosis (ALS) through more than one hundred causative mutations. The Rac1 GTPase regulates oxidant levels through its role in regulating NADPH oxidase (Nox) activity. Intriguingly, SOD1 was recently shown to bind Rac1 in a nucleotide and redox dependent manner to regulate Rac1 activity. Furthermore, two ALS-associated mutations in SOD1 were shown to render the SOD1/Rac1 interaction insensitive to redox regulation, hypothesized to cause increased oxidant levels via Nox hyperactivity. However, this premise is based on limited qualitative results obtained from only two bacterially-derived mutants. Unlike natural SOD1 and human SOD1 expressed in S. cerevisiae, E. coli-derived human SOD1 lacks both N-terminal acetylation and adequate metallation. In addition, we recently found that 40-50% of SOD1 in human erythrocytes is modified by glutathione - a key player in the cellular redox state. Glutathionylation promotes SOD1 dimer dissociation, which is a necessary initiating event for SOD1 aggregation and a focus of the parent proposal. Here, we aim to provide a quantitative description of the SOD1/Rac1 interaction, and its proposed relationship to ALS, using SOD1 isolated from human erythrocytes and S. cerevisiae. We will use NMR and other biophysical techniques to characterize and quantify the Rac1/SOD1 interaction in the presence and absence of modifications and ALS-associated mutations in SOD1. The recent discovery that SOD1 interacts with the Nox complex through Rac1 provides a potential link between SOD1 (dys)function and downstream events leading to cell death. It is therefore important to delineate how SOD1 modifications and familial ALS mutations affect the interactions with Rac1, which is the goal of this supplement.
The goal of the proposed work is to define a mechanism for the regulation of Rac1 by SOD1. Knowledge of this mechanism will lead to strategies for determining whether this new regulatory role of SOD1 is related to ALS. Furthermore if the Rac1/SOD1 the Rac1/SOD1 complex and contribute to ALS therapies.
|Li, Bo; Tunc-Ozdemir, Meral; Urano, Daisuke et al. (2018) Tyrosine phosphorylation switching of a G protein. J Biol Chem 293:4752-4766|
|Williams 2nd, Benfeard; Convertino, Marino; Das, Jhuma et al. (2017) Molecular Mechanisms of the R61T Mutation in Apolipoprotein E4: A Dynamic Rescue. Biophys J 113:2192-2198|
|Woods, Chanin T; Lackey, Lela; Williams, Benfeard et al. (2017) Comparative Visualization of the RNA Suboptimal Conformational Ensemble In Vivo. Biophys J 113:290-301|
|Dagliyan, Onur; Karginov, Andrei V; Yagishita, Sho et al. (2017) Engineering Pak1 Allosteric Switches. ACS Synth Biol 6:1257-1262|
|Zhu, Cheng; Mowrey, David D; Dokholyan, Nikolay V (2017) Computational Protein Design Through Grafting and Stabilization. Methods Mol Biol 1529:227-241|
|Mu, Xin; Choi, Seongil; Lang, Lisa et al. (2017) Physicochemical code for quinary protein interactions in Escherichia coli. Proc Natl Acad Sci U S A 114:E4556-E4563|
|Brodie, Nicholas I; Popov, Konstantin I; Petrotchenko, Evgeniy V et al. (2017) Solving protein structures using short-distance cross-linking constraints as a guide for discrete molecular dynamics simulations. Sci Adv 3:e1700479|
|Convertino, Marino; Dokholyan, Nikolay V (2016) Computational Modeling of Small Molecule Ligand Binding Interactions and Affinities. Methods Mol Biol 1414:23-32|
|Redler, Rachel L; Das, Jhuma; Diaz, Juan R et al. (2016) Protein Destabilization as a Common Factor in Diverse Inherited Disorders. J Mol Evol 82:11-6|
|Dokholyan, Nikolay V (2016) Controlling Allosteric Networks in Proteins. Chem Rev 116:6463-87|
Showing the most recent 10 out of 86 publications