Effective therapeutic strategies remain elusive for most age-related neurodegenerative disorders due to deficits in understanding of their multi-factorial pathologies. Small, soluble oligomers of disease-linked proteins have been proposed as cytotoxic agents in several such disorders, including Alzheimer's disease and amyotrophic lateral sclerosis (ALS). In the latter case, soluble non-native conformers of Cu, Zn superoxide dismutase (SOD1) appear before symptom onset and participate in numerous aberrant interactions with cellular components, supporting a primary role for these species in ALS pathogenesis. The blocking of surface patches that facilitate formation or deleterious interactions of soluble SOD1 oligomers thus holds therapeutic potential, but requires detailed structural and mechanistic insight that is not yet available. The ultimate goal of the proposed work is the determination of causes, mechanisms, and consequences of SOD1 misfolding and aggregation in ALS, particularly in early stages involving potentially toxic soluble misfolded states. Our recent findings of SOD1 destabilization by oxidative post-translational modifications suggest that SOD1 misfolding and aggregation are potential noxious factors even in the absence of destabilizing disease-associated mutations. The objective of the proposed work is to identify non-native structural features acquired by SOD1 as it misfolds and aggregates, and to identify determinants in the cellular environment (i.e. oxidative post-translational modifications) that drive these rearrangements. We hypothesize that both disease-linked mutations and oxidative modifications induce structural changes in SOD1 that promote oligomerization and expose surfaces that participate in non-native cellular interactions. Based on preliminary data identifying metastable soluble oligomers of wild type and mutant SOD1, as well as the destabilizing effect of a reversible oxidative modification (Cys-111 glutathionylation), we will test our hypothesis in three specific aims: 1) Characterize structural features of soluble SOD1 oligomers;2) Characterize the impact of oxidative modification on the SOD1 aggregation pathway;and 3) Identify non-native interacting surfaces that facilitate formation of soluble SOD1 oligomers. In the first two aims, we will use proven biophysical and biochemical techniques to assess changes in oligomeric state and surface hydrophobicity as wild type and mutant SOD1 aggregate. We will also probe metastable non-native oligomers with recently designed antibodies that specifically recognize misfolded SOD1 found in ALS patients, thereby identifying oligomers with structural similarity to potentially noxious species.
The second aim will ad- dress the contribution of a physiologically prevalent oxidative modification, Cys-111 glutathionylation, to non- native oligomerization by applying approaches used in aim 1 to the study of modified SOD1.
In aim 3, we will utilize innovative computational approaches to generate a structural model of a non-native SOD1 oligomer. By identifying exposed epitopes in soluble SOD1 oligomers as well as non-native interactions that stabilize them, these studies may reveal targets for preventing formation and aberrant interactions of these potentially toxic species.

Public Health Relevance

The proposed research addresses a critical public health concern by filling gaps in the current understanding of the mechanisms of protein aggregation and the connection of this process to neuronal death in neurodegenerative disorders. This work is relevant to the mission of the NIH not only because it contributes to fundamental knowledge regarding the link between protein aggregation and neurodegeneration, but also because it provides potential therapeutic targets for preventing noxious oligomerization of SOD1 in ALS.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM080742-06
Application #
8242523
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Wehrle, Janna P
Project Start
2007-04-01
Project End
2016-03-31
Budget Start
2012-06-01
Budget End
2013-03-31
Support Year
6
Fiscal Year
2012
Total Cost
$279,122
Indirect Cost
$89,122
Name
University of North Carolina Chapel Hill
Department
Biochemistry
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Li, Bo; Tunc-Ozdemir, Meral; Urano, Daisuke et al. (2018) Tyrosine phosphorylation switching of a G protein. J Biol Chem 293:4752-4766
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
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
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