Eucaryotic copper-zinc superoxide dismutases (CuZnSODs) are 32 kDa homodimeric metalloproteins that catalyze the conversion of superoxide radical to molecular oxygen and hydrogen peroxide. The enzyme is particularly abundant in spinal tissue and red blood cells in mammals. Approximately 60 different single site mutations in human CuZnSOD have individually been linked to an inherited (familial) form of amyotrophic lateral sclerosis (FALS or Lou Gehrig's disease). The disease is characterized by progressive paralysis resulting from motor neuron degeneration and death. Trangenic mouse, neuronal cell culture, and in-vitro studies suggest strongly that a copper-mediated toxic gain of function instead of a loss of superoxide disproportionation activity is responsible for disease onset in individuals heterozygous for any one of the FALS CuZnSOD mutations. Many of these human FALS mutants have been prepared in the laboratory and have been found to have aberrant metal binding properties relative to wild type CuZnSOD. The new properties of the FALS mutant proteins will likely be linked to the gain of the toxin function involved in FALS causation. A recently discovered class of molecules termed """"""""copper chaperones for SOD"""""""" (CCS) are responsible for sequestering reactive copper ion from the cellular environment and inserting it correctly into newly translated SOD apoprotein. Because the toxic gain of function of FALS mutant SODs appears to be copper-mediated, a search for inhibitors of the copper chaperone may represent a novel therapeutic avenue for FALS. The primary objectives of this proposal are to generate three-dimensional, atomic resolution structural information on the human and yeast FALS mutants and mutant protein analogs, their respective copper chaperones, and their complexes via the well-established tools of X-ray diffraction and analytical ultracentrifugation. Specifically, we shall: (1) compare and contrast the FALS structures with normal human CuZnSOD and yeast FALS mutant analog structures. (2) use X- radiation from synchroton sources tuned to copper and zinc absorption edges to determine the location and amount of these ions in the metal binding sites of remetallated and as-isolated FALS CuZnSODs, (3) illuminate the postulated interaction of peroxynitrite with normal and FALS mutant CuZnSOD proteins by determining their structures when exposed to peroxynitrite, (4) determine and analyze the structures of several species of CCS molecules alone and in complex with wild type and FALS mutant CuZnSODs, and elucidate the solution properties of these molecules and their complexes under different conditions using contemporary analytical ultracentrifugation methods. These studies will not only promote understanding of the molecular causes of FALS, but also will provide fundamental information on copper ion homeostasis and trafficking at the level of protein- protein interactions.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS039112-01A1
Application #
6130636
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Heemskerk, Jill E
Project Start
2000-05-05
Project End
2005-04-30
Budget Start
2000-05-05
Budget End
2001-04-30
Support Year
1
Fiscal Year
2000
Total Cost
$233,883
Indirect Cost
Name
University of Texas Health Science Center San Antonio
Department
Biochemistry
Type
Other Domestic Higher Education
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
Fetherolf, Morgan M; Boyd, Stefanie D; Taylor, Alexander B et al. (2017) Copper-zinc superoxide dismutase is activated through a sulfenic acid intermediate at a copper ion entry site. J Biol Chem 292:12025-12040
Sea, Kevin; Sohn, Se Hui; Durazo, Armando et al. (2015) Insights into the role of the unusual disulfide bond in copper-zinc superoxide dismutase. J Biol Chem 290:2405-18
Ivanova, Magdalena I; Sievers, Stuart A; Guenther, Elizabeth L et al. (2014) Aggregation-triggering segments of SOD1 fibril formation support a common pathway for familial and sporadic ALS. Proc Natl Acad Sci U S A 111:197-201
Bouldin, Samantha D; Darch, Maxwell A; Hart, P John et al. (2012) Redox properties of the disulfide bond of human Cu,Zn superoxide dismutase and the effects of human glutaredoxin 1. Biochem J 446:59-67
Leitch, Jeffry M; Li, Cissy X; Baron, J Allen et al. (2012) Post-translational modification of Cu/Zn superoxide dismutase under anaerobic conditions. Biochemistry 51:677-85
Seetharaman, Sai V; Winkler, Duane D; Taylor, Alexander B et al. (2010) Disrupted zinc-binding sites in structures of pathogenic SOD1 variants D124V and H80R. Biochemistry 49:5714-25
Seetharaman, Sai V; Taylor, Alexander B; Holloway, Stephen et al. (2010) Structures of mouse SOD1 and human/mouse SOD1 chimeras. Arch Biochem Biophys 503:183-90
You, Zheng; Cao, Xiaohang; Taylor, Alexander B et al. (2010) Characterization of a covalent polysulfane bridge in copper-zinc superoxide dismutase . Biochemistry 49:1191-8
Tiwari, Ashutosh; Liba, Amir; Sohn, Se Hui et al. (2009) Metal deficiency increases aberrant hydrophobicity of mutant superoxide dismutases that cause amyotrophic lateral sclerosis. J Biol Chem 284:27746-58
Seetharaman, Sai V; Prudencio, Mercedes; Karch, Celeste et al. (2009) Immature copper-zinc superoxide dismutase and familial amyotrophic lateral sclerosis. Exp Biol Med (Maywood) 234:1140-54

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