The Valentine program project seeks to determine the molecular basis of amyotrophic lateral sclerosis (ALS) caused by mutations in superoxide dismutase 1 (SODl). Having established that SODl proteins are amyloidogenic, four Projects propose novel experiments toward luiderstanding the nature of toxicity in ALS. The analytical core (Core A;PI: Whitelegge) provides services for physical, chemical and proteomic analysis of SODl-multimers and other extracts from human cells and transgenic arumals.
Specific Aim 1. Metal analysis: Incomplete or improper metallation of SODl has profotind effects upon the protein's properties and it is therefore critically important that levels of Cu and Zn are measured accurately in samples important to projects 1, 2, 3 &4. Inductively coupled plasma mass spectrometry (ICP-MS) as well as online size-exclusion chromatography ICP-MS will be used to measure metal levels with great sensitivity and reproducibility in whole tissues, isolated organelles and isolated proteins. Metal concentration, distribution, and oxidation state will be spatially imaged across tissue slices using synchrotron X-ray fluorescence microscopy (with Lisa Miller, Brookhaven National Laboratory).
Specific Aim 2. Protein multimer analysis: Non-native forms of SODl including monomeric and multimeric species appear key to toxicity in FALS. The diversity of structures that SODl can attain under different conditions, and the factors that influence its kinetics, will be characterized toward understanding the pathway to toxicity. Core A will use chromatography, mass spectrometry (including hydrogen-deuterium exchange and hydroxyl radical footprinting), synchrotron FTIR imaging, and other techniques to characterize the size and structure of multimer preparations and their heterogeneity. Soluble oligomers and fibrils produced in vitro (Proj. 1 &4), multimers from human embryoiuc stem cell motor neurons (HESCMNs; Proj. 2) and transgenic mice (Proj. 3) will be analyzed. Spedfic Aim 3. General and targeted proteomics: Measurements of protein identity and quantity are required for work proposed in Projects 1, 2,3 &4. Mass spectrometry protocols are established for a variety of measurements. Proteomics will be used to measure early cellular events occurring after HESC-MNs are exposed to defined SODl preparations (Projects 1 &2) toward defining the molecular basis of toxicity in FALS. We will use top-down high-resolution Fourier-transform mass spectrometry experiments and establish selected reaction monitoring protocols for accurate quantification of WT versus mutant SODl in mixed expression experiments (Projects 2 &3).

Public Health Relevance

Some families carry mutations in the enzyme Cu-Zn superoxide dismutase (SODl) that result in adult onset amyotrophic lateral sclerosis (ALS;motor neuron disease) that is most usually fatal within a few years. Our work aims to understand why very subtle alterations to SODl lead to ALS many years into life. Study of SODl structure and metals may identify new routes toward therapeutic intervention in progression of ALS.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Program Projects (P01)
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National Institute of Neurological Disorders and Stroke Initial Review Group (NSD)
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University of California Los Angeles
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Ming, Li-June; Valentine, Joan Selverstone (2014) Insights into SOD1-linked amyotrophic lateral sclerosis from NMR studies of Ni(2+)- and other metal-ion-substituted wild-type copper-zinc superoxide dismutases. J Biol Inorg Chem 19:647-57
Brown, Hilda H; Borchelt, David R (2014) Analysis of mutant SOD1 electrophoretic mobility by Blue Native gel electrophoresis; evidence for soluble multimeric assemblies. PLoS One 9:e104583
Ayers, Jacob; Lelie, Herman; Workman, Aron et al. (2014) Distinctive features of the D101N and D101G variants of superoxide dismutase 1; two mutations that produce rapidly progressing motor neuron disease. J Neurochem 128:305-14
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
Sheng, Yuewei; Abreu, Isabel A; Cabelli, Diane E et al. (2014) Superoxide dismutases and superoxide reductases. Chem Rev 114:3854-918
Ayers, Jacob I; Xu, Guilian; Pletnikova, Olga et al. (2014) Conformational specificity of the C4F6 SOD1 antibody; low frequency of reactivity in sporadic ALS cases. Acta Neuropathol Commun 2:55
Chan, Pik K; Chattopadhyay, Madhuri; Sharma, Shivani et al. (2013) Structural similarity of wild-type and ALS-mutant superoxide dismutase-1 fibrils using limited proteolysis and atomic force microscopy. Proc Natl Acad Sci U S A 110:10934-9
Rutherford, Nicola J; Lewis, Jada; Clippinger, Amy K et al. (2013) Unbiased screen reveals ubiquilin-1 and -2 highly associated with huntingtin inclusions. Brain Res 1524:62-73
Prudencio, Mercedes; Lelie, Herman; Brown, Hilda H et al. (2012) A novel variant of human superoxide dismutase 1 harboring amyotrophic lateral sclerosis-associated and experimental mutations in metal-binding residues and free cysteines lacks toxicity in vivo. J Neurochem 121:475-85
Lelie, Herman L; Liba, Amir; Bourassa, Megan W et al. (2011) Copper and zinc metallation status of copper-zinc superoxide dismutase from amyotrophic lateral sclerosis transgenic mice. J Biol Chem 286:2795-806

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