Amyotrophic lateral sclerosis (ALS) is a degenerative disorder of motor neurons in the spinal column, brainstem and motor cortex. 90% of ALS cases are sporadic (SALS). Of the remanining familial cases (FALS), 10-30% associate with mutations in the SOD1 gene encoding Cu,Zn- superoxide dismutase (SOD1). The mutations in SOD create a gain-of- peroxidase activity; decrease metal affinity; promote aggregation; and may imbue the SOD1 with the ability to catalyze toxic nitration reactions. These findings have led some to speculate that ALS patients suffer enhanced oxidative stress, perhaps as a result of bearing dysfunctional SOD1. Others have speculated that SALS may involve post-translational modifications to SOD1 that induce toxic functions; such modifications have not been heretofore identified. Our group has identified such a possible post-translational modification. We have been studying the cytotoxic lipid oxidation product 4-hydroxy-2- nonenal (HNE), which is known to be elevated in the AD brain and in the central nervous system of ALS patients. HNE and related alpha, beta-unsaturated aldehydes form covalent adducts with proteins and may act as heterobifunctional crosslinking agents. We have characterized HNE-reactive proteins in the AD brain using an antibody directed against HNE-protein adducts. We find that >95% of the HNE immunoreactivity in Western blots localizes to a single 32 kDa protein that we have identified as SOD1. We have developed an in vitro model system for studying HNE-SOD1 reactions, and we have begun to characterize sites of reactivity by MALDI-TOF-mass spectrometry. We now hypothesize that FALS-associated SOD1 will react more rapidly with HNE because of decreased distance between a key pair of lysine residues (Lys-9) in the interfacial region of the SOD1 homodimer, and that this reaction leads to protein aggregation and metal release from the enzyme. We seek funds to begin critically assessing this hypothesis. We will employ site-directed mutagenesis and an E. coli expression system to generate wild-type human SOD1 along with three mutant enzymes (G93A, G37R and G4V) that are associated with FALS. The enzymes will be characterized in vitro with respect to their rate of reaction with HNE and correlated properties, particularly copper release. In parallel work, a molecular modeling approach will be taken to study the effects of the mutations on the dimer interface and metal ligand binding sites. We hope these pilot studies will lead to more detailed understanding of the mechanism by which SOD1 is crosslinked by HNE in vivo, and perhaps eventually may lead to new therapeutic strategies for the treatment of ALS and/or AD.
