Tryptophan 32 Potentiates Aggregation and Cytotoxicity of a Copper/Zinc Superoxide Dismutase Mutant Associated with Familial Amyotrophic Lateral Sclerosis

Abstract

One familial form of the neurodegenerative disease, amyotrophic lateral sclerosis, is caused by gain-of-function mutations in the gene encoding copper/zinc superoxide dismutase (SOD-1). This study provides in vivo evidence that normally occurring oxidative modification to SOD-1 promotes aggregation and toxicity of mutant proteins. The oxidation of Trp-32 was identified as a normal modification being present in both wild-type enzyme and SOD-1 with the disease-causing mutation, G93A, isolated from erythrocytes. Mutating Trp-32 to a residue with a slower rate of oxidative modification, phenylalanine, decreased both the cytotoxicity of mutant SOD-1 and its propensity to form cytoplasmic inclusions in motor neurons of dissociated mouse spinal cord cultures. Amyotrophic lateral sclerosis (ALS)5 is an adult onset neurodegenerative disease in which two percent of all cases are caused by mutations in the gene encoding copper/zinc superoxide dismutase (SOD-1) (1). More than 100 mutations distributed throughout the structure of the protein introduce a toxic gain of function that decreases protein solubility, leading to aggregation and the formation of both detergent-insoluble SOD-1 (2, 3) and inclusions that are a histopathological hallmark of ALS (4 -8).The nature of the toxic gain of function imparted by familial ALS (fALS)-causing SOD-1 mutations is not completely understood (reviewed in Ref. 9). These mutations do, however, provide evidence that diverse and relatively minor changes in SOD-1 primary structure can cause fALS. Approximately ninety percent of ALS is sporadic, and it is plausible that posttranslational modification of wild-type proteins affects structural changes analogous to those caused by point mutation (10). Indeed, Lewy body-like inclusions in sporadic ALS are immunoreactive with antibodies to SOD-1 (11-13). It has also been demonstrated that oxidative post-translational modification of SOD-1 occurs in vivo with aging (14) and in association with the fALS (7, 15-17), Parkinson (18), and Alzheimer (18) neurodegenerative diseases. There is also considerable evidence that fALS-causing mutations predispose SOD-1 to post-translational modifications (19 -21), and that in vitro oxidative modification of SOD-1 induces aggregation (22-24).An understanding of how modifications that occur in vivo confer or potentiate toxic properties in proteins is lacking. Given the potential importance of post-translational modification of SOD-1, a study was designed to comprehensively characterize modifications that occur in vivo. Modification-prone residues in wild-type SOD-1 isolated from murine and human erythrocytes were identified and changed to residues that were less likely to be modified. Effects upon survival and aggregation were measured to determine whether preventing modification would attenuate the toxicity of SOD-1 with fALS-causing SOD-1 mutations. Tryptophan 32 (Trp-32) was shown to be modified in a significant fraction of as-isolated SOD-1. Herein we report that changing Trp-32 to a residue with a slower rate ...

Proteasomes remain intact, but show early focal alteration in their composition in a mouse model of amyotrophic lateral sclerosis

“…An antibody to human SOD1 (SOD100) did not label 20S/26S proteasomes, therefore, providing no evidence of stable interaction between SOD1 G93A and proteasomes being responsible for decreased proteasome activity in lumbar spinal cord. However, this experiment does not rule out the possibility that proteasomes are inhibited by post-translationally modified forms of mutant SOD1 or cleavage products not recognized by the antibody, even though SOD100 does recognize insoluble forms of SOD1 including SDS-resistant high molecular weight complexes that accumulate in spinal cord during the course of the disease as well as oxidatively modified mutant SOD1 (Taylor et al 2007). Our results complement the conclusion of Bennett et al (2005) that polyglutamine-expanded proteins or mutant rhodopsin do not impair the ubiquitin-proteasome system by clogging proteasomes.…”

Intermolecular transmission of superoxide dismutase 1 misfolding in living cells

“…W32 is the only tryptophan in the human protein and has previously been identified as a site of oxidative modification and a potentiator of aggregation (37). Moreover, W32 is highly solvent exposed, ranking in the 89th percentile for solvent exposure among nonredundant tryptophans in the Protein Data Bank.…”

A Common Property of Amyotrophic Lateral Sclerosis-associated Variants

“…Wild type (WT) SOD1 purified from human erythrocytes was purchased from Sigma and was fully metallated as indicated by native gel electrophoresis and MS of the intact protein. Metal content of the Sigma protein was analyzed by measuring the intact protein mass by performing electrospray ionization-MS out of pure, high pressure liquid chromatography (HPLC) grade water as described previously (24), affording the ratio of metallated versus apo SOD1. Differences in ionization efficiency of the apo versus metallated SOD1, as well as lower limits for the detection of apo SOD1 were accounted for by converting the metallated SOD1 to apo SOD1 using 0.1% formic acid.…”

“…Using a Fourier transform ion cyclotron resonance mass spectrometer (apex qe-94; Bruker Daltonics Inc., Billerica, MA), all of the proteins were checked for post-translational modifications via MS of the intact protein as described in Ref. 24. WT SOD1 (Sigma) was judged to consist of less than 12% non-native post-translationally modified SOD1, and the most modified fALS-SOD1 variant consisted of less than 30% non-native post-translationally modified proteins.…”