The rate and equilibrium constants for a multistep reaction sequence for the aggregation of superoxide dismutase in amyotrophic lateral sclerosis

Abstract

Mutation-induced aggregation of the dimeric enzyme Cu, Zn superoxide dismutase 1 (SOD1) has been implicated in the familial form of the disease amyotrophic lateral sclerosis, but the mechanism of aggregation is not known. Here, we show that in vitro SOD1 aggregation is a multistep reaction that minimally consists of dimer dissociation, metal loss from the monomers, and oligomerization of the apo-monomers:where Dholo, Mholo, Mapo, and A are the holo-dimer, holo-monomer, apo-monomer, and aggregate, respectively. Under aggregationpromoting conditions (pH 3.5), the rate and equilibrium constants corresponding to each step are: A myotrophic lateral sclerosis (ALS) involves selective motor neuron death in the brain and spinal cord (1-6), initiating a progressive paralysis in midlife. Point mutations in the cytoplasmic homodimeric enzyme Cu, Zn superoxide dismutase 1 [SOD1, molecular mass (M m ) Ϸ 32 kDa] were identified as the primary cause of Ϸ20% cases of the familial form of ALS (FALS) (7,8) in contrast with the sporadic form. Approximately 90 distinct FALS mutations are known (9). The toxic gain-of-function of the mutants is believed to be associated with either intracellular misfolding and aggregation or oxidative damage caused by mutant SOD1-catalyzed aberrant reactions, although the two scenarios may not be mutually exclusive (10, 11). The aggregation hypothesis is supported by the observations that in both mice and cell culture models death of motor neurons is preceded by formation of cytoplasmic aggregates containing mutant SOD1 (12-15), SOD1 knockout mice do not develop motor neuron disease (16), and even Cu-depleted SOD1 mutants cause the disease in mice (17). Although aggregation of SOD1 has also been found in some cases of sporadic ALS (18)(19)(20), the sporadic disease is believed to have a different molecular basis (1). FALS and SOD1-linked sporadic ALS may belong to the general class of protein conformational disorders in which perturbation of protein folding leads to a relatively higher population of misfolded or partially folded (21) protein molecules, which then aggregate into regular (22) structured fibrils. Toxicity may arise because of the saturation of the cellular chaperone machinery by misfolded or aggregated SOD1 molecules (23).ALS is an age-related disease occurring in midlife or later, and it is not surprising that the in vitro aggregation of SOD1 solutions under physiological conditions is slow. Therefore, it is common to study SOD1 aggregation by perturbing the environmental conditions, lowering the pH to 3.5 (24), adding mild denaturants such as trifluoroethanol, or using heat treatment (25), such that disease-like aggregates can be detected on experimental time scales. Aggregation-prone FALS mutations and͞or loss of metals also decrease SOD1 stability (26,27), indicating that exposure to nonphysiological conditions, such as low pH, mimics the effect of mutation by similarly lowering the barrier for SOD1 aggregation. Although the rate of aggregation can be enhanced in vitro, the molecula...

Simple but predictive protein models

“…Based on these results, we predicted that changes in pH that lead to titration of saltbridge residues should destabilize SOD1 and increase the population of folding intermediates. Recent experimental studies [40] and our observations of the pH-dependent destabilization of SOD1 [41] confirm this prediction.…”

Transient structural distortion of metal-free Cu/Zn superoxide dismutase triggers aberrant oligomerization

“…SOD1 undergoes complex posttranslational maturation, including formation of the intramolecular C57-C146 disulfide bond, binding of Zn, chaperone-assisted insertion of Cu, and dimerization (10,11). However, several lines of evidence indicate that it is apo-SOD1, not the metal-bound states, that plays a key role in aberrant oligomerization (11)(12)(13)(14)(15)(16)(17)(18)(19). In vitro studies of the aggregation kinetics have implicated the monomeric apo state as the origin for aggregation (12)(13)(14).…”

Disulfide cross-linked protein represents a significant fraction of ALS-associated Cu, Zn-superoxide dismutase aggregates in spinal cords of model mice

“…An enzymatically active form of SOD1 is stabilized by acquiring copper͞zinc ions and a conserved intramolecular disulfide bond and further exhibits a very high dimerization constant (K d Ϸ 1.0 ϫ 10 Ϫ10 M Ϫ1 ) (9). Although protein aggregation is generally triggered by unfolding͞misfolding of protein molecules, these posttranslational modifications confer high stability on the SOD1 polypeptide; holo-SOD1 is active even in 10 M urea, 4% SDS, or at 80°C (10).…”

“…In contrast, demetallation of SOD1 mutant proteins leads to structural destabilization, the degree of which exhibits the reverse correlation with the mean survival time after ALS diagnosis (11). Several groups, including our own, have proposed that conformational changes of the SOD1 dimer and͞or the dissociation into monomers facilitate protein aggregation (9,(12)(13)(14); in fact, stabilization of SOD1 dimer by small molecules is effective in reducing the protein aggregates in vitro (15). ALS-associated mutations, thus, would inhibit sufficient control of the posttranslational modifications of SOD1, which further leads to the protein destabilization, misfolding, and aggregation.…”