SOD1 is abundantly glutathionylated at cysteine-111 in human tissue.12
Because of the proximity of this tripeptide moiety to the dimer interface, we hypothesized that it introduces steric clashes that favor dimer dissociation and/or hinder association of modified monomers. To distinguish these kinetic effects, we estimate the equilibrium dissociation constant Kd
using size exclusion chromatography and measure the rate constant for dimer dissociation (koff
) with surface plasmon resonance. Since the equilibrium dissociation constant Kd
is equal to the ratio of the rate constants for dissociation and association, we can then deduce effects on dimer formation rate from these two parameters. Dimer dissociation precedes disulfide reduction and metal loss13
(), so the contribution of the latter processes and irreversible aggregation is minimal compared to the dimer dissociation reaction in our SEC ( and ) and SPR () experiments.
Figure 6 Summary of effects of Cys-111 glutathionylation on the stabilities of WT SOD1 and the FALS mutants I112T and A4V. Above, general schematic of SOD1 aggregation pathway. Below, effect of glutathionylation on dimer dissociation rate (koff: reaction 1), monomer (more ...)
Glutathionylation has a dramatic effect on wild type dimer stability at equilibrium. The SOD1 homodimer is exceptionally stable, having low nanomolar binding affnity.12,15
In agreement with these findings, the unmodified wild type enzyme is dimeric under the conditions of our assay (). In contrast, the Kd
is increased by several orders of magnitude, to approximately 10–20 μ
M, such that there is appreciable (~20%) dissociation at physiological con- centration (). Although GS-SOD1WT
is destabilized at equilibrium relative to the unmodified enzyme, the rate of dimer dissociation does not differ significantly as a result of modification (). We therefore conclude that glutathionylation destabilizes SOD1WT
dimers by decreasing kon
, the rate constant for monomer association. These results indicate a much greater destabilizing effect than we initially estimated for the glutathione modification.12
However, we previously estimated Kd
using an activity assay to quantify SOD1 dissociation. This method of Kd
estimation is predicated on the decreased activity of monomeric SOD1 due to loop disorder35
and is sensitive enough for use at low protein concentrations that are unobservable by A280
However, this method has the disadvantage of being an indirect measure of the oligomerization state and is susceptible to interference by factors unrelated to monomerization that influence the mobility of active site loops. It may be that altered loop mobilities caused by glutathionylation result in dismutase-active monomeric SOD1, which would cause an underestimation of Kd
using this method. SEC analysis, by contrast, is a simple and direct method for assessing the extent of dimer dissociation and clearly demonstrates the striking destabilization of SOD1WT
dimers by Cys-111 glutathionylation.
Glutathionylation also destabilizes SOD1A4V
dimers. The Kd
of this variant has previously been reported as 3 μ
which agrees with our calculated lower limit of 1 μ
M (). Modification by the glutathione moiety results in a significant shift toward monomeric SOD1 and an ~10-fold increase in Kd
(). As in the wild type, glutathionylation does not affect dissociation kinetics in SOD1A4V
, indicating that destabilization of GS-SOD1A4V
dimers occurs primarily through effects on kon
. Glutathionylation may hinder monomer association in SOD1WT
by sterically blocking the formation of certain interface contacts. Alternatively, glutathionylation at cysteine-111 may promote local structural rearrangements in these monomers that impede formation of interface contacts. The ~3.8 °C decrease in apparent melting temperature of GS-SOD1A4V
monomers () may provide evidence for this; however, it is also possible that structural differences exist that do not significantly alter secondary structural elements.
To our knowledge, the stability of the I112T mutant of SOD1 has not previously been studied experimentally. Although SEC peaks for SOD1I112T
show increased skewness compared to those of unmodified SOD1WT
, all appear to be unimodal and centered at the elution volume of dimeric SOD1 (). This difference in peak shape may reflect increased conformational flexibility of SOD1I112T
, resulting in a broader distribution of radii of gyration for the dimer. Alternatively, if this mutant has micromolar rather than nanomolar binding affinity, the peak could be skewed by the contribution of monomeric SOD1. Computational analysis of SOD1I112T
thermodynamics showed that this mutation has increased dimer stability compared to the wild type,1
supporting the interpretation that this variant is solely dimeric under the conditions of our assay. Regardless of the effect of the I112T mutation itself, changes in dimer stability resulting from glutathionylation are clearly minimal (). In contrast to wild type SOD1 and the A4V mutant, glutathionylation of SOD1I112T
dimers results in little to no effect on Kd
despite a modest but statistically signifi-cant increase in the dissociation rate constant (). Modification may exert opposing effects on this SOD1 mutant, destabilizing the dimer but facilitating the reassociation of modified monomers.
DMD simulations reveal a structural basis for the distinct effects of cysteine-111 glutathionylation on wild type and FALS mutant SOD1. SOD1WT
experience a net loss of both dimer interface area and Cα
interface contacts as a result of glutathionylation (), and these dimers are both destabilized exclusively by decreased kon
. Therefore, some losses in interface Cα
contacts may be indicative of structural changes that hinder monomer association (kon
) rather than directly impacting the rate constant for dissociation (koff
). In particular, a net loss of Cα
contacts specifically indicates backbone movements that separate the two monomers, rather than simple rearrangement of the residue side chains. The appearance of a significant smaller-interface population in the glutathionylated species of SOD1WT
during simulations () further indicates that this modification stabilizes a partially dissociated intermediate, as seen in ref 34
. In the SOD1I112T
dimer interface, glutathionylation results in a shift in interface composition rather than a net loss of Cα
contacts (); likewise, no smaller-interface population is observed for GS-SOD1I112T
(). For this variant, change in the dissociation constant Kd
is minimal even though koff
is increased. These trends raise the possibility that the identity, not quantity, of the residues participating in the dimer interface affects dissociation kinetics.
The late-onset nature of ALS suggests a connection to a natural process of aging that either allows the initiation of a previously suppressed pathology (e.g., SOD1 aggregation) or renders the organism less able to cope with an ongoing threat that was previously tightly regulated. While symptom onset occurs in midlife (>45 years) or later for the vast majority of ALS patients, disease duration is variable, even among patients with identical SOD1 mutations.2
Patients with the A4V mutation experience particularly aggressive motor function loss (<2 years average disease duration2
) while I112T is apparently incompletely penetrant (not all individuals with this allele develop ALS37
). The phenotypic heterogeneity of disease duration among those with identical SOD1 genotype implies that nongenetic environmental factors contribute significantly to mutant SOD1 pathogenicity.
Oxidative stress, manifested as a dysregulation of reactive oxygen species (ROS) or reactive nitrogen species (RNS), is one such process. The levels of ROS and RNS are normally tightly regulated by a variety of enzymes, such as SOD1, and small molecule or peptide redox couples. Glutathione, one such redox couple, is present at a high concentration in the cytosol (up to 12 mM38
) and protects against oxidative damage by acting as a reducing agent, as well as by reversibly modifying proteins to prevent permanent oxidation.10,11
Protein S-glutathionylation occurs more frequently under conditions of oxidative stress as a result of two mechanisms. In the first, thiyl radicals generated by oxidizing species react with reduced glutathione (GSH). Under oxidizing conditions, there also exists a greater proportion of cellular glutathione in the disulfide-linked oxidized form (GSSG), which modifies free cysteine residues by disulfide exchange.
SOD1 is an enzyme that directly interacts with oxidizing species, converting superoxide to hydrogen peroxide, and glutathionylation is a common modification of SOD1 in human tissue, including that of ALS patients.12,39
SOD1 is glutathionylated at a steady state level that likely reflects the immediate degree of oxidative stress occurring in the individual organism, rather than accumulating over the entire lifespan (discussed in ref 12
). Because a large fraction of SOD1 from a variety of healthy human donors is glutathionylated,12
SOD1 glutathionylation alone is unlikely to cause ALS. The substantial drop in glutathionylated wild type dimer stability to micromolar affinity () has not previously been observed even though this modification is prevalent in SOD1 from both human tissue and recombinant sources.12,39,40
Since enrichment of the glutathionylated protein by ion exchange is necessary to observe this destabilizing effect, it may be that the decreased kon
we report is only associated with formation of dimers of two modified subunits.
Given the central importance of dimer dissociation in the initiation of SOD1 aggregation,13,14
the high levels of glutathionylated SOD1 expected to be present in an oxidatively stressed motor neuron could trigger or exacerbate dysfunction by substantially increasing the monomer population. A prolonged shift in the monomer–dimer equilibrium, especially in harsh conditions, would result in increased populations of metal-free, misfolded, and aggregated SOD1 (). It has been observed that Cys-111 mediates mutant SOD1 aggregation in a cell culture model of ALS and that overexpression of glutaredoxin-1 (which reduces both protein–protein and protein–glutathione disulfides) or mutation of Cys-111 attenuate this toxic process.41
While initially interpreted as further evidence of the involvement of intermolecular disulfide bonds in aggregate formation,30,42
these data also support the hypothesis that destabilization caused by Cys-111 glutathionylation promotes aggregation and cell death in ALS. The A4V and I112T mutant SODs are affected differently by glutathionylation, suggesting differing sensitivities of these SOD1 variants to an oxidizing intracellular environment. These differences could explain some of the variability in disease progression among the over 140 mutations implicated in the familial form of the disease. Furthermore, the significant destabilization of both wild type SOD1 and the FALS mutant A4V by glutathione suggests that this modification could promote formation of non-native SOD1 oligomers in both sporadic and familial ALS cases. The modulation of SOD1 dimer stability by cysteine-111 glutathionylation, a post-translational modification linked to redox status, suggests a novel mechanism by which oxidative stress and SOD1 aggregation are interconnected in ALS pathology.