The impact of post-translational modifications on aggregation of Cu, Zn superoxide dismutase in amyotrophic lateral sclerosis

Aberrant conformers of disease-linked proteins have been proposed as cytotoxic agents in several late-onset neurodegenerative disorders, including Alzheimer's disease and amyotrophic lateral sclerosis (ALS). Mutations in the gene encoding Cu, Zn superoxide dismutase (SOD1) are present in a subset of familial ALS (FALS) cases; most of these mutations destabilize the protein, although typically by a small margin relative to SOD1's exceptionally high stability. Therefore, SOD1 with FALS-linked substitutions often misfolds and aggregates, adopting aberrant conformations that interact with numerous cellular components and disrupt their functioning, despite having a more stable folded state than would be expected for an aggregation-prone protein. This fact, together with the specific death of motor neurons late in life despite ubiquitous expression of mutant SOD1 since birth, implicates factors in the cellular environment as substantial contributors to the cytotoxicity of mutant SOD1 in FALS. One non-genetic factor likely to influence misfolding and aggregation of SOD1 in human tissue is its susceptibility to abundant post-translational modifications, including phosphorylation and numerous oxidative modifications. We find that reversible oxidative modification of Cys-111 by the glutathione tripeptide destabilizes the native SOD1 homodimer, increasing the equilibrium dissociation constant of the WT dimer from low nanomolar to the low micromolar range, and further destabilizes SOD1 containing a FALS-linked substitution within the dimer interface (A4V). Assessment of the effect of glutathionylation on dimer dissociation kinetics using surface plasmon resonance revealed that this modification causes minimal change in dimer dissociation rate; therefore, the increased Kd observed for glutathionylated WT and A4V SOD1 must result from slowed association of modified monomers. In addition to inducing dissociation of the native dimer, Cys-111 glutathionylation promotes the assembly of soluble non-native oligomers that contain an epitope specific to disease-relevant misfolded SOD1. Our findings suggest that soluble non-native SOD1 oligomers share structural similarity to pathogenic misfolded species found in ALS patients, and therefore represent potential cytotoxic agents and therapeutic targets in ALS. Furthermore, the induction of SOD1 misfolding and aggregation by glutathionylation represents a possible mechanism by which oxidative stress brought on by aging triggers the transition of SOD1 from its natively folded state to cytotoxic conformations.Doctor of Philosoph

Non-native Soluble Oligomers of Cu/Zn Superoxide Dismutase (SOD1) Contain a Conformational Epitope Linked to Cytotoxicity in Amyotrophic Lateral Sclerosis (ALS)

Soluble misfolded Cu/Zn superoxide dismutase (SOD1) is implicated in motor neuron death in amyotrophic lateral sclerosis (ALS); however, the relative toxicities of the various non-native species formed by SOD1 as it misfolds and aggregates are unknown. Here, we demonstrate that early stages of SOD1 aggregation involve the formation of soluble oligomers that contain an epitope specific to disease-relevant misfolded SOD1; this epitope, recognized by the C4F6 antibody, has been proposed as a marker of toxic species. Formation of potentially toxic oligomers is likely to be exacerbated by an oxidizing cellular environment, as evidenced by increased oligomerization propensity and C4F6 reactivity when oxidative modification by glutathione is present at Cys-111. These findings suggest that soluble non-native SOD1 oligomers, rather than native-like dimers or monomers, share structural similarity to pathogenic misfolded species found in ALS patients and therefore represent potential cytotoxic agents and therapeutic targets in ALS

Nonnative SOD1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis

Protein aggregation is a hallmark of neurodegenerative disease and is hypothesized to cause neuron death. Despite extensive study of disease-associated aggregating proteins, mechanisms of neuron death remain a mystery, and no cures or effective treatments yet exist. Here, we demonstrate the toxicity of a small aggregate of the Cu,Zn superoxide dismutase (SOD1) protein, associated with amyotrophic lateral sclerosis (ALS). We present an experimentally verified structural model of this toxic species and show that SOD1 mutants designed to promote formation of this aggregate increase cell death, providing a direct link between aggregate presence and neuron death. Knowledge of toxic species and the ability to manipulate their formation provides a valuable direction for pursuit of therapeutic strategies in ALS

Glutathionylation at Cys-111 Induces Dissociation of Wild Type and FALS Mutant SOD1 Dimers

Mutation of the ubiquitous cytosolic enzyme Cu/Zn superoxide dismutase (SOD1) is hypothesized to cause familial amyotrophic lateral sclerosis (FALS) through structural destabilization leading to misfolding and aggregation. Considering the late onset of symptoms as well as the phenotypic variability among patients with identical SOD1 mutations, it is clear that nongenetic factor(s) impact ALS etiology and disease progression. Here we examine the effect of Cys-111 glutathionylation, a physiologically prevalent post-translational oxidative modification, on the stabilities of wild type SOD1 and two phenotypically diverse FALS mutants, A4V and I112T. Glutathionylation results in profound destabilization of SOD1WT dimers, increasing the equilibrium dissociation constant Kd to ~10−20 μM, comparable to that of the aggressive A4V mutant. SOD1A4V is further destabilized by glutathionylation, experiencing an ~30-fold increase in Kd. Dissociation kinetics of glutathionylated SOD1WT and SOD1A4V are unchanged, as measured by surface plasmon resonance, indicating that glutathionylation destabilizes these variants by decreasing association rate. In contrast, SOD1I112T has a modestly increased dissociation rate but no change in Kd when glutathionylated. Using computational structural modeling, we show that the distinct effects of glutathionylation on different SOD1 variants correspond to changes in composition of the dimer interface. Our experimental and computational results show that Cys-111 glutathionylation induces structural rearrangements that modulate stability of both wild type and FALS mutant SOD1. The distinct sensitivities of SOD1 variants to glutathionylation, a modification that acts in part as a coping mechanism for oxidative stress, suggest a novel mode by which redox regulation and aggregation propensity interact in ALS

Serotonin-Induced Hypersensitivity via Inhibition of Catechol O-Methyltransferase Activity

Abstract The subcutaneous and systemic injection of serotonin reduces cutaneous and visceral pain thresholds and increases responses to noxious stimuli. Different subtypes of 5-hydroxytryptamine (5-HT) receptors are suggested to be associated with different types of pain responses. Here we show that serotonin also inhibits catechol O-methyltransferase (COMT), an enzyme that contributes to modultion the perception of pain, via non-competitive binding to the site bound by catechol substrates with a binding affinity comparable to the binding affinity of catechol itself (K i  = 44 μM). Using computational modeling, biochemical tests and cellular assays we show that serotonin actively competes with the methyl donor S-adenosyl-L-methionine (SAM) within the catalytic site. Binding of serotonin to the catalytic site inhibits the access of SAM, thus preventing methylation of COMT substrates. The results of in vivo animal studies show that serotonin-induced pain hypersensitivity in mice is reduced by either SAM pretreatment or by the combined administration of selective antagonists for β2- and β3-adrenergic receptors, which have been previously shown to mediate COMT-dependent pain signaling. Our results suggest that inhibition of COMT via serotonin binding contributes to pain hypersensitivity, providing additional strategies for the treatment of clinical pain conditions

Serotonin-Induced Hypersensitivity via Inhibition of Catechol O-Methyltransferase Activity

Abstract The subcutaneous and systemic injection of serotonin reduces cutaneous and visceral pain thresholds and increases responses to noxious stimuli. Different subtypes of 5-hydroxytryptamine (5-HT) receptors are suggested to be associated with different types of pain responses. Here we show that serotonin also inhibits catechol O-methyltransferase (COMT), an enzyme that contributes to modultion the perception of pain, via non-competitive binding to the site bound by catechol substrates with a binding affinity comparable to the binding affinity of catechol itself (Ki = 44 μM). Using computational modeling, biochemical tests and cellular assays we show that serotonin actively competes with the methyl donor S-adenosyl-L-methionine (SAM) within the catalytic site. Binding of serotonin to the catalytic site inhibits the access of SAM, thus preventing methylation of COMT substrates. The results of in vivo animal studies show that serotonin-induced pain hypersensitivity in mice is reduced by either SAM pretreatment or by the combined administration of selective antagonists for β2- and β3-adrenergic receptors, which have been previously shown to mediate COMT-dependent pain signaling. Our results suggest that inhibition of COMT via serotonin binding contributes to pain hypersensitivity, providing additional strategies for the treatment of clinical pain conditions.</p

Nonnative SOD1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis

Since the linking of mutations in the Cu,Zn superoxide dismutase gene (sod1) to amyotrophic lateral sclerosis (ALS) in 1993, researchers have sought the connection between SOD1 and motor neuron death. Disease-linked mutations tend to destabilize the native dimeric structure of SOD1, and plaques containing misfolded and aggregated SOD1 have been found in the motor neurons of patients with ALS. Despite advances in understanding of ALS disease progression and SOD1 folding and stability, cytotoxic species and mechanisms remain unknown, greatly impeding the search for and design of therapeutic interventions. Here, we definitively link cytotoxicity associated with SOD1 aggregation in ALS to a nonnative trimeric SOD1 species. We develop methodology for the incorporation of low-resolution experimental data into simulations toward the structural modeling of metastable, multidomain aggregation intermediates. We apply this methodology to derive the structure of a SOD1 trimer, which we validate in vitro and in hybridized motor neurons. We show that SOD1 mutants designed to promote trimerization increase cell death. Further, we demonstrate that the cytotoxicity of the designed mutants correlates with trimer stability, providing a direct link between the presence of misfolded oligomers and neuron death. Identification of cytotoxic species is the first and critical step in elucidating the molecular etiology of ALS, and the ability to manipulate formation of these species will provide an avenue for the development of future therapeutic strategies