Over 100 mutations in Cu/Zn-superoxide dismutase (SOD1) result in familial amyotrophic lateral sclerosis. Dimer dissociation is the first step in SOD1 aggregation, and studies suggest nearly every amino acid residue in SOD1 is dynamically connected to the dimer interface. Post-translational modifications of SOD1 residues might be expected to have similar effects to mutations, but few modifications have been identified. Here we show, using SOD1 isolated from human erythrocytes, that human SOD1 is phosphorylated at threonine 2 and glutathionylated at cysteine 111. A second SOD1 phosphorylation was observed and mapped to either Thr-58 or Ser-59. Cysteine 111 glutathionylation promotes SOD1 monomer formation, a necessary initiating step in SOD1 aggregation, by causing a 2-fold increase in the K d . This change in the dimer stability is expected to result in a 67% increase in monomer concentration, 315 nM rather than 212 nM at physiological SOD1 concentrations. Because protein glutathionylation is associated with redox regulation, our finding that glutathionylation promotes SOD1 monomer formation supports a model in which increased oxidative stress promotes SOD1 aggregation. Familial amyotrophic lateral sclerosis (FALS)4 is the hereditary form of amyotrophic lateral sclerosis, a fatal disease characterized by progressive motor neuron loss (1). A subset of FALS is caused by mutations in the gene encoding homodimeric Cu/Zn-superoxide dismutase (SOD1), which forms intraneuronal aggregates (2). Although SOD1 aggregation is involved in SOD1-mediated FALS, it is generally believed that the functional properties of the enzyme are not related to the toxic gain of function imparted by mutations in SOD1 (3). However, the discovery of roles for SOD1 in the regulation of the cellular phosphorylation balance (4) and redox state (5) provides additional avenues for connecting the cellular role of SOD1 to FALS. The classical studies of SOD1 were generally performed using bovine erythrocyte SOD1 or recombinant human SOD1. Although recombinant methods are widely used to produce SOD1 mutants, a disadvantage of studying recombinant SOD1 is the absence of potentially important post-translational modifications present in human tissues. The initial SOD1 crystal structure was solved using bovine erythrocyte SOD1 (6) and no structure of human erythrocyte SOD1 is available. Here we report results using human erythrocyte SOD1 rather than the recombinant enzyme and find that the native enzyme features a consistent pattern of post-translational modifications. Using a combination of "bottom-up" and "top-down" mass spectrometry (MS) approaches, we show that SOD1 isolated from human erythrocytes is post-translationally phosphorylated and glutathionylated. These modifications occur near the SOD1 dimer interface. Because monomer formation is thought to be the first intermediate leading to SOD1 aggregation (7, 8), we tested the dimer stability of modified SOD1 found, as expected, that glutathionylation promotes the formation of SOD1 monomer. EXPERIMEN...

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.neurodegeneration | protein aggregation | protein misfolding | ALS | structural modeling P rotein misfolding and aggregation are linked to cell death and disease progression in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). In these diseases and others, the formation of amyloid plaques, often observed post mortem, has long been thought to play a role in neurodegeneration, but toxicity has never been confirmed (1-3). Recent research has shown that small, soluble oligomers, rather than insoluble amyloids, are likely to be the cytotoxic species causing neurodegeneration (4-14). These small, soluble oligomers undergo aberrant interactions with cell machinery and activate cell death pathways, but their exact stoichiometry is not known and their properties have yet to be characterized. Recently, metastable soluble Cu,Zn superoxide dismutase (SOD1) oligomers have been identified that contain an epitope associated with disease-linked species of SOD1, mutants of which are implicated in a subset of ALS (15-18). Size exclusion chromatography (SEC) of these oligomers revealed a size range of two to four monomers, consistent with previous findings of potentially cytotoxic SOD1 oligomers (19)(20)(21).Knowledge of the structures of these species would not only allow for definitive testing of their toxicity but could potentially lead to an understanding of disease mechanism and therapeutic strategies against diseases for which no ...

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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.

A systematic and robust approach to generating complex protein nanomaterials would have broad utility. We develop a hierarchical approach to designing multi-component protein assemblies from two classes of modular building blocks: designed helical repeat proteins (DHRs) and helical bundle oligomers (HBs). We first rigidly fuse DHRs to HBs to generate a large library of oligomeric building blocks. We then generate assemblies with cyclic, dihedral, and point group symmetries from these building blocks using architecture guided rigid helical fusion with new software named WORMS. X-ray crystallography and cryo-electron microscopy characterization show that the hierarchical design approach can accurately generate a wide range of assemblies, including a 43 nm diameter icosahedral nanocage. The computational methods and building block sets described here provide a very general route to de novo designed protein nanomaterials.