Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes

Abstract

Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu 2+ binding site and defined cross-dimer copper-copper distance distributions. Importantly, we find that copper deficiency in these mutants promotes aggregation in a manner strikingly consistent with their clinical severities. G93 mutants seem to properly incorporate metal ions under physiological conditions when assisted by the copper chaperone but release copper under destabilizing conditions more readily than the WT enzyme. Altered intradimer flexibility in ALS mutants may cause differential metal retention and promote distinct aggregation trends observed for mutant proteins in vitro and in ALS patients. Combined biophysical and structural results test and link copper retention to the framework destabilization hypothesis as a unifying general mechanism for both SOD aggregation and ALS disease progression, with implications for disease severity and therapeutic intervention strategies. Lou Gehrig's disease | small-angle X-ray scattering | protein aggregation | protein conformation | ESR spectroscopy A LS is a lethal degenerative disease of the human motor system (1). Opportunities for improved understanding and clinical intervention arose from the discovery that up to 23.5% of familial ALS cases and 7% of spontaneous cases are caused by mutations in the superoxide dismutase 1 (SOD1) gene encoding human Cu, Zn SOD (2-4). SOD is a highly conserved (5), dimeric, antioxidant metalloenzyme that detoxifies superoxide radicals (6, 7), but overexpression of SOD1 ALS mutants is sufficient to cause disease in mice (8). Misfolded and/or aggregated SOD species are deposited within mouse neuronal and glial inclusions (9, 10), even before symptoms appear (11,12). Although human familial ALS has a symptomatic phenotype indistinguishable from sporadic cases (13), individual SOD1 mutations can result in highly variable disease progression and penetrance (14,15).Many nongeneral mechanisms, including loss of activity or gain of function, were postulated to explain the roles of SOD mutants in ALS (3,(16)(17)(18)(19). Recently, however, an initial hypothesis proposing that SOD manifests disease symptoms by framework dest...

Envisioning the dynamics and flexibility of Mre11-Rad50-Nbs1 complex to decipher its roles in DNA replication and repair

“…Identifying and characterizing the roles of metal ions is often challenging, in part because they may not be correctly incorporated into the protein in recombinant expression hosts, and may thus require purification from native biomass (Cvetkovic et al, 2010; Yannone et al, 2012). Even partial loss of metal ions impacts proper function and increases flexibility and misassembly, as seen for the stress response enzyme superoxide dismutase (Pratt et al, 2014) and the DNA repair and transcription helicase XPD (Fan et al, 2008). Biophysical techniques allowing the accurate identification and measurement of metal ions will be important in future research.…”

Structural, Functional, and Immunogenic Insights on Cu,Zn Superoxide Dismutase Pathogenic Virulence Factors from Neisseria meningitidis and Brucella abortus

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“…NmSOD mutants (E73A, K91Q, K91E, K91Q/K94Q, and K91E/K94E) were constructed in pJSK205 using a QuikChange sitedirected mutagenesis kit (Agilent) and purified as described above for WT NmSOD. Homo sapiens SOD (HsSOD) was prepared as described previously (55).…”

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Robust Production, Crystallization, Structure Determination, and Analysis of [Fe–S] Proteins: Uncovering Control of Electron Shuttling and Gating in the Respiratory Metabolism of Molybdopterin Guanine Dinucleotide Enzymes

“…Loss of such [Fe–S] clusters may cause domain disorders that decrease DNA-binding affinity and lead to functional defects. Indeed, metal site loss or defects may in general cause decreased fold stability and even human diseases, as seen, for example, for human superoxide dismutase (Pratt et al, 2014). …”