Many mutations confer upon copper/zinc superoxide dismutase-1 (SOD1) one or more toxic function(s) that impair motor neuron viability and cause familial amyotrophic lateral sclerosis (FALS). Using a conformation-specific antibody that detects misfolded SOD1 (C4F6), we demonstrate that oxidized WT-SOD1 and mutant-SOD1 share a conformational epitope that is not present in normal WT-SOD1. In a subset of human sporadic ALS (SALS) cases, motor neurons in the lumbosacral spinal cord displayed striking C4F6 immunoreactivity, denoting the presence of aberrant WT-SOD1 species. Recombinant, oxidized WT-SOD1 and WT-SOD1 immunopurified from SALS tissues inhibited kinesin-based fast axonal transport in a manner similar to FALS-linked mutant SOD1. Studies here suggest that WT-SOD1 can be pathogenic in SALS and identifies an SOD1-dependent pathogenic mechanism common to FALS and SALS.

A heterologously expressed form of the human Parkinson diseaseassociated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 Nterminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three-or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer structure is proposed, based on expected close association of the amphipathic central helices observed in the previously described micelle-associated "hairpin" structure of α-synuclein. T he protein α-synuclein (αSyn) is associated with the two most prevalent neurodegenerative diseases, Parkinson disease (PD) and Alzheimer's disease (AD). The presence of αSyn-rich aggregates (Lewy bodies) in neurons of the substantia nigra is the defining histopathological hallmark of PD, and is used to differentiate PD from other neurological disorders (1). Monogenic point mutations (A30P, A53T, and E46K) as well as gene duplication and triplication of the αSyn locus have been identified as causal factors of early onset familial PD; E46K has also been associated with Lewy body dementia, the second most common form of dementia after AD (2-4).αSyn is small (140 residues), and though the C-terminal region (∼residues 100-140) is highly acidic and expected to be disordered, the first 100 residues are predicted to be structured and to have α-helical propensity (SI Appendix, Fig. S1). Stable helical structures have been detected by circular dichroism (CD) and NMR when αSyn is incubated with detergent micelles and lipid vesicles (5, 6). Soluble αSyn is typically referred to as an "intrinsically disordered" protein (7,8). However, we herein report the biophysical characterization of a purified soluble form of αSyn that is oligomeric and fractionally occupies helical structures in the absence of micelles or vesicles. The αSyn construct used in our work is purified by use of an N-terminal GST affinity tag under mild conditions to preserve any native structure. After removal of the GST tag, a 10-residue N-terminal extension remains on the αSyn. However, the similarity of the 1 H, 15 N heteronuclear single-quantum coherence (HSQC) fingerprint of our αSyn construct (SI Appendix, Figs. S2 and S3) to those reported by other groups for αSyn suggests that the N-terminal extension does not change structural tendencies significantly. The αSyn construct described here is not toxic to membranes or cells, does not readily aggregate or ...

Iron-sulfur cluster biosynthesis in both prokaryotic and eukaryotic cells is known to be mediated by two highly conserved proteins, termed IscS and IscU in prokaryotes. The homodimeric IscS protein has been shown to be a cysteine desulfurase that catalyzes the reductive conversion of cysteine to alanine and sulfide. In this work, the time course of IscS-mediated Fe-S cluster assembly in IscU was monitored via anaerobic anion exchange chromatography. The nature and properties of the clusters assembled in discrete fractions were assessed via analytical studies together with absorption, resonance Raman, and Mössbauer investigations. The results show sequential cluster assembly with the initial IscU product containing one [2Fe-2S](2+) cluster per dimer converting first to a form containing two [2Fe-2S](2+) clusters per dimer and finally to a form that contains one [4Fe-4S](2+) cluster per dimer. Both the [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU are reductively labile and are degraded within minutes upon being exposed to air. On the basis of sequence considerations and spectroscopic studies, the [2Fe-2S](2+) clusters in IscU are shown to have incomplete cysteinyl ligation. In addition, the resonance Raman spectrum of the [4Fe-4S](2+) cluster in IscU is best interpreted in terms of noncysteinyl ligation at a unique Fe site. The ability to assemble both [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU supports the proposal that this ubiquitous protein provides a scaffold for IscS-mediated assembly of clusters that are subsequently used for maturation of apo Fe-S proteins.