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Aggregation of copper-zinc superoxide dismutase (SOD1) is a defining feature of familial ALS caused by inherited mutations in the sod1 gene, and misfolded and aggregated forms of wild-type SOD1 are found in both sporadic and familial ALS cases. Mature SOD1 owes its exceptional stability to a number of post-translational modifications as follows: formation of the intramolecular disulfide bond, binding of copper and zinc, and dimerization. Loss of stability due to the failure to acquire one or more of these modifications is proposed to lead to aggregation in vivo. Previously, we showed that the presence of apo-, disulfide-reduced SOD1, the most immature form of SOD1, results in initiation of fibrillation of more mature forms that have an intact Cys-57-Cys-146 disulfide bond and are partially metallated. In this study, we examine the ability of each of the above post-translational modifications to modulate fibril initiation and seeded growth. Cobalt or zinc binding, despite conferring great structural stability, neither inhibits the initiation propensity of disulfide-reduced SOD1 nor consistently protects disulfide-oxidized SOD1 from being recruited into growing fibrils across wild-type and a number of ALS mutants. In contrast, reduction of the disulfide bond, known to be necessary for fibril initiation, also allows for faster recruitment during seeded amyloid growth. These results identify separate factors that differently influence seeded growth and initiation and indicate a lack of correlation between the overall thermodynamic stability of partially mature SOD1 states and their ability to initiate fibrillation or be recruited by a growing fibril.

The fluorescence quenching and electron paramagnetic resonance (EPR) measurements on the octarepeat domain, along with a library of octarepeat-derived constructs and selective chelators in competition studies were used to determine the affinity for the three different copper-binding components of the prion protein (PrP). The findings show that the octarepeat domain is responsive to a remarkably wide copper concentration range covering approximately 5 orders of magnitude and the ability of the protein to quench copper redox activity at high occupancy, suggest that PrP might function to protect cells by scavenging excess copper.

Spectra obtained at high copper occupancy are reinvestigated and evidence for close copper-copper contacts consistent with a packing interaction in the copper loaded N-terminal octarepeat domain is revealed. The investigation provides the first complete characterization of all dominant copper coordination modes at pH 7.4.

Aggregation of copper-zinc superoxide dismutase (SOD1) is a defining feature of familial ALS caused by inherited mutations in the sod1 gene, and misfolded and aggregated forms of wildtype SOD1 are found in both sporadic and familial ALS cases. Mature SOD1 owes its exceptional stability to a number of posttranslational modifications as follows: formation of the intramolecular disulfide bond, binding of copper and zinc, and dimerization. Loss of stability due to the failure to acquire one or more of these modifications is proposed to lead to aggregation in vivo. Previously, we showed that the presence of apo-, disulfide-reduced SOD1, the most immature form of SOD1, results in initiation of fibrillation of more mature forms that have an intact Cys-57-Cys-146 disulfide bond and are partially metallated. In this study, we examine the ability of each of the above post-translational modifications to modulate fibril initiation and seeded growth. Cobalt or zinc binding, despite conferring great structural stability, neither inhibits the initiation propensity of disulfide-reduced SOD1 nor consistently protects disulfide-oxidized SOD1 from being recruited into growing fibrils across wild-type and a number of ALS mutants. In contrast, reduction of the disulfide bond, known to be necessary for fibril initiation, also allows for faster recruitment during seeded amyloid growth. These results identify separate factors that differently influence seeded growth and initiation and indicate a lack of correlation between the overall thermodynamic stability of partially mature SOD1 states and their ability to initiate fibrillation or be recruited by a growing fibril. [ABSTRACT FROM AUTHOR]

Oligopeptide repeats appear in many proteins that undergo conformational conversions to form amyloid, including the mammalian prion protein PrP and the yeast prion protein Sup35. Whereas the repeats in PrP have been studied more exhaustively, interpretation of these studies is confounded by the fact that many details of the PrP prion conformational conversion are not well understood. On the other hand, there is now a relatively good understanding of the factors that guide the conformational conversion of the Sup35 prion protein. To provide a general model for studying the role of oligopeptide repeats in prion conformational conversion and amyloid formation, we have substituted various numbers of the PrP octarepeats for the endogenous Sup35 repeats. The resulting chimeric proteins can adopt the [PSI+] prion state in yeast, and the stability of the prion state depends on the number of repeats. In vitro, these chimeric proteins form amyloid fibers, with more repeats leading to shorter lag phases and faster assembly rates. Both pH and the presence of metal ions modulate assembly kinetics of the chimeric proteins, and the extent of modulation is highly sensitive to the number of PrP repeats. This work offers new insight into the properties of the PrP octarepeats in amyloid assembly and prion formation, It also reveals new features of the yeast prion protein, and provides a level of control over yeast prion assembly that will be useful for future structural studies and for creating amyloid-based biomaterials. [ABSTRACT FROM AUTHOR]

Patients with amyotrophic lateral sclerosis (ALS) have evidence of chronic inflammation demonstrated by infiltration of the gray matter by inflammatory macrophages, IL17A-positive T cells, and mast cells. Increased serum levels of IL6 and IL17A have been detected in sporadic ALS (sALS) patients when compared to healthy controls. Herein we investigate, in peripheral blood mononuclear cells (PBMCs), the baseline transcription of genes associated with inflammation in sALS and control subjects and the impact of the IL6 receptor (IL6R) antibody (tocilizumab) on the transcription and/or secretion of inflammation factors (e.g. cytokines) stimulated by the apo-G37R superoxide dismutase (SOD1) mutant. At baseline, PBMCs of four sALS patients (Group 1) showed significantly increased expression of TLR2 and CD14; ALOX5, PTGS2 and MMP1; IL1α, IL1β, IL6, IL36G, IL8 and TNF; CCL3, CCL20, CXCL2, CXCL3 and CXCL5. In four other sALS patients (Group 2), most of the genes just mentioned were expressed at near control levels and a significant decrease in the expression of PPARG, PPARA, RARG, HDAC4 and KAT2B; IL6R, IL6ST and ADAM17; TNFRSF11A; MGAT2 and MGAT3; PLCG1; CXCL3 were detected. Apo-G37R SOD1 up regulated the transcription of cytokines (e.g. IL1α/β, IL6, IL8, IL36G), chemokines (e.g. CCL20; CXCL3, CXCL5), and enzymes (e.g. PTGS2 and MMP1). In vitro, tocilizumab down regulated the transcription of many inflammatory cytokines, chemokines, enzymes, and receptors, which were up regulated by pathogenic forms of SOD1. Tocilizumab also reduced the secretion of the pro-inflammatory cytokines IL1β, IL6, TNFα, GM-CSF, IFNγ, and IL17A by Group 1 PBMCs. Finally, sALS patients had significantly higher concentrations of IL6, sIL6R and C-reactive protein in the cerebrospinal fluid when compared to AD patients. This pilot study demonstrates that in vitro tocilizumab suppresses many factors that drive inflammation in sALS patients, with possible increased efficacy in Group 1 ALS patients.

Although the cause of neuronal degeneration in amyotrophic lateral sclerosis (ALS) remains hypothetical, there is evidence of spinal cord infiltration by macrophages and T cells. In post-mortem ALS spinal cords, 19.8 ± 4.8 % motor neurons, including caspase-negative and caspase-positive neurons, were ingested by IL-6- and TNF-α-positive macrophages. In ALS macrophages, in vitro aggregated superoxide dismutase-1 (SOD-1) stimulated in ALS macrophages expression of inflammatory cytokines, including IL-1β, IL-6, and TNF-α, through activation of cyclooxy-genase-2 (COX-2) and caspase-1. The lipid mediator resolvin D1 (RvD1) inhibited IL-6 and TNF-α production in ALS macrophages with 1,100 times greater potency than its parent molecule docosahexaenoic acid. ALS peripheral blood mononuclear cells (PBMCs) showed increased transcription of inflammatory cytokines and chemokines at baseline and after stimulation by aggregated wild-type SOD-1, and these cytokines were down regulated by RvD1. Thus the neurons are impacted by macrophages expressing inflammatory cytokines. RvD1 strongly inhibits in ALS macrophages and PBMCs cytokine transcription and production. Resolvins offer a new approach to suppression of inflammatory activation in ALS.

The structure and unfolding of metal-free (apo) human wild-type SOD1 and three pathogenic variants of SOD1 (A4V, G93R, and H48Q) that cause familial amyotrophic lateral sclerosis have been studied with amide hydrogen/deuterium exchange and mass spectrometry. The results indicate that a significant proportion of each of these proteins exists in solution in a conformation in which some strands of the beta-barrel (i.e. beta2) are well protected from exchange at physiological temperature (37 degrees C), whereas other strands (i.e. beta3 and beta4) appear to be unprotected from hydrogen/deuterium exchange. Moreover, the thermal unfolding of these proteins does not result in the uniform incorporation of deuterium throughout the polypeptide but involves the local unfolding of different residues at different temperatures. Some regions of the proteins (i.e. the "Greek key" loop, residues 104-116) unfold at a significantly higher temperature than other regions (i.e. beta3 and beta4, residues 21-53). Together, these results show that human wild-type apo-SOD1 and variants have a partially unfolded beta-barrel at physiological temperature and unfold non-cooperatively.