Familial amyotrophic lateral sclerosis (fALS) caused by mutations in copper-zinc superoxide dismutase (SOD1) is characterized by the presence of SOD1-rich inclusions in spinal cords. Similar inclusions observed in fALS transgenic mice have a fibrillar appearance suggestive of amyloid structure. Metal-free apo-SOD1 is a relatively stable protein and has been shown to form amyloid fibers in vitro only when it has been subjected to severely destabilizing conditions, such as low pH or reduction of its disulfide bonds. Here, by contrast, we show that a small amount of disulfide-reduced apo-SOD1 can rapidly initiate fibrillation of this exceptionally stable and highly structured protein under mild, physiologically accessible conditions, thus providing an unusual demonstration of a specific, physiologically relevant form of a protein acting as an initiating agent for the fibrillation of another form of the same protein. We also show that, once initiated, elongation can proceed via recruitment of either apo-or partially metallated disulfideintact SOD1 and that the presence of copper, but not zinc, ions inhibits fibrillation. Our findings provide a rare glimpse into the specific changes in a protein that can lead to nucleation and into the ability of amyloid nuclei to recruit diverse forms of the same protein into fibrils.amyloid ͉ amyotrophic lateral sclerosis ͉ neurodegeneration ͉ protein aggregation T he antioxidant metalloprotein copper-zinc superoxide dismutase (SOD1) is a 153-residue, ␤-rich, homodimeric protein that is abundantly present in the cytoplasm. Each subunit of the mature form contains a copper ion, a zinc ion, and a disulfide bond (1). In vitro studies have shown that the presence of the metal cofactors, copper and zinc, protect the disulfide bond from reduction, suggesting a possible explanation for the persistence of the disulfide bond in the reducing environment of the cytoplasm (2). More than 100 mutations in SOD1 have been linked to the familial form of amyotrophic lateral sclerosis (fALS), a fatal neurodegenerative disease caused by selective death of motor neurons. Neuronal death is attributed to a toxic gain of function by mutant SOD1, but the exact mechanism of toxicity is unknown. Mutations in SOD1 that have been identified in fALS patients occur in 74 positions that are well dispersed over the length of this 153-residue polypeptide (3).Proteinaceous aggregates have been found in the spinal cords of ALS patients, and immunomicroscopy has confirmed that the protein inclusions present in the spinal cords of SOD1-fALS patients are rich in SOD1 (4, 5). Transgenic mice expressing human SOD1 mutants that cause fALS share many symptoms with their human counterparts, including progressive motor neuron degeneration and the presence of detergent-resistant, SOD1-rich aggregates in their spinal cords (6, 7). These aggregates have recently been shown to consist primarily of full-length, unmodified SOD1 (6). The visible proteinaceous inclusions have a fibrillar appearance and bind thioflavin S, suggesting a...

The relative stabilities and structural properties of a representative set of 20 ALS-mutant Cu,Zn-superoxide dismutase apoproteins were examined by using differential scanning calorimetry and hydrogen-deuterium (H͞D) exchange followed by MS. Contrary to recent reports from other laboratories, we found that ALS-mutant apoproteins are not universally destabilized by the disease-causing mutations. For example, several of the apoproteins with substitutions at or near the metal binding region (MBR) (MBR mutants) exhibited melting temperatures (Tm) in the range 51.6°C to 56.2°C, i.e., similar to or higher than that of the WT apoprotein (Tm ‫؍‬ 52.5°C). The apoproteins with substitutions remote from the MBR (WT-like mutants) showed a wide range of Tms, 40.0°C to 52.4°C. The H͞D exchange properties of the mutants were also wideranging: the MBR mutant apoproteins exhibited H͞D exchange kinetics similar to the WT apoprotein, as did some of the more stable WT-like mutant apoproteins, whereas the less stable apoproteins exhibited significantly less protection from H͞D exchange than the WT apoprotein. Most striking were the three mutant apoproteins, D101N, E100K, and N139K, which have apparently normal metallation properties, and differ little from the WT apoprotein in either thermal stability or H͞D exchange kinetics. Thus, the ALS mutant Cu,Zn-superoxide dismutase apoproteins do not all share reduced global stability, and additional properties must be identified and understood to explain the toxicity of all of the mutant proteins.differential scanning calorimetry ͉ hydrogen-deuterium exchange ͉ protein stability ͉ protein aggregation ͉ neurodegenerative disease P rotein misfolding and aggregation have been linked to many diseases, including Alzheimer's disease, cystic fibrosis, transmissible spongiform encephalopathies, and ALS, but the pathways followed by pathogenic proteins from translation to disease-causing states are not completely understood (1-3). In some cases, partial or complete unfolding from the native state precedes protein aggregation, and thus the stability of a protein's native state may provide one measure of its propensity to aggregate. However, many familial protein misfolding diseases are caused by proteins that are not destabilized relative to their WT counterparts (4-6), implying that additional intrinsic or extrinsic factors may be required for protein aggregation.Our recent studies of a large number of ALS-mutant Cu,Znsuperoxide dismutase (SOD1) proteins have revealed that there is great diversity in the biophysical properties of these proteins (7-12). In contrast, Lindberg et al. (13) reported in 2002 that instability of the apoproteins of ALS-mutant SOD1 proteins is a ''common denominator'' among the nearly 100 known ALSlinked SOD1 mutations. More recently, Furukawa and O'Halloran (14) have reported that some of the destabilized mutant apoproteins studied by Lindberg et al. are further destabilized when the intrasubunit disulfide bond is reduced, again suggesting that protein destabilization is ...

The thermodynamics of zinc binding to metal-free (apo) human and bovine copper-zinc superoxide dismutases (SOD1) were measured using isothermal titration calorimetry. The apparent thermodynamics of zinc binding to the apoproteins were favorable (Ka > 108 M-1), with an observed stoichiometry of one zinc per homodimer. The change in heat capacity for the one-zinc binding event was large and negative (approximately -650 cal mol-1 K-1), suggestive of significant structural changes to the protein upon zinc binding. We further characterized the one-zinc derivative by circular dichroism and determined that this derivative had nearly the same secondary structure as the two-zinc derivative and that both are structurally distinct from the metal-free protein. In addition, we monitored the effect of zinc binding on hydrogen-deuterium exchange and accessibility of histidyl residues to modification by diethyl pyrocarbonate and observed that more than 50% protection was afforded by the binding of one zinc in both assays. Differential scanning calorimetry on the human SOD1 zinc derivatives also showed increased thermostability of the protein due to zinc binding. Further, the melting transitions observed for the one-zinc derivative closely resembled those of the two-zinc derivative. Finally, we observed that the quaternary structure of the protein is stabilized upon binding of one and two zinc ions in analytical ultracentrifugation experiments. Combined, these results suggest communication between the two monomers of SOD1 such that the binding of one zinc ion per homodimer has a more profound effect on the homodimeric protein structure than the binding of subsequent metal ions. The relevance of these findings to amyotrophic lateral sclerosis is discussed.

The mechanisms by which mutant variants of Cu/Zn-superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis are not clearly understood. Evidence to date suggests that altered conformations of amyotrophic lateral sclerosis mutant SOD1s trigger perturbations of cellular homeostasis that ultimately cause motor neuron degeneration. In this study we correlated the metal contents and disulfide bond status of purified wild-type (WT) and mutant SOD1 proteins to changes in electrophoretic mobility and surface hydrophobicity as detected by 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence. As-isolated WT and mutant SOD1s were copper-deficient and exhibited mobilities that correlated with their expected negative charge. However, upon disulfide reduction and demetallation at physiological pH, both WT and mutant SOD1s underwent a conformational change that produced a slower mobility indicative of partial unfolding. Furthermore, although ANS did not bind appreciably to the WT holoenzyme, incubation of metal-deficient WT or mutant SOD1s with ANS increased the ANS fluorescence and shifted its peak toward shorter wavelengths. This increased interaction with ANS was greater for the mutant SOD1s and could be reversed by the addition of metal ions, especially Cu 2؉ , even for SOD1 variants incapable of forming the disulfide bond. Overall, our findings support the notion that misfolding associated with metal deficiency may facilitate aberrant interactions of SOD1 with itself or with other cellular constituents and may thereby contribute to neuronal toxicity.The sequence of events by which more than 100 mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD1) 3 cause familial forms of amyotrophic lateral sclerosis (ALS) is unknown. Studies of purified SOD1 proteins and cellular or rodent models of SOD1-linked ALS suggest that impaired metal ion binding or misfolding of mutant SOD1 proteins in the cellular environment may be related to their toxicity (1-10). Available evidence suggests that partially unfolded mutant SOD1 species could contribute to motor neuron death by promoting abnormal interactions that produce cellular dysfunction (11-16).In previous studies we characterized physicochemical properties of 14 different biologically metallated ALS SOD1 mutants (17) and demonstrated altered thermal stabilities of these mutants compared with wild-type (WT) SOD1 (18). These "asisolated" SOD1 proteins, which contain variable amounts of copper and zinc, were broadly grouped into two classes based on their ability to incorporate and retain metal ions with high affinity. WT-like SOD1 mutants retain the ability to bind copper and zinc ions and exhibit dismutase activity similar to the normal enzyme, whereas metal binding region (MBR) mutants are significantly deficient in copper and/or zinc (17, 19). We also observed that ALS-associated SOD1 mutants were more susceptible than the WT enzyme to reduction of the intrasubunit disulfide bond between . The significance of these results is that even WT-like mutants, whic...

Background: Copper-zinc superoxide dismutase is a rare example of an intracellular protein with a disulfide bond. Results: Disulfide mutant C57S SOD1 has 10% of the enzymatic activity of wild type. Conclusion:The disulfide bond in SOD1 is not required for correct metal binding and enzymatic activity. Significance: The disulfide bond in SOD1 may play a role in SOD1-linked amyotrophic lateral sclerosis.

RDAVR is associated with significantly shorter ACC and CPB times than CAVR, although this difference did not translate into improved postoperative outcomes, early mortality, and all-cause mortality during follow-up. Care might be needed when implanting RD valves because they are associated with a higher incidence of PPM insertion, regardless of the RD valve type.

Bacteriophage T4 deoxycytidylate hydroxymethylase (EC 2.1.2.8), a homodimer of 246-residue subunits, catalyzes hydroxymethylation of the cytosine base in deoxycytidylate (dCMP) to produce 5-hydroxymethyldCMP. It forms part of a phage DNA protection system and appears to function in vivo as a component of a multienzyme complex called deoxyribonucleoside triphosphate (dNTP) synthetase. We have determined its crystal structure in the presence of the substrate dCMP at 1.6 Å resolution. The structure reveals a subunit fold and a dimerization pattern in common with thymidylate synthases, despite low (~20%) sequence identity. Among the residues that form the dCMP binding site, those interacting with the sugar and phosphate are arranged in a configuration similar to the deoxyuridylate binding site of thymidylate synthases. However, the residues interacting directly or indirectly with the cytosine base show a more divergent structure and the presumed folate cofactor binding site is more open. Our structure reveals a water molecule properly positioned near C-6 of cytosine to add to the C-7 methylene intermediate during the last step of hydroxymethylation. On the basis of sequence comparison and crystal packing analysis, a hypothetical model for the interaction between T4 deoxycytidylate hydroxymethylase and T4 thymidylate synthase in the dNTP-synthesizing complex has been built. Keywords: bacteriophage T4/deoxycytidylate hydroxymethylase/deoxyuridylate hydroxymethylase/ dNTP-synthesizing complex/thymidylate synthase

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