Palmitoylation of Superoxide Dismutase 1 (SOD1) Is Increased for Familial Amyotrophic Lateral Sclerosis-linked SOD1 Mutants

Mutations in Cu,Zn-superoxide dismutase (mtSOD1) cause familial amyotrophic lateral sclerosis (FALS), a neurodegenerative disease resulting from motor neuron degeneration. Here, we demonstrate that wild type SOD1 (wtSOD1) undergoes palmitoylation, a reversible post-translational modification that can regulate protein structure, function, and localization. SOD1 palmitoylation was confirmed by multiple techniques, including acyl-biotin exchange, click chemistry, cysteine mutagenesis, and mass spectrometry. Mass spectrometry and cysteine mutagenesis demonstrated that cysteine residue 6 was the primary site of palmitoylation. The palmitoylation of FALS-linked mtSOD1s (A4V and G93A) was significantly increased relative to that of wtSOD1 expressed in HEK cells and a motor neuron cell line. The palmitoylation of FALS-linked mtSOD1s (G93A and G85R) was also increased relative to that of wtSOD1 when assayed from transgenic mouse spinal cords. We found that the level of SOD1 palmitoylation correlated with the level of membrane-associated SOD1, suggesting a role for palmitoylation in targeting SOD1 to membranes. We further observed that palmitoylation occurred predominantly on disulfide-reduced as opposed to disulfide-bonded SOD1, suggesting that immature SOD1 is the primarily palmitoylated species. Increases in SOD1 disulfide bonding and maturation with increased copper chaperone for SOD1 expression caused a decrease in wtSOD1 palmitoylation. Copper chaperone for SOD1 overexpression decreased A4V palmitoylation less than wtSOD1 and had little effect on G93A mtSOD1 palmitoylation. These findings suggest that SOD1 palmitoylation occurs prior to disulfide bonding during SOD1 maturation and that palmitoylation is increased when disulfide bonding is delayed or decreased as observed for several mtSOD1s.     

Altered Thiol Chemistry in Human Amyotrophic Lateral Sclerosis-linked Mutants of Superoxide Dismutase 1

Neurodegenerative diseases share a common characteristic, the presence of intracellular or extracellular deposits of protein aggregates in nervous tissues. Amyotrophic Lateral Sclerosis (ALS) is a severe and fatal neurodegenerative disorder, which affects preferentially motoneurons. Changes in the redox state of superoxide dismutase 1 (SOD1) are associated with the onset and development of familial forms of ALS. In human SOD1 (hSOD1), a conserved disulfide bond and two free cysteine residues can engage in anomalous thiol/disulfide exchange resulting in non-native disulfides, a hallmark of ALS that is related to protein misfolding and aggregation. Because of the many competing reaction pathways, traditional bulk techniques fall short at quantifying individual thiol/disulfide exchange reactions. Here, we adapt recently developed single-bond chemistry techniques to study individual disulfide isomerization reactions in hSOD1. Mechanical unfolding of hSOD1 leads to the formation of a polypeptide loop held by the disulfide. This loop behaves as a molecular jump rope that brings reactive Cys-111 close to the disulfide. Using force-clamp spectroscopy, we monitor nucleophilic attack of Cys-111 at either sulfur of the disulfide and determine the selectivity of the reaction. Disease-causing mutations G93A and A4V show greatly altered reactivity patterns, which may contribute to the progression of familial ALS.     

Molecular Analyses of the Cu/Zn Superoxide Dismutase Gene in Patients with Familial Amyotrophic Lateral Sclerosis (ALS) in Japan

1. Amyotrophic lateral sclerosis (ALS) is a degenerative disorder characterized by selective damage to the neural system that mediates voluntary movement. Although the pathophysiologic process of ALS remains unknown, about 5 to 10% of cases are familial. According to genetic linkage studies, the familial ALS (FALS) gene has been mapped on chromosome 21 in some families and recent work identified some different missense mutations in the Cu/Zn superoxide dismutase gene in FALS families.2. We recently identified five mutations in six FALS families. The mutations identified in our FALS families are H46R, L84V, I104F, S134N, and V148I. The H46R mutation that locates in the active site of Cu/Zn SOD gene is associated with two Japanese families with very slow progression of ALS. On the other hand, the L84V mutation associated with a rapidly progressive loss of motor function with predominant lower motor neuron manifestations.3. In the family with the V148I, the phenotype of the patient varied very much among the affected members. One case had weakness of the lower extremities at first and died without bulbar paresis. The second case first noticed wasting of the upper limbs with bulbar symptoms, but the third had weakness of upper extremities without developing dysarthria nor dysphagia until death. These mutations account for 50% of all FALS families screened, although Cu/Zn SOD gene mutations are responsible for less than about 13–21% in the Western population.4. Our results indicate that the progression of disease with mutations of Cu/Zn SOD is well correlated with each mutation. The exact mechanism by which the abnormal Cu/Zn SOD molecules selectively affect the function of motor neurons is still unknown.     

The structure of holo and metal-deficient wild-type human Cu,Zn superoxide dismutase and its relevance to familial amyotrophic lateral sclerosis

Cu, Zn superoxide dismutase (SOD1) forms a crucial component of the cellular defence against oxidative stress. Zn-deficient wild-type and mutant human SOD1 have been implicated in the disease familial amyotrophic lateral sclerosis (FALS). We present here the crystal structures of holo and metal-deficient (apo) wild-type protein at 1.8A resolution. The P21 wild-type holo enzyme structure has nine independently refined dimers and these combine to form a "trimer of dimers" packing motif in each asymmetric unit. There is no significant asymmetry between the monomers in these dimers, in contrast to the subunit structures of the FALS G37R mutant of human SOD1 and in bovine Cu,Zn SOD. Metal-deficient apo SOD1 crystallizes with two dimers in the asymmetric unit and shows changes in the metal-binding sites and disorder in the Zn binding and electrostatic loops of one dimer, which is devoid of metals. The second dimer lacks Cu but has approximately 20% occupancy of the Zn site and remains structurally similar to wild-type SOD1. The apo protein forms a continuous, extended arrangement of beta-barrels stacked up along the short crystallographic b-axis, while perpendicular to this axis, the constituent beta-strands form a zig-zag array of filaments, the overall arrangement of which has a similarity to the common structure associated with amyloid-like fibrils.     

Disulfide Scrambling Describes the Oligomer Formation of Superoxide Dismutase (SOD1) Proteins in the Familial Form of Amyotrophic Lateral Sclerosis

Dominant mutations in Cu,Zn-superoxide dismutase (SOD1) are a cause of a familial form of amyotrophic lateral sclerosis. Wild-type SOD1 forms a highly conserved intra-molecular disulfide bond, whereas pathological SOD1 proteins are cross-linked via intermolecular disulfide bonds and form insoluble oligomers. A thiol-disulfide status in SOD1 will thus play a regulatory role in determining its folding/misfolding pathways; however, it remains unknown how pathogenic mutations in SOD1 affect the thiol-disulfide status to facilitate the protein misfolding. Here, we show that the structural destabilization of SOD1 scrambles a disulfide bond among four Cys residues in an SOD1 molecule. The disulfide scrambling produces SOD1 monomers with distinct electrophoretic mobility and also reproduces the formation of disulfide-linked oligomers. We have also found that the familial form of amyotrophic lateral sclerosis-causing mutations facilitate the disulfide scrambling in SOD1. Based upon our results, therefore, scrambling of the conserved disulfide bond will be a key event to cause the pathological changes in disease-associated mutant SOD1 proteins.     

Disulfide-Reduced ALS Variants of Cu, Zn Superoxide Dismutase Exhibit Increased Populations of Unfolded Species

Cu,Zn superoxide dismutase (SOD1) is a dimeric metal-binding enzyme responsible for the dismutation of toxic superoxide to hydrogen peroxide and oxygen in cells. Mutations at dozens of sites in SOD1 induce amyotrophic lateral sclerosis (ALS), a fatal gain-of-function neurodegenerative disease whose molecular basis is unknown. To obtain insights into effects of the mutations on the folded and unfolded populations of immature monomeric forms whose aggregation or self-association may be responsible for ALS, the thermodynamic and kinetic folding properties of a set of disulfide-reduced and disulfide-oxidized Zn-free and Zn-bound stable monomeric SOD1 variants were compared to properties of the wild-type (WT) protein. The most striking effect of the mutations on the monomer stability was observed for the disulfide-reduced metal-free variants. Whereas the WT and S134N monomers are > 95% folded at neutral pH and 37 °C, A4V, L38V, G93A, and L106V ranged from 50% to ∼ 90% unfolded. The reduction of the disulfide bond was also found to reduce the apparent Zn affinity of the WT monomer by 750-fold, into the nanomolar range, where it may be unable to compete for free Zn in the cell. With the exception of the S134N metal-binding variant, the Zn affinity of disulfide-oxidized SOD1 monomers showed little sensitivity to amino acid replacements. These results suggest a model for SOD1 aggregation where the constant synthesis of ALS variants of SOD1 on ribosomes provides a pool of species in which the increased population of the unfolded state may favor aggregation over productive folding to the native dimeric state.     

Dimerization,Oligomerization, and Aggregation of Human Amyotrophic Lateral Sclerosis Copper/Zinc Superoxide Dismutase 1 Protein Mutant Forms in Live Cells

More than 100 copper/zinc superoxide dismutase 1 (SOD1) genetic mutations have been characterized. These mutations lead to the death of motor neurons in ALS. In its native form, the SOD1 protein is expressed as a homodimer in the cytosol. In vitro studies have shown that SOD1 mutations impair the dimerization kinetics of the protein, and in vivo studies have shown that SOD1 forms aggregates in patients with familial forms of ALS. In this study, we analyzed WT SOD1 and 9 mutant (mt) forms of the protein by non-invasive fluorescence techniques. Using microscopic techniques such as fluorescence resonance energy transfer, fluorescence complementation, image-based quantification, and fluorescence correlation spectroscopy, we studied SOD1 dimerization, oligomerization, and aggregation. Our results indicate that SOD1 mutations lead to an impairment in SOD1 dimerization and, subsequently, affect protein aggregation. We also show that SOD1 WT and mt proteins can dimerize. However, aggregates are predominantly composed of SOD1 mt proteins.     

The Glycine-Alanine Dipeptide Repeat from C9orf72 Hexanucleotide Expansions Forms Toxic Amyloids Possessing Cell-to-Cell Transmission Properties

Hexanucleotide expansions, GGGGCC, in the non-coding regions of the C9orf72 gene were found in major frontotemporal lobar dementia and amyotrophic lateral sclerosis patients (C9FTD/ALS). In addition to possible RNA toxicity, several dipeptide repeats (DPRs) are translated through repeat-associated non-ATG-initiated translation. The DPRs, including poly(GA), poly(GR), poly(GP), poly(PR), and poly(PA), were found in the brains and spinal cords of C9FTD/ALS patients. Among the DPRs, poly(GA) is highly susceptible to form cytoplasmic inclusions, which is a characteristic of C9FTD/ALS. To elucidate DPR aggregation, we used synthetic (GA)₁₅ DPR as a model system to examine the aggregation and structural properties in vitro. We found that (GA)₁₅ with 15 repeats fibrillates rapidly and ultimately forms flat, ribbon-type fibrils evidenced by transmission electron microscopy and atomic force microscopy. The fibrils are capable of amyloid dye binding and contain a characteristic cross-β sheet structure, as revealed by x-ray scattering. Furthermore, using neuroblastoma cells, we demonstrated the neurotoxicity and cell-to-cell transmission property of (GA)₁₅ DPR. Overall, our results show the structural and toxicity properties of GA DPR to facilitate future DPR-related therapeutic development.     

Denaturational stress induces formation of zinc-deficient monomers of Cu,Zn superoxide dismutase: implications for pathogenesis in amyotrophic lateral sclerosis

Mutations in the Cu,Zn superoxide dismutase (SOD1) cause a subset of amyotrophic lateral sclerosis cases. SOD1 is a homodimer in which each monomer binds one copper atom and one zinc atom. Mutation is believed to increase the conformational flexibility of SOD1, giving rise to a misfolded SOD1 population with novel cytotoxic properties. While SOD1's metal ligands affect its stability greatly, little is known about the role these metals play in the folding, unfolding, and misfolding processes. Here, we present a method by which we were able to measure the rates of metal release during SOD1 unfolding in guanidine hydrochloride. Rates of dimer dissociation, measured by a time-resolved cross-linking assay, and conformational changes in SOD1's β-barrel core, monitored by tryptophan fluorescence intensity, were compared with the rates of copper release and zinc release. Correlations were observed across a range of denaturant concentrations, giving rise to a more detailed model of the SOD1 unfolding process than was previously available. According to this model, the major unfolding pathway involves simultaneous dimer dissociation and zinc release as an early step that is followed by a slow conformational change in the protein's core, which, in turn, is followed by rapid copper release. This model establishes a zinc-deficient, copper-loaded SOD1 monomer as a well-populated SOD1 unfolding intermediate and a species likely to be populated under conditions of denaturational stress. Because the cytotoxicity of zinc-deficient SOD1 has been demonstrated previously, this species is a good candidate for the cytotoxic species in SOD1-associated amyotrophic lateral sclerosis.     

Superoxide Dismutase from the Eukaryotic Thermophile Alvinella pompejana: Structures, Stability, Mechanism, and Insights into Amyotrophic Lateral Sclerosis

Prokaryotic thermophiles supply stable human protein homologs for structural biology; yet, eukaryotic thermophiles would provide more similar macromolecules plus those missing in microbes. Alvinella pompejana is a deep-sea hydrothermal-vent worm that has been found in temperatures averaging as high as 68 °C, with spikes up to 84 °C. Here, we used Cu,Zn superoxide dismutase (SOD) to test if this eukaryotic thermophile can provide insights into macromolecular mechanisms and stability by supplying better stable mammalian homologs for structural biology and other biophysical characterizations than those from prokaryotic thermophiles. Identification, cloning, characterization, X-ray scattering (small-angle X-ray scattering, SAXS), and crystal structure determinations show that A. pompejana SOD (ApSOD) is superstable, homologous, and informative. SAXS solution analyses identify the human-like ApSOD dimer. The crystal structure shows the active site at 0.99 Å resolution plus anchoring interaction motifs in loops and termini accounting for enhanced stability of ApSOD versus human SOD. Such stabilizing features may reduce movements that promote inappropriate intermolecular interactions, such as amyloid-like filaments found in SOD mutants causing the neurodegenerative disease familial amyotrophic lateral sclerosis or Lou Gehrig's disease. ApSOD further provides the structure of a long-sought SOD product complex at 1.35 Å resolution, suggesting a unified inner-sphere mechanism for catalysis involving metal ion movement. Notably, this proposed mechanism resolves apparent paradoxes regarding electron transfer. These results extend knowledge of SOD stability and catalysis and suggest that the eukaryote A. pompejana provides macromolecules highly similar to those from humans, but with enhanced stability more suitable for scientific and medical applications.     

Zinc binding modulates the entire folding free energy surface of human Cu,Zn superoxide dismutase

Over 100 amino acid replacements in human Cu,Zn superoxide dismutase (SOD) are known to cause amyotrophic lateral sclerosis, a gain-of-function neurodegenerative disease that destroys motor neurons. Supposing that aggregates of partially folded states are primarily responsible for toxicity, we determined the role of the structurally important zinc ion in defining the folding free energy surface of dimeric SOD by comparing the thermodynamic and kinetic folding properties of the zinc-free and zinc-bound forms of the protein. The presence of zinc was found to decrease the free energies of a peptide model of the unfolded monomer, a stable variant of the folded monomeric intermediate, and the folded dimeric species. The unfolded state binds zinc weakly with a micromolar dissociation constant, and the folded monomeric intermediate and the native dimeric form both bind zinc tightly, with subnanomolar dissociation constants. Coupled with the strong driving force for the subunit association reaction, the shift in the populations toward more well-folded states in the presence of zinc decreases the steady-state populations of higher-energy states in SOD under expected in vivo zinc concentrations (approximately nanomolar). The significant decrease in the population of partially folded states is expected to diminish their potential for aggregation and account for the known protective effect of zinc. The ∼ 100-fold increase in the rate of folding of SOD in the presence of micromolar concentrations of zinc demonstrates a significant role for a preorganized zinc-binding loop in the transition-state ensemble for the rate-limiting monomer folding reaction in this β-barrel protein.     

Oxidative stress,DNA damage,and the telomeric complex as therapeutic targets in acute neurodegeneration

Oxidative stress has been identified as an important contributor to neurodegeneration associated with acute CNS injuries and diseases such as spinal cord injury (SCI), traumatic brain injury (TBI), and ischemic stroke. In this review, we briefly detail the damaging effects of oxidative stress (lipid peroxidation, protein oxidation, etc.) with a particular emphasis on DNA damage. Evidence for DNA damage in acute CNS injuries is presented along with its downstream effects on neuronal viability. In particular, unchecked oxidative DNA damage initiates a series of signaling events (e.g. activation of p53 and PARP-1, cell cycle re-activation) which have been shown to promote neuronal loss following CNS injury. These findings suggest that preventing DNA damage might be an effective way to promote neuronal survival and enhance neurological recovery in these conditions. Finally, we identify the telomere and telomere-associated proteins (e.g. telomerase) as novel therapeutic targets in the treatment of neurodegeneration due to their ability to modulate the neuronal response to both oxidative stress and DNA damage.     

Functions and dysfunctions of nitric oxide in brain

Nitric oxide (NO) works as a retrograde neurotransmitter in synapses, allows the brain blood flow and also has important roles in intracellular signaling in neurons from the regulation of the neuronal metabolic status to the dendritic spine growth. Moreover NO is able to perform post-translational modifications in proteins by the S-nitrosylation of the thiol amino acids, which is a physiological mechanism to regulate protein function. On the other hand, during aging and pathological processes the behavior of NO can turn harmful when reacts with superoxide anion to form peroxynitrite. This gaseous compound can diffuse easily throughout the neuronal membranes damaging lipid, proteins and nucleic acids. In the case of proteins, peroxynitrite reacts mostly with the phenolic ring of the tyrosines forming nitro-tyrosines that affects dramatically to the physiological functions of the proteins. Protein nitrotyrosination is an irreversible process that also yields to the accumulation of the modified proteins contributing to the onset and progression of neurodegenerative processes such as Alzheimer's disease or Parkinson's disease.     

Lipid peroxidation and neurodegenerative disease

Lipid peroxidation is a complex process involving the interaction of oxygen-derived free radicals with polyunsaturated fatty acids, resulting in a variety of highly reactive electrophilic aldehydes. Since 1975, lipid peroxidation has been extensively studied in a variety of organisms. As neurodegenerative diseases became better understood, research establishing a link between this form of oxidative damage, neurodegeneration, and disease has provided a wealth of knowledge to the scientific community. With the advent of proteomics in 1995, the identification of biomarkers for neurodegenerative disorders became of paramount importance to better understand disease pathogenesis and develop potential therapeutic strategies. This review focuses on the relationship between lipid peroxidation and neurodegenerative diseases. It also demonstrates how findings in current research support the common themes of altered energy metabolism and mitochondrial dysfunction in neurodegenerative disorders.