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Delineating the Aggregation-Prone Hotspot Regions (Peptides) in the Human Cu/Zn Superoxide Dismutase 1.

ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a fatal, incurable neurodegenerative disease described by progressive degeneration of motor neurons. The most common familial form of ALS (fALS) has been associated with mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Mutation-induced misfolding and aggregation of SOD1 is often found in ALS patients. In this work, we probe the aggregation properties of peptides derived from the SOD1. To examine the source of SOD1 aggregation, we have employed a computational algorithm to identify four peptides from the SOD1 protein sequence that aggregates into a fibril. Aided by computational algorithms, we identified four peptides likely involved in SOD1 fibrillization. These four aggregation-prone peptides were ¹⁴VQGIINFE21, ³⁰KVWGSIKGL38, ¹⁰¹DSVISLS¹⁰⁷, and ¹⁴⁷GVIGIAQ¹⁵³. In addition, the formation of fibril propensities from the identified peptides was investigated through different biophysical techniques. The atomic structures of two fibril-forming peptides from the C-terminal SOD1 showed that the steric zippers formed by ¹⁰¹DSVISLS¹⁰⁷ and ¹⁴⁷GVIGIAQ¹⁵³ vary in their arrangement. We also discovered that fALS mutations in the peptide ¹⁴⁷GVIGIAQ¹⁵³ increased the fibril-forming propensity and altered the steric zipper's packing. Thus, our results suggested that the C-terminal peptides of SOD1 have a central role in amyloid formation and might be involved in forming the structural core of SOD1 aggregation observed in vivo.

SUBMITTER: Wahiduzzaman

PROVIDER: S-EPMC8675042 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Cholesterol secosterol aldehyde adduction and aggregation of Cu,Zn-superoxide dismutase: Potential implications in ALS.

Project description:Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by degeneration of upper and lower motor neurons. While the fundamental causes of the disease are still unclear, the accumulation of Cu,Zn-superoxide dismutase (SOD1) immunoreactive aggregates is associated with familial ALS cases. Cholesterol 5,6-secosterol aldehydes (Seco A and Seco B) are reported to contribute to neurodegenerative disease pathology by inducing protein modification and aggregation. Here we have investigated the presence of secosterol aldehydes in ALS SOD1-G93A rats and their capacity to induce SOD1 aggregation. Secosterol aldehydes were analyzed in blood plasma, spinal cord and motor cortex of ALS rats at the pre-symptomatic and symptomatic stages. Seco B was significantly increased in plasma of symptomatic ALS rats compared to pre-symptomatic animals, suggesting an association with disease progression. In vitro experiments showed that both Seco A and Seco B induce the formation of high molecular weight (HMW) SOD1 aggregates with amorphous morphology. SOD1 adduction to ?-alkynyl-secosterols analyzed by click assay showed that modified proteins are only detected in the HMW region, indicating that secosterol adduction generates species highly prone to aggregate. Of note, SOD1-secosterol adducts containing up to five secosterol molecules were confirmed by MALDI-TOF analysis. Interestingly, mass spectrometry sequencing of SOD1 aggregates revealed preferential secosterol adduction to Lys residues located at the electrostatic loop (Lys 122, 128 and 136) and nearby the dimer interface (Lys 3 and 9). Altogether, our results show that secosterol aldehydes are increased in plasma of symptomatic ALS rats and represent a class of aldehydes that can potentially modify SOD1 enhancing its propensity to aggregate.

| S-EPMC6106709 | biostudies-literature

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Project description:Amyotrophic lateral sclerosis has been linked to the gain of aberrant function of superoxide dismutase, Cu,Zn-SOD1 upon protein misfolding. The mechanism of SOD1 misfolding is thought to involve mutations leading to the loss of Zn, followed by protein unfolding and aggregation. We show that the removal of Zn from SOD1 may not lead to an immediate unfolding but immediately deactivates the enzyme through a combination of subtle structural and electronic effects. Using quantum mechanics/discrete molecular dynamics, we showed that both Zn-less wild-type (WT)-SOD1 and its D124N mutant that does not bind Zn have at least metastable folded states. In those states, the reduction potential of Cu increases, leading to the presence of detectable amounts of Cu(I) instead of Cu(II) in the active site, as confirmed experimentally. The Cu(I) protein cannot participate in the catalytic Cu(I)-Cu(II) cycle. However, even without the full reduction to Cu(I), the Cu site in the Zn-less variants of SOD1 is shown to be catalytically incompetent: unable to bind superoxide in a way comparable to the WT-SOD1. The changes are more radical and different in the D124N Zn-less mutant than in the Zn-less WT-SOD1, suggesting D124N being perhaps not the most adequate model for Zn-less SOD1. Overall, Zn in SOD1 appears to be influencing the Cu site directly by adjusting its reduction potential and geometry. Thus, the role of Zn in SOD1 is not just structural, as was previously thought; it is a vital part of the catalytic machinery.

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Cellular Redox Systems Impact the Aggregation of Cu,Zn Superoxide Dismutase Linked to Familial Amyotrophic Lateral Sclerosis.

Project description:Protein misfolding is implicated in neurodegenerative diseases such as ALS, where mutations of superoxide dismutase 1 (SOD1) account for about 20% of the inherited mutations. Human SOD1 (hSOD1) contains four cysteines, including Cys(57) and Cys(146), which have been linked to protein stability and folding via forming a disulfide bond, and Cys(6) and Cys(111) as free thiols. But the roles of the cellular oxidation-reduction (redox) environment in SOD1 folding and aggregation are not well understood. Here we explore the effects of cellular redox systems on the aggregation of hSOD1 proteins. We found that the known hSOD1 mutations G93A and A4V increased the capability of the thioredoxin and glutaredoxin systems to reduce hSOD1 compared with wild-type hSOD1. Treatment with inhibitors of these redox systems resulted in an increase of hSOD1 aggregates in the cytoplasm of cells transfected with mutants but not in cells transfected with wild-type hSOD1 or those containing a secondary C111G mutation. This aggregation may be coupled to changes in the redox state of the G93A and A4V mutants upon mild oxidative stress. These results strongly suggest that the thioredoxin and glutaredoxin systems are the key regulators for hSOD1 aggregation and may play critical roles in the pathogenesis of ALS.

| S-EPMC5016121 | biostudies-literature

Dynamical roles of metal ions and the disulfide bond in Cu, Zn superoxide dismutase folding and aggregation.

Project description:Misfolding and aggregation of Cu, Zn superoxide dismutase (SOD1) is implicated in neuronal death in amyotrophic lateral sclerosis. Each SOD1 monomer binds to 1 copper and 1 zinc ion and maintains its disulfide bond (Cys-57-Cys-146) in the reducing cytoplasm of cell. Mounting experimental evidence suggests that metal loss and/or disulfide reduction are important for initiating misfolding and aggregation of SOD1. To uncover the role of metals and the disulfide bond in the SOD1 folding, we systemically study the folding thermodynamics and structural dynamics of SOD1 monomer and dimer with and without metal binding and under disulfide-intact or disulfide-reduced environments in computational simulations. We use all-atom discrete molecular dynamics for sampling. Our simulation results provide dynamical evidence to the stabilizing role of metal ions in both dimer and monomer SOD1. The disulfide bond anchors a loop (Glu-49 to Asn-53) that contributes to the dimer interface. The reduction of the disulfide bond in SOD1 with metal ions depleted results in a flexible Glu-49-Asn-53 loop, which, in turn, disrupts dimer formation. Interestingly, the disulfide bond reduction does not affect the thermostability of monomer SOD1 as significantly as the metal ions do. We further study the structural dynamics of metal-free SOD1 monomers, the precursor for aggregation, in simulations and find inhomogeneous local unfolding of beta-strands. The strands protected by the metal-binding and electrostatic loops are found to unfold first after metal loss, leading to a partially unfolded beta-sheet prone to aggregation. Our simulation study sheds light on the critical role of metals and disulfide bond in SOD1 folding and aggregation.

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Project description:When replete with zinc and copper, amyotrophic lateral sclerosis (ALS)-associated mutant SOD proteins can protect motor neurons in culture from trophic factor deprivation as efficiently as wild-type SOD. However, the removal of zinc from either mutant or wild-type SOD results in apoptosis of motor neurons through a copper- and peroxynitrite-dependent mechanism. It has also been shown that motor neurons isolated from transgenic mice expressing mutant SODs survive well in culture but undergo apoptosis when exposed to nitric oxide via a Fas-dependent mechanism. We combined these two parallel approaches for understanding SOD toxicity in ALS and found that zinc-deficient SOD-induced motor neuron death required Fas activation, whereas the nitric oxide-dependent death of G93A SOD-expressing motor neurons required copper and involved peroxynitrite formation. Surprisingly, motor neuron death doubled when Cu,Zn-SOD protein was either delivered intracellularly to G93A SOD-expressing motor neurons or co-delivered with zinc-deficient SOD to nontransgenic motor neurons. These results could be rationalized by biophysical data showing that heterodimer formation of Cu,Zn-SOD with zinc-deficient SOD prevented the monomerization and subsequent aggregation of zinc-deficient SOD under thiol-reducing conditions. ALS mutant SOD was also stabilized by mutating cysteine 111 to serine, which greatly increased the toxicity of zinc-deficient SOD. Thus, stabilization of ALS mutant SOD by two different approaches augmented its toxicity to motor neurons. Taken together, these results are consistent with copper-containing zinc-deficient SOD being the elusive "partially unfolded intermediate" responsible for the toxic gain of function conferred by ALS mutant SOD.

| S-EPMC2962488 | biostudies-literature

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Project description:In most organisms, superoxide dismutases (SODs) are among the most effective antioxidant enzymes that regulate the reactive oxygen species (ROS) generated by oxidative energy metabolism. ROS are considered main proximate causes of aging. However, it remains unclear if SOD activities are associated with organismal longevity. The queens of eusocial insects, such as termites, ants, and honeybees, exhibit extraordinary longevity in comparison with the nonreproductive castes, such as workers. Therefore, the queens are promising candidates to study the underlying mechanisms of aging. Here, we found that queens have higher Cu/Zn-SOD activity than nonreproductive individuals of the termite Reticulitermes speratus. We identified three Cu/Zn-SOD sequences and one Mn-SOD sequence by RNA sequencing in R. speratus. Although the queens showed higher Cu/Zn-SOD activity than the nonreproductive individuals, there were no differences in their expression levels of the Cu/Zn-SOD genes RsSOD1 and RsSOD3A. Copper (Cu2+ and Cu+) is an essential cofactor for Cu/Zn-SOD enzyme activity, and the queens had higher concentrations of copper than the workers. These results suggest that the high Cu/Zn-SOD activity of termite queens is related to their high levels of the cofactor rather than gene expression. This study highlights that Cu/Zn-SOD activity contributes to extraordinary longevity in termites.

| S-EPMC5831368 | biostudies-literature

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Project description:In eukaryotic organisms, the largely cytosolic copper- and zinc-containing superoxide dismutase (Cu/Zn SOD) enzyme represents a key defense against reactive oxygen toxicity. Although much is known about the biology of this enzyme under aerobic conditions, less is understood regarding the effects of low oxygen levels on Cu/Zn SOD enzymes from diverse organisms. We show here that like bakers' yeast (Saccharomyces cerevisiae), adaptation of the multicellular Caenorhabditis elegans to growth at low oxygen levels involves strong downregulation of its Cu/Zn SOD. Much of this regulation occurs at the post-translational level where CCS-independent activation of Cu/Zn SOD is inhibited. Hypoxia inactivates the endogenous Cu/Zn SOD of C. elegans Cu/Zn SOD as well as a P144 mutant of S. cerevisiae Cu/Zn SOD (herein denoted Sod1p) that is independent of CCS. In our studies of S. cerevisiae Sod1p, we noted a post-translational modification to the inactive enzyme during hypoxia. Analysis of this modification by mass spectrometry revealed phosphorylation at serine 38. Serine 38 represents a putative proline-directed kinase target site located on a solvent-exposed loop that is positioned at one end of the Sod1p ?-barrel, a region immediately adjacent to residues previously shown to influence CCS-dependent activation. Although phosphorylation of serine 38 is minimal when the Sod1p is abundantly active (e.g., high oxygen level), up to 50% of Sod1p can be phosphorylated when CCS activation of the enzyme is blocked, e.g., by hypoxia or low-copper conditions. Serine 38 phosphorylation can be a marker for inactive pools of Sod1p.

| S-EPMC3264780 | biostudies-literature

Expression, purification and crystallization of Chaetomium thermophilum Cu,Zn superoxide dismutase.

Project description:Cu,Zn superoxide dismutase (Cu,ZnSOD) from the thermophilic fungus Chaetomium thermophilum was expressed in Pichia pastoris and purified. Crystals were grown in over 120 conditions but only those produced with 1.4 M sodium potassium phosphate pH 8.2 as precipitant were suitable for structural studies. Data were collected to 1.9 A resolution at 100 K from a single crystal using a synchrotron-radiation source. The crystals belonged to space group P6(1)/P6(5), with unit-cell parameters a=90.2, c=314.5 A and eight molecules in the asymmetric unit. Elucidation of the crystal structure will provide insights into the active site of the enzyme and a better understanding of the structure-activity relationship, assembly and thermal stability of Cu,ZnSODs.

| S-EPMC2935235 | biostudies-literature

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