Zn Superoxide Dismutase Research Articles

Pathogenic R163W Variant of the Copper Chaperone for Sod1 (Ccs) Functions as an Anti-chaperone.

The copper chaperone for Sod1 (Ccs) is a metallochaperone that plays a multifaceted role in the maturation of Cu,Zn superoxide dismutase (Sod1). The Ccs mutation R163W was identified in an infant with fatal neurological abnormalities. Based on a comprehensive structural and functional analysis, we developed the first data-driven model for R163W-related pathogenic phenotypes. The work here confirms previous findings that the substitution of arginine with tryptophan at this site, which is located adjacent to a conserved Zn binding site, creates an unstable Zn-deficient protein that loses its ability to efficiently activate Sod1. Intriguingly, R163W Ccs can reduce copper (i.e., Cu(II) → Cu(I)) bound in its Sod1-like domain (D2), and this novel redox event is accompanied by disulfide bond formation. The loss of Zn binding, along with the unusual ability to bind copper in D2, diverts R163W Ccs toward aggregation. The remarkably high affinity of D2 Cu(I) binding converts R163W from a Cu chaperone to a Cu scavenger that accelerates Sod1 deactivation (i.e., an Anti-chaperone). Overall, these findings present a first-of-its-kind molecular mechanism for Ccs dysfunction that leads to pathogenesis in humans.

Talaromyces marneffei Cu, Zn Superoxide Dismutase Recombinant Protein Expression in Pichia pastoris, Enzymatic Activity and Its Resistance to Oxidative Stress

Talaromyces marneffei is a dimorphic fungus that is known to cause a disease called talaromycosis, also known as penicilliosis, in immunocompromised individuals. The fungal pathogenicity and virulence factors remain unclear. Superoxide dismutase (SOD) is a neutralizing enzyme through reactive oxygen species generated by the host and has been proven to contribute to the virulence of many pathogenic bacteria and fungi. In this study, full-length sodA gene encoding T. marneffei Cu, Zn SOD was amplified, cloned into pPICzαB vector and successfully integrated into the Pichia pastoris yeast genome. The selected positive clone was induced for protein expression by methanol. An approximately 23 kDa molecular mass of secreted recombinant Cu, Zn SOD is enzymatically active which is like the native and standard enzyme. A rabbit polyclonal antibody raised against recombinant Cu, Zn SOD was proved to be reactive to the native enzyme by using Western blot analysis. pPICzαB/sodA also appeared to be more resistant than the control pPICzαB recombinant yeast in the oxidative stress conditions. This is the first study of the expression of recombinant T. marneffei Cu, Zn SOD protein and its enzyme activity determination. This enzyme is an important virulence factor and targeting this enzyme may be a promising strategy for developing new therapeutics.

Copper(II) Complexes with Carnosine Conjugates of Hyaluronic Acids at Different Dipeptide Loading Percentages Behave as Multiple SOD Mimics and Stimulate Nrf2 Translocation and Antioxidant Response in In Vitro Inflammatory Model.

A series of copper(II) complexes with the formula [Cu2+Hy(x)Car%] varying the molecular weight (MW) of Hyaluronic acid (Hy, x = 200 or 700 kDa) conjugated with carnosine (Car) present at different loading were synthesized and characterized via different spectroscopic techniques. The metal complexes behaved as Cu, Zn-superoxide dismutase (SOD1) mimics and showed some of the most efficient reaction rate values produced using a synthetic and water-soluble copper(II)-based SOD mimic reported to date. The increase in the percentage of Car moieties parallels the enhancement of the I50 value determined via the indirect method of Fridovich. The presence of the non-functionalized Hy OH groups favors the scavenger activity of the copper(II) complexes with HyCar, recalling similar behavior previously found for the copper(II) complexes with Car conjugated using β-cyclodextrin or trehalose. In keeping with the new abilities of SOD1 to activate protective agents against oxidative stress in rheumatoid arthritis and osteoarthritis diseases, Cu2+ interaction with HyCar promotes the nuclear translocation of erythroid 2-related factor that regulates the expressions of target genes, including Heme-Oxigenase-1, thus stimulating an antioxidant response in osteoblasts subjected to an inflammatory/oxidative insult.

Effects of copper exposure and recovery in juvenile yellowtail kingfish (Seriola lalandi): Histological, physiological and molecular responses

This study investigated the physiological responses to copper toxicity in liver and gill of juvenile yellowtail kingfish by exposing to 0 (control), 0.0672, 0.12, 0.214, 0.384, 0.672, 1.20, or 2.14 mg/L copper sulfate for 7 days and then recovered for 14 days. The histopathology, enzyme activities and differential gene expression were detected. The results indicate the safe concentration value of copper for juvenile yellowtail kingfish is 0.083 mg/L. Copper exposure caused structural damage to gill and liver, and could be disappeared or restored after the recovery period. Copper exposure enhanced the Cu,Zn superoxide dismutase (Cu/Zn-SOD) and glutathione peroxidase (GSH-Px) activities and the malondialdehyde (MDA) level in liver. However, with increasing copper concentrations, liver aspartate transaminase (AST) and gill Na+/K+-ATPase activities were at first enhanced and then inhibited. Serum lysozyme (LZM) and Cu/Zn-SOD activities were enhanced at low copper concentrations but inhibited at high concentrations, and the activities of alanine transaminase (ALT) and AST significantly increased at greater copper concentrations. After the recovery period, only liver Cu/Zn-SOD activity returned to control level. In contrast, liver GSH-Px activity and MDA levels and gill Na+/K+-ATPase activity, serum LZM and AST activities remained significantly higher than control, but liver AST activity, serum ALT, and Cu/Zn-SOD activities were significantly lower than control. We constructed the gene regulatory networks of molecular pathways in liver and gill in response to copper exposure. The DEGs were enriched mainly in the pathway of lipid metabolism in liver and pathway of oxidative phosphorylation in gill, indicating these respective pathways are involved in the response to copper stress in this species. The present results elucidate some histological, physiological, and molecular mechanisms underlying copper-exposure stresses in yellowtail kingfish, which could help guide the safe utilization of copper sulfate in disease control of farmed individuals.

Analysis of the value of copper erythrocyte concentration measurement in the diagnosis of copper deficiency in bovines

BackgroundA reliable and practical method for assessing Cu status in live animals is not available. Blood Cu levels may not accurately reflect the true Cu status of the herd, and can over-predict Cu status during stress and inflammation. On the other hand, assessment of liver Cu is the most reliable indicator of Cu stores, but it is an invasive procedure that requires specialized training. The aim of this study was to evaluate the usefulness of Cu levels in red blood cells to determine the Cu status, with special emphasis in their correlation with erythrocyte Cu, Zn superoxide dismutase enzyme activity (ESOD), in bovines with Cu deficiency induced by high molybdenum and sulfur levels in the diet. MethodsThree similar assays were performed, with a total of twenty eight calves. The Cu-deficient group (n = 15) received a basal diet supplemented with 11 mg of Mo/kg DM as sodium molybdate, and S as sodium sulfate. The control group (n = 13) received a basal diet supplemented with 9 mg of Cu/kg DM as copper sulfate.Samples of blood and liver were taken every 28–35 days. Cu levels were measured in liver (expressed as µg/g DM), plasma (expressed as µg/dl), and erythrocytes (expressed as µg/g Hb) by flame atomic absorption spectroscopy. Superoxide dismutase (SOD1) activity was determined in red blood cells and was expressed as IU/mg hemoglobin.InfoStat Statistical Software 2020 was used for the statistical analysis. Cu levels in plasma, red blood cells and liver, and ESOD activity were analyzed by ANOVA. The correlation between erythrocyte Cu levels and the rest of the parameters were analyzed by Pearson Correlation test. Unweighted Least Squares Linear Regression of SOD1 was developed. The autocorrelation between the monthly measurements was also determined by Durbin-Watson test and autocorrelation function. ResultsThe assays lasted 314–341 days, approximately. Levels indicative of Cu deficiency for bovines were detected at 224 days (23 ± 11.6 µg/g DM) for liver Cu concentration; and at 198 days (55 ± 10.4 µg/dl) for plasma Cu concentration, in Cu-deficient animals. Liver and plasma Cu values indicative of Cu deficiency were not observed in the control group.Pearson Correlation test indicated that all indices of Cu status used in this study were significantly correlated. The highest value was obtained between ESOD and red blood Cu (0.74). There was a significant correlation between red blood Cu and plasma Cu (0.65), and with hepatic Cu (0.57). ESOD activity showed a similar significant positive correlation with liver Cu concentrations and with plasma Cu (0.59 and 0.58, respectively). ConclusionThe extremely low levels of liver and plasma Cu, the ESOD activity, erythrocyte Cu levels, and the periocular achromotrichia observed in the Cu-deficient animals showed that the clinic phase of Cu deficiency was reached in this group. The ESOD activity and erythrocyte Cu levels showed a strong association, indicating that the values of erythrocyte Cu may serve as an effective tool in assessing Cu status and diagnose a long-term Cu deficiency in cattle.

Activities of Antioxidant Enzymes in Various Tissues of Male Fischer 344 Rats Are Altered by Food Restriction

The objective of this study was to determine how food restriction (40% restriction of food intake) altered the age-related changes in the activities of Cu,Zn superoxide dismutase, catalase and glutathione peroxidase in liver, brain cortex, heart, kidney and intestinal mucosa obtained from 6-, 16- and 26-mo-old male Fischer 344 rats. Food restriction increased the activity of one or more of the antioxidant enzymes in the liver, brain cortex, heart and kidney of the rats. However, the magnitude of the effect and the antioxidant enzyme(s) affected by food restriction varied from tissue to tissue, and food restriction had no significant effect on the activities of these enzymes in intestinal mucosa. Interestingly, the four tissues in which food restriction increased the activity of one or more of the antioxidant enzymes showed reduced lipid peroxidation as measured by thiobarbituric acid-reactive material. These data suggest that food restriction might enhance the survival of rodents by altering the levels of the antioxidant enzymes and hence reducing free radical damage.

Deletion of Sod1 in Motor Neurons Exacerbates Age-Related Changes in Axons and Neuromuscular Junctions in Mice.

Whole-body knock-out of Cu,Zn superoxide dismutase (Sod1KO) results in accelerated, age-related loss of muscle mass and function associated with neuromuscular junction (NMJ) breakdown similar to sarcopenia. In order to determine whether altered redox in motor neurons underlies this phenotype, an inducible neuron-specific deletion of Sod1 (i-mnSod1KO) was compared with wild-type (WT) mice of different ages (adult, mid-age, and old) and whole-body Sod1KO mice. Nerve oxidative damage, motor neuron numbers and structural changes to neurons and NMJ were examined. Tamoxifen-induced deletion of neuronal Sod1 from two months of age. No specific effect of a lack of neuronal Sod1 was seen on markers of nerve oxidation (electron paramagnetic resonance of an in vivo spin probe, protein carbonyl, or protein 3-nitrotyrosine contents). i-mnSod1KO mice showed increased denervated NMJ, reduced numbers of large axons and increased number of small axons compared with old WT mice. A large proportion of the innervated NMJs in old i-mnSod1KO mice displayed a simpler structure than that seen in adult or old WT mice. Thus, previous work showed that neuronal deletion of Sod1 induced exaggerated loss of muscle in old mice, and we report that this deletion leads to a specific nerve phenotype including reduced axonal area, increased proportion of denervated NMJ, and reduced acetyl choline receptor complexity. Other changes in nerve and NMJ structure seen in the old i-mnSod1KO mice reflect aging of the mice.

Effects of disinfectants, sponge densities, water circulation rates, and vibration frequency on the artificial incubation of redclaw crayfish embryos

Artificial incubation of embryos is helpful to large-scale production of redclaw crayfish. In this study, we applied a recirculating mechanical pulling device to incubate redclaw embryos, and evaluated the effects of eco-friendly disinfectants, sponge densities, water circulation rates, and vibration frequencies on hatching % and survival % of embryos. Each factor was tested separately from the others. (1) Of the five solutions tested, a disinfectant solution comprising an essential oil base solution (1 mg/L), Zn-superoxide dismutase (SOD) 10,000 U/mg, trehalose (30 mmol/L), bovine serum protein (200 mmol/L), and astaxanthin (30 mmol/L) (solution 4) resulted in the significantly highest hatching 89.54% and survival 62.22%, which were not significantly different from the formaldehyde-treated group, but significantly higher than other treatments. It could be used as an eco-friendly, effective disinfectant for the artificial incubation of redclaw embryos instead of using traditional formaldehyde. (2) Medium density sponges with a length of 4.5 cm helped improve the survival %, it was hypothesized that the low and high density sponges were not conducive to the attachment of the newly hatched juveniles, and 5.5 cm long sponge would cause hypoxia death of juveniles. (3) Hatching 94.83%, 94.08%, and 92.58%, and survival 65.66%, 66.33%, and 67.08%, at water circulation rates of 7.1, 4.8, and 2.1 cycles/h, respectively. There was no significant difference in hatching % and survival % among groups. (4) Hatching 94.08%, 89.17%, and 91.92%, and survival 73.92%, 61.75%, and 60.50%, at vibration frequencies of 11, 16, and 26 vibrations/min, respectively. The survival % of the group exposed to 11 vibrations/min was significantly higher than at the other two frequencies. The results may have application on a large scale to improve production.

A Reliable and Consistent Method to Quantify Percent Lethality and Life Span in Drosophila melanogaster.

Drosophila melanogaster is a classic model organism to study gene function as well as toxicological effects. To study gene function, the expression of a particular gene of interest is disrupted by using the widely explorable Drosophila genetic toolkit, whereas to study toxicological effects the flies are exposed to a particular toxicant through diet. These experiments often require the quantification of lethality from embryonic to adult stages, as well as the assessment of the life span in order to check the role of the gene/toxicant of interest in Drosophila. Here, we propose an experimental protocol that enables a consistent and rigorous assessment of lethality and life span of cadmium chloride (CdCl2)-exposed or genetically perturbed flies [downregulation and overexpression of the cytosolic Cu, Zn superoxide dismutase (SOD1) gene], consecutively. The protocol insists upon the requirement of one single experimental setup that is unique, distinctive, and cost-effective as it engages minimal laboratory equipment and resources. The described methods lead to the smooth observation of the embryos, their successive stagewise transition, and life span of the adult flies post eclosion. Additionally, these methods also facilitate the assessment of crawling and climbing behavioral parameters of the larvae and adults, respectively, and allow the calculation of lethal concentration (LC50) for the mentioned toxicant as well as median survival of the flies, which can be a determining factor in proceeding with further stages of experiments. Graphical abstract.

The 2-Oxoglutarate Carrier Is S-Nitrosylated in the Spinal Cord of G93A Mutant hSOD1 Mice Resulting in Disruption of Mitochondrial Glutathione Transport.

Mitochondrial oxidative stress and dysfunction are strongly implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Glutathione (GSH) is an endogenous antioxidant that exists as distinct cytosolic and mitochondrial pools. The status of the mitochondrial GSH pool is reliant on transport from the cytosol through the 2-oxoglutarate carrier (OGC), an inner membrane anion carrier. We have previously reported that the outer mitochondrial membrane protein, Bcl-2, directly binds GSH and is a key regulator of OGC-dependent mitochondrial GSH transport. Here, we show that G93A mutant SOD1 (Cu, Zn-superoxide dismutase) reduces the binding of GSH to Bcl-2 and disrupts mitochondrial GSH uptake in vitro. In the G93A mutant hSOD1 mouse model of ALS, mitochondrial GSH is significantly depleted in spinal cord of end-stage mice. Finally, we show that OGC is heavily S-nitrosylated in the spinal cord of end-stage mice and consequently, the GSH uptake capacity of spinal cord mitochondria isolated from these mutant mice is significantly diminished. Collectively, these findings suggest that spinal cord GSH depletion, particularly at the level of the mitochondria, plays a significant role in ALS pathogenesis induced by mutant SOD1. Furthermore, the depletion of mitochondrial GSH in the G93A mutant hSOD1 mouse model may be caused by the S-nitrosylation of OGC and the capacity of mutant SOD1 to disrupt the Bcl-2/GSH interaction, resulting in a disruption of mitochondrial GSH transport.

Therapeutic Administration of Oxcarbazepine Saves Cerebellar Purkinje Cells from Ischemia and Reperfusion Injury Induced by Cardiac Arrest through Attenuation of Oxidative Stress.

Research reports using animal models of ischemic insults have demonstrated that oxcarbazepine (a carbamazepine analog: one of the anticonvulsant compounds) extends neuroprotective effects against cerebral or forebrain injury induced by ischemia and reperfusion. However, research on protective effects against ischemia and reperfusion cerebellar injury induced by cardiac arrest (CA) and the return of spontaneous circulation has been poor. Rats were assigned to four groups as follows: (Groups 1 and 2) sham asphyxial CA and vehicle- or oxcarbazepine-treated, and (Groups 3 and 4) CA and vehicle- or oxcarbazepine-treated. Vehicle (0.3% dimethyl sulfoxide/saline) or oxcarbazepine (200 mg/kg) was administered intravenously ten minutes after the return of spontaneous circulation. In this study, CA was induced by asphyxia using vecuronium bromide (2 mg/kg). We conducted immunohistochemistry for calbindin D-28kDa and Fluoro-Jade B histofluorescence to examine Purkinje cell death induced by CA. In addition, immunohistochemistry for 4-hydroxy-2-nonenal (4HNE) was carried out to investigate CA-induced oxidative stress, and immunohistochemistry for Cu, Zn-superoxide dismutase (SOD1) and Mn-superoxide dismutase (SOD2) was performed to examine changes in endogenous antioxidant enzymes. Oxcarbazepine treatment after CA significantly increased the survival rate and improved neurological deficit when compared with vehicle-treated rats with CA (survival rates ≥ 63.6 versus 6.5%), showing that oxcarbazepine treatment dramatically protected cerebellar Purkinje cells from ischemia and reperfusion injury induced by CA. The salvation of the Purkinje cells from ischemic injury by oxcarbazepine treatment paralleled a dramatic reduction in 4HNE (an end-product of lipid peroxidation) and increased or maintained the endogenous antioxidant enzymes (SOD1 and SOD2). In brief, this study shows that therapeutic treatment with oxcarbazepine after CA apparently saved cerebellar neurons (Purkinje cells) from CA-induced neuronal death by attenuating oxidative stress and suggests that oxcarbazepine can be utilized as a therapeutic medicine for ischemia and reperfusion brain (cerebellar) injury induced by CA.

Cloning and expression of recombinant human superoxide dismutase 1 (hSOD1) in Bacillus subtilis 1012.

We aimed to clone and express the human Cu, Zn superoxide dismutase (hSOD1) in Bacillus subtilis 1012. Also, we investigated the expression level of hSOD1 under different induction conditions. As an essential member of the antioxidant defense system in vivo, hSOD1 has become a therapeutic agent against host diseases, such as oxygen toxicity, acute inflammation, and radiation injury. The recombinant hSOD1 was successfully secreted extracellularly into B. subtilis 1012. The expression conditions were optimized, including inoculum size, different media, temperatures, and inducer concentrations. Finally, the highest level of hSOD1 was produced as a soluble form in Super rich medium by 2% inoculum with 0.2 mM of IPTG at 37°C after the induction for 24h. Besides, 20g/L of lactose also displayed the same inductive effect on hSOD1 expression as that of IPTG (0.2 mM). Finally, the specific activity of purified hSOD1 was determined to be 1625U/mg in the presence of 800μM of Cu2+ and 20μM of Zn2+. We propose that the B.subtilis 1012-hSOD1 strain system has great potential in future industrial applications.

Native Mass Spectrometry Coupled to Spectroscopic Methods to Investigate the Effect of Soybean Isoflavones on Structural Stability and Aggregation of Zinc Deficient and Metal-Free Superoxide Dismutase.

The deficiency or wrong combination of metal ions in Cu, Zn-superoxide dismutase (SOD1), is regarded as one of the main factors causing the aggregation of SOD1 and then inducing amyotrophic lateral sclerosis (ALS). A ligands-targets screening process based on native electrospray ionization ion mobility mass spectrometry (ESI-IMS-MS) was established in this study. Four glycosides including daidzin, sophoricoside, glycitin, and genistin were screened out from seven soybean isoflavone compounds and were found to interact with zinc-deficient or metal-free SOD1. The structure and conformation stability of metal-free and zinc-deficient SOD1 and their complexes with the four glycosides was investigated by collision-induced dissociation (CID) and collision-induced unfolding (CIU). The four glycosides could strongly bind to the metal-free and copper recombined SOD1 and enhance the folding stability of these proteins. Additionally, the ThT fluorescence assay showed that these glycosides could inhibit the toxic aggregation of the zinc-deficient or metal-free SOD1. The competitive interaction experiments together with molecular docking indicate that glycitin, which showed the best stabilizing effects, binds with SOD1 between β-sheet 6 and loop IV. In short, this study provides good insight into the relationship between inhibitors and different SOD1s.

Structural analysis of the overoxidized Cu/Zn-superoxide dismutase in ROS-induced ALS filament formation

Eukaryotic Cu, Zn-superoxide dismutase (SOD1) is primarily responsible for cytotoxic filament formation in amyotrophic lateral sclerosis (ALS) neurons. Two cysteine residues in SOD1 form an intramolecular disulfide bond. This study aims to explore the molecular mechanism of SOD1 filament formation by cysteine overoxidation in sporadic ALS (sALS). In this study, we determined the crystal structure of the double mutant (C57D/C146D) SOD1 that mimics the overoxidation of the disulfide-forming cysteine residues. The structure revealed the open and relaxed conformation of loop IV containing the mutated Asp57. The double mutant SOD1 produced more contagious filaments than wild-type protein, promoting filament formation of the wild-type SOD1 proteins. Importantly, we further found that HOCl treatment to the wild-type SOD1 proteins facilitated their filament formation. We propose a feasible mechanism for SOD1 filament formation in ALS from the wild-type SOD1, suggesting that overoxidized SOD1 is a triggering factor of sALS. Our findings extend our understanding of other neurodegenerative disorders associated with ROS stresses at the molecular level.

Expression, purification, and characterization of cell-permeable fusion antioxidant enzyme sensitive to matrix metalloproteinases-2/9

Antioxidant enzymes fused with cell-penetrating peptides could enter cells and protect cells from irradiation damage. However, the unselective transmembrane ability of cell-penetrating peptide may also bring antioxidant enzymes into tumor cells, thus protecting tumor cells and consequently reducing the efficacy of radiotherapy. There are active matrix metalloproteinase (MMP)-2 or MMP-9 in most tumor cellular microenvironments. Therefore, a fusion protein containing an MMP-2/9 cleavable substrate peptide X, a cell-penetrating peptide R9, a glutathione S-transferase (GST), and a human Cu, Zn superoxide dismutase (SOD1), was designed and named GST-SOD1-X-R9. In the tumor microenvironment, GST-SOD1-X-R9 would lose its cell-penetrating peptide and could not enter tumor cells due to the cleavage of substrate X by active MMP-2/9, thereby achieving selected entering normal cells. The complete nucleotide sequence of SOD1-X-R9 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1. The pGEX4T-1-SOD1-X-R9 recombinant plasmid was obtained, and soluble expression of the fusion protein was achieved. GST-SOD1-X-R9 was purified by ammonium sulfate precipitation and GST affinity chromatography. The molecular weight of the fusion protein was approximately 47 kDa, consistent with the theoretical value. The SOD and GST activities were 2 954 U/mg and 328 U/mg, respectively. Stability test suggested that almost no change in either SOD activity or GST activity of GST-SOD1-X-R9 was observed under physiological conditions. The fusion protein could be partially digested by collagenase Ⅳ in solution. Subsequently, the effect of MMP-2/9 activity on transmembrane ability of the fusion protein was tested using 2D and 3D cultured HepG2 cells. Little extracellular MMP-2 activity of HepG2 cells was observed under 2D culture condition. While under the 3D culture model, the size and the MMP-2 activity of the HepG2 tumor spheroid increased daily. GST-SOD1-R9 proteins showed the same transmembrane efficiency in 2D cultured HepG2 cells, but the transmembrane efficiency of GST-SOD1-X-R9 in 3D cultured HepG2 spheres was reduced remarkably. This study provided a basis for further investigating the selectively protective effect of GST-SOD1-X-R9 against oxidative damage in normal cells.

Cryo-EM structure of an amyloid fibril formed by full-length human SOD1 reveals its conformational conversion

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease. Misfolded Cu, Zn-superoxide dismutase (SOD1) has been linked to both familial and sporadic ALS. SOD1 fibrils formed in vitro share toxic properties with ALS inclusions. Here we produced cytotoxic amyloid fibrils from full-length apo human SOD1 under reducing conditions and determined the atomic structure using cryo-EM. The SOD1 fibril consists of a single protofilament with a left-handed helix. The fibril core exhibits a serpentine fold comprising N-terminal segment (residues 3–55) and C-terminal segment (residues 86–153) with an intrinsic disordered segment. The two segments are zipped up by three salt bridge pairs. By comparison with the structure of apo SOD1 dimer, we propose that eight β-strands (to form a β-barrel) and one α-helix in the subunit of apo SOD1 convert into thirteen β-strands stabilized by five hydrophobic cavities in the SOD1 fibril. Our data provide insights into how SOD1 converts between structurally and functionally distinct states.

Broiler responses to copper levels and sources: growth, tissue mineral content, antioxidant status and mRNA expression of genes involved in lipid and protein metabolism

BackgroundFive hundred 8-d old male broilers Cobb500 were randomly allotted into 10 treatments in factorial arrangement with 5 Cu levels (0, 4, 8, 12, and 16 mg/kg), and 2 sources (Cu proteinate, CuPro and Cu sulphate, CuSO4.5H2O) for a 10-d-experiment.ResultsFeed conversion ratio (FCR) was better (P < 0.05) in CuPro fed chicks compared with CuSO4.5H2O group. Average daily feed intake (ADFI) decreased linearly (P < 0.05) as dietary Cu increased. A quadratic response (P < 0.05) to Cu levels was found for FCR, being optimized at 9.87 and 8.84 mg Cu/kg in CuPro and CuSO4.5H2O diets, respectively. Copper supplementation linearly increased liver Cu content (P < 0.05) and tended to linearly increase (P = 0.07) phosphorus (P) and copper in tibia. Manganese and zinc were higher (P < 0.05) in tibia of CuPro fed birds. Broilers fed CuPro exhibited lower liver iron (P < 0.05) content, lower activities of Cu, Zn superoxide dismutase (CuZnSOD) in breast muscle and liver, and glutathione peroxidase in liver. Glutathione peroxidase reduced linearly (P < 0.05) with CuPro levels and increased linearly (P < 0.05) with CuSO4.5H2O levels and were lower (P < 0.05) in all CuPro levels in breast muscle. Breast muscle malondialdehyde concentration tended to be higher (P = 0.08) in broilers fed CuSO4.5H2O. Copper levels linearly increased (P < 0.05) metallothionein (MT) and malate dehydrogenase (MDH) expression in liver, and six-transmembrane epithelial antigen of the prostate-1 (STEAP-1) in the intestine. Copper elicited a quadratic response (P < 0.050) in AKT-1 and mammalian target of rapamycin (mTOR) in breast muscle, CuZnSOD in liver and antioxidant 1 copper chaperone (ATOX 1) in intestine. Broilers fed CuPro exhibited higher mRNA expression of mTOR in muscle breast and lower CuZnSOD in liver and ATOX 1 in intestine. Interaction (P < 0.05) between levels and sources was found in mRNA expression for GSK-3β, MT, and CuZnSOD in breast muscle, FAS and LPL in liver and MT and CTR1 in intestine.ConclusionsCuPro showed beneficial effects on feed conversion and bone mineralization. Organic and inorganic Cu requirements are 9.87 and 8.84 mg Cu/kg, respectively.

Unfolding and aggregation of oxidized metal-deficient superoxide dismutase and isoflavone inhibition based on ion mobility mass spectrometry and ThT fluorescence assay

Structurally abnormal Cu, Zn-superoxide dismutase (SOD1) is considered one of the causes of amyotrophic lateral sclerosis. The misfolding and neurotoxic aggregation of SOD1 can be induced by mutations, metal deficiency, and post-translational modification. Here, we revealed the risk of oxidation damage on zinc-deficient SOD1 by native mass spectrometry coupled with ion mobility. The copper ions were found to be released in the early period of oxidation which may be the result of oxidation on its binding site. On the other hand, zinc-deficient SOD1 showed a faster and deeper dissociation tendency than SOD1 with no metal ions. The results of collision-induced unfolding indicated that the oxidized zinc-deficient SOD1 is more easily to be turned into totally unfolded conformation. ThT fluorescence also showed stronger aggregation of oxidized zinc-deficient SOD1. Compared with DTT-induced reduction, oxidized zinc-deficient SOD1 acted differently in dimer dissociation, conformation stability, and aggregation, suggesting that the conserved intramolecular disulfide bonds were influenced little during oxidation. Additionally, we explored glycitin, an isoflavone glycoside, to prevent the oxidation of metal-deficient SOD1 and inhibit the unfolding and aggregation of oxidized metal-deficient SOD1.

Toxic SOD1 trimers are off-pathway in the formation of amyloid-like fibrils in ALS

Accumulation of insoluble amyloid fibrils is widely studied as a critical factor in the pathology of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. Misfolded Cu, Zn superoxide dismutase (SOD1) was the first protein linked to ALS, and non-native SOD1 trimeric oligomers were recently linked to cytotoxicity, while larger oligomers were protective to cells. The balance between trimers and larger aggregates in the process of SOD1 aggregation is, thus, a critical determinant of potential therapeutic approaches to treat ALS. However, it is unknown whether these trimeric oligomers are a necessary intermediate for larger aggregate formation or a distinct off-pathway species competing with fibril formation. Depending on the on- or off-pathway scenario of trimer formation, we expect drastically different therapeutic approaches. Here, we show that the toxic SOD1 trimer is an off-pathway intermediate competing with protective fibril formation. We design mutant SOD1 constructs that remain in a trimeric state (super-stable trimers) and show that stabilizing the trimeric SOD1 prevents formation of fibrils in vitro and in a motor neuron-like cell model (NSC-34). Using size exclusion chromatography, we track the aggregation kinetics of purified SOD1 and show direct competition of trimeric SOD1 with larger oligomer and fibril formation. Finally, we show the trimer is structurally independent of both larger soluble oligomers and insoluble fibrils using circular dichroism spectroscopy and limited proteolysis.

Herbicide Roundup shows toxic effects in nontarget organism Drosophila.

Glyphosate-based herbicide Roundup, as the most employed herbicide used for multiple purposes in agriculture, adversely affects nontarget organisms. We tested the effects of Roundup applied at larval and adult stages. Roundup caused developmental delay and increased larvae mortality. Roundup treatment reduced hemolymph glucose and glycogen levels in adult flies of both sexes at the highest concentration tested. Sex-dependent diverse effects were found in catalase and Cu,Zn superoxide dismutase (Cu,Zn-SOD) activities. Decreased aconitase activity, contents of thiols, and lipid peroxides were found after larval Roundup exposure. Furthermore, chronic exposure to adult flies decreased appetite, body weight, and shortened lifespan. Thus, our results suggest that high concentrations of Roundup are deleterious to both larvae and adults, resulting in a shift of the metabolism and antioxidant defense system in Drosophila melanogaster.