Engineering a monobody specific to monomeric Cu/Zn-superoxide dismutase associated with amyotrophic lateral sclerosis.

Misfolding of mutant Cu/Zn-superoxide dismutase (SOD1) has been implicated in familial form of amyotrophic lateral sclerosis (ALS). A natively folded SOD1 forms a tight homodimer, and the dimer dissociation has been proposed to trigger the oligomerization/aggregation of SOD1. Besides increasing demand for probes allowing the detection of monomerized forms of SOD1 in various applications, the development of probes has been limited to conventional antibodies. Here, we have developed Mb(S4) monobody, a small synthetic binding protein based on the fibronectin type III scaffold, that recognizes a monomeric but not dimeric form of SOD1 by performing combinatorial library selections using phage and yeast-surface display methods. Although Mb(S4) was characterized by its excellent selectivity to the monomeric conformation of SOD1, the monomeric SOD1/Mb(S4) complex was not so stable (apparent Kd ~ µM) as to be detected in conventional pull-down experiments. Instead, the complex of Mb(S4) with monomeric but not dimeric SOD1 was successfully trapped by proximity-enabled chemical crosslinking even when reacted in the cell lysates. We thus anticipate that Mb(S4) binding followed by chemical crosslinking would be a useful strategy for in vitro and also ex vivo detection of the monomeric SOD1 proteins.

Electrochemical biosensing platform based on AuNWs/rGO-CMC-PEDOT:PSS composite for the detection of superoxide anion released from living cells.

Detection of superoxide anion (O2·-) levels holds significant importance for the diagnosis and even clinical treatments of oxidative stress-related diseases. Herein, we prepared a composite electrode material to encapsulate copper-zinc superoxide dismutase (SOD1) for biosensing of O2·-. The sensing material consists of gold nanowires (AuNWs), reduced graphene oxide (rGO), carboxymethyl cellulose (CMC) and PEDOT:PSS. CMC provides abundant -COOH to bind SOD1, with a high adsorption coverage of 1.499 × 10-9 mol cm-2 on the sensor surface. rGO and PEDOT endow the composite with significant conductivity, whereas PSS has antifouling capability. Moreover, AuNWs exhibit excellent electrical conductivity and a high aspect ratio, which promotes electron transfer, and ultimately enhances the catalytic performance of the enzyme. Meanwhile, SOD1(Cu2+) catalyzes the dismutation of O2·- to O2 and H2O2, and H2O2 is then electrochemically oxidized to generate amperometric signals for determination of O2·-. The sensor demonstrates outstanding detection performance for O2·- with a low detection limit of 2.52 nM, and two dynamic ranges (14.30 nM-1.34 µM and 1.34 µM-42.97 µM) with corresponding sensitivity of 0.479 and 0.052 µA µM-1cm-2, respectively. Additionally, the calculated apparent Michaelis constant (Kmapp) of 1.804 µM for SOD1 demonstrates the outstanding catalytic activity and the surface-immobilized enzyme's substrate affinity. Furthermore, the sensor shows the capability to dynamically detect the level of O2·- released from living HepG2 cells. This study provides an inovative design to obtain a biocompatible electrochemical sensing platform with plenty of immobilization sites for biomolecules, large surface area, high conductivity and flexibility.

Evaluating protein cross-linking as a therapeutic strategy to stabilize SOD1 variants in a mouse model of familial ALS.

Mutations in the gene encoding Cu-Zn superoxide dismutase 1 (SOD1) cause a subset of familial amyotrophic lateral sclerosis (fALS) cases. A shared effect of these mutations is that SOD1, which is normally a stable dimer, dissociates into toxic monomers that seed toxic aggregates. Considerable research effort has been devoted to developing compounds that stabilize the dimer of fALS SOD1 variants, but unfortunately, this has not yet resulted in a treatment. We hypothesized that cyclic thiosulfinate cross-linkers, which selectively target a rare, 2 cysteine-containing motif, can stabilize fALS-causing SOD1 variants in vivo. We created a library of chemically diverse cyclic thiosulfinates and determined structure-cross-linking-activity relationships. A pre-lead compound, "S-XL6," was selected based upon its cross-linking rate and drug-like properties. Co-crystallographic structure clearly establishes the binding of S-XL6 at Cys 111 bridging the monomers and stabilizing the SOD1 dimer. Biophysical studies reveal that the degree of stabilization afforded by S-XL6 (up to 24°C) is unprecedented for fALS, and to our knowledge, for any protein target of any kinetic stabilizer. Gene silencing and protein degrading therapeutic approaches require careful dose titration to balance the benefit of diminished fALS SOD1 expression with the toxic loss-of-enzymatic function. We show that S-XL6 does not share this liability because it rescues the activity of fALS SOD1 variants. No pharmacological agent has been proven to bind to SOD1 in vivo. Here, using a fALS mouse model, we demonstrate oral bioavailability; rapid engagement of SOD1G93A by S-XL6 that increases SOD1G93A's in vivo half-life; and that S-XL6 crosses the blood-brain barrier. S-XL6 demonstrated a degree of selectivity by avoiding off-target binding to plasma proteins. Taken together, our results indicate that cyclic thiosulfinate-mediated SOD1 stabilization should receive further attention as a potential therapeutic approach for fALS.

The role of the terminal cysteine moiety in a metallopeptide mimicking the active site of the NiSOD enzyme.

Superoxide dismutase (SOD) enzymes are pivotal in regulating oxidative stress. In order to model Ni containing SOD enzymes, the results of the thermodynamic, spectroscopic and SOD activity studies on the complexes formed between nickel(II) and a NiSOD related peptide, CysCysAspLeuProCysGlyValTyr-NH2 (wtCC), are reported. Cysteine was introduced to replace the first histidine residue in the amino acid sequence of the active site of the NiSOD enzyme. The novel peptide exhibits 3 times higher metal binding affinity compared to the native NiSOD fragment. This is due to the presence of the first cysteine in the coordination sphere of nickel(II). At physiological pH, the (NH2,S-,S-,S-) coordinated complex is the major species. This coordination mode is altered when one thiolate group is replaced by an amide nitrogen of the peptide backbone above pH 7.5. The nickel complexes of wtCC exhibit similar SOD activity to that of the complex formed with the active site fragment of the native NiSOD. The reaction between the complexes and the superoxide anion was studied by the sequential stopped-flow method. These studies revealed that the nickel(II) complex is always in excess over the nickel(III) complex during the dismutation process. However, the nickel(III) species is also involved in a relatively fast degradation process. This unambiguously proves that a protective mechanism must be operative in the NiSOD enzyme which prevents the oxidation of the sulfur atom of cysteine in the presence of O2-. The results provide new possibilities for the use of NiSOD mimics in bio- and industrial catalytic processes.

Unveiling local and global conformational changes and allosteric communications in SOD1 systems using molecular dynamics simulation and network analyses.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a serious neurodegenerative disorder affecting nerve cells in the brain and spinal cord that is caused by mutations in the superoxide dismutase 1 (SOD1) enzyme. ALS-related mutations cause misfolding, dimerisation instability, and increased formation of aggregates. The underlying allosteric mechanisms, however, remain obscure as far as details of their fundamental atomistic structure are concerned. Hence, this gap in knowledge limits the development of novel SOD1 inhibitors and the understanding of how disease-associated mutations in distal sites affect enzyme activity. METHODS: We combined microsecond-scale based unbiased molecular dynamics (MD) simulation with network analysis to elucidate the local and global conformational changes and allosteric communications in SOD1 Apo (unmetallated form), Holo, Apo_CallA (mutant and unmetallated form), and Holo_CallA (mutant form) systems. To identify hotspot residues involved in SOD1 signalling and allosteric communications, we performed network centrality, community network, and path analyses. RESULTS: Structural analyses showed that unmetallated SOD1 systems and cysteine mutations displayed large structural variations in the catalytic sites, affecting structural stability. Inter- and intra H-bond analyses identified several important residues crucial for maintaining interfacial stability, structural stability, and enzyme catalysis. Dynamic motion analysis demonstrated more balanced atomic displacement and highly correlated motions in the Holo system. The rationale for structural disparity observed in the disulfide bond formation and R143 configuration in Apo and Holo systems were elucidated using distance and dihedral probability distribution analyses. CONCLUSION: Our study highlights the efficiency of combining extensive MD simulations with network analyses to unravel the features of protein allostery.

Direct relationship between dimeric form and activity in the acidic copper-zinc superoxide dismutase from lemon.

The copper-zinc superoxide dismutase (CuZnSOD) from lemon (SOD_CL) is active in an acidic environment and resists proteolytic degradation. The enzyme occurs as a dimer, which has an indirect effect on the enzyme activity as the monomer retains only ∼35% of the activity. Here, the crystal structure of SOD_CL at 1.86 Šresolution is reported that may explain this peculiarity. The crystal belonged to space group P21, with unit-cell parameters a = 61.11, b = 74.55, c = 61.69 Å, ß = 106.86°, and contained four molecules in the asymmetric unit. The overall structure of SOD_CL resembles that of CuZnSOD from plants. The structure of SOD_CL shows a unique arrangement of surface loop IV that connects the dimer interface and the active site, which is located away from the dimer-interface region. This arrangement allows direct interaction between the residues residing in the dimer interface and those in the active site. The arrangement also includes Leu62 and Gln164, which are conserved in cytoplasmic CuZnSOD. This supports the classification of SOD_CL as a cytoplasmic CuZnSOD despite sharing the highest amino-acid sequence homology with CuZnSODs from spinach and tomato, which are chloroplastic.

Polyphenolic flavonoid compounds act as the inhibitory potential of aggregation process: Implications for the prevention and therapeutics against FALS-associated D101G SOD1 mutant.

Aggregation of proteins is a biological phenomenon caused by misfolded proteins. Human superoxide dismutase (hSOD1) misfolding and aggregation underlie the neurological illness amyotrophic lateral sclerosis (ALS). The most significant contributing factor to ALS is genetic point mutations in SOD1. particularly, D101G mutant is the most harmful because it significantly reduces the life expectancy of patients. Subsequently, the use of natural polyphenolic flavonoids is strongly recommended to reduce the amyloidogenic behavior of protopathic proteins. In this study, using computational parameters such as protein-ligand interaction and molecular dynamics (MD) simulation analyses, we are trying to identify a pharmacodynamically promising flavonoid compound that can effectively inhibit the pathogenic behavior of the D101G mutant. Epigallocatechin-gallate (EGCG), Hesperidin, Isorhamnetin, and Diosmetin were identified as potential leads in a preliminary screening of flavonoids to anti-amyloid action. The results of MD showed that the binding of flavonoids to D101G mutant caused changes in stability, hydrophobicity of protein, and flexibility, as well as significantly led to the restoration of lost hydrogen bonds. Secondary structure analysis showed that protein destabilization and the increased propensity of ß-sheet caused by the mutation were restored to the wild-type state upon binding of flavonoids. Besides, to differentiate aggregation, we elucidated alterations in the free energy landscape (FEL) and dynamic cross-correlation matrix (DCCM) of WT-SOD1 and mutant (unbound /bound) states. Among flavonoids, Epigallocatechin-gallate and Hesperidin had the most therapeutic efficacy against the D101G mutant. Therefore, Epigallocatechin-gallate and Hesperidin promise considerable therapeutic potential to develop highly effective inhibitors in reducing fatal and irreversible ALS.

Structural and solution speciation studies on selected [Cu(NN)(OO)] complexes and an investigation of their biomimetic activity, ROS generation and their cytotoxicity in normoxic, hypoxic and anoxic environments in MCF-7 breast cancer-derived cells.

Reactive oxygen species(ROS) generation with subsequent DNA damage is one of the principle mechanisms of action assigned to copper-based anticancer complexes. The efficacy of this type of chemotherapeutic may be reduced in the low oxygen environment of tumours. In this study the cytotoxicity of three complexes, [Cu(dips)(phen)] (1), [Cu(ph)(phen)]·2H2O (2) and [Cu(ph)(bpy)]·H2O (3) (disp: 3,5-diisopropylsalicylate, phen: 1,10- phenanthroline, ph: phthalate, bpy: 2,2'-bipyridyl) were assessed for anticancer activity in the breast-cancer derived MCF-7 line under normoxic, hypoxic and anoxic conditions. In an immortalised keratinocyte HaCaT cell line, the cytotoxicity of complexes 2 and 3 was significantly reduced under both normoxic and hypoxic conditions, whilst the cytotoxicity of complex 1 was increased under hypoxic conditions. The ability of the complexes to generate ROS in the MCF-7 cell line was evaluated as was their ability to act as superoxide dismutase(SOD) and catalase mimics using a yeast cell assay. ROS generation was significant for complexes 2 and 3, less so for complex 1 though all three complexes had SOD mimetic ability. Given the ternary nature of the complexes, solution speciation studies were undertaken but were only successful for complex 3, due to solubility issues with the other two complexes. The concentration distribution of various species, formed in aqueous solution, was evaluated as a function of pH and confirmed that complex 3 is the dominant species at physiological pH in the mM concentration range. However, as its concentration diminishes, it experiences a progressive dissociation, leading to the formation of binary complexes of bpy alongside unbound phthalate.

Mn Single-Atom Nanozymes with Superior Loading Capability and Superb Superoxide Dismutase-like Activity for Bioassay.

Single-atom nanozymes (SANs) with highly exposed active sites and remarkable catalytic activity have shown noteworthy practicability in heterogeneous catalysis-based bioassay. Nevertheless, most of them were reported with peroxidase-like activity and ordinary loading capability. It is still a challenge to prepare high-loading SANs with desirable superoxide dismutase (SOD)-like activity. In this work, Mn SAN was successfully confined in the frameworks of Prussian blue analogues formed on Ti3C2 MXene sheets with the assistance of massive surfactants, which show a superior loading efficiency of 13.5 wt % (typically <2.0 wt %). The prepared Mn SAN exhibits desirable superoxide radical anion elimination capability because of its SOD-like activity. Moreover, due to the wide-spectrum absorption behavior of the carriers, Mn SAN shows a synergistically quenching efficiency up to 98.89% on the emission of the reactive oxygen species-mediated chemiluminescent (CL) system. Inspired by these features, a CL quenching method was developed on a lateral flow test strip platform by utilizing Mn SAN as a signal quencher and acetamiprid as a model analyte. The method for detecting acetamiprid shows a detection range of 1.0-10,000 pg mL-1 and a limit of detection of 0.3 pg mL-1. Its accuracy has been validated by detecting acetamiprid in medicinal herbs with acceptable recoveries. This work opens an avenue for preparing SANs with a surfactant-assisted protocol and pioneers the study of SANs with SOD-like activity in bioassay.

Structure of a superoxide dismutase from a tardigrade: Ramazzottius varieornatus strain YOKOZUNA-1.

Superoxide dismutase (SOD) is an essential and ubiquitous antioxidant protein that is widely present in biological systems. The anhydrobiotic tardigrades are some of the toughest micro-animals. They have an expanded set of genes for antioxidant proteins such as SODs. These proteins are thought to play an essential role in oxidative stress resistance in critical situations such as desiccation, although their functions at the molecular level have yet to be explored. Here, crystal structures of a copper/zinc-containing SOD (RvSOD15) from an anhydrobiotic tardigrade, Ramazzottius varieornatus strain YOKOZUNA-1, are reported. In RvSOD15, one of the histidine ligands of the catalytic copper center is replaced by a valine (Val87). The crystal structures of the wild type and the V87H mutant show that even though a histidine is placed at position 87, a nearby flexible loop can destabilize the coordination of His87 to the Cu atom. Model structures of other RvSODs were investigated and it was found that some of them are also unusual SODs, with features such as deletion of the electrostatic loop or ß3 sheet and unusual metal-binding residues. These studies show that RvSOD15 and some other RvSODs may have evolved to lose the SOD function, suggesting that gene duplications of antioxidant proteins do not solely explain the high stress tolerance of anhydrobiotic tardigrades.

Coordination Construction of the Dual Superoxide Dismutase Catalytic Center Nanozyme: Synergistic Contribution of the Site, Structure, and Electron Transfer Pathway.

Superoxide dismutase (SOD) mimics are limited by a single active center, and their performance is difficult to achieve the activity level of natural SOD. Herein, we exhibit the coordination construction of different SOD active centers (Cu and Mn) and structural regulation of framework carbonization in MOFs. The obtained catalytic activity and excellent biocompatibility are comparable to Cu/Zn-SOD. The improvement of catalytic performance was attributed to the bimetallic sites' synergistic catalysis (enhancing the substrate affinity and accelerating the reaction process) on the one hand and the contribution of framework carbonization on the other hand, which not only regulate the relative position and valence of metal nodes but improve the spatial adaptability of the reaction and reduce the reaction barrier, and the increased conductivity of the framework accelerates the electron transfer process in the reaction. The excellent biocompatibility results from the fixing effect of the carbonized framework on the metal nodes. Mn/Cu-C-N2 was encapsulated in a chitosan film as an antioxidant compared with a pure chitosan film; the anthocyanin content of blueberries increased 2 times after being stored at room temperature for 7 days, and the content was 83% of the fresh blueberries, providing exciting potential for biological applications limited by the performance of SOD nanozymes.

A Series of Ni Complexes Based on a Versatile ATCUN-Like Tripeptide Scaffold to Decipher Key Parameters for Superoxide Dismutase Activity.

The cellular level of reactive oxygen species (ROS) has to be controlled to avoid some pathologies, especially those linked to oxidative stress. One strategy for designing antioxidants consists of modeling natural enzymes involved in ROS degradation. Among them, nickel superoxide dismutase (NiSOD) catalyzes the dismutation of the superoxide radical anion, O2•-, into O2 and H2O2. We report here Ni complexes with tripeptides derived from the amino-terminal CuII- and NiII-binding (ATCUN) motif that mimics some structural features found in the active site of the NiSOD. A series of six mononuclear NiII complexes were investigated in water at physiological pH with different first coordination spheres, from compounds with a N3S to N2S2 set, and also complexes that are in equilibrium between the N-coordination (N3S) and S-coordination (N2S2). They were fully characterized by a combination of spectroscopic techniques, including 1H NMR, UV-vis, circular dichroism, and X-ray absorption spectroscopy, together with theoretical calculations and their redox properties studied by cyclic voltammetry. They all display SOD-like activity, with a kcat ranging between 0.5 and 2.0 × 106 M-1 s-1. The complexes in which the two coordination modes are in equilibrium are the most efficient, suggesting a beneficial effect of a nearby proton relay.

Superoxide Dismutase-like Activity of Zeolitic Imidazolate Framework Nanoparticles Comprising Biomimetic Imidazolato-bridged CuZn Units.

Nanoparticles exhibiting enzymatic functions have garnered considerable attention due to their structural robustness and the profusion of active sites that can be introduced to a single nanosized particle. Here we report that nanosized mixed-metal zeolitic imidazolate frameworks (ZIFs) show a superoxide dismutase (SOD)-like catalytic activity. We chose a ZIF composed of copper and zinc ions and 2-methylimidazole, CuZn-ZIF-8, in which the Cu and Zn ions are bridged by an imidazolato ligand. This coordination geometry closely mimics the active site of CuZn superoxide dismutase (CuZnSOD). The CuZn-ZIF-8 nanoparticles exhibit potent SOD-like activity, attributed to their porous nature and numerous copper active sites, and also possess exceptional recyclability.

Scavenging of Superoxide in Aprotic Solvents of Four Isoflavones That Mimic Superoxide Dismutase.

Isoflavones are plant-derived natural products commonly found in legumes that show a large spectrum of biomedical activities. A common antidiabetic remedy in traditional Chinese medicine, Astragalus trimestris L. contains the isoflavone formononetin (FMNT). Literature reports show that FMNT can increase insulin sensitivity and potentially target the peroxisome proliferator-activated receptor gamma, PPARγ, as a partial agonist. PPARγ is highly relevant for diabetes control and plays a major role in Type 2 diabetes mellitus development. In this study, we evaluate the biological role of FMNT, and three related isoflavones, genistein, daidzein and biochanin A, using several computational and experimental procedures. Our results reveal the FMNT X-ray crystal structure has strong intermolecular hydrogen bonding and stacking interactions which are useful for antioxidant action. Cyclovoltammetry rotating ring disk electrode (RRDE) measurements show that all four isoflavones behave in a similar manner when scavenging the superoxide radical. DFT calculations conclude that antioxidant activity is based on the familiar superoxide σ-scavenging mode involving hydrogen capture of ring-A H7(hydroxyl) as well as the π-π (polyphenol-superoxide) scavenging activity. These results suggest the possibility of their mimicking superoxide dismutase (SOD) action and help explain the ability of natural polyphenols to assist in lowering superoxide concentrations. The SOD metalloenzymes all dismutate O2•- to H2O2 plus O2 through metal ion redox chemistry whereas these polyphenolic compounds do so through suitable hydrogen bonding and stacking intermolecular interactions. Additionally, docking calculations suggest FMNT can be a partial agonist of the PPARγ domain. Overall, our work confirms the efficacy in combining multidisciplinary approaches to provide insight into the mechanism of action of small molecule polyphenol antioxidants. Our findings promote the further exploration of other natural products, including those known to be effective in traditional Chinese medicine for potential drug design in diabetes research.

Multienzyme-Mimicking Au@Cu2O with Complete Antioxidant Capacity for Reactive Oxygen Species Scavenging.

Most enzyme catalysts are unable to achieve effective oxidation resistance because of the monotonous mimicking function or production of secondary reactive oxygen species (ROS). Herein, the Au@Cu2O heterostructure with multienzyme-like activities is deigned, which has significantly improved antioxidant capacity compared with pure Cu2O for the scavenging of highly cell-damaging secondary ROS, i.e.,·OH. Experiments and theoretical calculations show that the heterostructure exhibits a built-in electric field and lattice mismatch at the metal-semiconductor interface, which facilitate to generate abundant oxygen vacancies, redox couples, and surface electron deficiency. On the one hand, the presence of rich oxygen vacancies and redox couple can enhance the adsorption and activation of oxygen-containing ROS (including O2·- and H2O2). On the other hand, the electron transfer between the electron-deficient Au@Cu2O surface and electron donor would promote peroxide-like activity and avoid producing ·OH. Importantly, endogenous ·OH could be eliminated in both acidic and neutral conditions, which is no longer limited by the volatile physiological environment. Therefore, Au@Cu2O can simulate superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and glutathione peroxidase (GPx) to form a complete antioxidant system. The deigned nanoenzyme is explored in the real sample world such as A549 cells and zebrafish. This work provides theoretical and practical strategies for the construction of a complete antioxidant enzyme system.

Exploring the Folding Mechanism of Dimeric Superoxide Dismutase.

The Cu/Zn Human Superoxide Dismutase (SOD1) is a dimeric metalloenzyme whose genetic mutations are directly related to amyotrophic lateral sclerosis (ALS), so understanding its folding mechanism is of fundamental importance. Currently, the SOD1 dimer formation is studied via molecular dynamics simulations using a simplified structure-based model and an all-atom model. Results from the simplified model reveal a mechanism dependent on distances between monomers, which are limited by constraints to mimic concentration dependence. The stability of intermediates (during the int state) is significantly affected by this distance, as well as by the presence of two folded monomers prior to dimer formation. The kinetics of interface formation are also highly dependent on the separation distance. The folding temperature of the dimer is about 4.2% higher than that of the monomer, a value not too different from experimental data. All-atom simulations on the apo dimer give binding free energy between monomers similar to experimental values. An intermediate state is evident for the apo form at a separation distance between monomers slightly larger than the native distance which has little formed interface between monomers. We have shown that this intermediate is stabilized by non-native intra- and intercontacts.

Overcoming Cytosolic Delivery Barriers of Proteins Using Denatured Protein-Conjugated Mesoporous Silica Nanoparticles.

Intracellular delivery of therapeutic proteins has increased advantages over current small-molecule drugs and gene therapies, especially in therapeutic efficacies for a broad spectrum of diseases. Hence, developing the protein therapeutics approach provides a needed alternative. Here, we designed a mesoporous silica nanoparticle (MSN)-mediated protein delivery approach and demonstrated effective intracellular delivery of the denatured superoxide dismutase (SOD) protein, overcoming the delivery challenges and achieving higher enzymatic activity than native SOD-conjugated MSNs. The denatured SOD-conjugated MSN delivery strategy provides benefits of reduced size and steric hindrance, increased protein flexibility without distorting its secondary structure, exposure of the cell-penetrating peptide transactivator of transcription for enhanced efficient delivery, and a change in the corona protein composition, enabling cytosolic delivery. After delivery, SOD displayed a specific activity around threefold higher than in our previous reports. Furthermore, the in vivo biosafety and therapeutic potential for neuron therapy were evaluated, demonstrating the biocompatibility and the effective antioxidant effect in Neuro-2a cells that protected neurite outgrowth from paraquat-induced reactive oxygen species attack. This study offers an opportunity to realize the druggable possibility of cytosolic proteins using MSNs.

Novel cells integrated biosensor based on superoxide dismutase on electrospun fiber scaffolds for the electrochemical screening of cellular stress.

A novel electrochemical biosensor was developed to monitor fibroblast cells stress levels for the first time in situ under external stimuli based on the recognition of superoxide anion released upon cell damage. The biosensor comprised metallized polycaprolactone electrospun fibers covered with zinc oxide for improved cell adhesion and signal transduction, whilst stable bioconjugates of mercaptobenzoic acid-functionalized gold nanoparticles/superoxide dismutase were employed as recognition bioelements. Biosensors were first tested and optimized for in situ generated superoxide detection by fixed potential amperometry at +0.3 V, with minimal interferences from electroactive species in cell culture media. L929 fibroblast cells were then implanted on the optimized biosensor surface and the biosensor morphologically characterized by scanning electron microscopy (SEM) and fluorescence microscopy, which illustrated the network-type pattern of fibroblasts adjacent to the fiber scaffold. Fibroblast stress was induced by zymosan and monitored at the cells integrated biosensor using fixed potential amperometry (CA) with a sensitivity of 26 nA cm-2 µg mL-1 zymosan and electrochemical impedance spectroscopy (EIS), with similar sensitivity of the biosensor considering the Rs and Z' parameters of around 0.13 Ω cm2 µg-1 mL and high correlation factors R2 of 0.9994. The obtained results underline the applicability of the here developed biosensor for the electrochemical screening of the fibroblast cells stress. The concept in using low-cost biocompatible polymeric fibers as versatile scaffolds for both enzyme immobilization and cell adhesion, opens a new path in developing biosensors for the in-situ investigation of a variety of cellular events.

Amyotrophic lateral sclerosis disease-related mutations disrupt the dimerization of superoxide dismutase 1 - A comparative molecular dynamics simulation study.

More than 150 genes are involved in amyotrophic lateral sclerosis (ALS), with superoxide dismutase 1 (SOD1) being one of the most studied. Mutations in SOD1 gene, which encodes the enzyme SOD1 is the second most prevalent and studied cause of familial ALS. SOD1 is a ubiquitous, homodimeric metalloenzyme that forms a critical component of the cellular defense against reactive oxygen species. Several mutations in the SOD1 enzyme cause misfolding, dimerization instability, and increased aggregate formation in ALS. However, there is a lack of information on the dimerization of SOD1 monomers and the mechanistic underpinnings on how the pathogenic mutations disrupt the dimerization mechanism. Here, we presented microsecond-scale molecular dynamics (MD) simulations to unravel how interface-based mutations compromise SOD1 dimerization and provide mechanistic understanding into the corresponding process using WT and three interface-based mutant systems (A4V, T54R, and I113T). Structural stability analysis showed that the mutant systems displayed disparate variations in the catalytic sites which may directly alter the stability and activity of the SOD1 enzyme. Based on the dynamic network analysis and principal component analysis, it has been identified that the mutations weakened the correlated motions along the dimer interface and altered the protein conformational behavior, thus weakening the stability of dimer formation. Moreover, the simulation results identified crucial residues such as G51, D52, G114, I151, and Q153 in establishing the dimerization interaction network, which were weakened or absent in the presence of interfacial mutants. Surface potential analysis on mutant systems also displayed changes in the dimerization potential, thus showing the unfavorable dimer formation. Furthermore, network analysis identified the hotspot residues necessary for SOD1 signal transduction which were surprisingly found in the catalytic sites rather than the anticipated dimerization interface.

Application of Spectroscopic Methods for the Identification of Superoxide Dismutases in Cyanobacteria.

Superoxide dismutases (SODs) belong to the group of metalloenzymes that remove superoxide anion radicals and they have been identified in three domains of life: Bacteria, Archaea and Eucarya. SODs in Synechocystis sp. PCC 6803, Gloeobacter violaceus CCALA 979, and Geitlerinema sp. ZHR1A were investigated. We hypothesized that iron (FeSOD) and/or manganese (MnSOD) dominate as active forms in these cyanobacteria. Activity staining and three different spectroscopic methods of SOD activity bands excised from the gels were used to identify a suitable metal in the separated samples. FeSODs or enzymes belonging to the Fe-MnSOD superfamily were detected. The spectroscopic analyses showed that only Fe is present in the SOD activity bands. We found FeSOD in Synechocystis sp. PCC 6803 while two forms in G. violaceus and Geitlerinema sp. ZHR1A: FeSOD1 and FeSOD2 were present. However, no active Cu/ZnSODs were identified in G. violaceus and Geitlerinema sp. ZHR1A. We have shown that selected spectroscopic techniques can be complementary to the commonly used method of staining for SOD activity in a gel. Furthermore, the occurrence of active SODs in the cyanobacteria studied is also discussed in the context of SOD evolution in oxyphotrophs.