Form Of Manganese Superoxide Dismutase Research Articles

Cryotrapping peroxide in the active site of human mitochondrial manganese superoxide dismutase crystals for neutron diffraction.

Structurally identifying the enzymatic intermediates of redox proteins has been elusive due to difficulty in resolving the H atoms involved in catalysis and the susceptibility of ligand complexes to photoreduction from X-rays. Cryotrapping ligands for neutron protein crystallography combines two powerful tools that offer the advantage of directly identifying hydrogen positions in redox-enzyme intermediates without radiolytic perturbation of metal-containing active sites. However, translating cryogenic techniques from X-ray to neutron crystallography is not straightforward due to the large crystal volumes and long data-collection times. Here, methods have been developed to visualize the evasive peroxo complex of manganese superoxide dismutase (MnSOD) so that all atoms, including H atoms, could be visualized. The subsequent cryocooling and ligand-trapping methods resulted in neutron data collection to 2.30 Å resolution. The P6122 crystal form of MnSOD is challenging because it has some of the largest unit-cell dimensions (a = b = 77.8, c = 236.8 Å) ever studied using high-resolution cryo-neutron crystallography. The resulting neutron diffraction data permitted the visualization of a dioxygen species bound to the MnSOD active-site metal that was indicative of successful cryotrapping.

The role of S126 in the Staphylococcus equorum MnSOD activity and stability

The dimeric form of manganese superoxide dismutase is instrumental for activity because each of the monomers provides amino acid residues participating in the enzymatic reaction. Hence, preventing dissociation of the dimer would maintain the enzymatic activity in detrimental conditions e.g. high temperature. To prevent dissociation of the dimer, a disulphide (S-S) bond was introduced at the dimer interface. In the wild type structure, S126 interacts with S126 of the other monomer. In the presented work, a mutant was designed with an S126C substitution. The crystal structure of the S126C mutant showed that only 50–70% of monomers formed the S-S bond. This observed imperfect S-S bonding was likely caused by photolytic S-S bond breakage mediated by the neighbouring tryptophan residue. In the wild type, S126 is located facing W163 and forms a water-mediated hydrogen bond with E164; W163 and E164 are crucial in the enzyme’s activity. The replacement of S126 by a cysteine residue lowered the activity of the enzyme by ~70%. S126 has never been considered to play a role in the enzyme’s activity or stability, thus the finding showed the importance of this residue.

Abstract SY20-02: The SIRT3-MnSOD-Ac axis in carcinogenesis and tumor resistance

Abstract ABSTRACT The molecular mechanism by which acetylation regulates manganese superoxide dismutase (MnSOD) activity, and its oncogenic and/or physiological significance, is unclear. Here we show that expression of the MnSOD-K68 acetylation (Ac) mimic mutant (MnSODK68Q) transformed wild-type primary mouse embryonic fibroblasts (pMEFs) when co-infected with either c-Myc or Ras, as measured by in vitro transformation assays and xenograft formation. Immortalized MnSOD-/- mouse embryonic fibroblasts (MEFs) infected with lenti-MnSODK68Q also exhibited an in vitro transformation permissive phenotype. In addition, MCF7 and T47D cells expressing MnSODK68Q demonstrated increased in vitro and xenograft oncogenic properties. These findings collectively identify MnSODK68Q as a tumor promoter. MCF7, T47D, and MnSOD-/- cells expressing MnSODK68Q displayed a monomeric form of MnSOD, distinct from the well-established homotetramer, and MnSOD-K68-Ac functions as a peroxidase, determined using a bacterial system for site-specific incorporation of N-(ε)-acetyl-l-lysine. Finally, tamoxifen resistant MCF7 and T47D cells, and a subgroup of human breast malignancies, displayed a MnSOD-K68-Ac signature. Citation Format: David R. Gius, Yueming Zhu. The SIRT3-MnSOD-Ac axis in carcinogenesis and tumor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY20-02.

A Gel Formulation Containing a New Recombinant Form of Manganese Superoxide Dismutase: A Clinical Experience Based on Compassionate Use-Safety of a Case Report.

Background. We report a case of bilateral posterior subcapsular cataracts (PSCs) in a 24-year-old man with an allergic conjunctivitis history caused by a long-term therapy with glucocorticoids. Case Presentation. The patient showed a visual acuity of 9/10 for both eyes. He followed a therapy with ketotifen and bilastine for four years. During the last six months before our evaluation, he was treated with chloramphenicol and betamethasone, interrupted for onset of cataracts and increased intraocular pressure. We treated him with ophthalmic gel preparation containing a new recombinant form of manganese superoxide dismutase (rMnSOD) at a concentration of 12.5 μg/mL, only for the right eye, while left eye was treated with standard protocol of Bendazac-lysine g 0.5. Conclusion. This case report shows the protective effects of rMnSOD versus PSC disease, probably due to the capacity of rMnSOD of countering free radical species.

Differential Regulation of Proteins and a Possible Role for Manganese Superoxide Dismutase in Bioluminescence of <i>Panellus stipticus</i> Revealed by Suppression Subtractive Hybridization

Suppression subtractive hybridization (SSH) was employed to investigate bioluminescence in Panellus stipticus (Bull.) P. Karst. by detecting proteins differentially expressed in bioluminescent and luminescent strains. Comparisons of luminescent and non-luminescent monokaryon cultures of North American strains revealed differences in transcript levels of proteins responsible for post-translational modification (PTM) of enzymes. A similar comparison of a luminescent strain of P. stipticus from North America with a non-luminescent European strain revealed the presence of extracellular manganese superoxide dismutase (MnSOD) in the luminescent form, in addition to proteins involved in PTM. The application of MnSOD-specific inhibitors to luminescent mycelium resulted in the rapid loss of luminescence. The relevance to luminescence of proteins involved in PTM is discussed, together with a possible role for MnSOD that considers the potential for SODs to form stable complexes with catechols revealed in previously published research. In light of the recent discovery that hispidine may be the precursor of fungal luciferin, we consider a hypothetical mechanism for fungal luminescence in which the ο-hydroquinone moiety of a hispidine derivative ligates with the extracellular form of MnSOD producing a semiquinone-radical complex, with the resultant semiquinonato complex potentially reacting with molecular oxygen or other reactive oxygen species to produce sufficiently excited intermediates to emit light on relaxation.

Study on the Protective Effect of a New Manganese Superoxide Dismutase on the Microvilli of Rabbit Eyes Exposed to UV Radiation.

We present a study on the protective effects against UV radiation of a gel formulation containing a new recombinant form of manganese superoxide dismutase on the conjunctiva and corneal epithelia of rabbit eyes. The integrity of the microvilli of both ocular tissues has been considered as an indicator of the health of the tissues. Samples, collected by impression cytology technique, were added of 80 µL of a gel formulation containing superoxide dismutase (2.0 µg/mL) and irradiated with UV rays for 30 minutes and were evaluated with scanning electron microscopy. Wilcoxon test was used to verify the possible occurrence of statistically significant differences between damage for treated and nontreated tissues. Application of gel produces a significant reduction of damage by UV irradiation of ocular epithelia; both epithelia present a significant reduction of damaged microvilli number if treated with the superoxide dismutase gel formulation: the p values (differences between damage found for treated and nontreated both ocular tissues) for conjunctiva and cornea samples were p ≪ 0.01 and p ≪ 0.0001, respectively, at confidence level of 95%. The administration of this gel formulation before UV exposure plays a considerable protective role in ocular tissues of rabbit eye with a significant reduction of the damage.

MnSOD activity regulates hydroxytyrosol-induced extension of chronological lifespan

Chronological lifespan (CLS) is defined as the duration of quiescence in which normal cells retain the capacity to reenter the proliferative cycle. This study investigates whether hydroxytyrosol (HT), a naturally occurring polyphenol found in olives, extends CLS in normal human fibroblasts (NHFs). Quiescent NHFs cultured for a long duration (30-60days) lose their capacity to repopulate. Approximately 60% of these cells exit the cell cycle permanently; a significant increase in the doubling time of the cell population was observed. CLS was extended in quiescent NHFs that were cultured in the presence of HT for 30-60days. HT-induced extension of CLS was associated with an approximately 3-fold increase in manganese superoxide dismutase (MnSOD) activity while there was no change in copper-zinc superoxide dismutase, catalase, or glutathione peroxidase protein levels. Quiescent NHFs overexpressing a dominant-negative mutant form of MnSOD failed to extend CLS. HT suppressed age-associated increase in mitochondrial ROS levels. Results from spectroscopy assays indicate that HT in the presence of peroxidases can undergo catechol-semiquinone-quinone redox cycling generating superoxide, which in a cellular context can activate the antioxidant system, e.g., MnSOD expression. These results demonstrate that HT extends CLS by increasing MnSOD activity and decreasing age-associated mitochondrial reactive oxygen species accumulation.

Using Elevated Temperature as an Assay to Determine the Importance of Various SODs and the Life Extending Properties of Various Manganese Salts in C. elegans

C. elegans have a similar aging mechanism as humans, including the presence of superoxide scavenging enzymes, SODs (Superoxide Dismutase). This study focused only on two out of five different forms of SODs, a manganese SOD (SOD-3) and a copper-zinc SOD (SOD-4), to uncover the most important one for combating oxidative stress. In addition, this current study extended on the previous findings that ionic manganese supplementation provides life-span extending benefit, to uncover a preferred chemical form of manganese that has superior efficacy compared to previously utilized MnSO4. The various worm strains were grown as a synchronous population from eggs at 22°C for 48 hours. They were then subjected to thermal stress at 35°C, which elevates free radical levels in vivo to a lethal limit. The results showed that a form of manganese SOD (SOD-3) is vital in combating oxidative stress, that MnSO4 supplementation rescued the reduced thermotolerance seen in SOD-3 knockout worms and that MnSO4 is the preferential form for worm supplementation studies compared to other simple salts based on thermotolerance assay. Future studies will include manganese containing complexes, some of which are known to have antioxidant properties, to examine if simple manganese salts can be as efficacious as the complexes using both thermotolerance and life-span as assays.

Mild Hypoxia Promotes Survival and Proliferation of SOD2-Deficient Astrocytes via c-Myc Activation

Mouse astrocytes deficient in the mitochondrial form of manganese superoxide dismutase (SOD2) do not survive in culture under atmospheric air with 20% oxygen (O2), which is a common condition for cell cultures. Seeding the cells and maintaining them under mild hypoxic conditions (5% O2) circumvents this problem and allows the cells to grow and become confluent. Previous studies from our laboratory showed that this adaptation of the cells was not attributable to compensation by other enzymes of the antioxidant defense system. We hypothesized that transcriptional activity and upregulation of genes other than those with an antioxidant function are involved. Our present study shows that c-Myc was significantly induced and that it inhibited p21 and induced proteins such as cyclin-dependent kinases, cyclin D, and cyclin E, which are involved in the cell cycle process, along with phosphorylation of the retinoblastoma protein and Cdc2 (cell division cycle 2). These mechanisms contribute to cell proliferation. Small interfering RNA of c-Myc, however, blocked proliferation of SOD2 homozygous (SOD2-/-) astrocytes under mild hypoxia consisting of 5% O2, whereas it did not affect the growth of wild-type astrocytes. Our results indicate that c-Myc plays a critical role in hypoxia-induced proliferation and survival of SOD2-/- astrocytes by overcoming injury caused by oxidative stress.

Mimicking the reduced, oxidized and azide inhibited form of manganese superoxide dismutase by mononuclear Mn compounds utilizing tridentate ligands

The manganese complexes [Mn II(Hbmimpm) 2(NO 3)](NO 3) · Et 2O (1), [Mn III(bmimpm) 2(OAc)] · 2CH 2Cl 2 (2), and [Mn III(bmiapm) 2(OAc)] · MeOH · H 2O · CH 2Cl 2 (3) containing the new ligands Bis(1-methylimidazol-2-yl)-(4-methoxyphen-1-yl)methanol (Hbmimpm) and Bis[(1-methylimidazol-2-yl)](2-aminophenyl)methanol (Hbmiapm) were synthesized. They are good structural models for the reduced ( 1) and oxidized ( 2, 3) form of manganese superoxide dismutase. All complexes were characterized by spectroscopic methods and X-ray structure analysis. Compounds 1 and 2 crystallize in the monoclinic space group P2 1/ c whereas complex 3 crystallizes in the monoclinic space group P2 1/ n. The coordination sphere around the manganese cores is distorted octahedral with two corresponding tridentate ligands representing the protein ligands and one nitrate ( 1) or acetate ( 2, 3) ion occupying two cis positions. Similar to the enzyme the Mn(III) complex 2 reacts with sodium azide. The obtained microcrystalline azide adduct was characterized by UV–Vis and IR spectroscopy.