A model for gain of function in superoxide dismutase

Studies have found that mutant, misfolded superoxide dismutase [Cu–Zn] (SOD1) can convert wild type SOD1 (wtSOD1) in a prion-like fashion, and that misfolded wtSOD1 can be propagated by release and uptake of protein aggregates. In developing a prion-like mechanism for this propagation of SOD1 misfolding we have previously shown how enervation of the SOD1 electrostatic loop (ESL), caused by the formation of transient non-obligate SOD1 oligomers, can lead to an experimentally observed gain of interaction (GOI) that results in the formation of SOD1 amyloid-like filaments. It has also been shown that freedom of ESL motion is essential to catalytic function. This work investigates the possibility that restricting ESL mobility might not only compromise superoxide catalytic activity but also serve to promote the peroxidase activity of SOD1, thus implicating the formation of SOD1 oligomers in both protein misfolding and in protein oxidation.     

The essential requirement for superoxide radical and nitric oxide formation for normal physiological function and healthy aging

Contrary to the dogma that superoxide anion and hydrogen peroxide formation are highly deleterious to cell function and healthy aging, we suggest this premise is flawed. Superoxide anion and hydrogen peroxide formation are essential to normal cellular function; they constitute a second messenger system absolutely required for the regulation of the metabolome. Embraced within this regulation is the modulation of cellular redox poise, bioenergy output, gene expression and cell differentiation. A key component in the overall process is coenzyme Q10 whose prooxidant function through the formation of superoxide anion and hydrogen peroxide is a major factor in the overall processes. The free radical gas, nitric oxide (similarly to superoxide anion), functions in the regulation of a wide range of cell systems. As part of the normal physiological process, superoxide anion and NO function separately and interactively as second messengers. Superoxide anion and nitric oxide play an intrinsic role in the regulated ordered turnover of proteins, rather than randomly cause protein damage and their inactivation. The proposition that metabolic free radical formation is unequivocally deleterious to cell function is rebutted; their toxicity as primary effectors in the aging process has been overemphasized. The concept that a dietary supplement of high concentrations of small-molecule antioxidants is a prophylactic/amelioration therapy for the aging process and age-associated diseases is questioned as to its clinical validity.     

MPTP, MPP+ and mitochondrial function

1-Methyl-4-phenylpyridinium (MPP+), the putative toxic metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), inhibited NAD(H)-linked mitochondrial oxidation at the level of Complex I of the electron transport system. MPTP and MPP+ inhibited aerobic glycolysis in mouse striatal slices, as measured by increased lactate production; MPTP-induced effects were prevented by inhibition of monoamine oxidase B activity. Several neurotoxic analogs of MPTP also form pyridinium metabolites via MAO; these MPP+ analogs were all inhibitors of NAD(H)-linked oxidation by isolated mitochondria. 2'-Methyl-MPTP, a more potent neurotoxin in mice than MPTP, was also more potent than MPTP in inducing lactate accumulation in mouse brain striatal slices. Overall, the studies support the hypothesis that compromise of mitochondrial oxidative capacity is an important factor in the mechanisms underlying the toxicity of MPTP and similar compounds.     

A sheep model for MPTP induced Parkinson-like symptoms

Administration of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) causes behaviors reminiscent of idiopathic Parkinson's disease in man and other primates, but development of such symptomology has not been reported to date in other species. We now report a sheep model which responds to administration of low levels of the compound with well defined, apparently permanent symptomology very similar to that seen in primates. Histological examination indicates drug dependent destruction of the substantia nigra which, in sheep, lacks the high levels of neuromelanin present in primates. Following infusion of either MPTP or MPP+, only the metabolite MPP+ was detected in serum with this metabolite demonstrating a very long half life. The rapid disappearance of MPTP suggests that its potency will be directly related to a function of body size and inversely related to heart rate.     

Aging enhances pressure-induced arterial superoxide formation

The purpose of this study was to investigate the mechanisms that regulate superoxide (O(2)(*-)) production as a function of an acute elevation of intravascular pressure and age. Mesenteric arteries isolated from young (6 mo) and aged (24 mo) male Fischer 344 rats were used. O(2)(*-) production in vessels in response to 80 (normal pressure, NP) and 180 (high pressure, HP) mmHg was determined by the superoxide dismutase-inhibitable nitroblue tetrazolium (NBT) reduction assay. In vessels exposed to NP, O(2)(*-) production was significantly higher in aged than in young vessels (32.7 +/- 7.0 vs. 15.4 +/- 2.4 nmol.mg(-1).30 min(-1)). HP enhanced O(2)(*-) production in vessels of both groups, but the enhancement was significantly greater in aged than in young vessels (63.4 +/- 6.7 vs. 32.7 +/- 4.3 nmol.mg(-1).30 min(-1)). Apocynin (100 micromol/l) attenuated HP-induced increases in O(2)(*-) production in both groups, whereas allopurinol (100 micromol/l) and N(omega)-nitro-L-arginine methyl ester (100 mumol/l) inhibited the response only in aged vessels. Confocal microscopy showed increases in O(2)(*-) in response to HP in endothelial and smooth muscle layers of both groups, with much greater fluorescent staining in aged than in young rats and in the endothelium than in smooth muscle cells. No significant changes in NAD(P)H oxidase gene and protein expressions were observed in vessels of the two groups. Upregulation of protein expression of xanthine oxidase was detected in aged vessels. We conclude that NAD(P)H oxidase contributes importantly to HP-induced enhanced O(2)(*-) production in vessels of both young and aged rats, whereas xanthine oxidase and nitric oxide synthase-dependent O(2)(*-) production also contribute to the enhancement in mesenteric arteries of aged rats.     

The mitochondrial site of superoxide formation

Ubiquinone and cytochrome b566 have both been postulated to cause mitochondrial O2 formation by autoxidation of their reduced forms. The present investigation was made to evaluate capabilities of the two candidates to transfer electrons to molecular oxygen out of sequence of the normal pathway of respiration. The results show that electron transfer from ubisemiquinone to oxygen depends on the availability of protons. In agreement with this finding autoxidation of redox cycling ubiquinone could not be observed due to its location in an aprotic environment of the mitochondrial membrane. However, O2 release from mitochondria was found to be related to the existence of low potential cytochrome b566. The transfer of this b type cytochrome to more positive values caused a concomitant decrease and finally inhibition of univalent electron transfer to oxygen out of sequence. Our findings suggest a role of cytochrome b 566 in mitochondrial O2 formation. A contribution by ubiquinone is unlikely as long as protons are deprived from penetrating into the domain where ubiquinone is operating.     

Adriamycin stimulated superoxide formation in submitochondrial particles.

Adriamycin (doxorubicin), an anticancer agent, stimulated the formation of superoxide in submitochondrial particles isolated from bovine heart. Superoxide formation was detected by oxygen uptake, by the cooxidation of epinephrine to adrenochrome and by the reduction of acetylated cytochrome c. These processes were sensitive to superoxide dismutase (SOD). Rotenone-insensitive oxidation of NADH by the mitochondrial respiratory chain in the presence of oxygen caused the formation of approx 4 nmol of superoxide per min/mg of protein. Adriamycin at a concentration of 400 micron stimulated the rate of superoxide formation 6-fold to 25 nmol.min-1.mg-1, but this was not a maximum rate. Approximately 50 micron adriamycin was estimated to be sufficient for obtaining one-half maximal stimulation. Hydrogen peroxide accumulated as a final reaction product. Measurements of the relative catalase activity of blood-free tissues of rabbits and rats indicated that heart contained 2 to 4% of the catalase activity of liver or kidney. An enhanced production of superoxide and hydrogen peroxide and the relatively low catalase content of heart tissue may be factors in the cardiotoxicity induced by adriamycin chemotherapy if a similar reaction occurs in vivo.     

Montmorillonite: A multifunctional mineral catalyst for the prebiological formation of phosphate esters

Reaction of diiminosuccinonitrile (DISN) with 3'-AMP in the presence of alkali- and alkaline earth-montmorillonites results in the formation of 2',3'-cAMP in aqueous solution. Little or no 2', 3'-cAMP is produced when metal ion concentrations equivalent to that of the metal ion associated with the homoionic clays are used instead of mobntmorillionite. Yields comparable to those obtained with DISN are obtained when diaminomaleonitrile (DAMN) is used in place of DISN as the condensing agent. DAMN, a compound which is more stable than DISN in aqueous solution, is oxidized to DISN on the surface of the clay by Fe+3 in the clay lattice. DISN, the true condensing agent, is thus generated in the presence of the bound 3'-AMP on the montmorillonite surface. The montmorillonite catalyzes the DISN-mediated formation of 2', 3'-cAMP and this product, which binds much less strongly than does the 3'-AMP, is desorbed from the clay surface. This research established that the montmorillonite performs four different functions in its role as catalyst: (1) Binding one of the substrate molecules (3'-AMP) (2) Activating the second substrate (DAMN) (3) Catalyzing the formation of 2', 3'-cAMP (4) Releasing the reaction product so another substrate molecules can bind to the montmorillonite.     

Evidence for formation of superoxide and formate radicals in Methanobacterium formicicum.

Using spin labeling and spin trapping techniques in combination with electron paramagnetic resonance spectrometry, we have detected the formation of superoxide by whole cells of Methanobacterium formicicum under aerobic conditions in the presence and absence of sodium formate. Rates of superoxide generation have been estimated. The formation of additional free radical species, including formate, was observed. Production of these and other free radicals resulted in lipid peroxidation and concomitant cell damage.     

The function of superoxide dismutase during the enzymatic formation of the free radical of ribonucleotide reductase

An enzyme system from Escherichia coli activates an inactive form of ribonucleotide reductase by transforming a tyrosine residue of the enzyme into a cationic free radical. The process requires NAD(P)H, a flavin, dithiothreitol, and oxygen and at least three proteins. After purification to near homogeneity two of the proteins were identified as superoxide dismutase and NAD(P)H:flavin oxidoreductase (Fontecave, M., Eliasson, R., and Reichard, P. (1987) J. Biol. Chem. 262, 12325-12331). The nature of the third protein, provisionally named Fraction b, is unknown. The flavin reductase is believed to reduce the ferric iron center of the ribonucleotide reductase as a prerequisite for radical generation. Here we demonstrate that the flavin reductase under aerobic conditions generates superoxide anions which inactivate ribonucleotide reductase. Superoxide dismutase protects the enzyme or a sensitive intermediate formed during the generation of the tyrosyl radical from the harmful effects of superoxide. Hydrogen peroxide, formed by superoxide dismutase, is also harmful. In this case, catalase present in Fraction b might protect the system. Fraction b has, however, an additional unknown function in the overall process of radical generation.     

A protective function of superoxide dismutase during respiratory chain activity.

(1) Aerobic incubation of heart muscle submitochondrial particles in phosphate buffer after treatment with NADH causes a progressive and substantial inhibition of the NADH oxidation system. Succinate oxidation remains almost unaffected by NADH treatment. (2) The loss of NADH oxidase activity is due to an inhibition of the respiratory chain-linked NADH dehydrogenase. This inhibition of the enzyme is very similar to that caused by combination of the organic mercurial mersalyl with NADH dehydrogenase. (3) The inhibition of NADH oxidation is largely prevented by compounds that are known to react with superoxide ions (02-.), including superoxide dismutase, cytochrome c, tiron and Mn2+. EDTA also has a protective effect, but a number of other metal chelating agents, and several proteins, including catalase, are without effect. (4) It is concluded that the inhibition of NADH oxidation of NADH oxidation by superoxide ions or by mersalyl is reversible and is therefore not due to the loss of oxidoreduction components from the respiratory chain or to an irreversible change in protein conformation. (6) The function of mitochondrial superxide dismutase is discussed in relation to the key role of NADH dehydrogenase in energy-conserving reactions and the formation of hydrogen peroxide during mitochondrial oxidations.     

Integration of superoxide formation and cristae morphology for mitochondrial redox signaling

The mitochondrial network provides the central cell’s energetic and regulatory unit, which besides ATP and metabolite production participates in cellular signaling through regulated reactive oxygen species (ROS) production and various protein/ion fluxes. The inner membrane forms extensive folds, called cristae, i.e. cavities enfolded from and situated perpendicularly to its inner boundary membrane portion, which encompasses an inner cylinder within the outer membrane tubule. Mitochondrial cristae ultramorphology reflects various metabolic, physiological or pathological states. Since the mitochondrion is typically a predominant superoxide source and generated ROS also serve for the creation of information redox signals, we review known relationships between ROS generation within the respiratory chain complexes of cristae and cristae morphology. Notably, it is emphasized that cristae shape is governed by ATP-synthase dimers, MICOS complexes, OPA1 isoforms and the umbrella of their regulation, and also dependent on local protonmotive force (electrical potential component) in cristae. Cristae are also affected by redox-sensitive kinases/phosphatases or p66SHC. ATP-synthase dimers decrease in the inflated intracristal space, diminishing pH and hypothetically having minimal superoxide formation. Matrix-released signaling superoxide/H₂O₂ is predominantly integrated along mitochondrial tubules, whereas the diffusion of intracristal signaling ROS species is controlled by crista junctions, the widening of which enables specific retrograde redox signaling such as during hypoxic cell adaptation. Other physiological cases of H₂O₂ release from the mitochondrion include the modulation of insulin release in pancreatic β-cells, enhancement of insulin signaling in peripheral tissues, signaling by T-cell receptors, retrograde signaling during the cell cycle and cell differentiation, specifically that of adipocytes.     

A dual function for Pex3p in peroxisome formation and inheritance

Saccharomyces cerevisiae Pex3p has been shown to act at the ER during de novo peroxisome formation. However, its steady state is at the peroxisomal membrane, where its role is debated. Here we show that Pex3p has a dual function: one in peroxisome formation and one in peroxisome segregation. We show that the peroxisome retention factor Inp1p interacts physically with Pex3p in vitro and in vivo, and split-GFP analysis shows that the site of interaction is the peroxisomal membrane. Furthermore, we have generated PEX3 alleles that support peroxisome formation but fail to support recruitment of Inp1p to peroxisomes, and as a consequence are affected in peroxisome segregation. We conclude that Pex3p functions as an anchor for Inp1p at the peroxisomal membrane, and that this function is independent of its role at the ER in peroxisome biogenesis.     

Aggregate formation in Cu,Zn superoxide dismutase-related proteins

Aggregation of Cu,Zn superoxide dismutase (SOD1) protein is a pathologic hallmark of familial amyotrophic lateral sclerosis linked to mutations in the SOD1 gene, although the structural motifs within mutant SOD1 that are responsible for its aggregation are unknown. Copper chaperone for SOD1 (CCS) and extracellular Cu,Zn superoxide dismutase (SOD3) have some sequence identity with SOD1, particularly in the regions of metal binding, but play no significant role in mutant SOD1-induced disease. We hypothesized that it would be possible to form CCS- or SOD3-positive aggregates by making these molecules resemble mutant SOD1 via the introduction of point mutations in codons homologous to a disease causing G85R SOD1 mutation. Using an in vitro assay system, we found that expression of wild type human CCS or a modified intracellular wild type SOD3 does not result in significant aggregate formation. In contrast, expression of G168R CCS or G146R SOD3 produced aggregates as evidenced by the presence of high molecular weight protein complexes on Western gels or inclusion bodies on immunofluorescence. CCS- and SOD3-positive inclusions appear to be ubiquitinated and localized to aggresomes. These results suggest that proteins sharing structural similarities to mutant SOD1 are also at risk for aggregate formation.     

The duality of LysU, a catalyst for both Ap4A and Ap3A formation

Heat shock inducible lysyl-tRNA synthetase of Escherichia coli (LysU) is known to be a highly efficient diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) synthase. However, we use an ion-exchange HPLC technique to demonstrate that active LysU mixtures actually have a dual catalytic activity, initially producing Ap4A from ATP, before converting that tetraphosphate to a triphosphate. LysU appears to be an effective diadenosine 5',5'-P1,P3-triphosphate (Ap3A) synthase. Mechanistic investigations reveal that Ap3A formation requires: (a) that the second step of Ap4A formation is slightly reversible, thereby leading to a modest reappearance of adenylate intermediate; and (b) that phosphate is present to trap the intermediate (either as inorganic phosphate, as added ADP, or as ADP generated in situ from inorganic phosphate). Ap3A forms readily from Ap4A in the presence of such phosphate-based adenylate traps (via a 'reverse-trap' mechanism). LysU is also clearly demonstrated to exist in a phosphorylated state that is more physically robust as a catalyst of Ap4A formation than the nonphosphorylated state. However, phosphorylated LysU shows only marginally improved catalytic efficiency. We note that Ap3A effects have barely been studied in prokaryotic organisms. By contrast, there is a body of literature that describes Ap3A and Ap4A having substantially different functions in eukaryotic cells. Our data suggest that Ap3A and Ap4A biosynthesis could be linked together through a single prokaryotic dual 'synthase' enzyme. Therefore, in our view there is a need for new research into the effects and impact of Ap3A alone and the intracellular [Ap3A]/[Ap4A] ratio on prokaryotic organisms.     

NADH- and NADPH-dependent formation of superoxide radicals in liver nuclei

A method for the quantitation of the superoxide radical generation rate (V) in murine liver nuclei by the oxidation of 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine O2-. radicals with the formation of a stable nitroxyl radical recorded by the EPR method, has been developed. It was shown that NADP- and NADPH-dependent superoxide radical generation is suppressed by superoxide dismutase (approximately by 90%). The Km values for NADH and NADPH are 1.5 x 10(-6) and 4.4 x 10(-7) M, respectively; the maximal rate (0.2 nmol.min-1.mg protein-1) is equal for both substrates. Cyanide (greater than 2 mM) causes a practically complete inhibition of the O2-. generation by both substrates. It is suggested that there exists a single readily autooxidized site of O2-. generation by both substrates for NADH- and NADPH-dependent site of the electron transport chain in nuclear membranes.