Oxidation-reduction reaction

Oxidative damage is an inevitable consequence of aerobic respiration. Methionine
sulfoxide reductases (Msr) are a group of enzymes that function to repair oxidized
methionine residues in both free methionine and methionine in proteins. MsrA was the
first of these enzymes to be discovered and is the most thoroughly studied. It is thought to
play a role in both the aging process and probably several neurodegenerative diseases. I
recently obtained a strain of Drosophila that was reported to have a P-element transposon
located within Exon 2 (part of the open reading frame) of the eip71cd gene, which is the
Drosophila homolog of MsrA. Thus, the transposon insertion should disrupt expression
of the msrA gene. I did a series of experiments to "jump out" the P-element in an effort
to recover two types of isogenic strains. The first would be a null mutation of the MsrA
gene created by deletion of flanking genomic DNA when the P-element excised from the
chromosome. The second would be a precise excision of the P-element, which would
restore the genetic locus to its original structure. This study looks at the effect of a null
mutant of the MsrA gene on aging and resistance to oxidative stress.

Mo2o3 (Et2dtc) 2 (THF) 2I2, readily reduces various oxides. The Mo vio2+2 product of this reaction oxidizes TPP to triphenylphosphine oxide. The transient Mo(iv) species formed in the later reaction rapidly and irreversibly reacts with excess Mo vio2+2 to form the original Mo2 o3 4+ complex. These reactions can be also be coupled to provide catalytic oxygen transfer from PNO to TPP. This catalytic cycle can be monitored using a reverse phase high pressure liquid chromatography method that will also be discussed. The oxides chosen ranged from pyridine-N-oxide to the biological substrates: diphenylsufoxide, DMSO, nicotinamide-N-oxide, and biotin-S-oxide. Since Mo2o3 (Et2dtc) 2 (THF) 2I2 has the ability to abstract oxygen from these biologically significant substrates, it may result in the reconsideration of the role of Mo(V) complexes in catalytic cycles.

Aging is a multifactoral biological process of progressive and deleterious changes partially attributed to a build up of oxidatively damaged biomolecules resulting from attacks by free radicals. Methionine sulfoxide reductases (Msrs) are enzymes that repair oxidized methionine (Met) residues found in proteins. Oxidized Met produces two enantiomers, Met-S-(o) and Met-R-(o), reduced by MsrA and MsrB respectively. Unlike other model organisms, our MsrA null fly mutant did not display increased sensitivity to oxidative stress or shortened lifespan, suggesting that in Drosophila, having either a functional copy of either Msr is sufficient. Here, two Msr mutant types were phenotypically assayed against isogenic controls. Results suggest that only the loss of both MsrA and MsrB produces increased sensitivity to oxidative stress and shortened lifespan, while locomotor defects became more severe with the full Msr knockout fly.

Harman's theory of aging proposes that a buildup of damaging reactive oxygen species (ROS) is one of the primary causes of the deleterious symptoms attributed to aging. Cellular defenses in the form of antioxidants have evolved to combat ROS and reverse damage; one such group is the methionine sulfoxide reductases (Msr), which function to reduce oxidized methionine. MsrA reduces the S enantiomer of methionine sulfoxide, Met-S-(o), while MsrB reduces the R enantiomer, Met-R-(o). The focus of this study was to investigate how the absence of one or both forms of Msr affects locomotion in Drosophila using both traditional genetic mutants and more recently developed RNA interference (RNAi) strains. Results indicate that lack of MsrA does not affect locomotion. However, lack of MsrB drastically reduces rates of locomotion in all age classes. Furthermore, creation of an RNAi line capable of knocking down both MsrA and MsrB in progeny was completed.

Drosophila melanogaster encounter periods of increased temperature or decreased oxygen in its native environment. One consequence of these environmental stresses is increased production of reactive oxygen species that damage major molecules within cells. Another consequence is that flies fall into a protective coma where biological functions are minimized to conserve energy expenditures. This biological phenomenon is called spreading depression. The overarching aim of this project is to determine if methionine sulfoxide reductases affect entrance or exit from the protective coma induced by acute thermal stress. The data revealed that complete deficiency of Msr in young flies causes a faster induction of the coma. In both young and old flies, Msr does not affect average recovery time but does affect the pattern of recovery from coma. Entrance into the coma is age dependent with young flies maintaining activity longer than before entering into the coma as compared to old flies.

Sulindac is a known NSAID that has also been shown to have anti-cancer activity that is not related to its ability to inhibit COX 1 and 2. During the past 15 years there have been a large number of studies attempting to elucidate its mechanism of action. Our laboratory has shown that sulindac can both protect normal cells and enhance the killing of cancer cells under oxidative stress from H2O2 and TBHP. However, except for mitochondrial dysfunction and ROS production, the mechanism by which sulindac sensitized the cancer cells to oxidative stress remains unknown. Results of this research project suggest that the effect of sulindac and oxidative stress not only involves mitochondrial ROS production, but also aspects of the preconditioning response. In normal cells this leads to survival by a preconditioning pathway, likely involving PKCε. . However, cancer cells react by initiating a pathway leading to apoptosis involving PKCδ.

Mitochondria are a major source of reactive oxygen species and are particularly vulnerable to oxidative stress. Mitochondrial dysfunction, methionine oxidation, and oxidative stress are thought to play a role in both the aging process and several neurodegenerative diseases. Two major classes of methionine sulfoxide reductases, designated MsrA and MsrB are enzymes that function to repair the enatiomers of methionine sulfoxide, met-(o)-S and met-(o)- R, respectively. This study focuses on the effect of Msr deficiencies on mitochondrial function by utilizing mutant alleles of MsrA and MsrB. The data show that loss of only one form of Msr in the mitochondria does not completely impair the function of the mitochondria. However, loss of both Msr proteins within the mitochondria leads to an increased ROS production and a diminished energy output of the mitochondria. These results support the hypothesis that Msr plays a key role in proper mitochondrial function.

The enzyme Methionine sulfoxide reductase A (MsrA) repairs oxidized proteins, and may act as a scavenger of reactive oxygen species (ROS), making it a potential therapeutic target for age-related neurodegenerative diseases. The anoxia-tolerant turtle offers a unique model to observe the effects of oxidative stress on a system that maintains neuronal function following anoxia and reoxygenation, and that ages without senescence. MsrA is present in both the mitochondria and cytosol, with protein levels increasing respectively 3- and 4-fold over 4 hours of anoxia, and remaining 2-fold higher than basal upon reoxygenation. MsrA was knocked down in neuronally-enriched cell cultures via RNAi transfection. Propidium iodide staining showed no significant cell death during anoxia, but this increased 7-fold upon reoxygenation, suggesting a role for MsrA in ROS suppression during reperfusion. This is the first report in any system of MsrA transcript and protein levels being regulated by oxygen levels.

The methionine sulfoxide reductase (Msr) enzymes catalyze the reduction of methionine sulfoxide (Met(O)) to methionine. The Msr enzymes protect cells against oxidative stress and may have a role in aging. The MsrA family of enzymes reduces stereospecifically the S epimer of free and protein-bound Met(O) while the MsrB family reduces the R epimer of Met(O) in proteins. It has been generally accepted, primarily from studies on MsrA, that the biological reductant for the Msr enzymes is thioredoxin (Trx), although high levels of dithiothreitol (DTT) can be used as the reductant in vitro. In contrast, certain MsrB enzymes show less than 10% of the activity with Trx as compared to DTT. This raises the possibility that in animal cells Trx may not be the direct hydrogen donor for the MsrB enzymes. Studies with bovine liver extracts have shown that thionein, the apoprotein of metallothionein, can function as a reductant for the Msr proteins. Certain selenium compounds such as selenocystamine and selenocystine can also serve as potent reducing agents for the Msr enzymes. Since an increased activity of Msr enzymes can reduce the level of oxidative damage in tissues, compounds that could activate Msr may have therapeutic potential. A high-throughput screening assay has been developed to screen large chemical libraries to find activators of MsrA, as well as specific inhibitors that could be useful research tools. This study will be done in collaboration with The Scripps Florida Research Institute. Sulindac was originally developed as a non-steroidal anti-inflammatory drug but has also shown efficacy in the treatment of certain cancers. The S epimer of sulindac is known to be reduced by MsrA, but the enzymes responsible for reduction of the R epimer are not known.