Oxidative stress is a long-hypothesized cause of diverse neurological and psychiatric disorders but the pathways by which physiological redox perturbations may detour healthy brain development and aging are unknown. We reported recently (Foley et al., Neurochem Res 39:2030-2039, 2014) that two-electron oxidations, to disulfides, of protein vicinal thiols can vary markedly in association with more modest oxidations of the glutathione redox couple in brains from healthy adolescent rats whereas levels of protein S-glutathionylation were low and unchanged. Here, we demonstrate that the selective oxidations of protein vicinal thiols, occurring only in the more oxidized brains under study, were linked specifically to a peroxide stress as evidenced by increased oxidations, to disulfides, of the presumed catalytic vicinal thiols of peroxiredoxins 1 and 2. Moreover, we identify the catalytic subunit(s) of Na, K-ATPase, tubulins, glyceraldehyde-3-phosphate dehydrogenase, and protein phosphatase 1, all of which can modulate glutamate neurotransmission and the vulnerability of neurons to excitotoxicity, as non-peroxidase proteins exhibiting prominent oxidations of vicinal thiols. The two-electron pathway, demonstrated here, linking physiological redox perturbations in otherwise healthy brains to protein determinants of excitotoxicity, suggests an alternative to free radical pathways by which oxidative stress may impact brain development and aging.
Reversible oxidations of protein thiols have emerged as alternatives to free radical-mediated oxidative damage with which to consider the impacts of oxidative stress on cellular activities but the scope and pathways of such oxidations in tissues, including the brain, have yet to be fully defined. We report here a characterization of reversible oxidations of glutathione and protein thiols in extracts from rat brains, from two sources, which had been (1) frozen quickly after euthanasia to preserve in vivo redox states and (2) subjected to alkylation upon tissue disruption to trap reduced thiols. Brains were defined, relatively, as Reduced and Moderately Oxidized based on measured ratios of reduced (GSH) to oxidized (GSSG) glutathione. Levels of protein disulfides formed by the cross-linking of closely-spaced (vicinal) protein thiols, but not protein S-glutathionylation, were higher in extracts from the Moderately Oxidized brains compared to the Reduced brains. Moreover, the oxidized vicinal thiol proteome contains proteins that impact cellular energetics, signaling, neurotransmission, and cytoskeletal dynamics among others. These findings argue that kinetically-competent pathways for reversible, two-electron oxidations, of protein vicinal thiols can be activated in healthy brains in response to physiological oxidative stresses. We propose that such oxidations may link oxidative stress to adaptive, but also potentially deleterious, changes in neural cell activities in otherwise healthy brains.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral agent that is responsible for the coronavirus disease-2019 (COVID-19) pandemic. One of the live virus vaccine candidates Merck and Co., Inc. was developing to help combat the pandemic was V590. V590 was a live-attenuated, replication-competent, recombinant vesicular stomatitis virus (rVSV) in which the envelope VSV glycoprotein (G protein) gene was replaced with the gene for the SARS-CoV-2 spike protein (S protein), the protein responsible for viral binding and fusion to the cell membrane. To assist with product and process development, a quantitative Simple Western (SW) assay was successfully developed and phase-appropriately qualified to quantitate the concentration of S protein expressed in V590 samples. A strong correlation was established between potency and S-protein concentration, which suggested that the S-protein SW assay could be used as a proxy for virus productivity optimization with faster data turnaround time (3 h vs 3 days). In addition, unlike potency, the SW assay was able to provide a qualitative profile assessment of the forms of S protein (S protein, S1 subunit, and S multimer) to ensure appropriate levels of S protein were maintained throughout process and product development. Finally, V590 stressed stability studies suggested that time and temperature contributed to the instability of S protein demonstrated by cleavage into its subunits, S1 and S2, and aggregation into S multimer. Both of which could potentially have a deleterious effect on the vaccine immunogenicity.
Improvement in this laboratory of a previously‐described method, exploiting the high affinity of vicinal thiols for phenylarsine oxide (PAO), has led to the development of a procedure for the efficient capture of proteins containing vicinal thiols that have been oxidized, in tissues, to disulfide bonds. Using this enhanced PAO‐affinity method, we have found that about 50% of the glycolytic enzyme triosephosphate isomerase (TPI) contains oxidized vicinal thiols, amidst a very low oxidation of total protein vicinal thiols, in brains from adult rats. Results of preliminary experiments employing a glutathione redox buffer argue that the vicinal thiols of TPI do not have an unusually high propensity for oxidation and support the view presented here that these thiols are highly oxidized in the brains under study because TPI is enriched in a population of cells that is experiencing substantial oxidative stress. We hypothesize that the observed TPI‐associated oxidative stress may result from increased rates of glucose metabolism by glycolysis at the expense of the antioxidant‐generating pentose phosphate pathway in TPI‐enriched cells. Our findings support the notion that protein thiols may serve as intrinsic sensors of the redox environments in which they reside and that the thiol redox states of cell type‐specific and metabolic state‐associated proteins may shed new light on the origins and, possibly, the pathological triggers of oxidative stress in complex tissues.
Oxidative crosslinking of protein thiols via disulfide bonds, promoted by non‐radical peroxides, offers alternatives to traditional, oxygen radical‐centered, perspectives with which to consider the impacts of oxidative stress on brain functions. Protein disulfides, involving the bridging of closely‐spaced (vicinal) thiol pairs, as well as protein‐glutathione mixed disulfides occur readily in cells exposed to hydrogen peroxide but the scope, pathways, and relationships to health and disease of protein thiol oxidations in the brain remain to be fully established. We demonstrated recently that (i) brains of healthy adolescent rats, from different sources, could be distinguished by ratios of oxidized to reduced glutathione and (ii) moderate increases in these ratios were associated with marked elevations in the levels of total protein disulfides but not mixed disulfides. We report here that the more oxidized brains were characterized also by increases in the protein levels of thioredoxin (Trx)‐dependent peroxiredoxin (Prx) peroxidases as well as the extents of oxidations of the catalytic vicinal thiols of these enzymes. Moreover, among the non‐Prx proteins that were oxidized most prominently were glyceraldehyde‐3‐phosphate dehydrogenase and the catalytic subunit(s) of the Na+, K+‐ATPase, critical enzymatic determinants of glucose utilization and neuronal excitability, respectively. We hypothesize that radical‐free oxidations of protein vicinal thiol motifs, controlled by the Trx/Prx system, may facilitate short‐term adaptation to oxidative stress but that prolonged oxidations, i.e., protein disulfide stress, may derail healthy brain aging by increasing the vulnerability of neurons to excitotoxic insults.
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