Intrachain disulfide bond formation among the cysteine thiols of SNAP-25, a component of the SNARE protein complex required for neurotransmitter release, has been hypothesized to link oxidative stress and inhibition of synaptic transmission. However, neither the availability in vivo of SNAP-25 thiols, which are known targets of S-palmitoylation, nor the tendency of these thiols to form intrachain disulfide bonds is known. We have examined, in rat brain extracts, both the availability of closely spaced, or vicinal, thiol pairs in SNAP-25 and the propensity of these dithiols toward disulfide bond formation using a method improved by us recently that exploits the high chemoselectivity of phenylarsine oxide (PAO) for vicinal thiols. The results show for the first time that a substantial fraction of soluble and, to a lesser extent, particulate SNAP-25 contain non-acylated PAO-binding thiol pairs and that these thiols in soluble SNAP-25 in particular have a high propensity toward disulfide bond formation. Indeed, disulfide bonds were detected in a small fraction of soluble SNAP-25 even under conditions designed to prevent or greatly limit protein thiol oxidation during experimental procedures. These results provide direct experimental support for the availability, in a subpopulation of SNAP-25, of vicinal thiols that may confer on one or more isoforms of this family of proteins a sensitivity to oxidative stress.
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.
Postsurgical trigeminal neuralgia (TN), although rare, can lead to significant hemodynamic perturbations by triggering the trigeminocardiac reflex (TCR). The combination can lead to diagnostic as well as management challenges for clinicians. We present the case of a patient with a parotid abscess, which developed as a complication of his otolaryngologic surgery, and which led to repeated episodes of symptomatic bradycardia associated with cardiovascular collapse. This case highlights the importance of heightened awareness, early diagnosis, and timely treatment of postsurgical neuropathic pain syndromes to avoid life-threatening complications.
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.
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