Protein Vicinal Thiol Oxidations in the Healthy Brain: Not So Radical Links between Physiological Oxidative Stress and Neural Cell Activities

Abstract

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.

Glutathionylation: a regulatory role of glutathione in physiological processes

“…In recent years, research has focused on glutathionylation and its relation with neurodegenerative diseases and neurophysiology (Table 2), particularly on the major transcription factors and regulatory molecules such as histones (186) in the brain (183)(184)(185)(186). Carvalho In brain cancers, posttranslational glutathionylation on the Cys 145 of O 6 -methylguanine-DNA methyltransferase (MGMT) has promising therapeutic effects (188).…”

Protein vicinal thiols as intrinsic probes of brain redox states in health, aging, and ischemia

“…In particular, dithiol-disulfide cycling underpins the catalytic preservation of redox homeostasis by redoxin enzymes, including thioredoxin peroxidases (peroxiredoxins) (Holmgren et al 2005 ), and have been connected, more generally, to redox buffering and regulation. Indeed, findings by us (Foley et al 2014 ) and others (Beer et al 2004 ; Hansen et al 2009 ) argue that protein disulfides form more readily than do mixed disulfides with glutathione (i.e., S-glutathionylation) under physiological conditions, consistent with the notion that protein thiols may be as crucial for cellular redox buffering as is glutathione (Hansen et al 2009 ).…”

“…However, the extraordinarily high reactivities of the peroxidatic thiols of Prxs with hydrogen peroxide can present challenges to trapping the in vivo redox states of these enzymes (Peskin et al 2007 ). Indeed, we have observed unusually high extents of oxidations of Prx-2 and Prx-1 from brain under experimental conditions considered sufficient to trap the in vivo redox states of thiols on non-peroxidase proteins and on glutathione (Foley et al 2014 , 2016 ). Thus, we propose that measures of redox states of vicinal thiols on proteins other than peroxidases may prove advantageous as reporters of redox perturbations occurring in tissues, in vivo.…”

Simultaneous Tracing of Protein Vicinal Dithiols in Live Cells Using an “Off-on” Fluorescent Probe

“…7 The high sensitivity and rapid reactivity of vicinal dithiols on VDPs empowers them with the ability to work as a redox buffer, indicating that they may play a key role in the maintenance of redox homeostasis and signalling pathways, 5,8 and ultimately maintain the viable microenvironment of living cells. [9][10] For instance, the alternation of VDPs in response to the change of ROS levels in the surrounding environment suggests that protein vicinal dithiols may be involved in cellular signaling systems, 9 particularly in regulating the mitochondria where multiple core metabolic machineries utilizing redox reactions exist, [11][12][13] such as calcium release from mitochondria 14 and tuning of the mitochondrial membrane sensitivity. 12,15 Another important role of vicinal dithiols is stabilizing the structures of VDPs, which prevent and manage protein misfolding and aggregation.…”