RedoxiFluor: A microplate assay to quantify protein thiol redox state in percentages and moles

An accessible, time- and cost-efficient microplate assay to quantify protein thiol redox state in percentages and moles relative to the thiol proteome (i.e., context) and other targets (i.e., array mode) would be invaluable for understanding how protein thiols regulate essential biological processes. RedoxiFluor achieves several key benefits (i.e., percentages, moles, context, array mode) in a microplate format. After robustly validating RedoxiFluor, comparative analysis reveals that key benefits are intractable to other immunological techniques. Moles is an unprecedented achievement. Proof-of-concept studies illuminating fundamental redox principles (i.e., specificity, context, and heterogeneity) through measurement alone demonstrate how RedoxiFluor can advance understanding. For example, target specific protein thiol redox state changes are: (1) context specific (i.e., redox stimulus dependent); (2) selective (i.e., redox stimuli oxidise select targets); and (3) heterogenous (i.e., target responses vary markedly). RedoxiFluor is a powerful new tool for advancing a far-reaching and influential field: protein thiol redox biology.

The Effectiveness of Glutathione Redox Status as a Possible Tumor Marker in Colorectal Cancer

The role of oxidative stress (OS) in cancer is a matter of great interest due to the implication of reactive oxygen species (ROS) and their oxidation products in the initiation of tumorigenesis, its progression, and metastatic dissemination. Great efforts have been made to identify the mechanisms of ROS-induced carcinogenesis; however, the validation of OS byproducts as potential tumor markers (TMs) remains to be established. This interventional study included a total of 80 colorectal cancer (CRC) patients and 60 controls. By measuring reduced glutathione (GSH), its oxidized form (GSSG), and the glutathione redox state in terms of the GSSG/GSH ratio in the serum of CRC patients, we identified significant changes as compared to healthy subjects. These findings are compatible with the effectiveness of glutathione as a TM. The thiol redox state showed a significant increase towards oxidation in the CRC group and correlated significantly with both the tumor state and the clinical evolution. The sensitivity and specificity of serum glutathione levels are far above those of the classical TMs CEA and CA19.9. We conclude that the GSSG/GSH ratio is a simple assay which could be validated as a novel clinical TM for the diagnosis and monitoring of CRC.

Late-life restoration of mitochondrial function reverses cardiac dysfunction in old mice

Diastolic dysfunction is a prominent feature of cardiac aging in both mice and humans. We show here that 8-week treatment of old mice with the mitochondrial targeted peptide SS-31 (elamipretide) can substantially reverse this deficit. SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts. Improved diastolic function was concordant with increased phosphorylation of cMyBP-C Ser282 but was independent of titin isoform shift. Late-life viral expression of mitochondrial-targeted catalase (mCAT) produced similar functional benefits in old mice and SS-31 did not improve cardiac function of old mCAT mice, implicating normalizing mitochondrial oxidative stress as an overlapping mechanism. These results demonstrate that pre-existing cardiac aging phenotypes can be reversed by targeting mitochondrial dysfunction and implicate mitochondrial energetics and redox signaling as therapeutic targets for cardiac aging.

Regulatory thiol oxidation in chloroplast metabolism, oxidative stress response and environmental signaling in plants

The antagonism between thiol oxidation and reduction enables efficient control of protein function and is used as central mechanism in cellular regulation. The best-studied mechanism is the dithiol-disulfide transition in the Calvin Benson Cycle in photosynthesis, including mixed disulfide formation by glutathionylation. The adjustment of the proper thiol redox state is a fundamental property of all cellular compartments. The glutathione redox potential of the cytosol, stroma, matrix and nucleoplasm usually ranges between −300 and −320 mV. Thiol reduction proceeds by short electron transfer cascades consisting of redox input elements and redox transmitters such as thioredoxins. Thiol oxidation ultimately is linked to reactive oxygen species (ROS) and reactive nitrogen species (RNS). Enhanced ROS production under stress shifts the redox network to more positive redox potentials. ROS do not react randomly but primarily with few specific redox sensors in the cell. The most commonly encountered reaction within the redox regulatory network however is the disulfide swapping. The thiol oxidation dynamics also involves transnitrosylation. This review compiles present knowledge on this network and its central role in sensing environmental cues with focus on chloroplast metabolism.

Immunological Techniques to Assess Protein Thiol Redox State: Opportunities, Challenges and Solutions

To understand oxidative stress, antioxidant defense, and redox signaling in health and disease it is essential to assess protein thiol redox state. Protein thiol redox state is seldom assessed immunologically because of the inability to distinguish reduced and reversibly oxidized thiols by Western blotting. An underappreciated opportunity exists to use Click PEGylation to realize the transformative power of simple, time and cost-efficient immunological techniques. Click PEGylation harnesses selective, bio-orthogonal Click chemistry to separate reduced and reversibly oxidized thiols by selectively ligating a low molecular weight polyethylene glycol moiety to the redox state of interest. The resultant ability to disambiguate reduced and reversibly oxidized species by Western blotting enables Click PEGylation to assess protein thiol redox state. In the present review, to enable investigators to effectively harness immunological techniques to assess protein thiol redox state we critique the chemistry, promise and challenges of Click PEGylation.