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

2021 ◽  
Author(s):  
Ahmet Tuncay ◽  
Anna Noble ◽  
Matthew Guille ◽  
James Cobley
Keyword(s):  
Redox State ◽  
Specific Protein ◽  
Microplate Assay ◽  
Protein Thiol ◽  
Redox Biology ◽  
Protein Thiols ◽  
Thiol Redox State ◽  
Thiol Redox ◽  
Cost Efficient ◽  
Context Specific

Abstract 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.

ALISA: A microplate assay to measure protein thiol redox state

2021 ◽  
Vol 174 ◽  
pp. 272-280 ◽  
Author(s):  
Anna Noble ◽  
Matthew Guille ◽  
James N. Cobley
Keyword(s):  
Redox State ◽  
Microplate Assay ◽  
Protein Thiol ◽  
Thiol Redox State ◽  
Thiol Redox

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

Antioxidants ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 315 ◽  
Author(s):  
James Nathan Cobley ◽  
Holger Husi
Keyword(s):  
Western Blotting ◽  
Redox State ◽  
Antioxidant Defense ◽  
Low Molecular Weight ◽  
Protein Thiol ◽  
Transformative Power ◽  
Thiol Redox State ◽  
Health And Disease ◽  
Thiol Redox ◽  
Cost Efficient

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.

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

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ying Ann Chiao ◽  
Huiliang Zhang ◽  
Mariya Sweetwyne ◽  
Jeremy Whitson ◽  
Ying Sonia Ting ◽  
...  
Keyword(s):  
Redox State ◽  
Late Life ◽  
Protein Thiol ◽  
Cardiac Aging ◽  
Mitochondrial Oxidative Stress ◽  
Viral Expression ◽  
Mitochondrial Ros ◽  
Thiol Redox State ◽  
Thiol Redox ◽  
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.

scholarly journals Nucleoredoxin-Dependent Targets and Processes in Neuronal Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Claudia Urbainsky ◽  
Rolf Nölker ◽  
Marcel Imber ◽  
Adrian Lübken ◽  
Jörg Mostertz ◽  
...  
Keyword(s):  
Redox State ◽  
Redox Regulation ◽  
Neuroblastoma Cells ◽  
Neuronal Cell ◽  
Biological Processes ◽  
Quantitative Mass Spectrometry ◽  
Mixed Disulfide ◽  
Protein Thiols ◽  
Thiol Redox State ◽  
Thiol Redox

Nucleoredoxin (Nrx) is an oxidoreductase of the thioredoxin family of proteins. It was shown to act as a signal transducer in some pathways; however, so far, no comprehensive analysis of its regulated substrates and functions was available. Here, we used a combination of two different strategies to fill this gap. First, we analyzed the thiol-redox state of the proteome of SH-SY5Y neuroblastoma cells depleted of Nrx compared to control cells using a differential thiol-labeling technique and quantitative mass spectrometry. 171 proteins were identified with an altered redox state; 161 of these were more reduced in the absence of Nrx. This suggests functions of Nrx in the oxidation of protein thiols. Second, we utilized the active site mutant Cys208Ser of Nrx, which stabilizes a mixed disulfide intermediate with its substrates and therefore trapped interacting proteins from the mouse brain (identifying 1710 proteins) and neuronal cell culture extracts (identifying 609 proteins). Profiling of the affected biological processes and molecular functions in cells of neuronal origin suggests numerous functions of Nrx in the redox regulation of metabolic pathways, cellular morphology, and signal transduction. These results characterize Nrx as a cellular oxidase that itself may be oxidized by the formation of disulfide relays with peroxiredoxins.

Measuring Mitochondrial Protein Thiol Redox State

2010 ◽  
pp. 123-147 ◽  
Author(s):  
Raquel Requejo ◽  
Edward T. Chouchani ◽  
Thomas R. Hurd ◽  
Katja E. Menger ◽  
Mark B. Hampton ◽  
...  
Keyword(s):  
Redox State ◽  
Mitochondrial Protein ◽  
Protein Thiol ◽  
Thiol Redox State ◽  
Thiol Redox

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

2020 ◽  
Author(s):  
Ying Ann Chiao ◽  
Huiliang Zhang ◽  
Mariya Sweetwyne ◽  
Jeremy Whitson ◽  
Ying Sonia Ting ◽  
...  
Keyword(s):  
Redox State ◽  
Late Life ◽  
Protein Thiol ◽  
Cardiac Aging ◽  
Mitochondrial Oxidative Stress ◽  
Viral Expression ◽  
Mitochondrial Ros ◽  
Thiol Redox State ◽  
Thiol Redox ◽  
Old Mice

AbstractDiastolic 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.

scholarly journals The thiol redox state of lymphoid organs is modified by immunization: Role of different immune cell populations

2008 ◽  
Vol 38 (9) ◽  
pp. 2419-2425 ◽  
Author(s):  
Patrizia Castellani ◽  
Giovanna Angelini ◽  
Laura Delfino ◽  
Andrea Matucci ◽  
Anna Rubartelli
Keyword(s):  
Redox State ◽  
Immune Cell ◽  
Lymphoid Organs ◽  
Cell Populations ◽  
Thiol Redox State ◽  
Thiol Redox
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