Quantitative display of the redox status of proteins with maleimide‐polyethylene glycol tagging

Lee, Yu-Jung; Chang, Geen-Dong

doi:10.1002/elps.201800335

Cited by 11 publications

(6 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Lysates were then treated with DTT to reduce disulfide bonds and incubated with m-PEG to label the reduced cysteine residues. Samples were subjected to SDS-PAGE and blotted with anti-tau antibody to detect m-PEG-tagged tau through mobility shift in immunoblots ( 25 , 26 ). To evaluate the efficiency of m-PEG tagging and oxidation of samples during the experiments, samples were also prepared without alkylation, which should expose all cysteines for tagging if there is no oxidation during the experiment (alkylation [−]).…”

Section: Resultsmentioning

confidence: 99%

Disulfide bond formation in microtubule-associated tau protein promotes tau accumulation and toxicity in vivo

Saito

Chiku

Oka

et al. 2021

Human Molecular Genetics

View full text Add to dashboard Cite

Accumulation of microtubule-associated tau protein is thought to cause neuron loss in a group of neurodegenerative diseases called tauopathies. In diseased brains, tau molecules adopt pathological structures that propagate into insoluble forms with disease-specific patterns. Several types of posttranslational modifications in tau are known to modulate its aggregation propensity in vitro, but their influence on tau accumulation and toxicity at the whole-organism level has not been fully elucidated. Herein, we utilized a series of transgenic Drosophila models to compare systematically the toxicity induced by five tau constructs with mutations or deletions associated with aggregation, including substitutions at seven disease-associated phosphorylation sites (S7A and S7E), deletions of PHF6 and PHF6* sequences (ΔPHF6 and ΔPHF6*), and substitutions of cysteine residues in the microtubule binding repeats (C291/322A). We found that substitutions and deletions resulted in different patterns of neurodegeneration and accumulation, with C291/322A having a dramatic effect on both tau accumulation and neurodegeneration. These cysteines formed disulfide bonds in mouse primary cultured neurons and in the fly retina, and stabilized tau proteins. Additionally, they contributed to tau accumulation under oxidative stress. We also found that each of these cysteine residues contributes to the microtubule polymerization rate and microtubule levels at equilibrium, but none of them affected tau binding to polymerized microtubules. Since tau proteins expressed in the Drosophila retina are mostly present in the early stages of tau filaments self-assembly, our results suggest that disulfide bond formation by these cysteine residues could be attractive therapeutic targets.

show abstract

Section: Resultsmentioning

confidence: 99%

Disulfide bond formation in microtubule-associated tau protein promotes tau accumulation and toxicity in vivo

Saito

Chiku

Oka

et al. 2021

Human Molecular Genetics

View full text Add to dashboard Cite

show abstract

“…Non-reducing immunoblotting quantifies the oxidation of some proteins, such as protein kinase G (Burgoyne et al, 2007). For the many proteins that fail to exhibit endogenous oxidationinduced mobility shifts, cysteines can be derivatised with mobility-shifting polyethylene glycol (PEG)-payloads (Burgoyne et al, 2013;Cobley et al, , 2019aLee and Chang, 2019;Leeuwen et al, 2017;Makmura et al, 2001). These assays quantify cysteine redox proteoforms (Cobley, 2023b).…”

Section: Immunologicalmentioning

confidence: 99%

Ten “Cheat Codes” for Measuring Oxidative Stress in Humans

Cobley,

Margaritelis,

Chatzinikolaou

et al. 2024

Preprint

View full text Add to dashboard Cite

Formidable and often seemingly insurmountable conceptual, technical, and methodological challenges hamper the measurement of oxidative stress in humans. For instance, fraught and flawed methods, such as the thiobarbituric acid reactive substances assay kits for lipid peroxidation, rate-limit progress. To advance translational redox research, we present ten comprehensive “cheat codes” for measuring oxidative stress in humans. The cheat codes include analytical approaches to assess reactive oxygen species, antioxidants, biomarkers of oxidative damage and redox regulation. They provide essential conceptual, technical, and methodological information inclusive of curated “do” and “don’t” guidelines. Given the biochemical complexity of oxidative stress, we present a research question-grounded decision tree guide for selecting the most appropriate cheat code (s) to implement in a prospective human experiment. Worked examples demonstrate the benefits of the decision tree-based cheat code selection tool. The ten cheat codes define an invaluable resource for measuring oxidative stress in humans.

show abstract

“…A disulfide bond between cysteines 302 and 550 could cause folding of dMIC60 protein. To explore this possibility, we treated 15-day-old fly whole-body lysates with either the reducing agent, DTT, which opens disulfide bonds and linearizes proteins, or the non-reducing agent, PEG-MAL, which reacts to and tags only free cysteines leading to an upward mass shift of 5 kDa per cysteine but cannot access to oxidized cysteines 27 . dMIC60 protein migrated faster under the non-reducing condition (PEG-MAL) than the reducing condition (DTT) in SDS-PAGE (Fig.…”

Section: Mic60 Protein Undergoes An Oxidative Conformational Changementioning

confidence: 99%

A mitochondrial membrane-bridging machinery mediates signal transduction of intramitochondrial oxidation

Conradson

Bharat

et al. 2021

Nat Metab

View full text Add to dashboard Cite

Mitochondria are the main site for generating reactive oxygen species, which are key players in diverse biological processes. However, the molecular pathways of redox signal transduction from the matrix to the cytosol are poorly defined. Here we report an inside-out redox signal of mitochondria. Cysteine oxidation of MIC60, an inner mitochondrial membrane protein, triggers the formation of disulfide bonds and the physical association of MIC60 with Miro, an outer mitochondrial membrane protein. The oxidative structural change of this membrane-crossing complex ultimately elicits cellular responses that delay mitophagy, impair cellular respiration, and cause oxidative stress. Blocking the MIC60-Miro interaction or reducing either protein, genetically or pharmacologically, extends lifespan and health-span of healthy fruit flies, and benefits multiple models of Parkinson's disease and Friedreich's Ataxia. Our discovery provides a molecular basis for common treatment strategies against oxidative stress.

show abstract

Quantitative display of the redox status of proteins with maleimide‐polyethylene glycol tagging

Cited by 11 publications

References 49 publications

Disulfide bond formation in microtubule-associated tau protein promotes tau accumulation and toxicity in vivo

Disulfide bond formation in microtubule-associated tau protein promotes tau accumulation and toxicity in vivo

Ten “Cheat Codes” for Measuring Oxidative Stress in Humans

A mitochondrial membrane-bridging machinery mediates signal transduction of intramitochondrial oxidation

Contact Info

Product

Resources

About