Protein Redox State Monitoring Studies of Thiol Reactivity.

Yuichiro J Suzuki,Yuki Tatenaka,Yuya Ohuchi,Takashi Shimomura,Tinatin I Brelidze,Lucia Marcocci

doi:10.3390/antiox8050143

Abstract

Protein cysteine thiol status is a major determinant of oxidative stress and oxidant signaling. The -SulfoBiotics- Protein Redox State Monitoring Kit provides a unique opportunity to investigate protein thiol states. This system adds a 15-kDa Protein-SHifter to reduced cysteine residues, and this molecular mass shift can be detected by gel electrophoresis. Even in biological samples, Protein-SHifter Plus allows the thiol states of specific proteins to be studied using Western blotting. Peroxiredoxin 6 (Prx6) is a unique one-cysteine peroxiredoxin that scavenges peroxides by utilizing conserved Cysteine-47. Human Prx6 also contains an additional non-conserved cysteine residue, while rat Prx6 only has the catalytic cysteine. In cultured cells, cysteine residues of Prx6 were found to be predominantly fully reduced. The treatment of human cells with hydrogen peroxide (H2O2) formed Prx6 with one cysteine reduced. Since catalytic cysteine becomes oxidized in rat cells by the same H2O2 treatment and treating denatured human Prx6 with H2O2 results in the oxidation of both cysteines, non-conserved cysteine may not be accessible to H2O2 in human cells. We also found that untreated cells contained Prx6 multimers bound through disulfide bonds. Surprisingly, treating cells with H2O2 eliminated these Prx6 multimers. In contrast, treating cell lysates with H2O2 promoted the formation of Prx6 multimers. Similarly, treating purified preparations of the recombinant cyclic nucleotide-binding domain of the human hyperpolarization-activated cyclic nucleotide-modulated channels with H2O2 promoted the formation of multimers. These studies revealed that the cellular environment defines the susceptibility of protein cysteines to H2O2 and determines whether H2O2 acts as a facilitator or a disrupter of disulfide bonds.

Highlights

The redox state of protein cysteine residues plays a crucial role in cell regulation and is often the major target of oxidative stress and oxidant signaling conferred by reactive oxygen species [1,2,3,4,5,6,7]
We investigated the capability of the -SulfoBiotics- Protein Redox State Monitoring Kit to report on the cysteine redox state of two structurally and functionally unrelated human proteins, Peroxiredoxin 6 (Prx6) and cyclic nucleotide-binding domain (CNBD) of HCN4 channels, one of which (CNBD) we have the ability to purify with affinity and size-exclusion chromatography
H2O2 dramatically decreased the 40 band. These results suggest that, perhaps due to the three-dimensional kDa Prx6 band. These results suggest that, perhaps due to the structure of Prx6 defined by the cellular environment, oxidation of non-conserved cysteine with H2 O2 three-dimensional structure of Prx6 defined by the cellular environment, oxidation of non-conserved is blocked in the human Prx6 protein

Summary

Introduction

Various labeling probes allow for the determination of the global protein thiol status in the cells [8]. The examination of the thiol states of specific cellular proteins has been difficult. In this regard, Dojindo Molecular Technologies, Inc. has developed a novel approach that allows easy determination of the cysteine redox states within the proteins of interest. Monitoring Kit system, a 15-kDa Protein-SHifter is added to every reduced cysteine residue, allowing. Antioxidants 2019, 8, 143 for the defining of protein redox states using gel electrophoresis while examining the mobility shift. In the improved “Plus” kit, the bulky Protein-SHifter is eliminated by UV light after gel electrophoresis, making the system suitable for Western blotting. We investigated the capability of the -SulfoBiotics- Protein Redox State Monitoring Kit to report on the cysteine redox state of two structurally and functionally unrelated human proteins, Prx and cyclic nucleotide-binding domain (CNBD) of HCN4 channels, one of which (CNBD) we have the ability to purify with affinity and size-exclusion chromatography

Methods

Discussion

Conclusion