Experimental Approaches for Investigating Disulfide-Based Redox Relays in Cells

Abstract

Reversible oxidation of cysteine residues within proteins occurs naturally during normal cellular homeostasis and can increase during oxidative stress. Cysteine oxidation often leads to the formation of disulfide bonds, which can impact protein folding, stability, and function. Work in both prokaryotic and eukaryotic models over the past five decades has revealed several multiprotein systems that use thiol-dependent oxidoreductases to mediate disulfide bond reduction, formation, and/or rearrangement. Here, I provide an overview of how these systems operate to carry out disulfide exchange reactions in different cellular compartments, with a focus on their roles in maintaining redox homeostasis, transducing redox signals, and facilitating protein folding. Additionally, I review thiol-independent and thiol-dependent approaches for interrogating what proteins partner together in such disulfide-based redox relays. While the thiol-independent approaches rely either on predictive measures or standard procedures for monitoring protein−protein interactions, the thiol-dependent approaches include direct disulfide trapping methods as well as thiol-dependent chemical cross-linking. These strategies may prove useful in the systematic characterization of known and newly discovered disulfide relay mechanisms and redox switches involved in oxidant defense, protein folding, and cell signaling.

A Review of Temporomandibular Joint (TMJ) Synovitis and the Important Role of the Nuclear Protein, High Mobility Group Box 1 (HMGB1)

“…These oxidative changes can affect the formation of disulfide bonds, folding of the protein molecule, stability, and function. 29 The HMGB1 molecule also contains many other potential active amino acid residues (Lysine, Methionine and Tyrosine) with the potential for Post Translational Modification (PTM) of these residues by reactive oxygen species (REDOX).…”

“…These oxidative changes can affect the formation of disulfide bonds, folding of the protein molecule, stability, and function. 29…”

Reappraisal of Oxidized HMGB1 As a Mediator and Biomarker

“…Reactive, reversible modifications sensitive to the redox environment pose a challenge for sample preparation, requiring care that endogenous modifications are not lost, while ensuring that artifactual modifications are not created. Despite these challenges, technologies sufficient for robust characterization of oxidized proteoforms exist and may aid in the development of specific biomarkers (reviewed in [ 92 ]). Derivatization and subsequent analysis by western blotting, LC–MS/MS or other methods can provide oxidation state information.…”

Cysteine Redox Chemistry in Peptide Self-Assembly to Modulate Hydrogelation

“…Nature makes wide use of cysteine (Cys) thiol reactivity to direct protein assembly into functional structures [ 1 ], especially through its oxidation to disulfide bridges [ 2 ]. Other types of thiol reactivity in naturally occurring systems include metal coordination, for instance in metalloproteins, also towards catalytic function [ 3 , 4 ].…”