The reversible oxidation of cysteine by reactive oxygen species (ROS) is both a mechanism for cellular protein signaling as well as a cause of cellular injury and death through the generation of oxidative stress. The study of cysteine oxidation is complicated by the methodology currently available to isolate and enrich oxidized-cysteine containing proteins. We sought to simplify this process by reducing the time needed to process samples and reducing sample loss and contamination risk.
We accomplished this by eliminating precipitation steps needed for the protocol by (a) introducing an in-solution NEM-quenching step prior to reduction and (b) replacing soluble dithiothreitol reductant with a series of newly-developed high-capacity polyacrylamide-based solid-phase reductants that could be easily separated from the lysate through centrifugation. These modifications, collectively called resin-assisted reduction and capture (RARC), reduced the time needed to perform the RAC method from 2-3 days to 4-5 hours, while the overall quality and quantity of previously-oxidized cysteines captured was increased.
In order to demonstrate the RARC method’s utility in studying complex cellular oxidants, the optimized methodology was used to study cysteine oxidation caused by the redox-active metals arsenic, cadmium, and chromium. As(III), Cr(VI), and Cd(II) were all found to increase cysteine oxidation significantly, with As(III) and Cd(II) inducing more oxidation than Cr(VI) following a 24-hour exposure to cytotoxic concentrations. Label-free proteomic analysis and western blotting of RARC-isolated oxidized proteins found a high degree of commonality between the proteins oxidized by these metals, with cytoskeletal, translational, stress response, and metabolic proteins all being oxidized. Several previously-unreported redox-active cysteines were also identified.
These results indicate that cysteine oxidation by As(III), Cr(VI), and Cd(II) may play a significant role in these metals’ cytotoxicity and demonstrates the utility of the RARC method as a strategy for studying reversible cysteine oxidation by oxidants in oxidative signaling and disease. The RARC method is a simplification and improvement upon the current state of the art which decreases the barrier of entry to studying cysteine oxidation, allowing more researchers to study this modification. We predict that the RARC methodology will be critical in expanding our understanding of reactive cysteines in cellular function and disease.
| Identifer | oai:union.ndltd.org:uky.edu/oai:uknowledge.uky.edu:toxicology_etds-1022 |
| Date | 01 January 2018 |
| Creators | Hitron, John Andrew |
| Publisher | UKnowledge |
| Source Sets | University of Kentucky |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations--Toxicology and Cancer Biology |
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