Abstract
The formation and reduction of disulfide bonds through thiol-disulfide exchange is an important cellular mechanism for the regulation of protein activity and plays a crucial role in redox signaling. Thiol-disulfide exchange on key regulatory cysteine residues offers an efficient and reversible mechanism for regulatory control in response to alterations in the intracellular redox environment. Cysteines are one of the most rarely used amino acids in proteins, but when conserved in proteins, they usually play critical roles in protein structure, function, or regulation. Diagonal electrophoresis is a relatively simple technique to analyze the formation of protein disulfides by sequential nonreducing/reducing electrophoresis. Proteins that do not form disulfides migrate identically in both dimensions and form a diagonal after the second dimension; proteins that contained intra-chain disulfides lie above the diagonal, while proteins involved in inter-disulfides fall below the diagonal. This technique allows for the detection and subsequent identification of proteins with inter-disulfides and has been applied in combination with mass spectrometry to identify proteins involved in redox signaling cascades.
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References
Sobotta MC, Liou W, Stocker S, Talwar D, Oehler M, Ruppert T, Scharf AN, Dick TP (2015) Peroxiredoxin-2 and STAT3 form a redox relay for H2O2 signaling. Nat Chem Biol 11:64–70
Marino SM, Gladyshev VN (2010) Cysteine function governs its conservation and degeneration and restricts its utilization on protein surfaces. J Mol Biol 404:902–916
Jones DP (2008) Radical-free biology of oxidative stress. Am J Physiol Cell Physiol 295:C849–C868
Mates JM, Segura JA, Alonso FJ, Marquez J (2008) Intracellular redox status and oxidative stress: implications for cell proliferation, apoptosis, and carcinogenesis. Arch Toxicol 82:273–299
Calabrese V, Sultana R, Scapagnini G, Guagliano E, Sapienza M, Bella R, Kanski J, Pennisi G, Mancuso C, Stella AM, Butterfield DA (2006) Nitrosative stress, cellular stress response, and thiol homeostasis in patients with Alzheimer's disease. Antioxid Redox Signal 8:1975–1986
O'Neill HM, Holloway GP, Steinberg GR (2013) AMPK regulation of fatty acid metabolism and mitochondrial biogenesis: implications for obesity. Mol Cell Endocrinol 366:135–151
Gil-del Valle L, de la CML, Toledo A, Vilaro N, Tapanes R, Otero MA (2005) Altered redox status in patients with diabetes mellitus type I. Pharmacol Res 51:375–380
Winterbourn CC (2014) Are free radicals involved in thiol-based redox signaling? Free Radic Biol Med 80:164–170
Poole LB (2014) The basics of thiols and cysteines in redox biology and chemistry. Free Radic Biol Med 80:148–157
Hansen RE, Roth D, Winther JR (2009) Quantifying the global cellular thiol-disulfide status. Proc Natl Acad Sci U S A 106:422–427
Sommer A, Traut RR (1974) Diagonal polyacrylamide-dodecyl sulfate gel electrophoresis for the identification of ribosomal proteins crosslinked with methyl-4-mercaptobutyrimidate. Proc Natl Acad Sci U S A 71:3946–3950
Cumming RC, Andon NL, Haynes PA, Park M, Fischer WH, Schubert D (2004) Protein disulfide bond formation in the cytoplasm during oxidative stress. J Biol Chem 279:21749–21758
Brennan JP, Wait R, Begum S, Bell JR, Dunn MJ, Eaton P (2004) Detection and mapping of widespread intermolecular protein disulfide formation during cardiac oxidative stress using proteomics with diagonal electrophoresis. J Biol Chem 279:41352–41360
Mata-Cabana A, Florencio FJ, Lindahl M (2007) Membrane proteins from the cyanobacterium Synechocystis sp. PCC 6803 interacting with thioredoxin. Proteomics 7:3953–3963
McDonagh B, Sheehan D (2007) Effect of oxidative stress on protein thiols in the blue mussel Mytilus edulis: proteomic identification of target proteins. Proteomics 7:3395–3403
Acknowledgments
This work was supported by the NUI Galway Department of Physiology and School of Medicine.
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Saraswat, R., McDonagh, B. (2019). Diagonal Electrophoresis for the Detection of Proteins Involved in Disulfide Bonds. In: Kurien, B., Scofield, R. (eds) Electrophoretic Separation of Proteins. Methods in Molecular Biology, vol 1855. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8793-1_23
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DOI: https://doi.org/10.1007/978-1-4939-8793-1_23
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