Improved Red Fluorescent Redox Indicators for Monitoring Cytosolic and Mitochondrial Thioredoxin Redox Dynamics

Overview

Journal Biochemistry

Specialty Biochemistry

Date 2022 Feb 8

PMID 35133140

Authors

Yu Pang

Hao Zhang

Hui-Wang Ai

Affiliations

Soon will be listed here.

Abstract

Thioredoxin (Trx) is one of the major thiol-dependent antioxidants in living systems. The study of Trx functions in redox biology was impeded by the lack of practical tools to track Trx redox dynamics in live cells. Our previous work developed TrxRFP1, the first genetically encoded fluorescent indicator for Trx redox. In this work, we report an improved fluorescent indicator, TrxRFP2, for tracking the redox of Trx1, which is primarily cytosolic and nuclear. Furthermore, because mitochondria specifically express Trx2, we have created a new genetically encoded fluorescent indicator, MtrxRFP2, for the redox of mitochondrial Trx. We characterized MtrxRFP2 as a purified protein and used subcellularly localized MtrxRFP2 to image mitochondrial redox changes in live cells.

Citing Articles

Engineering and Characterization of 3-Aminotyrosine-Derived Red Fluorescent Variants of Circularly Permutated Green Fluorescent Protein.

Zhang H, Tian X, Zhang J, Ai H Biosensors (Basel). 2024; 14(1).

PMID: 38275307 PMC: 10813706. DOI: 10.3390/bios14010054.

References

Spyrou G, Enmark E, Miranda-Vizuete A, Gustafsson J . Cloning and expression of a novel mammalian thioredoxin. J Biol Chem. 1997; 272(5):2936-41. DOI: 10.1074/jbc.272.5.2936. View

Huang Q, Zhou H, Zhang H, Huang Y, Hinojosa-Kirschenbaum F, Fan P . Thioredoxin-2 inhibits mitochondrial reactive oxygen species generation and apoptosis stress kinase-1 activity to maintain cardiac function. Circulation. 2015; 131(12):1082-97. PMC: 4374031. DOI: 10.1161/CIRCULATIONAHA.114.012725. View

Nakamura H, Nakamura K, Yodoi J . Redox regulation of cellular activation. Annu Rev Immunol. 1997; 15:351-69. DOI: 10.1146/annurev.immunol.15.1.351. View

Fan Y, Ai H . Development of redox-sensitive red fluorescent proteins for imaging redox dynamics in cellular compartments. Anal Bioanal Chem. 2016; 408(11):2901-11. DOI: 10.1007/s00216-015-9280-3. View

Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O . Highly accurate protein structure prediction with AlphaFold. Nature. 2021; 596(7873):583-589. PMC: 8371605. DOI: 10.1038/s41586-021-03819-2. View

Hanson G, Aggeler R, Oglesbee D, Cannon M, Capaldi R, Tsien R . Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J Biol Chem. 2004; 279(13):13044-53. DOI: 10.1074/jbc.M312846200. View

Gibson D, Young L, Chuang R, Venter J, Hutchison 3rd C, Smith H . Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 2009; 6(5):343-5. DOI: 10.1038/nmeth.1318. View

Steinegger M, Soding J . MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets. Nat Biotechnol. 2017; 35(11):1026-1028. DOI: 10.1038/nbt.3988. View

Pang Y, Zhang H, Ai H . Genetically Encoded Fluorescent Redox Indicators for Unveiling Redox Signaling and Oxidative Toxicity. Chem Res Toxicol. 2021; 34(8):1826-1845. PMC: 8383716. DOI: 10.1021/acs.chemrestox.1c00149. View

10.

Ren X, Zou L, Zhang X, Branco V, Wang J, Carvalho C . Redox Signaling Mediated by Thioredoxin and Glutathione Systems in the Central Nervous System. Antioxid Redox Signal. 2017; 27(13):989-1010. PMC: 5649126. DOI: 10.1089/ars.2016.6925. View

11.

Matthias L, Yam P, Jiang X, Vandegraaff N, Li P, Poumbourios P . Disulfide exchange in domain 2 of CD4 is required for entry of HIV-1. Nat Immunol. 2002; 3(8):727-32. DOI: 10.1038/ni815. View

12.

Taniguchi Y, Mori K, Yodoi J . A novel promoter sequence is involved in the oxidative stress-induced expression of the adult T-cell leukemia-derived factor (ADF)/human thioredoxin (Trx) gene. Nucleic Acids Res. 1996; 24(14):2746-52. PMC: 145999. DOI: 10.1093/nar/24.14.2746. View

13.

Mirdita M, Schutze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M . ColabFold: making protein folding accessible to all. Nat Methods. 2022; 19(6):679-682. PMC: 9184281. DOI: 10.1038/s41592-022-01488-1. View

14.

Starkov A, Andreyev A, Zhang S, Starkova N, Korneeva M, Syromyatnikov M . Scavenging of H2O2 by mouse brain mitochondria. J Bioenerg Biomembr. 2014; 46(6):471-7. PMC: 4634880. DOI: 10.1007/s10863-014-9581-9. View

15.

Ungerstedt J, Du Y, Zhang H, Nair D, Holmgren A . In vivo redox state of human thioredoxin and redox shift by the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). Free Radic Biol Med. 2012; 53(11):2002-7. DOI: 10.1016/j.freeradbiomed.2012.09.019. View

16.

Holmgren A . Thioredoxin. Annu Rev Biochem. 1985; 54:237-71. DOI: 10.1146/annurev.bi.54.070185.001321. View

17.

Benhar M, Shytaj I, Stamler J, Savarino A . Dual targeting of the thioredoxin and glutathione systems in cancer and HIV. J Clin Invest. 2016; 126(5):1630-9. PMC: 4855928. DOI: 10.1172/JCI85339. View

18.

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T . Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012; 9(7):676-82. PMC: 3855844. DOI: 10.1038/nmeth.2019. View

19.

Ostergaard H, Henriksen A, Hansen F, Winther J . Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein. EMBO J. 2001; 20(21):5853-62. PMC: 125700. DOI: 10.1093/emboj/20.21.5853. View

20.

Gutscher M, Pauleau A, Marty L, Brach T, Wabnitz G, Samstag Y . Real-time imaging of the intracellular glutathione redox potential. Nat Methods. 2008; 5(6):553-9. DOI: 10.1038/nmeth.1212. View