Glutathione-dependent Thioredoxin Reduction and Lipoamide System Support In-vitro Mammalian Ribonucleotide Reductase Catalysis: a Possible Antioxidant Redundancy

Overview

Journal Mol Biol Rep

Specialty Molecular Biology

Date 2022 Jun 2

PMID 35655055

Authors

Ajanta Chatterji

Kumar Sachin

Rajib Sengupta

Affiliations

Soon will be listed here.

Abstract

Background: The thioredoxin system (Trx), comprising of Trx, Thioredoxin reductase (TrxR) and NADPH aids in donating hydrogen group to support Ribonucleotide reductase (RNR) catalysis during de-novo DNA biosynthesis. However, it has been observed that inhibiting TrxR does not affect the viability of cancer cells that are susceptible to pharmacological glutathione (GSH) depletion. This prompted us to study the potential antioxidant redundancies that might prolong RNR activity.

Methods: To study the RNR activity assay, the RNR complex was reconstituted by mixing purified mouse recombinant RNR subunits and the conversion of [ H] CDP into [ H] dCDP was monitored. In the assay system, either purified Trx and GSH or Lipoamide system was supplemented as reducing agents to support RNR catalysis.

Results: Herein, we have found that GSH-dependent Trx reduction supports mammalian class I RNR catalysis in absence of TrxR in the system. Our data also presents the first report that the LAM system is capable of supporting in-vitro RNR activity in the complete absence of either Trx or Grx systems.

Conclusions: We conclude that GSH-mediated Trx reduction and LAM systems support basal level RNR activity in vitro; in absence of TrxR and complete redoxin systems respectively and hypothesize that potential redundancy between the various antioxidant systems might synergize in sustaining RNR activity.

Citing Articles

Mitochondrial Glutathione in Cellular Redox Homeostasis and Disease Manifestation.

Chen T, Wang H, Chang C, Lee S Int J Mol Sci. 2024; 25(2).

PMID: 38279310 PMC: 10816320. DOI: 10.3390/ijms25021314.

References

Lu J, Holmgren A . The thioredoxin antioxidant system. Free Radic Biol Med. 2013; 66:75-87. DOI: 10.1016/j.freeradbiomed.2013.07.036. View

Sengupta R, Holmgren A . Thioredoxin and thioredoxin reductase in relation to reversible S-nitrosylation. Antioxid Redox Signal. 2012; 18(3):259-69. DOI: 10.1089/ars.2012.4716. View

Du Y, Zhang H, Lu J, Holmgren A . Glutathione and glutaredoxin act as a backup of human thioredoxin reductase 1 to reduce thioredoxin 1 preventing cell death by aurothioglucose. J Biol Chem. 2012; 287(45):38210-9. PMC: 3488090. DOI: 10.1074/jbc.M112.392225. View

Sengupta R, Holmgren A . Thioredoxin and glutaredoxin-mediated redox regulation of ribonucleotide reductase. World J Biol Chem. 2014; 5(1):68-74. PMC: 3942543. DOI: 10.4331/wjbc.v5.i1.68. View

Eklund H, Eriksson M, Uhlin U, Nordlund P, Logan D . Ribonucleotide reductase--structural studies of a radical enzyme. Biol Chem. 1997; 378(8):821-5. View

Mandal P, Schneider M, Kolle P, Kuhlencordt P, Forster H, Beck H . Loss of thioredoxin reductase 1 renders tumors highly susceptible to pharmacologic glutathione deprivation. Cancer Res. 2010; 70(22):9505-14. DOI: 10.1158/0008-5472.CAN-10-1509. View

Bansal A, Simon M . Glutathione metabolism in cancer progression and treatment resistance. J Cell Biol. 2018; 217(7):2291-2298. PMC: 6028537. DOI: 10.1083/jcb.201804161. View

Biewenga G, Dorstijn M, Verhagen J, Haenen G, Bast A . Reduction of lipoic acid by lipoamide dehydrogenase. Biochem Pharmacol. 1996; 51(3):233-8. DOI: 10.1016/0006-2952(95)02124-8. View

Arner E, Nordberg J, Holmgren A . Efficient reduction of lipoamide and lipoic acid by mammalian thioredoxin reductase. Biochem Biophys Res Commun. 1996; 225(1):268-74. DOI: 10.1006/bbrc.1996.1165. View

10.

Zhao L, Liu Z, Jia H, Feng Z, Liu J, Li X . Lipoamide Acts as an Indirect Antioxidant by Simultaneously Stimulating Mitochondrial Biogenesis and Phase II Antioxidant Enzyme Systems in ARPE-19 Cells. PLoS One. 2015; 10(6):e0128502. PMC: 4452644. DOI: 10.1371/journal.pone.0128502. View

11.

Persson H, Svensson A, Brunk U . Alpha-lipoic acid and alpha-lipoamide prevent oxidant-induced lysosomal rupture and apoptosis. Redox Rep. 2002; 6(5):327-34. DOI: 10.1179/135100001101536472. View

12.

Hou Y, Li X, Peng S, Yao J, Bai F, Fang J . Lipoamide Ameliorates Oxidative Stress via Induction of Nrf2/ARE Signaling Pathway in PC12 Cells. J Agric Food Chem. 2019; 67(29):8227-8234. DOI: 10.1021/acs.jafc.9b02680. View

13.

Mann G, Graslund A, Ochiai E, Ingemarson R, Thelander L . Purification and characterization of recombinant mouse and herpes simplex virus ribonucleotide reductase R2 subunit. Biochemistry. 1991; 30(7):1939-47. DOI: 10.1021/bi00221a030. View

14.

Avval F, Holmgren A . Molecular mechanisms of thioredoxin and glutaredoxin as hydrogen donors for Mammalian s phase ribonucleotide reductase. J Biol Chem. 2009; 284(13):8233-40. PMC: 2659180. DOI: 10.1074/jbc.M809338200. View

15.

Chatterji A, Sengupta R . Cellular S-denitrosylases: Potential role and interplay of Thioredoxin, TRP14, and Glutaredoxin systems in thiol-dependent protein denitrosylation. Int J Biochem Cell Biol. 2020; 131:105904. DOI: 10.1016/j.biocel.2020.105904. View

16.

Chatterji A, Holmgren A, Sengupta R . Ribonucleotide reductase: In-vitro S-glutathionylation of R2 and p53R2 subunits of mammalian class I ribonucleotide reductase protein. Mol Biol Rep. 2021; 48(11):7621-7626. DOI: 10.1007/s11033-021-06721-2. View

17.

Sengupta R, Coppo L, Mishra P, Holmgren A . Glutathione-glutaredoxin is an efficient electron donor system for mammalian p53R2-R1-dependent ribonucleotide reductase. J Biol Chem. 2019; 294(34):12708-12716. PMC: 6709626. DOI: 10.1074/jbc.RA119.008752. View

18.

Bucurenci N, Sakamoto H, Briozzo P, Palibroda N, Serina L, Sarfati R . CMP kinase from Escherichia coli is structurally related to other nucleoside monophosphate kinases. J Biol Chem. 1996; 271(5):2856-62. DOI: 10.1074/jbc.271.5.2856. View

19.

Slabaugh M, Mathews C . Vaccinia virus-induced ribonucleotide reductase can be distinguished from host cell activity. J Virol. 1984; 52(2):501-6. PMC: 254551. DOI: 10.1128/JVI.52.2.501-506.1984. View