Ajanta Chatterji1, Kumar Sachin2, Rajib Sengupta3,4. 1. Amity Institute of Biotechnology Kolkata, Amity University Kolkata, Action Area II, Kadampukur village, Rajarhat, Newtown, 700135, Kolkata, West Bengal, India. 2. Dept of Biosciences, Swami Rama Himalayan University, 248016, Jolly Grant, Dehradun, India. 3. Amity Institute of Biotechnology Kolkata, Amity University Kolkata, Action Area II, Kadampukur village, Rajarhat, Newtown, 700135, Kolkata, West Bengal, India. rsengupta@kol.amity.edu. 4. Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden. rsengupta@kol.amity.edu.
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 [3 H] CDP into [3 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.
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 [3 H] CDP into [3 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.
Authors: Pankaj Kumar Mandal; Manuela Schneider; Pirkko Kölle; Peter Kuhlencordt; Heidi Förster; Heike Beck; Georg W Bornkamm; Marcus Conrad Journal: Cancer Res Date: 2010-11-02 Impact factor: 12.701