PYCR1 and PYCR2 Interact and Collaborate with RRM2B to Protect Cells from Overt Oxidative Stress
Abstract
Ribonucleotide reductase small subunit B (RRM2B) is a stress response protein that protects normal human fibroblasts from oxidative stress. However, the underlying mechanism that governs this function is not entirely understood. To identify factors that interact with RRM2B and mediate anti-oxidation function, large-scale purification of human Flag-tagged RRM2B complexes was performed. Pyrroline-5-carboxylate reductase 1 and 2 (PYCR1, PYCR2) were identified by mass spectrometry analysis as components of RRM2B complexes. Silencing of both PYCR1 and PYCR2 by expressing short hairpin RNAs induced defects in cell proliferation, partial fragmentation of the mitochondrial network, and hypersensitivity to oxidative stress in hTERT-immortalized human foreskin fibroblasts (HFF-hTERT). Moderate overexpression of RRM2B, comparable to stress-induced level, protected cells from oxidative stress. Silencing of both PYCR1 and PYCR2 completely abolished anti-oxidation activity of RRM2B, demonstrating a functional collaboration of these metabolic enzymes in response to oxidative stress.
Additional Information
© 2016 Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0 Received: 01 October 2015. accepted: 26 November 2015. Published: 06 January 2016. We thank Geoffrey T Smith (Proteome Exploration Laboratory, California Institute of Technology, Pasadena, CA, USA) for technical assistance in mass spectrometry analysis; Dr. Xiwei Wu (City of Hope, Duarte, CA, USA) for bioinformatic analysis of candidate RRM2B-associating proteins; Dr. Jeffrey S. Dome for providing HFF-hTERT cell line, Dr. David Chan and Dr. Prashant Mishra (California Institute of Technology) for providing pclbw-cox4-Dsred vector and advice on mitochondrial morphology studies, Dr. Brain Armstrong and Tina Patel (City of Hope) for their assistance with the confocal fluorescence microscopy assays; Lucy Brown (City of Hope) for FACS analysis; Dr. John R. Gray (St. Jude Children's Research Hospital, Memphis, TN, USA), and Dr. Richard C. Mulligan (Harvard University, Cambridge, MA) for providing the retroviral and lentiviral helper plasmids; Dr. David Baltimore (California Institute of Technology) for providing the 293T cells for retroviral and lentiviral production; Alexander Sy and Dr. Lixin Yang for technical assistance; Dr. Frank Luh for editing manuscript and members of the Yen laboratory for their helpful suggestions during the course of this work. This project was supported by NIH grant 5R01CA127541, Gordon and Betty Moore Foundation Grant GBMF775 (M.J.S. and S. Hess) and the Beckman Institute (M.J.S. and S. Hess). Mabel Bin-Er Lee & Michelle Tang: These authors contributed equally to this work. Contributions: M.-L.K. designed and performed the experiments, analyzed and interpreted the data and wrote the paper; M.B.-E.L., M.T., W.d.B., S. Hu, C.-M.C. and C.A.C performed experiments; M.J.S. and S. Hess performed mass spectrometry analysis; M.S. and W.J. performed targeted metabolite analysis; L.S. performed computational prediction of RRM2B/PYCR1 complex and Y.Y. supervised the project. The authors declare no competing financial interests.Attached Files
Published - srep18846.pdf
Supplemental Material - srep18846-s1.pdf
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Additional details
- PMCID
- PMC4702135
- Eprint ID
- 63569
- Resolver ID
- CaltechAUTHORS:20160112-084402668
- 5R01CA127541
- NIH
- GBMF775
- Gordon and Betty Moore Foundation
- Caltech Beckman Institute
- Created
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2016-01-12Created from EPrint's datestamp field
- Updated
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2021-11-10Created from EPrint's last_modified field