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Vitamin B6 conjugation to nuclear corepressor RIP140 and its role in gene regulation

Nature Chemical Biology volume 3, pages 161–165 (2007)Cite this article

Abstract

Pyridoxal 5′-phosphate (PLP), the biologically active form of vitamin B6, is an important cofactor in amino acid metabolism1, and supplementary vitamin B6 has protective effects in many disorders2,3,4,5. Other than serving as a cofactor, it can also modulate the activities of steroid hormone receptors6,7,8,9 and transcription factors10. However, the molecular basis of this modulation is unclear. Here, we report that mouse nuclear receptor interacting protein 140 (RIP140) can be modified by PLP conjugation. We mapped the modification site to Lys613 by LC-ESI-MS/MS analysis. This modification enhanced its transcriptional corepressive activity and its physiological function in adipocyte differentiation. We attribute this effect to increased interaction of RIP140 with histone deacetylases and nuclear retention of RIP140. This study uncovers a new physiological role of vitamin B6 in gene regulation by PLP conjugation to a transcriptional coregulator, which represents a new function of an old form11 of protein post-translational modification that has important biological consequences.

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Figure 1: RIP140 is conjugated to PLP.
Figure 2: RIP140 is modified by PLP in mammalian cells.
Figure 3: PLP conjugation enhances RIP140 interaction with HDAC3.
Figure 4: PLP modulates the biological activities of RIP140.
Figure 5: PLP conjugation affects the subcellular distribution of RIP140.

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Acknowledgements

This work was supported by US National Institutes of Health grants DA11190, DA11806, DK54733, DK60521 and K02-DA13926 to L.-N.W. We also thank the staffs of the Mass Spectrometry Consortium for the Life Sciences, Department of Biochemistry, Molecular Biology and Biophysics at the University of Minnesota, St. Paul for recording the mass spectra, and the staff in L.-N.W.'s lab for their help.

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Authors and Affiliations

  1. Department of Pharmacology, Molecular Biology and Biophysics, University of Minnesota Medical School, 6-120 Jackson Hall, 321 Church Street SE, Minneapolis, 55455, Minnesota, USA

    M D Mostaqul Huq, Nien-Pei Tsai, Ya-Ping Lin & Li-Na Wei

  2. Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Medical School, 6-120 Jackson Hall, 321 Church Street SE, Minneapolis, 55455, Minnesota, USA

    LeeAnn Higgins

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Contributions

M.D.M.H. conceived of and directed the project, conducted overall experiments, interpreted data and prepared the manuscript. N.P.T. and Y.P.L. prepared antibodies and conducted site-directed mutagenesis. L.A.H. recorded mass and assisted in interpretation of mass data and text editing. L.-N.W. provided overall guidance and direction in experimentation, interpretation of data and manuscript preparation and provided all financial support.

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Correspondence to Li-Na Wei.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Chemistry of PLP conjugation to lysine and the proposed MS fragmentation. (PDF 189 kb)

Supplementary Fig. 2

The expanded MS/MS spectra (225-240 m/z) of the precursor ions of RIP140 peptide (608-630 aa) from insect cells. (PDF 148 kb)

Supplementary Fig. 3

Mapping of PLP modification at Lys-613. (PDF 201 kb)

Supplementary Fig. 4

The expanded MS/MS spectra (225-240 m/z) of the precursor ions from RIP140 peptide (606-630 aa). (PDF 147 kb)

Supplementary Fig. 5

Identification of PLP modification site at Lys613 on RIP140 in vitro by LC-ESI-MS/MS analysis. (PDF 177 kb)

Supplementary Fig. 6

ESI-MS/MS of analysis of in vitro PLP modified synthetic peptide. (PDF 263 kb)

Supplementary Fig. 7

PLP conjugation of RIP140 and its biological impacts. (PDF 223 kb)

Supplementary Methods

Expression, purification and mass spectrometry of RIP140 and ESI-MS/MS analysis of in vitro PLP-conjugated synthetic peptide. (PDF 104 kb)

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Huq, M., Tsai, NP., Lin, YP. et al. Vitamin B6 conjugation to nuclear corepressor RIP140 and its role in gene regulation. Nat Chem Biol 3, 161–165 (2007). https://doi.org/10.1038/nchembio861

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