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The N-end rule pathway as a nitric oxide sensor controlling the levels of multiple regulators


The conjugation of arginine to proteins is a part of the N-end rule pathway of protein degradation. Three amino (N)-terminal residues—aspartate, glutamate and cysteine—are arginylated by ATE1-encoded arginyl-transferases. Here we report that oxidation of N-terminal cysteine is essential for its arginylation. The in vivo oxidation of N-terminal cysteine, before its arginylation, is shown to require nitric oxide. We reconstituted this process in vitro as well. The levels of regulatory proteins bearing N-terminal cysteine, such as RGS4, RGS5 and RGS16, are greatly increased in mouse ATE1-/- embryos, which lack arginylation. Stabilization of these proteins, the first physiological substrates of mammalian N-end rule pathway, may underlie cardiovascular defects in ATE1-/- embryos. Our findings identify the N-end rule pathway as a new nitric oxide sensor that functions through its ability to destroy specific regulatory proteins bearing N-terminal cysteine, at rates controlled by nitric oxide and apparently by oxygen as well.

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Figure 1: N-terminal cysteine must be oxidized before its arginylation.
Figure 2: Strongly increased levels of RGS4, RGS5 and RGS16 proteins in ATE1 -/- embryos.
Figure 3: Decreasing NO concentration in vivo stabilizes RGS4 and RGS16.
Figure 4: In vitro reconstitution of nitric oxide-dependent arginylation of RGS4.


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We thank G. Enikolopov, T. Michurina and J. M. Encinas for NOS1-/- mice; A. Mülsch for DNIC-[GSH]2; M. Shahgholi and J. Zhou for MS analyses of peptides; F. Rusnak and G. Hathaway for protein sequencing; J. Racs and S. Horvath for peptide synthesis; L. del Carmen Sandoval, B. W. Kennedy and S. Pease for advice and assistance with mouse mutants; Y. T. Kwon for [UBR1-/-UBR2-/-] cells; R. Baker for plasmids that enabled the USP2-based ubiquitin fusion technique; G. Eriani and F. Du for gifts of other plasmids; Z. Xia for USP2 enzyme; R. Roberts for use of his laboratory equipment; and E. Graciet and C. Brower for comments on the manuscript. Purification of mouse ATE1-1 was performed at CalTech's Protein Expression Center by I. K. Nangiana and the late P. Snow. We dedicate this paper to the memory of Dr Snow. This work was supported by grants from the NIH and the Ellison Medical Foundation to A.V.

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Correspondence to Alexander Varshavsky.

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Supplementary information

Supplementary Notes

Contains Supplementary Methods, Supplmentary Discussion, Supplementary Figure Legends and additional references. (DOC 147 kb)

Supplementary Figure S1

N-terminal cysteine must be oxidized before its arginylation by S. cerevisiae R transferase. (PDF 1215 kb)

Supplementary Figure S2

Northern hybridization and immunoblotting with antibody to ATE1. (PDF 591 kb)

Supplementary Figure S3

RT–PCR of mRNAs encoding NO synthases. (PDF 1964 kb)

Supplementary Figure S4

Levels of RGS4 in NOS1-/- mice. (PDF 197 kb)

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Hu, RG., Sheng, J., Qi, X. et al. The N-end rule pathway as a nitric oxide sensor controlling the levels of multiple regulators. Nature 437, 981–986 (2005).

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