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Published October 13, 2005 | Supplemental Material
Journal Article Open

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.

Additional Information

© 2005 Nature Publishing Group. Received 26 May; accepted 15 July 2005. 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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October 20, 2023