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J. Biol. Chem., Vol. 280, Issue 21, 20365-20374, May 27, 2005

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Lys⁶-modified Ubiquitin Inhibits Ubiquitin-dependent Protein Degradation^*

Fu Shang, Gejing Deng, Qing Liu, Weimin Guo, Arthur L. Haas¶, Bernat Crosas||, Daniel Finley||, and Allen Taylor

From the Laboratory for Nutrition and Vision Research, Jean Mayer United States Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts 02111, the ^¶Department of Biochemistry and Molecular Biology, Louisiana State University School of Medicine, New Orleans, Louisiana 70112, and the ^||Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02138

Ubiquitin plays essential roles in various cellular processes; therefore, it is of keen interest to study the structure-function relationship of ubiquitin itself. We investigated the modification of Lys⁶ of ubiquitin and its physiological consequences. Mass spectrometry-based peptide mapping and N-terminal sequencing demonstrated that, of the 7 Lys residues in ubiquitin, Lys⁶ was the most readily labeled with sulfosuccinimidobiotin. Lys⁶-biotinylated ubiquitin was incorporated into high molecular mass ubiquitin conjugates as efficiently as unmodified ubiquitin. However, Lys⁶-biotinylated ubiquitin inhibited ubiquitin-dependent proteolysis, as conjugates formed with Lys⁶-biotinylated ubiquitin were resistant to proteasomal degradation. Ubiquitins with a mutation of Lys⁶ had similar phenotypes as Lys⁶-biotinylated ubiquitin. Lys⁶ mutant ubiquitins (K6A, K6R, and K6W) also inhibited ATP-dependent proteolysis and caused accumulation of ubiquitin conjugates. Conjugates formed with K6W mutant ubiquitin were also resistant to proteasomal degradation. The dominant-negative effect of Lys⁶-modified ubiquitin was further demonstrated in intact cells. Overexpression of K6W mutant ubiquitin resulted in accumulation of intracellular ubiquitin conjugates, stabilization of typical substrates for ubiquitin-dependent proteolysis, and enhanced susceptibility to oxidative stress. Taken together, these results show that Lys⁶-modified ubiquitin is a potent and specific inhibitor of ubiquitin-mediated protein degradation.

Received for publication, December 21, 2004 , and in revised form, March 21, 2005.

^* This work was supported in part by National Institutes of Health Grants EY11717 (to F. S.), EY13250 (to A. T.), and GM34009 (to A. L. H.) and by the United States Department of Agriculture under Agreement 58-1950-9-001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Lab. for Nutrition and Vision Research, Jean Mayer USDA HNRCA, Tufts University, 711 Washington St., Boston, MA 02111. Tel.: 617-556-3158; Fax: 617-556-3344; E-mail: fu.shang{at}tufts.edu.

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