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ARS Home » Northeast Area » Boston, Massachusetts » Jean Mayer Human Nutrition Research Center On Aging » Research » Publications at this Location » Publication #166344
Title: LYS-6-MODIFIED UBIQUITIN INHIBITS UBIQUITIN-DEPENDENT PROTEIN DEGRADATION

Author
item SHANG, FU - TUFTS-HNRCA
item DENG, GEJING - TUFTS-HNRCA
item LIU, QING - TUFTS-HNRCA
item GUO, WEIMIN - TUFTS-HNRCA
item HAAS, ARTHUR - LOUISIANA STATE UNIV
item CROSAS, BERNAT - HARVARD MEDICAL SCHOOL
item FINLEY, DAVID - HARVARD MEDICAL SCHOOL
item TAYLOR, ALLEN - TUFTS-HNRCA

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/24/2005
Publication Date: 3/24/2005
Citation: Shang, F., Deng, G., Liu, Q., Guo, W., Haas, A.L., Crosas, B., Finley, D., Taylor, A. 2005. Lys-6-modified ubiquitin inhibits ubiquitin-dependent protein degradation. Journal of Biological Chemistry. [published online]. 280(21) 20365-20374. Available at http://www.jbc.org/cgi/content/full/280/21/20365

Interpretive Summary: Ubiquitin is a small protein that can be attached to other proteins and can affect a variety of cellular processes. Therefore it is of keen interest to study the structure-function relationship of ubiquitin itself. One of the most important functions of ubiquitin attachment is the targeting of proteins to degradation via the proteasome. Ubiquitin is added to the lysines of target proteins and contains 7 lysine residues itself. Using mass spectrometry-based peptide mapping, we observed that the lysine at position 6 of ubiquitin was most often modified. Changing the amino acid structure at position 6 in ubiquitin by attaching biotin, or mutating it to alanine, arginine or trytophan altered the ability of ubiquitin to target proteins for degradation while not affecting it’s ability to be attached to those target proteins. Thus our results show that ubiquitin modified at position 6 is a potent and specific inhibitor of ubiquitin-mediated protein degradation.

Technical Abstract: Ubiquitin plays an essential role in various cellular processes and therefore it is of keen interest to study the structure-function relationship of ubiquitin itself. This study investigated the modification of K6 of ubiquitin and its physiological consequences. Mass spectrometry-based peptide mapping and N-terminal sequencing demonstrated that, among the 7 K residues in ubiquitin, K6 was the most readily labeled with sulfo-NHS-biotin. The K6-biotinylated ubiquitin was incorporated into high mass ubiquitin conjugates as efficiently as unmodified ubiquitin. However, K6-biotinylated ubiquitin inhibited ubiquitin-dependent proteolysis, as conjugates formed with K6-biotinylated ubiquitin were resistant to proteasomal degradation. Ubiquitins with a mutation of the K6 residue had similar phenotypes as K6-biotinylated ubiquitin. K6-mutated (K6A, K6R and K6W) ubiquitins 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 K6-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 the ubiquitin-dependent proteolysis, and enhanced susceptibility to oxidative stress. Taken together, these results show that K6-modified ubiquitin is a potent and specific inhibitor of ubiquitin-mediated protein degradation.