Purchase this article with an account.
Allen Taylor, Ke Liu, Min-Lee Chang, Brigit Riley, Thomas Shaler, Kristie Rose, Kevin Schey, Fu Shang; Expression of K6W-mutant ubiquitin in human lens epithelial cells (HLEC) alters conformation and linkages of poly-ubiquitin chains. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4964.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Expression of ubiquitin in which lysine 6 is replaced by tryptophan (K6W-Ub) in HLEC or in lens in vivo perturbs the function of the ubiquitin-proteasome pathway (UPP) and results in accumulation of ubiquitin conjugates and typical substrates of the UPP. The objective of this work was to investigate the mechanism by which K6W-Ub impairs the function of the UPP.
HLEC were infected with adenoviruses that encode wild type ubiquitin (wt-Ub) or K6W-Ub. Ub conjugates were also formed in HLEC lysate with recombinant wt-Ub or K6W-Ub. Ub conjugates were isolated and characterized by Western blot with linkage-specific antibodies. Ubiquitinated peptides were isolated after trypsin digestion using a Gly-Gly antibody and analyzed by quantitative mass spectrometric analysis.
There are 7 lysines in ubiquitin, and thus 7 possible linkages for one ubiquitin links to another ubiquitin in polyubiquitin chains. When probed with linkage specific antibodies, it appeared that levels of K48-linked poly-Ub chain were dramatically reduced in conjugates formed with K6W-Ub. The levels of K11-linked poly-Ub chain also apparently decreased whereas levels of K63-linked poly-Ub chain apparently increased in conjugates formed with K6W-Ub as compared to conjugates that were formed with wt-Ub. To more accurately measure the effects of K6W-Ub on linkages of poly-Ub chains in the conjugates, poly-Ub linkages were quantitatively analyzed by mass spectrometry. Surprisingly, the mass spectrometry data showed that formation of K48-linked conjgautes was equivalent with K6W-Ub vs. wt-Ub. In comparison, levels of K63-linked chain did not change. Levels of K27-linked chains, K29-linked chains, K33-linked chains as well as K27/K29 linked fork chains increased when K6W-Ub was expressed.
These data suggest that K6W-Ub forms K48-linked poly-Ub chains, but that the conjugates have altered the conformation such that there is reduced affinity of K48 specific antibody for these conjugates. The altered conformation of K48-linked poly-Ub chains, together with the increase in formation of K27-, K29- K33-, and K27/K29-linked fork chains, may contribute to the resistance of K6W-Ub conjugates to proteasomal degradation and the accumulation of these conjugates in cells in which K6W-Ub is expressed.
This PDF is available to Subscribers Only