Several publications indicate that the mutation of Gly75 and Gly76 of ubiquitin to Ala protects ubiquitin-fusion-proteins against deubiquitination.
However, in our case, the protein of interest, eventhough expressd as a genetic fusion to Ub(G75/76A), was only partially protected against deubiquitination. Still, the major part of it was deubiquitinated within S. cerevisiae cells.
Are there any other suggestions (like e.g. other residues than Ala used for the exchange of the Gly residues) to render a Ub-fusion protein stable against deubiquitination?
Try a proline as the immediate next residue after the Ub Gly76? I think it's still the case that DUB/UBP struggle to cleave Ub from a conjugate/fusion if the residue immediately following the Ub Gly76 in the conjugate is a proline. You should be able to do this easily enough as a genetic fusion. Maybe combine G75,76A with a proline immediately C-terminal to these residues - at P77?
I think human Unph (and the mouse homologue) can cleave adjacent to proline (J Biol Chem. 1997 Dec 19;272(51):32280-5) but is there a functional homologue in yeast? Varshavsky's stuff (Science. 1986 Oct 10;234(4773):179-86) using Ub-Pro-beta-Gal as a model substrate in yeast would tend to support the fact that DUBs in yeast are very poor at deconjugating linear ub-fusions if proline is immediately adjacent to Ub Gly76.
Can't think of anything else!
Hi Harald,
perhaps you can try the point mutation of the Ub C-terminal residue Leu73 to Phe or Tyr. It was observed in 2012 that this mutation is resistant to cleavage by DUBs.
Zhao et al., ACS Chem Biol. 2012 Dec 21;7(12):2027-35. doi: 10.1021/cb300339p.
Best, Antje
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