Nonhydrolyzable Ubiquitin−Isopeptide Isosteres as Deubiquitinating Enzyme Probes.

Abstract

We demonstrate that oxime ligation is an efficient, straightforward, and generally applicable strategy for generating nonhydrolyzable ubiquitin (Ub)-isopeptide isosteres. We synthesized nonhydrolyzable K48- and K63-linked Ub-isopeptide isosteres to investigate the selectivity of deubiquitinating enzymes for specific linkages employing surface plasmon resonance spectroscopy. The results indicate that deubiquitinating enzymes specifically recognize the local peptide sequence flanking Ub-branched lysine residues in target proteins. The described strategy allows the systematic investigation of sequence requirements for substrate selectivity of deubiquitinating enzymes.

Generation of nonhydrolyzable ubiquitin–histone mimics

“…Several mimics of the native isopeptide bond have been introduced that are resistant to DUB cleavage, such as oxime or triazole linkages [24–26]. The approach we used to generate nonhydrolyzable Ub-histone mimics was derived from a strategy previously employed to generate diUb [27] and ubiquitinated PCNA [28] analogs.…”

Towards Elucidation of the Role of Ubiquitination in the Pathogenesis of Parkinson’s Disease with Semisynthetic Ubiquitinated α‐Synuclein

“…Several of these methods are based on the attachment of ubiquitin through non-native isopeptide bonds to generate enzymatically stable Ub conjugates of peptides or recombinant proteins. [4][5][6][7][8][9] The site-specific ubiquitination of peptides and proteins through the formation of a native isopeptide bond is now possible as a result of studies by Muir and co-workers, Liu and co-workers, and our research group. [10][11][12][13] We recently developed a highly efficient and chemoselective method to facilitate the site-specific ubiquitination of peptides and proteins.…”

Chemical Methods for Encoding and Decoding of Posttranslational Modifications

“…More recently, similar strategies have been developed that take advantage of the incorporation of azide- or alkyne-containing amino acids (Eger et al, 2011; Rösner et al, 2015; Sommer et al, 2011). Additional methods based on the use of nonnatural linkages continue to be developed and applied, including propanone (Yin et al, 2000) and oxime (Shanmugham et al, 2010) linkages.…”