Recent progress in dissecting ubiquitin signals with chemical biology tools

Activity-based profiling of cullin-RING ligase networks by conformation-specific probes

“…Utilizing binders to recognize an activating PTM, or a conformation unique to active complexes, provides an alternative approach to select active complexes from amongst the broader cellular pool of constituent molecules. While the ubiquitin system has classically been probed by small-molecules reacting with enzyme catalytic cysteines, such as deubiquitinating enzymes and E3 ligases in the HECT, RBR, and RCR families [70][71][72][73][74] , we show utility of conformation-specific affinity probes to ensnare E3 complexes lacking residues easily targeted by reactive chemical moieties. Our approach selectively targeting a site-specific modification -and in so doing capturing activated forms of numerous CRL complexes with multiple cullin backbones, from across mammalian species, without prior engineering of the cellular system of interest and with a generally applicable proteomic platform -enables new insights into dynamic E3 ligase and targeted protein degradation pathways.…”

Rapid reconstitution of ubiquitinated nucleosome using a non-denatured histone octamer ubiquitylation approach

“…NDHOU approach aligns with prior strategies in producing a covalent bond akin to the native isopeptide linkage, with the DBA crosslinker proving more effective for ubiquitination than DCA [ 16 , 26 ]. Additionally, our approach uniquely facilitates the modification of histone octamers under non-denaturing conditions, a departure from the predominantly denaturing conditions of existing methods [ 19 , 20 ]. However, it is unfortunately limitation of our NDHOU method to add multiple ubiquitin to the octamer at same or different histones in one time due to the complexity of the products after cross-linking, which poses challenges during the purification stage.…”

“…Nonetheless, it remains unknown whether ubiquitination can be efficiently carried out on histone octamers in a soluble state in vitro. Moreover, in the context of histone ubiquitylation, the specificity of the reaction typically targets the ε-amino of lysine residues on histones and the G76 position on ubiquitin to form isopeptide bonds [19,20]. To facilitate this reaction in vitro, chemical approaches have been devised that utilize the sulfhydryl group of cysteine, employing hydrazide-, disulfide-, or 1,3-dichloroacetone (DCA)-derived isopeptide bond analogs [16,[20][21][22][23][24][25][26][27][28][29][30][31][32][33].…”

“…Moreover, in the context of histone ubiquitylation, the specificity of the reaction typically targets the ε-amino of lysine residues on histones and the G76 position on ubiquitin to form isopeptide bonds [19,20]. To facilitate this reaction in vitro, chemical approaches have been devised that utilize the sulfhydryl group of cysteine, employing hydrazide-, disulfide-, or 1,3-dichloroacetone (DCA)-derived isopeptide bond analogs [16,[20][21][22][23][24][25][26][27][28][29][30][31][32][33]. While disulfide-based ubiquitination modifications are unstable under reducing conditions, DCA-mediated ubiquitination of histones is stable, resistant to deubiquitination enzymes, and favorable for structural studies due to the bond's close resemblance to native isopeptide bonds.…”

Revealing the extracellular function of HMGB1 N-terminal region acetylation assisted by a protein semi-synthesis approach

“…Notably, a variety of proteins with complicated modications (e.g., glycosylation) are becoming available by means of chemical synthesis. [65][66][67][68][69][70][71] However, the advantages of chemical protein synthesis have not been fully realized since the process can be relatively burdensome and is oen difficult to initiate in a biological laboratory. Alternatively, chemical ligation enabled protein semi-synthesis, which harnesses the advantages of both organic synthesis and recombinant protein technology, presents a more feasible approach for biologists (small synthetic peptide parts will be more affordable or synthetically accessible by themselves).…”