Nonenzymatic Rubylation and Ubiquitination of Proteins for Structural and Functional Studies

Abstract

Uncovering the mechanisms that allow conjugates of ubiquitin (Ub) and/or Ub‐like (UBL) proteins such as Rub1 to serve as distinct molecular signals requires the ability to make them with native connectivity and defined length and linkage composition. A novel, effective, and affordable strategy for controlled chemical assembly of fully natural UBL–Ub, Ub–UBL, and UBL–UBL conjugates from recombinant monomers is presented. Rubylation of Ub and Rub1 and ubiquitination of Rub1 was achieved without E2/E3 enzymes. New residue‐specific information was obtained on the interdomain contacts in naturally‐occurring K48‐linked Rub1–Ub and Ub–Rub1, and K29‐linked Rub1–Ub heterodimers, and their recognition by a K48‐linkage‐specific Ub receptor. The disassembly of these heterodimers by major deubiquitinating enzymes was examined and it was discovered that some deubiquitinases also possess derubylase activity. This unexpected result suggests possible crosstalk between Ub and Rub1/Nedd8 signaling pathways.

The use of unnatural amino acids to study and engineer protein function

“…Heterodimers of ubiquitin with Rub1, a ubiquitin-like protein homologous to Nedd8, have been produced using GOPAL and characterized by NMR and as substrate for deubiquitinases [35].…”

Profiling DUBs and Ubl-specific proteases with activity-based probes

“…Best studied is the crosstalk between Ub and SUMO signaling, which includes the identification of ubiquitinated SUMO and SUMOylated Ub (Hendriks et al, 2014;Hendriks & Vertegaal, 2016;. Furthermore, ubiquitinated Nedd8 and crosstalk between Ub and Nedd8 signaling pathways have also been reported (Leidecker, Matic, Mahata, Pion, & Xirodimas, 2012;Singh, Sundar, & Fushman, 2014), as the existence of ISGylated ubiquitin (Fan et al, 2015). However, these so-called heterogeneous chains have so far remained largely unstudied, and their functions remain unknown (Swatek & Komander, 2016).…”

Rub1/NEDD8, a ubiquitin-like modifier, is also a ubiquitin modifier

“…Since Rub1/NEDD8 lack a lysine residue at position 63, the direct outcome is on K63linked chains, however we observed an effect on other linkages as well, probably due to the different ability of E2 enzymes to modify Rub1. We conclude that although the two proteins are quite similar (Fig 1), sharing a similar 3-D fold and key residues on their surface that can be recognized by similar receptors or processing enzymes (16,61,92), Rub1 and Ub diverge in their potential to polymerize or form elongated chains.…”

“…Apparently, residue 72 is the key to be recognized by E1 (either UAE or NAE), however, some ubiquitin E2 conjugating enzymes do recognize residues other than 72 and therefore Rub1 that does get activated by UAE is distributed differently than ubiquitin in the ubiquitin landscape. Moreover, some deubiquitinases also recognize unique surface of Ub and hence cleave Ub-Rub1 or Rub1-Ub conjugates differentially (92), in effect guaranteeing a distinct fate for mixed UBL-Ub polymers over polyubiquitin (although CSN or proteasome are able to remove Rub1 from Ub (16)). In this manner, introduction of Rub1 mutated at position 72 is a new tool for specifically affecting the Ub system without greatly affecting the Rub1 signaling system because it is inert to NAE.…”