Mechanistic Studies on Activation of Ubiquitin and Di-ubiquitin-like Protein, FAT10, by Ubiquitin-like Modifier Activating Enzyme 6, Uba6

Abstract

Background:The Uba6 pathway and its components play an important role in a variety of biological processes. Results: The mechanism of how Uba6 activates two distinct substrates, ubiquitin and FAT10, was characterized. Conclusion: Uba6 was shown to use a similar mechanism for activating both substrates with a greater affinity for FAT10. Significance: Relative levels of ubiquitin and FAT10 could regulate the Uba6 pathway in cells.

Mechanistic Study of Uba5 Enzyme and the Ufm1 Conjugation Pathway

“…1A). Release of 2 eq of AMP has been experimentally confirmed for Uba1 and Uba6 in previous studies (27,28). For Uba5, however, approximately 1 eq of AMP per monomer was produced in reactions with Ufm1 (Table 1).…”

“…Homogeneous Time-resolved Fluorescence (HTRF)-based UBa5-Ufc1 Transthiolation Assay-The HTRF assay was performed using a similar protocol previously developed for other E1 enzymes (24,25,27,28). Biotinylated Ufc1 and FLAGtagged Ufm1 were used for detection.…”

“…To measure the rate of adenylate formation, similar conditions were used as above except that reactions were quenched at varying time points. The reaction mixtures were analyzed, and the signals on the TLC plates were quantified as described previously (27,28).…”

The ubiquitin-like modifier FAT10 in cancer development

“…The FAT10 E1 activating enzyme UBA6 acts as a bi-specific E1 enzyme, because it is able to activate both, FAT10 and ubiquitin (Chiu et al, 2007;Pelzer et al, 2007). In doing so, UBA6 is able to distinguish between the two modifiers and binds FAT10 with a higher affinity than ubiquitin, although the adenylation and transthiolation reaction is slower for FAT10 than for ubiquitin (Gavin et al, 2012). The activated FAT10 is then transferred onto the active site cysteine of the likewise bi-specific E2 conjugating enzyme UBA6-specific E2 enzyme 1 (USE1, also called UBE2Z) which directly undergoes auto-FAT10ylation in cis, mainly on Lys323 Gu et al, 2007;Jin et al, 2007).…”

“…Potential therapeutic avenues aiming at FAT10-inducing pathways such as NF-B/STAT3 signaling as well as targeting the FAT10 conjugation pathway seem to hold promise in particular because the genetic ablation of FAT10 in mice does not cause severe phenotypes. A possible starting point for targeting the FAT10 conjugation pathway might be an inhibitor of the FAT10 E1 activating enzyme UBA6, developed by Gavin and colleagues, which was already successfully tested to inhibit ubiquitin activation by UBA6 (Gavin et al, 2012). However, since UBA6 is a bispecific E1 activating enzyme for FAT10 and ubiquitin, this option must be handled with care to minimize putative side effects.…”