UBE1L2, a Novel E1 Enzyme Specific for Ubiquitin*

Abstract

UBE1 is known as the human ubiquitin-activating enzyme (E1), which activates ubiquitin in an ATP-dependent manner. Here, we identified a novel human ubiquitin-activating enzyme referred to as UBE1L2, which also shows specificity for ubiquitin. The UBE1L2 sequence displays a 40% identity to UBE1 and also contains an ATP-binding domain and an active site cysteine conserved among E1 family proteins. UBE1L2 forms a covalent link with ubiquitin in vitro and in vivo, which is sensitive to reducing conditions. In an in vitro polyubiquitylation assay, recombinant UBE1L2 could activate ubiquitin and transfer it onto the ubiquitin-conjugating enzyme UbcH5b. Ubiquitin activated by UBE1L2 could be used for ubiquitylation of p53 by MDM2 and supported the autoubiquitylation of the E3 ubiquitin ligases HectH9 and E6-AP. The UBE1L2 mRNA is most abundantly expressed in the testis, suggesting an organspecific regulation of ubiquitin activation.

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

“…These results suggest that FAT10 is exclusively activated by Uba6. Consistent with earlier studies (13)(14)(15), ubiquitin was activated by both Uba6 and UAE (data not shown).…”

“…sc-67203) and Uba6 (Millennium). 15 N-Labeled FAT10 and ubiquitin internal standards were generated by expressing FAT10 or ubiquitin pDEST14 vectors into in Rosetta/DE3 cells (EMD Biosciences, Inc., Darmstadt, Germany) supplemented with BioExpress cell growth media (U-15 N, 98%, Cambridge Isotope Laboratories, Inc., Andover, MA). Proteins were purified using HiTrap SP column and Superdex 75 column (GE Healthcare).…”

“…Recently, Uba6 was identified as a second E1 in mammals that can efficiently activate ubiquitin (13)(14)(15). Interestingly, Uba6 was also found to be uniquely capable of activating a second UBL called FAT10 (13).…”

The ubiquitin-like modifier FAT10 in cancer development

“…Activation and conjugation of FAT10 to its substrate proteins is performed most probably by a three step enzymatic cascade, as described for ubiquitin. 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).…”

“…Moreover, inhibitors of the proteasome are already in use or under further investigation for the development of cancer therapies (Chen and Dou, 2010;Tu et al, 2012). Ubiquitylation as well as poly-ubiquitylation is achieved by an enzymatic cascade where first ubiquitin is adenylated at its C-terminal diglycine residue by one of its two ubiquitin E1 activating enzymes UBE1 (Ciechanover et al, 1981) or UBA6 (Jin et al, 2007;Pelzer et al, 2007) and then transferred onto the active site-cysteine of the respective E1 enzyme to form a thioester bond. In a second step, ubiquitin is transferred to the active site cysteine of a cognate ubiquitin conjugating enzyme (E2) by a transthiolation reaction.…”