Abstract
PafA, the prokaryotic ubiquitin-like protein (Pup) ligase, catalyzes the Pup modification of bacterial proteins and targets the substrates for proteasomal degradation. It has been reported that that M. smegmatis PafA can be poly-pupylated. In this study, the mechanism of PafA self-pupylation is explored. We found that K320 is the major target residue for the pupylation of PafA. During the self-pupylation of PafA, the attachment of the first Pup to PafA is catalyzed by the other PafA molecule through an intermolecular reaction, while the formation of the polymeric Pup chain is carried out in an intramolecular manner through the internal ligase activity of the already pupylated PafA. Among the three lysine residues, K7, K31 and K61, in M. smegmatis Pup, K7 and K31 are involved in the formation of the poly-Pup chain in PafA poly-pupylation. Poly-pupylation of PafA can be reversibly regulated by depupylase Dop. The polymeric Pup chain formed through K7/K31 linkage is much more sensitive to Dop than the mono-Pup directly attached to PafA. Moreover, self-pupylation of PafA is involved in the regulation of its stability in vivo in a proteasome-dependent manner, suggesting that PafA self-pupylation functions as a mechanism in the auto-regulation of the Pup-proteasome system.
Highlights
In eukaryotic cells, ubiquitin is post-translationally attached to many cellular proteins as a single moiety or in the form of polymeric chains
For protein expression in E. coli, the gene encoding M. smegmatis proteasomal accessory factor A (PafA) or deaminase of Pup (Dop) with N-terminal His6-tag was cloned into plasmid pACYCDuet-1 (Novagen), the gene encoding M. smegmatis PupE with N-terminal His6-tag was cloned into plasmid pET-28a (Novagen), and the gene encoding M. tuberculosis PanB with N-terminal His6-tag was cloned into plasmid pET21cc (Novagen)
The prokaryotic ubiquitin-like protein (Pup) protein used has a glutamate at its C-terminus, termed PupE, which can be directly conjugated to the lysine residues in target proteins by PafA
Summary
Ubiquitin is post-translationally attached to many cellular proteins as a single moiety or in the form of polymeric chains. The attachment of the ubiquitin chain, termed poly-ubiquitination, targets substrate proteins to undergo 26S proteasome-mediated degradation [1, 2]. Ubiquitination is carried out by three enzymes, namely, ubiquitin-activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase enzyme (E3). The E3 ligase is responsible for transferring ubiquitin from E2 to target substrates, endowing the ubiquitin-proteasome system (UPS) with its high substrate specificity [3]. A typical feature of most eukaryotic E3 ligases is targeting for degradation via self-catalyzed ubiquitination or through ubiquitin modification mediated by an external ligase, which plays a critical role in the regulation of the ubiquitin system [4].
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