The Mechanism of Mycobacterium smegmatis PafA Self-Pupylation

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.

Pupylation of PafA or Pup inhibits components of the Pup‐Proteasome System

“…To better understand how and why PafA is multipupylated, we examined the effect of the different Pup mutants on PafA autopupylation. Consistent with recent in vitro findings [9,20], a ladder of pupylated PafA was formed in the presence of wild-type Pup ( Fig. S7).…”

“…Consistent with monopupylation of PafA at multiple sites, the RRR mutant of Pup (which is unable to form polyPup chains) formed a distinctive ladder of PafA conjugates. Interestingly, although Msm PafA is predicted to contain 9 surface exposed lysine residues, the conjugation of Pup was largely restricted to three sites in PafA, presumably K320 (identified by Zhang and colleagues [9]) and two additional residues. Next, we examined the pupylation profile of PafA in the presence of the different single-and double-lysine mutants of Pup (Fig.…”

The Effect of Ubiquitin Like Protein-Proteasome System on the Drug Resistance of Isoniazid Mono-Resistant Mycobacterium tuberculosis

“…Posttranslational modification of target proteins with the help of Pup, not only made a substrate be degraded by the proteasome but also regulated the enzyme activity of PPS itself (19). It has been reported that PafA could be polypupylated by itself and PafA accumulated in the strain with the deletion of Mpa or prcBA (21). The results suggest that the pupylation of Mpa or PafA intends to maintain the stability of PPS.…”

Bacterial Proteasomes: Mechanistic and Functional Insights

“…Another major difference between the Ub and Pup systems is that while polyubiquitylation is generally required for substrate recognition and degradation in eukaryotes (70), polypupylation has been observed only in vitro or in bacteria overproducing Pup (71)(72)(73). While we cannot rule out the occurrence of polypupylation in a natural setting, the fusion of a single Pup to a protein is sufficient to target it for efficient proteasomal degradation (68).…”