Dop functions as a depupylase in the prokaryotic ubiquitin-like modification pathway

Area of Science:

• Molecular biology
• Bacteriology
• Protein biochemistry

Background:

• Post-translational modification with prokaryotic ubiquitin-like protein (Pup) is a bacterial process analogous to eukaryotic ubiquitination.
• Mycobacterial pupylation involves a two-step enzymatic pathway: deamidation of Pup by Dop, followed by ligation to substrates via PafA.

Purpose of the Study:

• To investigate novel functions of the Dop enzyme within the mycobacterial pupylation system.
• To elucidate the role of Dop in the Pup proteasome pathway.

Main Methods:

• In vitro enzymatic assays to assess Dop's depupylase activity.
• In vivo studies using mycobacterial systems to confirm Dop's function in protein degradation.
• Biochemical analysis of the interaction between Dop, Pup, and the proteasome.

Main Results:

• Dop functions as a depupylase, specifically cleaving the isopeptide bond to remove Pup from modified proteins.
• Depupylation activity of Dop was observed both in vitro and in vivo.
• The mycobacterial proteasomal ATPase Mpa enhances Dop's depupylation activity through its unfolding function.

Conclusions:

• Dop possesses a dual function as both a deamidase and a depupylase in the mycobacterial pupylation system.
• Dop's depupylase activity is an integral part of the Pup proteasome pathway, contributing to protein turnover.
• The interplay between Dop and Mpa highlights a coordinated mechanism for regulating protein modification and degradation in bacteria.