Related Experiment Video

Updated: Mar 24, 2026

Preparation of Mycobacterium Tuberculosis Culture Filtrate to Understand TB Pathogenesis
07:32

Preparation of Mycobacterium Tuberculosis Culture Filtrate to Understand TB Pathogenesis

Published on: March 28, 2025

1.3K

The Mechanism of Mycobacterium smegmatis PafA Self-Pupylation

Xuejie Chen1, Chandan Li1, Li Wang1

  • 1The Key Laboratory of Cell Proliferation and Regulation Biology of Ministry of Education, Institute of Cell Biology, College of Life Sciences, Beijing Normal University, Beijing 100875, China.

Plos One
|March 9, 2016
PubMed

Insights

PafA self-pupylation involves intermolecular and intramolecular mechanisms, regulated by specific lysine residues and depupylase Dop. This process impacts PafA stability and auto-regulates the bacterial Pup-proteasome system.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Biochemistry

Background:

  • PafA is a prokaryotic ubiquitin-like protein (Pup) ligase essential for bacterial protein modification and proteasomal degradation.
  • M. smegmatis PafA undergoes poly-pupylation, a process whose mechanism requires elucidation.

Purpose of the Study:

  • To investigate the mechanism of PafA self-pupylation.
  • To identify key residues and regulatory factors involved in PafA poly-pupylation.
  • To understand the functional implications of PafA self-pupylation in vivo.

Main Methods:

  • Site-directed mutagenesis to identify target lysine residues.
  • In vitro pupylation assays to study intermolecular and intramolecular reactions.
  • Analysis of poly-Pup chain formation and regulation by depupylase Dop.
  • In vivo stability assays in a proteasome-dependent manner.

Main Results:

  • K320 is the primary site for PafA pupylation.
  • Self-pupylation initiates intermolecularly and elongates intramolecularly.
  • M. smegmatis Pup residues K7 and K31 are crucial for poly-Pup chain formation.
  • Depupylase Dop reversibly regulates poly-pupylation, with K7/K31 chains being more sensitive.
  • PafA self-pupylation enhances its stability in vivo via proteasomal degradation.

Conclusions:

  • PafA self-pupylation is a complex, regulated process involving specific lysine residues and Dop.
  • This auto-pupylation mechanism contributes to the stability and auto-regulation of the bacterial Pup-proteasome system.

More Related Videos

In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination07:58

In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination

Published on: January 2, 2026

589
Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens10:39

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Published on: June 12, 2013

14.1K

Related Concept Videos

The Proteasome02:18

The Proteasome

Eukaryotic cells can degrade proteins through several pathways. One of the most important amongst these is the ubiquitin-proteasome pathway. It helps the cell eliminate the misfolded, damaged, or unwarranted cytoplasmic proteins in a highly specific manner.
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. A series of enzymes carry out the ubiquitination of the target proteins - E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...
10.5K
The Proteasome01:13

The Proteasome

Eukaryotic cells can degrade proteins through several pathways. One of the most important among these is the ubiquitin-proteasome pathway. It helps the cell eliminate the misfolded, damaged, or unwarranted cytoplasmic proteins in a highly specific manner.
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. This involves participation of a series of enzymes including— E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...
2.0K
The Proteasome02:18

The Proteasome

5.0K
The Proteasome Structure01:17

The Proteasome Structure

The ubiquitin-proteasome pathway is a well-known mechanism utilized by eukaryotic cells to remove cytoplasmic proteins that are misfolded, damaged, or no longer needed. In this pathway, the protein that needs to be eliminated undergoes a process called ubiquitination, where a chain of ubiquitin molecules is attached to the 48th lysine residue of the target protein. This ubiquitin modification helps the proteasome distinguish between a target protein and a healthy protein.
The proteasome is an...
2.1K
Amino Acid Catabolism01:18

Amino Acid Catabolism

Microorganisms rely on proteins as an essential carbon and energy source, particularly in environments with limited polysaccharides or lipids. However, proteins are too large to cross the plasma membrane unaided, necessitating enzymatic degradation. Microbes secrete extracellular proteases and peptidases that hydrolyze proteins into peptides, which can then be transported across the membrane. Once inside the cell, intracellular proteases degrade these peptides into free amino acids, which...
1.6K
Export of Misfolded Proteins out of the ER01:32

Export of Misfolded Proteins out of the ER

After folding, the ER assesses the quality of secretory and membrane proteins. The correctly folded proteins are cleared by the calnexin cycle for transport to their final destination, while misfolded proteins are held back in the ER lumen. The ER chaperones attempt to unfold and refold the misfolded proteins but sometimes fail to achieve the correct native conformation. Such terminally misfolded proteins are then exported to the cytosol by ER-associated degradation or ERAD pathway for...
5.5K