title: 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.
authors.affiliation: The 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.
authors.forename: Xuejie
authors.initials: X
authors.lastname: Chen
authors.affiliation: The 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.
authors.forename: Chandan
authors.initials: C
authors.lastname: Li
authors.affiliation: The 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.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.affiliation: The 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.|Department of Bacteriology and Immunology, Beijing Tuberculosis and Thoracic Tumor Research Institute/Beijing Chest Hospital, Capital Medical University, Tongzhou District, Beijing, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Bacteriology and Immunology, Beijing Tuberculosis and Thoracic Tumor Research Institute/Beijing Chest Hospital, Capital Medical University, Tongzhou District, Beijing, China.
authors.forename: Chuanyou
authors.initials: C
authors.lastname: Li
authors.affiliation: The 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.
authors.forename: Junjie
authors.initials: J
authors.lastname: Zhang
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002853:Chromatography, Liquid; D008025:Ligases / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D020102:Mycobacterium smegmatis / Q000502:physiology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D059748:Proteolysis; D053719:Tandem Mass Spectrometry; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; D008025:Ligases; D008239:Lysine