title: Targeted substrate degradation by an engineered double RING ubiquitin ligase.
abstract: Recognition of the substrates by ubiquitin ligases is crucial for substrate specificity in the ubiquitin-proteasome proteolytic pathway. In the present study, we designed a double RING finger ubiquitin ligase to direct the ubiquitin machinery to a specific substrate. The engineered ligase contains the RING finger domains of both BRCA1 and BARD1 linked to a substrate recognition site PCNA, which is known to interact with cyclin-dependent kinase inhibitor p57. The double RING finger ubiquitin ligase formed a homo-oligomer complex and exhibited significant ligase activity. Co-transfection of the ligase reduced the expression of transfected p57 to the background level in a proteasome-dependent manner and restored the colony formation ability of U2OS cells that is otherwise inhibited by overexpressed p57. The results indicate the ability of the engineered double RING ubiquitin ligase to target the intended substrate. By redesigning the substrate recognition site, expression of engineered double RING ubiquitin ligases may provide a useful tool for removing many different gene products at the protein level.
authors.affiliation: Department of Surgery, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawaski 216-8511, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Oyake
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nishikawa
authors.forename: Izumi
authors.initials: I
authors.lastname: Koizuka
authors.forename: Mamoru
authors.initials: M
authors.lastname: Fukuda
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Ohta
mesh_terms: D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D006868:Hydrolysis; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D015202:Protein Engineering; D013379:Substrate Specificity
chemical_list: D009097:Multienzyme Complexes; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases