title: DUB-resistant ubiquitin to survey ubiquitination switches in mammalian cells.
abstract: The ubiquitin-modification status of proteins in cells is highly dynamic and maintained by specific ligation machineries (E3 ligases) that tag proteins with ubiquitin or by deubiquitinating enzymes (DUBs) that remove the ubiquitin tag. The development of tools that offset this balance is critical in characterizing signaling pathways that utilize such ubiquitination switches. Herein, we generated a DUB-resistant ubiquitin mutant that is recalcitrant to cleavage by various families of DUBs both in vitro and in mammalian cells. As a proof-of-principle experiment, ectopic expression of the uncleavable ubiquitin stabilized monoubiquitinated PCNA in the absence of DNA damage and also revealed a defect in the clearance of the DNA damage response at unprotected telomeres. Importantly, a proteomic survey using the uncleavable ubiquitin identified ubiquitinated substrates, validating the DUB-resistant ubiquitin expression system as a valuable tool for interrogating cell signaling pathways.
authors.affiliation: Department of Biochemistry & Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA.
authors.forename: Miklós
authors.initials: M
authors.lastname: Békés
authors.forename: Keiji
authors.initials: K
authors.lastname: Okamoto
authors.forename: Sarah B
authors.initials: SB
authors.lastname: Crist
authors.forename: Mathew J
authors.initials: MJ
authors.lastname: Jones
authors.forename: Jessica R
authors.initials: JR
authors.lastname: Chapman
authors.forename: Bradley B
authors.initials: BB
authors.lastname: Brasher
authors.forename: Francesco D
authors.initials: FD
authors.lastname: Melandri
authors.forename: Beatrix M
authors.initials: BM
authors.lastname: Ueberheide
authors.forename: Eros Lazzerini
authors.initials: EL
authors.lastname: Denchi
authors.forename: Tony T
authors.initials: TT
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D004249:DNA Damage; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D064570:Ubiquitin-Specific Proteases / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D018809:Proliferating Cell Nuclear Antigen; D025801:Ubiquitin; D064570:Ubiquitin-Specific Proteases