title: Method for measuring the activity of deubiquitinating enzymes in cell lines and tissue samples.
abstract: The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, techniques for studying the regulatory mechanism of this system are essential to elucidating the cellular and molecular processes of the aforementioned diseases. The use of hemagglutinin derived ubiquitin probes outlined in this paper serves as a valuable tool for the study of this system. This paper details a method that enables the user to perform assays that give a direct visualization of deubiquitinating enzyme activity. Deubiquitinating enzymes control proteasomal degradation and share functional homology at their active sites, which allows the user to investigate the activity of multiple enzymes in one assay. Lysates are obtained through gentle mechanical cell disruption and incubated with active site directed probes. Functional enzymes are tagged with the probes while inactive enzymes remain unbound. By running this assay, the user obtains information on both the activity and potential expression of multiple deubiquitinating enzymes in a fast and easy manner. The current method is significantly more efficient than using individual antibodies for the predicted one hundred deubiquitinating enzymes in the human cell.
authors.affiliation: Department of Neuroscience, University of Minnesota; Institute for Translational Neuroscience, University of Minnesota.
authors.forename: Percy
authors.initials: P
authors.lastname: Griffin
authors.affiliation: Department of Obstetrics, Gynecology, and Women's Heath, University of Minnesota.
authors.forename: Ashley
authors.initials: A
authors.lastname: Sexton
authors.affiliation: Department of Obstetrics, Gynecology, and Women's Heath, University of Minnesota.
authors.forename: Lauren
authors.initials: L
authors.lastname: Macneill
authors.affiliation: Department of Obstetrics, Gynecology, and Women's Heath, University of Minnesota; Masonic Cancer Center, University of Minnesota.
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Iizuka
authors.affiliation: Department of Neuroscience, University of Minnesota; Institute for Translational Neuroscience, University of Minnesota.
authors.forename: Michael K
authors.initials: MK
authors.lastname: Lee
authors.affiliation: Department of Obstetrics, Gynecology, and Women's Heath, University of Minnesota; Masonic Cancer Center, University of Minnesota; mbazzaro@umn.edu.
authors.forename: Martina
authors.initials: M
authors.lastname: Bazzaro
mesh_terms: D045744:Cell Line, Tumor; D006367:HeLa Cells; D006801:Humans; D011499:Protein Processing, Post-Translational; D025801:Ubiquitin / Q000032:analysis / Q000378:metabolism*; D064570:Ubiquitin-Specific Proteases / Q000032:analysis* / Q000378:metabolism*
chemical_list: D025801:Ubiquitin; D064570:Ubiquitin-Specific Proteases