title: Synthetic Uncleavable Ubiquitinated Proteins Dissect Proteasome Deubiquitination and Degradation, and Highlight Distinctive Fate of Tetraubiquitin.
abstract: Various hypotheses have been proposed regarding how chain length, linkage type, position on substrate, and susceptibility to deubiquitinases (DUBs) affect processing of different substrates by proteasome. Here we report a new strategy for the chemical synthesis of ubiquitinated proteins to generate a set of well-defined conjugates bearing an oxime bond between the chain and the substrate. We confirmed that this isopeptide replacement is resistant to DUBs and to shaving by proteasome. Analyzing products generated by proteasomes ranked how chain length governed degradation outcome. Our results support that (1) the cleavage of the proximal isopeptide bond is not a prerequisite for proteasomal degradation, (2) by overcoming trimming at the proteasome, tetraUb is a fundamentally different signal than shorter chains, and (3) the tetra-ubiquitin chain can be degraded with the substrate. Together these results highlight the usefulness of chemistry to dissect the contribution of proteasome-associated DUBs and the complexity of the degradation process.
authors.affiliation: Schulich Faculty of Chemistry, Technion-Israel Institute of Technology , 3200008 Haifa, Israel.
authors.forename: Sumeet K
authors.initials: SK
authors.lastname: Singh
authors.affiliation: Department of Biology Technion-Israel Institute of Technology , 3200008 Haifa, Israel.
authors.forename: Indrajit
authors.initials: I
authors.lastname: Sahu
authors.affiliation: Schulich Faculty of Chemistry, Technion-Israel Institute of Technology , 3200008 Haifa, Israel.
authors.forename: Sachitanand M
authors.initials: SM
authors.lastname: Mali
authors.affiliation: Schulich Faculty of Chemistry, Technion-Israel Institute of Technology , 3200008 Haifa, Israel.
authors.forename: Hosahalli P
authors.initials: HP
authors.lastname: Hemantha
authors.affiliation: Department of Biology Technion-Israel Institute of Technology , 3200008 Haifa, Israel.
authors.forename: Oded
authors.initials: O
authors.lastname: Kleifeld
authors.affiliation: Department of Biology Technion-Israel Institute of Technology , 3200008 Haifa, Israel.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Glickman
authors.affiliation: Schulich Faculty of Chemistry, Technion-Israel Institute of Technology , 3200008 Haifa, Israel.
authors.forename: Ashraf
authors.initials: A
authors.lastname: Brik
mesh_terms: D000072017:Deubiquitinating Enzymes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015394:Molecular Structure; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism*; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D057149:Ubiquitinated Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*
chemical_list: D025801:Ubiquitin; D057149:Ubiquitinated Proteins; D000072017:Deubiquitinating Enzymes; D046988:Proteasome Endopeptidase Complex