title: The de novo synthesis of ubiquitin: identification of deubiquitinases acting on ubiquitin precursors.
abstract: Protein ubiquitination, a major post-translational modification in eukaryotes, requires an adequate pool of free ubiquitin. Cells maintain this pool by two pathways, both involving deubiquitinases (DUBs): recycling of ubiquitin from ubiquitin conjugates and processing of ubiquitin precursors synthesized de novo. Although many advances have been made in recent years regarding ubiquitin recycling, our knowledge on ubiquitin precursor processing is still limited, and questions such as when are these precursors processed and which DUBs are involved remain largely unanswered. Here we provide data suggesting that two of the four mammalian ubiquitin precursors, UBA52 and UBA80, are processed mostly post-translationally whereas the other two, UBB and UBC, probably undergo a combination of co- and post-translational processing. Using an unbiased biochemical approach we found that UCHL3, USP9X, USP7, USP5 and Otulin/Gumby/FAM105b are by far the most active DUBs acting on these precursors. The identification of these DUBs together with their properties suggests that each ubiquitin precursor can be processed in at least two different manners, explaining the robustness of the ubiquitin de novo synthesis pathway.
authors.affiliation: 1] Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Portugal [2] Organelle Biogenesis and Function Group, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
authors.forename: Cláudia P
authors.initials: CP
authors.lastname: Grou
authors.affiliation: 1] Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Portugal [2] Organelle Biogenesis and Function Group, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
authors.forename: Manuel P
authors.initials: MP
authors.lastname: Pinto
authors.affiliation: 1] Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Portugal [2] Organelle Biogenesis and Function Group, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
authors.forename: Andreia V
authors.initials: AV
authors.lastname: Mendes
authors.affiliation: Mass Spectrometry Centre, UI-QOPNA, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Domingues
authors.affiliation: 1] Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Portugal [2] Organelle Biogenesis and Function Group, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal [3] Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal.
authors.forename: Jorge E
authors.initials: JE
authors.lastname: Azevedo
mesh_terms: D000818:Animals; D000072017:Deubiquitinating Enzymes / Q000235:genetics / Q000378:metabolism*; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008099:Liver / Q000378:metabolism; D051379:Mice; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D012269:Ribosomal Proteins / Q000378:metabolism; D025801:Ubiquitin / Q000096:biosynthesis* / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes / Q000378:metabolism; D054875:Ubiquitination; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*
chemical_list: D011498:Protein Precursors; C408062:RPS27a protein, human; D012269:Ribosomal Proteins; C521757:UBA6 protein, human; C526917:UCHL3 protein, mouse; D025801:Ubiquitin; D014452:Ubiquitins; C045677:ubiquitin precursor; D010450:Endopeptidases; C582107:OTULIN protein, human; D000072017:Deubiquitinating Enzymes; D043222:Ubiquitin Thiolesterase; C404328:Usp9x protein, mouse; C091939:ubiquitin isopeptidase; D044764:Ubiquitin-Activating Enzymes