title: Identification of ubiquitin-specific protease 9X (USP9X) as a deubiquitinase acting on ubiquitin-peroxin 5 (PEX5) thioester conjugate.
abstract: Peroxin 5 (PEX5), the peroxisomal protein shuttling receptor, binds newly synthesized peroxisomal matrix proteins in the cytosol and promotes their translocation across the organelle membrane. During the translocation step, PEX5 itself becomes inserted into the peroxisomal docking/translocation machinery. PEX5 is then monoubiquitinated at a conserved cysteine residue and extracted back into the cytosol in an ATP-dependent manner. We have previously shown that the ubiquitin-PEX5 thioester conjugate (Ub-PEX5) released into the cytosol can be efficiently disrupted by physiological concentrations of glutathione, raising the possibility that a fraction of Ub-PEX5 is nonenzymatically deubiquitinated in vivo. However, data suggesting that Ub-PEX5 is also a target of a deubiquitinase were also obtained in that work. Here, we used an unbiased biochemical approach to identify this enzyme. Our results suggest that ubiquitin-specific protease 9X (USP9X) is by far the most active deubiquitinase acting on Ub-PEX5, both in female rat liver and HeLa cells. We also show that USP9X is an elongated monomeric protein with the capacity to hydrolyze thioester, isopeptide, and peptide bonds. The strategy described here will be useful in identifying deubiquitinases acting on other ubiquitin conjugates.
authors.affiliation: 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.forename: Tânia
authors.initials: T
authors.lastname: Francisco
authors.forename: Tony A
authors.initials: TA
authors.lastname: Rodrigues
authors.forename: Marta O
authors.initials: MO
authors.lastname: Freitas
authors.forename: Manuel P
authors.initials: MP
authors.lastname: Pinto
authors.forename: Andreia F
authors.initials: AF
authors.lastname: Carvalho
authors.forename: Pedro
authors.initials: P
authors.lastname: Domingues
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Wood
authors.forename: José E
authors.initials: JE
authors.lastname: Rodríguez-Borges
authors.forename: Clara
authors.initials: C
authors.lastname: Sá-Miranda
authors.forename: Marc
authors.initials: M
authors.lastname: Fransen
authors.forename: Jorge E
authors.initials: JE
authors.lastname: Azevedo
mesh_terms: D000818:Animals; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000502:physiology; D004952:Esters / Q000378:metabolism; D005260:Female; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology; D008297:Male; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity / Q000502:physiology; D025801:Ubiquitin / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D004952:Esters; C000614264:PEX5 protein, human; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; C506234:USP9X protein, human; D025801:Ubiquitin; C554839:USP9X protein, rat; D043222:Ubiquitin Thiolesterase