title: Deubiquitylation of Protein Cargo Is Not an Essential Step in Exosome Formation.
abstract: Exosomes, derived from multivesicular bodies (MVBs), contain proteins and genetic materials from their cell of origin and are secreted from various cells types, including kidney epithelial cells. In general, it is thought that protein cargo is ubiquitylated but that ubiquitin is cleaved by specific deubiquitylases during the process of cargo incorporation into MVBs. Here, we provide direct evidence that, in vivo, deubiquitylation is not essential. Ubiquitin was detected within human MVBs and urinary exosomes by electron microscopy. Of the >6000 proteins identified in human urinary exosomes was mass spectrometry, 15% were ubiquitylated with various topologies (Lys63>Lys48> Lys11>Lys6>Lys29>Lys33>Lys27). A significant preference for basic amino acids upstream of ubiquitylation sites suggests specific ubiquitylation motifs. The current studies demonstrate that, in vivo, deubiquitylation of proteins is not necessary for their incorporation into MVBs and highlight that urinary exosomes are an enriched source for studying ubiquitin modifications in physiological or disease states.
authors.affiliation: From the ‡Department of Biomedicine and Center for Interactions of Proteins in Epithelial Transport, Aarhus University, Denmark;
authors.forename: Alyssa R
authors.initials: AR
authors.lastname: Huebner
authors.affiliation: From the ‡Department of Biomedicine and Center for Interactions of Proteins in Epithelial Transport, Aarhus University, Denmark;
authors.forename: Lei
authors.initials: L
authors.lastname: Cheng
authors.affiliation: §Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand;
authors.forename: Poorichaya
authors.initials: P
authors.lastname: Somparn
authors.affiliation: ¶Epithelial Systems Biology Laboratory, NHLBI, National Institutes of Health, Bethesda, MD 20892-1603.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Knepper
authors.affiliation: From the ‡Department of Biomedicine and Center for Interactions of Proteins in Epithelial Transport, Aarhus University, Denmark; trairak@gmail.com robert.a.fenton@biomed.au.dk.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Fenton
authors.affiliation: From the ‡Department of Biomedicine and Center for Interactions of Proteins in Epithelial Transport, Aarhus University, Denmark; §Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; trairak@gmail.com robert.a.fenton@biomed.au.dk.
authors.forename: Trairak
authors.initials: T
authors.lastname: Pisitkun
mesh_terms: D000328:Adult; D020816:Amino Acid Motifs; D004847:Epithelial Cells / Q000378:metabolism*; D055354:Exosomes / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology* / Q000378:metabolism; D013058:Mass Spectrometry; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods; D054875:Ubiquitination; D014452:Ubiquitins / Q000378:metabolism* / Q000652:urine; D014556:Urine / Q000737:chemistry*; D055815:Young Adult
chemical_list: D011506:Proteins; D014452:Ubiquitins