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.
Highlights
Exosomes are extracellular nanovesicles (20 –100 nm) that are secreted from various cells types in the body [1]
Ubiquitin Is Detected in Both multivesicular bodies (MVBs) and in Urinary Exosomes—Detection of ubiquitylated proteins in the low-density membrane fractions from urine supports a mechanism where ubiquitylated proteins are recruited to MVBs, where a proportion are sequestered into internal luminal vesicles (ILVs) without prior deubiquitylation before release into the urinary space as exosomes
In general [22], it is thought that protein cargo to be incorporated into urinary exosomes is ubiquitylated, recognized by the endosomal-sorting complex required for transport (ESCRT) apparatus on MVBs, and deubiquitylated by deubiquitylases during the process of cargo incorporation into MVBs (8 –11)
Summary
Urine Collection and Exosome Isolation—Urine was collected from healthy volunteers ages 19 – 45 years following the Danish guidelines for collection of biological materials according to the Act on Research Ethics Review of Health Research Projects, Act number 593 of 14 July 2011, section 14(3). Immunoblots of samples were generated by standard techniques and probed with a ubiquitin (P4D1) mouse monoclonal primary antibody (#3936, Cell Signaling Technology, Danvers, MA) at 1:250 dilution, followed by chemiluminescence detection. 50 g of proteins were separated by SDS-PAGE, stained with Coomassie (Imperial Protein Stain, Pierce) and fractionated into 32 pieces before in-gel digestion and mass spectrometry (MS) sample preparation as described [2]. Due to the higher charge states of the enriched ubiquitylated peptides, the full MS scan range of 300 to 1500 m/z was selected and the additional exclusion of ϩ2 precursor ions was implemented. Conserved domain analysis was performed by the Batch Web CD-Search Tool (http://www.ncbi.nlm.nih.gov/Structure/ bwrpsb/bwrpsb.cgi) [18]. Novelty of ubiquitylated sites were determined by comparison with ubiquitylation datasets
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