Proteomics

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Proteomic profiling of ubiquitylated proteins in urinary exosomes


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. Protein mass spectrometry identified >6000 proteins in human urinary exosomes, 15% of which were ubiquitylated with various topologies (K63>K48> K11>K6>K29>K33>K27). Ubiquitylated proteins involved in transcriptional regulation or solute transport were significantly enriched. Non-biased analysis of over-represented motifs uncovered a significant preference for basic amino acids upstream of the ubiquitylation site. The current studies demonstrate that in human epithelial cells, deubiquitylation of protein cargo is not necessary for MVB formation and exosome secretion and highlight that urinary exosomes are an enriched source for studying ubiquitin modifications in physiological or disease states.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Urine

SUBMITTER: Trairak Pisitkun  

LAB HEAD: Trairak Pisitkun

PROVIDER: PXD002645 | Pride | 2016-02-22

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
Exo01.mgf Mgf
Exo01.raw Raw
Exo01_105min.mgf Mgf
Exo01_105min.raw Raw
Exo02.mgf Mgf
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Publications

Deubiquitylation of Protein Cargo Is Not an Essential Step in Exosome Formation.

Huebner Alyssa R AR   Cheng Lei L   Somparn Poorichaya P   Knepper Mark A MA   Fenton Robert A RA   Pisitkun Trairak T  

Molecular & cellular proteomics : MCP 20160216 5


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 uri  ...[more]

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