Ubiquitin escapes the cell

As scientists begin to unravel ubiquitin signalling, a web of increasing complexity is unfolding for this signalling mechanism. Ubiquitinated proteins can be monoubiquitinated, multi-monoubiquitinated or polyubiquitinated. Polyubiquitination can take a range of topologies, dependent upon the lysine residue used for growth of the chains. Ubiquitin can itself be phosphorylated and cross-talk between ubiquitin and the kinase pathway and ubiquitin and SUMO has been revealed. Each of these is associated with different potential fates for the target protein.

Ubiquitinated proteins are secreted from cells in nanovesicles. But why?
Ubiquitinated proteins are secreted from cells in nanovesicles. But why?

Now a group of researchers from Denmark, Thailand and the US have peered into exosomes, tiny nanovesicles secreted from cells, to discover that ubiquitinated proteins can also leave the cell inside these packages, and that these ubiquitinated proteins possess an array of novel ubiquitination sites.

Exosomes are derived from the multi-vesicular body, which is a late endosomal compartment that can fuse with the lysosome for degradation of the contents of its lumen or with the cell wall for secretion. These small secretory vesicles can operate as a mechanism for intercellular communication and function in cancer progression, immune activation or evasion and the spread of infection. As part of this study researchers obtained urine samples from healthy volunteers and examined the exosomes secreted from epithelial cells in all segments of the kidney tubule and urinary tract. As the exosomes secreted into urine contain proteins and mRNA, the contents of these vesicles can provide information on the physiological state of the cells, including any disease states.

ESCRT is a protein located on the outer membrane of the multi-vesicular body that contains multiple ubiquitin binding domains. It has been thought that ubiquitination serves as a mechanism to package cargo proteins into exosomes for secretion: proteins bound for secretion in exosomes are ubiquitinated in the cytosol, associate with ESCRT for translocation to the multi-vesicular body and then are deubiquitinated by DUBs as they are loaded into internal vesicular vesicles for secretion. However, the team of researchers show that ubqiuitinated proteins are present within exosomes secreted into human urine. In fact, the presence of ubiquitinated proteins within urinary exosomes was so high, that analysis by mass spectrometry showed ubiquitin itself was the third most abundant protein in the exosomal fraction of urine samples.

The proteome of urinary exosomes revealed 5041 different proteins present within these vesicles, 619 of which were ubiquitinated. Among the ubiquitinated cargo proteins transcription factors were highly represented, as were proteins associated with ion or solute transport, though the reason for secretion of these proteins in their ubiquitinated form remains unknown.

Detailed analysis of the protein ubiquitination sites within urinary exosomal proteins showed that as many as 88% of the ubiquitination sites identified may be entirely novel. The researchers estimated that approximately 43% of the ubqituinated proteins were monoubiquitinated, 3% multi-monoubiquitinated and 54% polyubiquitinated or multi-monoubiquitinated in the presence of other post-translational modifications. A huge diversity in the polyubiquitin linkages found on the cargo proteins, with all seven different lysine linkages identified within the exosomes. K63 linkages were the most abundant, accounting for over half of all polyubiquitin linkages observed. This was followed by K48 and K11 linkages which together make up another 40%.

While it raises a lot of questions about the function of ubiquitin signalling in exosomes, this new study reveals that ubiquitinated proteins can be packaged into exosomes for secretion and offers the cell biology community a resource of numerous new ubiquitination sites for study. The team also suggest that the study of exosomal proteins may provide a non-invasive approach to the study of ubiquitin modifications in health and disease. The team have catalogued the full list of proteins and all associated information, and this is publicly available at http://interpretdb.au.dk/database/UbExo.  It seems that far from acting as a mere marker for protein degradation ubiquitin is applying new regulatory roles within the cell.