Ubiquitinated Proteins in Exosomes Secreted by Myeloid-Derived Suppressor Cells

Abstract

We provide evidence at the molecular level that ubiquitinated proteins are present in exosomes shed by myeloid-derived suppressor cells (MDSC). Ubiquitin was selected as a post-translational modification of interest because it is known to play a determinant role in the endosomal trafficking that culminates in exosome release. Enrichment was achieved by two immunoprecipitations, first at the protein level and subsequently at the peptide level. Fifty ubiquitinated proteins were identified by tandem mass spectrometry filtering at a 5% spectral false discovery rate and using the conservative requirement that glycinylglycine-modified lysine residues were observed in tryptic peptides. Thirty five of these proteins have not previously been reported to be ubiquitinated. The ubiquitinated cohort spans a range of protein sizes and favors basic pI values and hydrophobicity. Five proteins associated with endosomal trafficking were identified as ubiquitinated, along with pro-inflammatory high mobility group protein B1 and proinflammatory histones.

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

“…However, the vesicle size distribution by transmission electron microscopy and Nanosight analysis, coupled with the immunogold detection of ubiquitin in membrane-bound vesicles Ͻ100 nm in diameter, strongly supports our conclusions of ubiquitylated proteins within exosomes. Furthermore, although this is the first study of ubiquitylated proteins from exosomes derived directly from human subjects, detection of ubiquitin in exosomes isolated from culture media of myeloidderived suppressor cells (23), human B-cells (HLA-DR15), or mouse immature splenic dendritic cells (D1) (24) further supports a model where deubiquitylation of protein cargo does not always occur during MVB formation. In addition to ubiquitylated cargo, our mass spectrometry data identified numerous nonubiquitylated peptides in low-density membrane fractions of human urine.…”

Post-translational add-ons mark the path in exosomal protein sorting

“…1). For example, myeloid-derived suppressor cells (MDSC) secrete exosomes enriched in ubiquitinated proteins, including endosomal trafficking proteins and histones [74]. Moreover, high molecular weight complexes of non-classical human leukocyte antigen-G (HLA-G) are ubiquitinated, but not glycosylated, in exosomes obtained from ascitic and pleural exudates of patients [75].…”

“…Of these, 21% are transmembrane proteins, underscoring that deubiquitination is not mandatory for the incorporation of protein cargo into MVBs and exosomes [84]. Although, ubiquitination-specific motifs remain unidentified, this PTM has preference for basic * More proteins idenƟfied in a high-throughput screening approach, proteomics [74,84,101,108,120]. # Mycobacterial proteins detected in exosomes from human and mouse cell lines.…”

Extracellular vesicle microRNAs mediate skeletal muscle myogenesis and disease

“…While the mechanisms of extracellular formation and secretion are not well-defined, evidence indicates that such vesicles possess the capability of ‘communicating’ with neighboring or distant cells by fusing with the plasma membrane and subsequently delivering their cargo, which consists of various molecules including proteins, mRNAs, and miRNAs (30,31). Moreover, transported miRNAs are capable of targeting mRNAs in recipient cells (30,32).…”