Sumoylation regulates protein cargo in small extracellular vesicles

Recent studies describe a new mechanism of intercellular communication mediated by secreted extracellular vesicles (EVs). Exosomes are small EVs (sEVs) released to the extracellular environment by the fusion of the endosomal pathway-related multivesicular bodies (containing intraluminal vesicles) with the plasma membrane. Their diameter varies between 30 to 200 nm, although the presence of small vesicles in the same size range, but of other biological origin (such as the plasma membrane), cannot be discarded in biochemical sEV preparations. sEVs contain a molecular cargo consisting of lipids, proteins and nucleic acids. However, the loading mechanisms for these molecules have not been completely elucidated. In that line, the post translational modification SUMO (Small Ubiquitin-like Modifier) has been shown to impact the molecular cargo of sEVs. SUMO modification consists in covalent conjugation of K residues of target proteins with SUMO-1 or SUMO-2 proteins. This has been shown to constitute a sEV destination signal for selected proteins. Considering that protein SUMOylation determines the formation of multiprotein complexes through interactions of SUMO with SUMO interacting motifs (SIMs), SUMOylation of sEV proteins might define important changes in their protein cargo. Based on preliminary experiments supporting a critical role for SUMO conjugation in the biogenesis of sEVs we propose the following hypothesis: “SUMOylation regulates the protein cargo of sEVs and determines functional consequences in target cells”. To answer our hypothesis, SUMO-1 or SUMO-2 were expressed in HeLa cells. In cells expressing SUMO-2, an increase in cell proliferation was observed while the derived sEVs showed a tendency of increased protein load. In turn, HeLa cells incubated with sEVs derived from SUMO-2 expressing cells showed an increase in mitochondrial activity. To get a better insight into the underlying mechanisms, we analyzed the proteomics of these vesicles and found clusters of proteins related to cell cycle, protein synthesis and metabolism. Furthermore, sEVs derived from HeLa cells treated with 2-D08 (SUMOylation inhibitor) showed a decrease of protein load in the derived sEVs, while these vesicles provoke a decrease of cell proliferation in target cells. In turn, astrocytes are an essential cell type of the central nervous system with homeostatic functions. The sEVs derived from astrocytes transfected with SUMO-1 or SUMO-2 contained an increased protein cargo per vesicle. sEVs derived from SUMO-2 expressing astrocytes evoked an increase in cell proliferation in astrocyte target cells. By mass spectrometry, we detected clusters of proteins related with cell cycle, metabolism and protein synthesis. In astrocytes treated with 2-D08 we observed an increase of the number of released sEVs and a decreased protein cargo in these sEVs, similar to HeLa cells. To change the physiological environment of astrocytes, they were treated with the stress hormone corticosterone. We found an increase of proteins conjugated with SUMO-2 and these sEVs contained an augmented protein cargo. Furthermore, to test whether astrocyte-derived sEVs obtained in these experimental conditions have a consequence on neuronal functions, we incubated neurons with sEVs from astrocytes treated with corticosterone or 2-D08. We found increased protein synthesis in neurons incubated with sEVs derived from astrocytes that had been exposed to corticosterone. No difference was found in protein synthesis in neurons incubated with sEVs from astrocytes treated with 2-D08 compared to neurons without sEVs, suggesting that the inhibition of SUMOylation prevents the loading of proteins relevant for protein synthesis in sEVs.
Finally, we performed in vivo experiments with rats exposed to stress by movement restriction. After one stress session, the left insula of these animals showed an increase of SUMO-1. Taken together, our results show that SUMOylation contributes importantly to determine the protein cargo of sEVs, and this has important consequences on the modulation of target cell functions. This work contributes to a better understanding of SUMOylation regulated sEV protein loading and the possible impact of this mechanism in stress conditions.