SUMOylation regulates protein cargo in Astrocyte-derived small extracellular vesicles

  1. Anllely Fernández
  2. Maxs Méndez
  3. Octavia Santis
  4. Katherine Corvalan
  5. Maria-Teresa Gomez
  6. Peter Landgraf
  7. Thilo Kahne
  8. Alejandro Rojas-Fernandez
  9. Ursula Wyneken

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Abstract

Recent studies have described a new mechanism of intercellular communication mediated by various types of extracellular vesicles (EVs). In particular, 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. sEVs contain a molecular cargo consisting of lipids, proteins, and nucleic acids. However, the loading mechanisms for this complex molecular cargo have not yet been completely elucidated. In that line, the post translational modification SUMO (Small Ubiquitin-like Modifier) has been shown to impact the incorporation of select proteins into sEVs. We therefore decided to investigate whether SUMOylation is a mechanism that defines protein loading to sEVs. In order to investigate the role of SUMOylation in cargo loading into sEVs, we utilized astrocytes, an essential cell type of the central nervous system with homeostatic functions, to study the impact of SUMOylation on the protein cargo of sEVs. Following SUMO overexpression, achieved by transfection of SUMO plasmids or experimental conditions that modulate SUMOylation in primary astrocyte cultures, we detected proteins related to cell division, translation, and transcription by mass-spectrometry. In astrocyte cultures treated with the general SUMOylation inhibitor 2-D08 (2′,3′,4′-trihydroxy-flavone, 2-(2,3,4-Trihydroxyphenyl)-4H-1-Benzopyran-4-one) we observed an increase in the number of sEVs and a decreased amount of protein cargo within them. In turn, in astrocytes treated with the stress hormone corticosterone, we found an increase of SUMO-2 conjugated proteins and sEVs from these cells contained an augmented protein cargo. In this case, the proteins detected with mass-spectrometry were mostly proteins related to protein translation. To test whether astrocyte-derived sEVs obtained in these experimental conditions could modulate protein synthesis in target cells, we incubated primary neurons with astrocyte-derived sEVs. sEVs from corticosterone-treated astrocytes stimulated protein synthesis while no difference was found with sEVs derived from 2-D08-treated astrocytes. Our results show that SUMO conjugation plays a fundamental role in defining the protein cargo of sEVs impacting the physiological function of target cells.

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  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/15084675.

    This review resulted from the graduate-level course "How to Read and Evaluate Scientific Papers and Preprints" from the University of São Paulo, which aimed to provide students with the opportunity to review scientific articles, develop critical and constructive discussions on the endless frontiers of knowledge, and understand the peer review process.

    The work is of great relevance in the field of extracellular vesicles (EVs), as it explores the role of the SUMO (Small Ubiquitin-like Modifier) pathway in the packaging of post-translational proteins in these structures. The data corroborate other studies that seek to understand the packaging mechanisms of extracellular vesicles that can influence the function of vesicles and, consequently, their interaction with target cells, such as in astrocytes, enriching the knowledge of how these pathways are involved in the construction of EVs and modifications, in this case protein modifications.

    One detail that caught my attention was the terms used and I would like to comment on a few things: Extracellular vesicles are complex structures that carry information in cellular communication, being responsible for the exchange of molecules between cells, including proteins, lipids, and genetic material. Their function in cellular communication depends on the specific composition of each vesicle, which can vary depending on the type of cell that releases it, for example in the modulation of biological processes, such as the immune response. In addition, the field of study with EV is being increasingly explored, precisely because these small structures are mediators in such important functions in target cells. However, it is still an area that is expanding and many terms, nomenclatures, and methodologies are still being discussed. Therefore, the use of more general terms involving location, group, and nomenclatures can be strategic to avoid having data invalidated in the future. For example, today the nomenclature "microvesicles" has been invalidated, "exosomes" discouraged and "extracellular vesicles" is well accepted according to MISEV (Minimal Information for Studies of Extracellular Vesicles)3. Another suggestion is to be cautious when declaring the position of the components of these structures if there is no data presented that confirms whether it is inside, on the surface or if it is transmembrane in these released structures. I suggest that the authors perform an assay that really shows the location of the protein.

    The viability data should be presented in a graph to support the integrity of the extracellular vesicles obtained. Another control that I believe should be incorporated is the graph of the populations (size and quantity) by NTA obtained from a portion of the sample of extracellular vesicles used in the functional assay, with the corresponding results. Thus, the quantity of extracellular vesicles used can be calculated and also corroborates that the quantity of vesicles does not interfere with the effect on astrocytes, but rather on the proteins involved in SUMOylation.

    Finally, when performing functional assays with extracellular vesicles, it is important to incorporate rigorous controls to validate the results. The viability of the cells subjected to vesiculation can also be assessed using rezasurin, and data on the size and quantity of EVs can be presented using NTA (Nanoparticle Tracking Analysis) graphs to ensure that the samples used in the functional assays followed a methodology for good purification and preservation of the integrity of these vesicles. Since storage at -80°C can compromise the bilayer of the EV membrane, the results obtained for size and quantity may change, as well as the form of communication with the target cell of the assay. Immunofluorescence assays can complement the data and elucidate how these EVs interact with the target cell: membrane-membrane contact, fusion between membranes or internalization of these structures. These controls will allow a more accurate assessment of EV functionality and contribute to the understanding of their mechanisms of action. In addition, they can also open new research possibilities, such as the evaluation of the impact of EVs formed by the SUMO pathway in neurodegenerative diseases, such as Alzheimer's, where cellular communication and protein accumulation play key roles in disease progression. Studies in the literature mention disease progression related to the accumulation of beta-amyloid protein and other factors still under investigation, such as changes in the protein profile and receptors of microglia.

    Congratulations on your work! Here are some works that may be of interest to the group.

    1. The biology and function of extracellular vesicles in immune response and immunity Immunity, Volume 57, Issue 8, 1752 - 1768

    2. Challenges and directions in the study of cell-cell communication by extracellular vesicles.

    3. Nat Rev Mol Cell Biol, doi: 10.1038/s41580-022-00460-3 (2022)

    4. Welsh JA, et al.  Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. J Extracell Vesicles. doi: 10.1002/jev2.12451.

    5. Exploiting the biogenesis of extracellular vesicles for bioengineering and therapeutic cargo loading. doi: 10.1016/j.ymthe.2023.02.013.

    6. Anti-inflammatory clearance of amyloid-β by a chimeric Gas6 fusion protein. doi: 10.1038/s41591-022-01926-9.

    Competing interests

    The author declares that they have no competing interests.

  2. Version published to 10.1101/2020.09.15.298554v1 on bioRxiv