Background: The multifunctional protein Alix is a bona fide extracellular vesicle (EV) regulator. Skeletal muscle (SkM) cells can release Alix-positive nano-sized EVs directly from their plasma membrane, offering a new paradigm for understanding how myofibres communicate within skeletal muscle and other organs. S-palmitoylation is a reversible lipid post-translational modification (PTM) that is involved in different biological processes, such as the trafficking of membrane proteins and stabilization of protein interaction. Methods: Here, we have evaluated the extent to which S-palmitoylation is functionally linked to Alix and EVs by: (i) a comparative analysis of publicly available palmitoyl- and exosome-proteome data sets and (ii) altering protein palmitoylation, using a specific inhibitor (2-Br-Palmitate; 2BP) and evaluating S-palmitoylation of Alix as well as its subcellular distribution and interaction in SkM cells. Results: We found a higher percentage of S-palmitoylated proteins in exosomes, compared to all the other cellular compartments. This finding suggests that this PTM could be a distinctive signature for exosomal proteins. By coupling bioinformatic observation with biochemical analyses, we have also determined that endogenous Alix undergoes S-palmitoylation. In particular, exosomal Alix is palmitoylated to a larger extent than cellular Alix, and the inhibition of palmitoylation altered its subcellular localization. Furthermore, endogenous Alix interacts with CD9, and S-palmitoylation supports this interaction, as it also does for tetraspanin complexes in the tetraspanin enriched microdomains. Summary/Conclusion: Thus, we propose that S-palmitoylation might regulate the proper function of Alix in facilitating interactions among exosome-specific regulators in SkM-derived exosome biogenesis. Essential discoveries related to SkM-derived EVs may help in designing engineered exosomes which can be employed in the tissue regeneration field, e.g. to help in recovery from muscle atrophy and/or injury. Funding: The research leading to these results has been funded by the Italian Ministry for Education, University, and Research in the framework of the Flagship Project NanoMAX.
S-palmitoylation is a post-translational modification of Alix that regulates its interaction with the CD9 tetraspanin
Antonella Bongiovanni
2018
Abstract
Background: The multifunctional protein Alix is a bona fide extracellular vesicle (EV) regulator. Skeletal muscle (SkM) cells can release Alix-positive nano-sized EVs directly from their plasma membrane, offering a new paradigm for understanding how myofibres communicate within skeletal muscle and other organs. S-palmitoylation is a reversible lipid post-translational modification (PTM) that is involved in different biological processes, such as the trafficking of membrane proteins and stabilization of protein interaction. Methods: Here, we have evaluated the extent to which S-palmitoylation is functionally linked to Alix and EVs by: (i) a comparative analysis of publicly available palmitoyl- and exosome-proteome data sets and (ii) altering protein palmitoylation, using a specific inhibitor (2-Br-Palmitate; 2BP) and evaluating S-palmitoylation of Alix as well as its subcellular distribution and interaction in SkM cells. Results: We found a higher percentage of S-palmitoylated proteins in exosomes, compared to all the other cellular compartments. This finding suggests that this PTM could be a distinctive signature for exosomal proteins. By coupling bioinformatic observation with biochemical analyses, we have also determined that endogenous Alix undergoes S-palmitoylation. In particular, exosomal Alix is palmitoylated to a larger extent than cellular Alix, and the inhibition of palmitoylation altered its subcellular localization. Furthermore, endogenous Alix interacts with CD9, and S-palmitoylation supports this interaction, as it also does for tetraspanin complexes in the tetraspanin enriched microdomains. Summary/Conclusion: Thus, we propose that S-palmitoylation might regulate the proper function of Alix in facilitating interactions among exosome-specific regulators in SkM-derived exosome biogenesis. Essential discoveries related to SkM-derived EVs may help in designing engineered exosomes which can be employed in the tissue regeneration field, e.g. to help in recovery from muscle atrophy and/or injury. Funding: The research leading to these results has been funded by the Italian Ministry for Education, University, and Research in the framework of the Flagship Project NanoMAX.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.