Daniele P Romancino1, Valentina Buffa1, Stefano Caruso2, Ines Ferrara1, Samuele Raccosta3, Antonietta Notaro1, Yvan Campos4, Rosina Noto3, Vincenzo Martorana3, Antonio Cupane5, Agata Giallongo1, Alessandra d'Azzo4, Mauro Manno3, Antonella Bongiovanni6. 1. Institute of Biomedicine and Molecular Immunology (IBIM), National Research Council (CNR) of Italy, Palermo, Italy. 2. UMR-1162, Functional Genomics of Solid Tumors, Inserm, Paris 1162, France. 3. Institute of Biophysics (IBF), National Research Council (CNR) of Italy, Palermo, Italy. 4. Department of Genetics, St. Jude Children's Research Hospital, Memphis, TN, USA. 5. Department of Physics and Chemistry, University of Palermo, Italy. 6. Institute of Biomedicine and Molecular Immunology (IBIM), National Research Council (CNR) of Italy, Palermo, Italy. Electronic address: bongiovanni@ibim.cnr.it.
Abstract
BACKGROUND: Virtually all cell types have the capacity to secrete nanometer-sized extracellular vesicles, which have emerged in recent years as potent signal transducers and cell-cell communicators. The multifunctional protein Alix is a bona fide exosomal regulator and skeletal muscle cells can release Alix-positive nano-sized extracellular vesicles, offering a new paradigm for understanding how myofibers communicate within skeletal muscle and with other organs. S-palmitoylation is a reversible lipid post-translational modification, involved in different biological processes, such as the trafficking of membrane proteins, achievement of stable protein conformations, and stabilization of protein interactions. METHODS: Here, we have used an integrated biochemical-biophysical approach to determine whether S-palmitoylation contributes to the regulation of extracellular vesicle production in skeletal muscle cells. RESULTS: We ascertained that Alix is S-palmitoylated and that this post-translational modification influences its protein-protein interaction with CD9, a member of the tetraspanin protein family. Furthermore, we showed that the structural organization of the lipid bilayer of the small (nano-sized) extracellular vesicle membrane with altered palmitoylation is qualitatively different compared to mock control vesicles. CONCLUSIONS: We propose that S-palmitoylation regulates the function of Alix in facilitating the interactions among extracellular vesicle-specific regulators and maintains the proper structural organization of exosome-like extracellular vesicle membranes. GENERAL SIGNIFICANCE: Beyond its biological relevance, our study also provides the means for a comprehensive structural characterization of EVs.
BACKGROUND: Virtually all cell types have the capacity to secrete nanometer-sized extracellular vesicles, which have emerged in recent years as potent signal transducers and cell-cell communicators. The multifunctional protein Alix is a bona fide exosomal regulator and skeletal muscle cells can release Alix-positive nano-sized extracellular vesicles, offering a new paradigm for understanding how myofibers communicate within skeletal muscle and with other organs. S-palmitoylation is a reversible lipid post-translational modification, involved in different biological processes, such as the trafficking of membrane proteins, achievement of stable protein conformations, and stabilization of protein interactions. METHODS: Here, we have used an integrated biochemical-biophysical approach to determine whether S-palmitoylation contributes to the regulation of extracellular vesicle production in skeletal muscle cells. RESULTS: We ascertained that Alix is S-palmitoylated and that this post-translational modification influences its protein-protein interaction with CD9, a member of the tetraspanin protein family. Furthermore, we showed that the structural organization of the lipid bilayer of the small (nano-sized) extracellular vesicle membrane with altered palmitoylation is qualitatively different compared to mock control vesicles. CONCLUSIONS: We propose that S-palmitoylation regulates the function of Alix in facilitating the interactions among extracellular vesicle-specific regulators and maintains the proper structural organization of exosome-like extracellular vesicle membranes. GENERAL SIGNIFICANCE: Beyond its biological relevance, our study also provides the means for a comprehensive structural characterization of EVs.
Authors: Joseph P Flemming; Brianna L Hill; Mohammed W Haque; Jessica Raad; Claudine S Bonder; Larry A Harshyne; Ulrich Rodeck; Adam Luginbuhl; James K Wahl; Kenneth Y Tsai; Peter J Wermuth; Andrew M Overmiller; Mỹ G Mahoney Journal: J Extracell Vesicles Date: 2020-07-13