Abstract
Fetal growth restriction is characterized by impaired fetal growth and dysregulated lipid metabolism. Extracellular vesicles have been proved playing a crucial role in transporting biomolecules from mother to fetus. However, the mechanisms underlying cargo sorting and loading into trophoblastic extracellular vesicles remain elusive. The focus of our study is to examine how the essential fatty acid regulator, peroxisome proliferator-activated receptor gamma (PPARγ), is sorted and loaded into extracellular vesicles originating from trophoblasts. In this study, proteomic analysis was performed on placenta derived extracellular vesicles from normal and fetal growth restriction pregnancies. Interactions between PPARγ and COPI subunit were evaluated using co-immunoprecipitation and bioinformatics simulation. Molecular dynamics simulations were conducted to identify critical binding sites between β'-COP, a subunit of COPI, and PPARγ. lentivirus-mediated knockout and overexpression were employed to elucidate the role of β'-COP in PPARγ loading into extracellular vesicles. We have demonstrated that PPARγ protein levels were significantly decreased in fetal growth restriction placental extracellular vesicles. β'-COP subunit directly interacted with PPARγ in trophoblasts, mediating its sorting into early endosomes and multivesicular bodies for extracellular vesicle incorporation. Knockout of β'-COP impaired PPARγ loading into extracellular vesicles. Molecular dynamics simulations identified critical binding sites for β'-COP and PPARγ. Mutation of these sites significantly weakened the β'-COP-PPARγ interaction and reduced PPARγ levels in trophoblastic extracellular vesicles. In conclusion, β'-COP mediates sorting and loading of PPARγ into trophoblastic extracellular vesicles. This study provide insights into regulating extracellular vesicles cargo loading and potential strategies for targeted cargo delivery from maternal to fetus.