The Function And Mechanism Of ACE2 Palmitoylation And Engineering Extracellular Vesicles As COVID-19 Therapy

With the completion of vaccine development and comprehensive vaccination,as well as the reduced pathogenicity of dominant variants,global prevention and control of COVID-19 have been largely liberalized.However,in the post-epidemic period,SARS-CoV-2 may continue to cause localized infections with distinct periodicity.This phenomenon is believed to be attributed to the gradual waning of antibody titers and neutralizing efficacy conferred by vaccination or initial infection,thereby diminishing the protective immunity.Furthermore,it is crucial to emphasize that many specific mutations within the S protein of SARS-CoV-2,such as L452 R,E484Q,and P681 R,have been shown to significantly enhance viral transmissibility and immune evasion capabilities.Recurrent infections with the SARS-CoV-2 may result in varying degrees of immune impairment and long-COVID syndrome in the general population,while posing significant health threats to immunocompromised populations,as well as to the elderly undergoing immune senescence and suffering from underlying chronic conditions.Therefore,it is still necessary to explore and develop convenient,effective and precise strategies for the diagnosis and treatment of COVID-19.In this study,we have identified that ACE2,the crucial receptor required for cell invasion by SARS-CoV-2,is significantly regulated by palmitoylation modification for both its membrane localization and extracellular vesicles(EVs)targeting.Mass spectrometry analysis revealed that palmitoylation modifications occured at two major cysteine residues,Cys141 and Cys498.Further investigation using an si RNA library screening identified ZDHHC3 as the palmitoyltransferase specific for ACE2,and LYPLA1 as the specific de-palmitoylase.Considering the important regulatory role of palmitoylation on ACE2 membrane targeting and EV targeting,we designed and constructed a fusion protein,PM-ACE2,with a plasma membrane(PM)targeting sequence derived from the neuronal growth cone protein GAP43.This PM sequence undergoes robust palmitoylation modifications.Then we demonstrated that PM-ACE2 exhibits higher levels of palmitoylation and increased membrane localization.Moreover,PM-ACE2 showed increased and more widespread enrichment in extracellular vesicles.Therefore,we utilized mesenchymal stem cells to produce extracellular vesicles loaded with a substantial amount of PM-ACE2.Then we validate the neutralizing and blocking effects of PM-ACE2-EVs on viral infection,as well as the immunoprotective effects on lungs of h ACE2 transgenic mice using pseudovirus and authentic SARS-CoV-2.In summary,we have proposed that palmitoylation modification strictly regulates the membrane localization and EV-targeted transport of ACE2.We identified the major modification sites and specific enzymes involved,thus further elucidating the regulatory mechanisms of palmitoylation during SARS-CoV-2 infection.Moreover,we have established a standardized procedure for producing engineered extracellular vesicles enriched with ACE2 on their surface and demonstrated the potent neutralizing efficacy of these engineered EVs against SARS-CoV-2 in vitro and in vivo.This provides a novel antiviral strategy for COVID-19 treatment and offers new insights for the design of drugs to combat emerging unknown viruses.