Application Of Nanobodies To Modify E3-ligase Mediated EIF4E2-ISGylation And Investigation Of Its Function

Genes that are induced to be expressed when cells are stimulated by interferon are called IFN-stimulated genes(ISGs).Interferon-Stimulated Gene 15(ISG15)was one of the first ISGs to be identified.ISG15 affects various diseases such as cancer,stroke,inflammatory and neurodegenerative diseases by mediating ISGylation modifications.Experimental activation of ISGylation is generally accomplished by drug activation such as lipopolysaccharide and transfection of ISGylation modification systems,but drug activation is pathologically stressful ISGylation activation;cotransfection of ISGylation modification system plasmids also faces the problem of low efficiency of multiple plasmid transfection,and these two methods are global activation of These two methods activate ISGylation globally and cannot specifically activate ISGylation modification at a certain target site.In this paper,we have developed chimeric tools that activate target-specific ISGylation by screening for highly specific nanobodies that replace the substrate recognition region of E3 ligase.The eukaryotic translation initiation factor eIF4E2 is a homologous protein of eIF4E that regulates protein translation in response to stresses such as hypoxia.eIF4E2 is also capable of ISGylation modifications,and ISGylation modifications of eIF4E2 can enhance its cap-binding ability and thus participate in translation regulation.However,there is a lack of research on whether eIF4E2 ISGylation is involved in the regulation of certain physiological processes or the development of diseases.Ischemic stroke is a type of cerebrovascular disease with a high mortality and disability rate.When stroke occurs,a microenvironment of hypoxia and inflammation is created in the area of brain damage.eIF4E2,a translation initiator that responds to hypoxia,may be associated with the development of stroke.We therefore used eIF4E2 as a research target and obtained chimeric tools that specifically activate eIF4E2 ISGylation by screening to nanobodies specifically targeting eIF4E2 to modify the E3 ligase of ISGylation by phage and yeast surface display techniques.Microglia are a major group of cells in the central nervous system that are rapidly activated in response to brain injury and can exert anti-inflammatory or pro-inflammatory effects by altering their phenotype.In this study,we subjected mouse microglia BV2 to Oxygen glucose deprivation/re-oxygenation(OGD/R)treatment to simulate brain cell injury caused by cerebral ischemia,and used chimeras to activate eIF4E2 ISGylation in BV2 cells to investigate The results show that eIF4E2 ISGylation is an important component of stroke.The results show that activation of eIF4E2 ISGylation reduces OGD/R-induced reactive oxygen species(ROS)production and inhibits M1-type polarization in microglia BV2,while promoting M2-type polarization.We also examined the changes of inflammatory factors in BV2 cells after OGD/R treatment and eIF4E2 ISGylation chimera intervention by real-time fluorescence quantitative PCR.The results showed that OGD/R treatment increased the expression of pro-inflammatory factors IL-1β and IL-6 in BV2 cells,while decreasing the expression of anti-inflammatory factors TGFβ,IL-4 and IL-10,while eIF4E2 ISGylation was able to reverse this phenomenon in BV2 cells after OGD/R treatment.We therefore hypothesize that eIF4E2 ISGylation can reduce the production of ROS and pro-inflammatory factors by inhibiting the M1type polarization of BV2 cells after OGD/R treatment,while eIF4E2 ISGylation can also increase the expression of inflammatory factors to promote the M2-type polarization of BV2 cells,thus possibly exerting a neuroprotective effect.