Disruption Of Nuclear Pore Complexes And Nucleocytoplasmic Transport By OPTN-E50K Mutation

Nucleocytoplasmic transport,mediated by nuclear pore complexes(NPCs),is a highly regulated process that ensures the efficient transport of macromolecules,such as RNA and proteins,between the nucleus and cytoplasm.The transport of these molecules is essential for various cellular processes,including gene expression,cell signaling,and protein degradation.Abnormal nucleocytoplasmic transport is a common pathological feature of many neurodegenerative diseases,including amyotrophic lateral sclerosis(ALS),Huntington’s disease(HD),Alzheimer’s disease(AD),and Parkinson’s disease(PD).The mislocalization of nuclear pore proteins,such as nucleoporins,due to their aggregation in cells is one of the primary causes of nucleocytoplasmic transport defects in neurodegenerative diseases.Protein aggregates and other pathological hallmarks,such as inclusion bodies and Lewy bodies,have been observed in the brains of patients with neurodegenerative diseases.These aggregates can cause nucleoporin mislocalization and aggregation,leading to defects in nucleocytoplasmic transport.Glaucoma is the second leading cause of blindness worldwide characterized by the degeneration of retinal ganglion cells(RGCs)and their axons,leading to irreversible vision loss.Elevated intraocular pressure(IOP)is a primary risk factor for glaucoma,but other factors,such as genetics,vascular factors,and immune dysregulation,also contribute to its pathogenesis.The exact mechanisms underlying RGC death in glaucoma remain unclear,but several pathways have been implicated.Oxidative stress,excitotoxicity,neuroinflammation,and mitochondrial dysfunction have all been shown to play a role in RGC degeneration.These pathways can be triggered by elevated IOP,but they can also be activated by other factors,such as genetic mutations or aging.Several genetic mutations have been identified that increase the risk of developing glaucoma,including mutations in the optineurin(OPTN)gene,which is associated with normal-tension glaucoma.Multiple mutations in the optineurin gene have been identified in both familial and sporadic cases of primary open-angle glaucoma(POAG).Optineurin is involved in multiple cellular processes,including vesicular trafficking,autophagy,and regulation of NF-κB signaling.Optineurin E50 K is a genetic variant that has been strongly associated with the development of primary open-angle glaucoma.The E50 K variant is in the N-terminal coiled-coil domain of OPTN and is believed to impair its ability to bind to and regulate multiple interacting partners.This disruption of OPTN function may contribute to the degeneration of retinal ganglion cells and their axons that characterizes POAG.However,the precise molecular mechanisms underlying the pathogenic effects of OPTN E50 K are still not fully understood.Investigating the role of OPTN E50 K in the pathogenesis of POAG may offer insights into the molecular mechanisms underlying the disease and provide new avenues for the development of targeted therapies.Protein-protein interaction is the basis of cellular process and one of the core issues in the field.Proximity-dependent biotin identification(Bio ID)is widely used in the study of protein-protein interaction and becomes a valuable tool to identify protein-protein interaction in cells.This technique relies on the use of engineered biotin ligases that can selectively biotinylate proteins that are in close proximity to a target protein of interest(POI).When expressed in cells,the proteins interacting with POI,or the adjacent proteins could be labeled by excessive biotin.These biotinylated proteins were then purified by streptavidin beads and identified by mass spectrometry.By coupling this approach with mass spectrometry-based proteomics,it is possible to identify the proteins that interact with the target protein in a spatially and temporally controlled manner.Proximitydependent biotin identification has been successfully applied to study various biological processes,including signal transduction,chromatin remodeling,and membrane trafficking.Its high sensitivity and specificity make it a valuable tool for uncovering novel protein-protein interactions and elucidating the mechanisms underlying cellular function.In this study,we aim to utilize Bio ID method to identify the proteins that interact with optineurin.These interactors will be further validated using cell biology and molecular biology techniques.By constructing a complete optineurin interaction network and identifying candidate interactors,we aim to uncover a new molecular biological mechanism of glaucoma caused by OPTN-E50 K,providing a theoretical basis and potential therapeutic targets for the pathogenesis of OPTN-E50 K.To achieve this,we first cloned myc-Bio ID-OPTN plasmids to express a biotin ligase fused with OPTN-WT or its E50 K mutant in Neuro-2A neuroblastoma cells.Our results showed that the fusion of OPTN and Bio ID did not affect the normal localization of OPTN in cells.Western blot analysis showed that biotinylated proteins were enriched in myc-Bio ID-OPTN transfected cells.Overexpressing OPTN constructs showed a biotinylated pattern compared to the control group,indicating that biotinylated proteins were enriched in myc-Bio ID-OPTN transfected cells.The biotinylated proteins were identified by mass spectrometry,and the complete protein-protein interaction of OPTN-WT and OPTN-E50 K was established by bioinformatics analysis.Our bioinformatics analysis showed that OPTN-E50 K aggregates are enriched with the components of nuclear pore complexes and nucleocytoplasmic transport.The proteomics results were validated by fluorescence microscopy.To test whether OPTN interacts with nucleoporin complex and its component nucleoporin OPTN-WT or OPTN-E50 K were transfected with EGFP-Nups.Results showed that OPTN-E50 K led to changes in the morphology and localization of Nup50,Nup62,Nup98,Nup214,and Ranbp2,which all belong to the phenylalanine-glycine repeat(FG)nucleoporin family.Next,the endogenous FG-Nups were stained with m Ab414 antibody,which showed that OPTN-E50 K interacts with endogenous FG-Nups from the nuclear pore complexes.The FG Nups dissociated from NPC and entered the cytoplasm to produce abnormal aggregation.To examine the effect of OPTN-E50 K on transmembrane nuclear pore protein OPTN and POM121 were transfected into N2 a cells.POM121 localization was also destroyed by overexpressing m Cherry-OPTN-E50 K.We used anti-Lamin B antibody to detect the integrity of the nuclear Lamina and found that the nuclear membrane invaginated and misfolded,indicating that the cytoplasmic OPTN-E50 K aggregation would damage the nuclear morphology.To understand effect of OPTN function,we evaluated the nucleocytoplasmic transport function utilizing immunofluorescence staining and fluorescence in situ hybridization.To examine whether the nuclear protein entry function is affected by E50 K,we co-expressed EGFP or EGFP-OPTN or EGFP-OPTN-E50 K with fluorescent reporter protein NES-td Tomato-NLS in N2 a cells.The results showed that overexpression of OPTN led to the accumulation of reporter protein in the cytoplasm.To study whether OPTN affects m RNA transport,fluorescence in situ hybridization was used to quantitatively study the distribution of RNA.The results showed that the aggregation of OPTN in the cytoplasm caused poly(A)RNA retention in the nucleus,suggesting that nuclear RNA output was impaired.To elucidate the underlying mechanism of impaired m RNA transport,the critical proteins involved in m RNA transport,THOC2 and GLE1,were examined via immunofluorescence.The results showed that OPTN-E50 K coaggregated with THOC2 and GLE1,thereby compromising their regular function.This discovery introduces a novel perspective into the mechanism of OPTN-induced impaired RNA output function.Overall,we identified the composition of OPTN-E50 K aggregates by the proximity-dependent biotin identification method.Our results reveal that OPTN-E50 K interacts with various components of the nuclear pore complex.Additionally,the aggregated OPTN in the cytoplasm was observed to interfere with the regular nuclear-cytoplasmic transport,thereby potentially contributing to the pathogenesis of glaucoma.These findings provide novel insights into the biological mechanisms underlying glaucoma and suggest potential targets for therapeutic interventions.