The Role Of Neddylation In The Regulation Of Protein Subcellular Translocation Between Cytoplasm And Nucleus

Background:Nedd8 is a ubiquitin-like protein that can covalently modify its substrates with the participation of E1 activating enzyme,E2 conjugating enzyme,and substrate-specific E3 ligase.This process is called Neddylation.The process by which Nedd8 is dissociated from the substrate is called de-Neddylation.This process requires the participation of de-Neddylation enzymes,JAB1(Jun Activation Domain-Binding Protein)or NEDP1(Nedd8-Specific Protease 1).JAB1 mainly mediates the de-Neddylation process of Cullin proteins,while NEDP1 participates in the de-Neddylation process of non-Cullin family.The main substrates of Nedd8 are Cullins,which serve as the scaffolds for Cullin Ring ubiquitin ligase.When Cullin is modified by Neddylation,the Cullin Ring E3 ligase will be activated,promoting the ubiquitination degradation process.Recently,more and more non-Cullin family proteins were found to be substrates of Nedd8,such as P53,MDM2,Smurfl,Smurf2 and so on.The Nedd8 pathway deletion has embryonic lethal effects in many species such as mice and nematodes.Nedd8 is highly expressed in oral squamous cell carcinoma and colorectal cancer.And the Nedd8 pathway is defective in neurodegenerative diseases such as Alzheimer's disease,Huntington's disease and Parkinson's disease.The proper localization of proteins within cells is of great significance for their biological functions.Post-translational modification of proteins can affect the subcellular localization of proteins,and more and more studies have found that ubiquitination has a regulatory effect on the subcellular localization of proteins.For example,the monoubiquitination of P53 promotes its export from the nucleus to the cytoplasm,anchoring P53 to the endoplasmic reticulum,and promotes apoptosis.The interaction between Neddylation and ubiquitination is very common.However,the current research on Neddylation is very limited.How exactly Nedd8 affects the subcellular localization of proteins is also worth further investigation.To answer this question,we tried to investigate the effect of Neddylation on the localization of proteins in the nucleus or cytoplasm using subcellular proteomics techniques that targeting Nedd8.Objective:To investigate the effect of Neddylation on protein subcellular localization in the nucleus and cytoplasm.Methods:Construct a subcellular protein quantification system targeting Nedd8:Wild-type or NEDP1 knocked out 293T cells were transfected with the Flag-Nedd8 or vector plasmid,these two cell lines were further subcellularly lysed to obtain cytoplasmic and nuclear protein respectively.Finally,quantitative analysis of Nedd8-related proteins in the nucleus and cytoplasm of these two cell lines were performed to obtain potential Nedd8 substrates and translocated proteins.The nucleus and cytoplasmic proteins were obtained by subcellular lysis technology;the Neddylation modification of proteins was detected by WB;the subcellular localization of TAK1 was detected by immunofluorescence technology.Results:1.After the NEDP1 knockout,the overall level,the nucleus and the cytoplasm levels of Neddylation were significantly up-regulated.2.Subcellular lysed proteins have specific cytoplasmic and nuclear distribution characteristics.Subcellular proteomics to predict the subcellular locations and the annotations of subcellular location of these proteins are consistent.3.By subcellular quantitative mass spectrometry data processing and screening,81 potential translocation proteins were obtained in this experimental system.4.TAK1,a translocated candidate in response to Neddylation,was proved to undergo Neddylation,and the nuclear TAK1 accumulated after being neddylatedConclusions:1.Quantitative proteomics at the subcellular level can effectively analyze proteins subcellular localization and their translocation between subcellular compartments.2.Increased Neddylation promotes nuclear-cytoplasmic translocation of proteins.3.TAK1 is a novel substrate of Nedd8.Neddylation of TAK1 promotes its transport from the cytoplasm to the nucleus.