Establishment And Evaluation Of A TurboID Adjacent Protein Labeling System For Targeted Identification Of FLCN Interacting Proteins

Folliculin(FLCN)is a tumor suppressor gene that has been shown in deleted or mutation to cause Birt-Hogg-Dubé(BHD)syndrome,causing symptoms such as polycytumors in the kidneys,skin and lungs.TurboID is a biotin-adjacent protein labeling technology that efficiently medibiotinylation of endogenous proteins.Based on Bio ID technology,the biotinyase Bir A can be started with the addition of exogenous biotin,which has the advantages of simple operation and non-toxicity to cells.To explore the unknown function of FLCN,this study obtained more clues from the combination of mass spectrometry identification by screening FLCN interaction protein network using TurboID technology.First,the TurboID-FLCN adjacent protein labeling system was constructed.Second,we have designed different biotin addition time gradients to optimal labeling time.Then,Cell lysates were purified using streptavidin affinity media for biotin-labeled FLCN adjacent proteins.After SDS-PAGE isolation and Western blot identification,the purified biotinylated protein samples were subjected to LC-M S/MS identification and bioinformatics analysis and screening.Finally,co-immunoprecipitation and cellular immunofluorescence experiments of the selected FLCN interacting candidates of interest were performed to verify the protein interaction relationship.The results showed that we successfully constructed and realized the operation of TurboID-FLCN adjacent protein labeling system in HEK293 T cells,determined 4 h as the best biotin labeling time for this system.And the mass spectrometry identification results showed 1483 proteins in TurboID-FLCN and and 496 in Control groups under 4 h,which the number of proteins belonging to FLCN interacting proteins was 31 and 13,respectively.After comparing the TurboID-FLCN group with the control group,removing the background proteins and screening for the specific peptide number difference of the proteins in the identified results,33 unique interacting proteins were identified in the TurboID-FLCN group.The results of further GO and KEGG signaling pathway enrichment analysis showed that,In the GO-enriched cell composition analysis,TurboID-FLCN histones were significantly enriched in the cytoplasm,nucleus,nucleolus,nuclear chromosome,ribosome,and spliceosomal complexes.In the molecular function analysis,Including RNA binding,ATP binding,protein binding,nucleotide binding,m RNA binding,RNA helicase activity,ATPase activity and other functions.Of the biological processes,Protein accumulate significantly in translation,viral transcription,m RNA catabolism,m RNA splicing and processing,and protein transport.KEGG enrichment analysis revealed that ribosome versus ribosome biogenesis,RNA transport,spliceosome,Signaling pathways,such as protein export and DNA replication.Combined with GO analysis,we found that among 33 unique proteins,DDX 3 X not only gathered in nuclear,microtubule organization center,cytoskeleton,and centrosome with FLCN,but also exercise protein binding function to participate in many biological processes.However,the combined KEGG analysis found that EEF 2 is enriched in AMPK signaling pathway,which is of great significance for mining the molecular mechanism of FLCN in AMPK signaling pathway.Taken together,we selected DDX 3 X,EEF 2 as FLCN candidate interaction proteins,using Co-IP experiments and immunofluorescence experiment successfully verified the interaction with FLCN.This study further confirmed that TurboID can be used as an effective neighboring protein marker technology and can play a key role in exploring the direction of FLCN interacting protein.In addition,the two FLCN interacting proteins identified by the screen,EEF 2 and DDX 3 X,provide important clues to the further exploration of FLCN genes,including the regulatory network in which they are involved.Based on this study,it is speculated that,(1)FLCN activates EEF 2 K by interacting with EEF 2,which will disactivates EEF 2 and reduces the translation and synthesis of proteins during the excessive proliferation of tumor cells,then ultimately prevents tumor suppression.(2)After FLCN interacts with DDX 3 X,it suppresses its ability to activate the carcinogenic factor β-catenin and then suppresses tumor development.This will be further verified in the subsequent study of the research group.