LUBAC Mediated Validation Of RabGEF1 Linear Ubiquitination

Research background: Protein ubiquitination is an important post-translational modification that participates in a variety of cellular biological activities.Ubiquitin is a small-molecule protein that can be modified by binding to substrate proteins in a single or aggregated form,and thus ubiquitination can be divided into monoubiquitination and polyubiquitination.Ubiquitination is a complex process catalyzed by a cascade of ubiquitin-activating enzymes(E1),ubiquitin-conjugating enzymes(E2),and ubiquitin ligases(E3)Ubiquitin molecules are covalently bound to substrate molecules to regulate protein function.Ubiquitin has seven lysine(Lys)residues(Lys6,Lys11,Lys27,Lys29,Lys33,Lys48 and Lys63),and previous studies have found that ubiquitination is modified by one of these seven Lys residues.In recent years,a new ubiquitination modification was discovered through the formation of methionine(Met)at the N-terminus of ubiquitin,called linear ubiquitin modification(linear ubiquitin modification).Linear ubiquitination assembly complex(LUBAC)is the only known ubiquitin ligase that can catalyze linear ubiquitination so far.It consists of catalytic subunit HOIP and two regulatory subunits SHARPIN and HOIL-1L.composed.Current studies have found that linear ubiquitination modification is involved in the activation of nuclear factor-κb(NF-κB)and mitogen-activated protein kinases(MAPKs),inflammasome activation in macrophages,selective autophagy,interferon signaling,etc.With regard to important roles in innate and adaptive immunity,loss of function of any one of the subunits in LUBAC can cause inflammation,immunodeficiency and even embryonic death in mouse models or humans.However,there are not many substrates identified so far.In this study,the linear ubiquitin chain assembly complex LUBAC was used as the entry point to explore new linear ubiquitinated substrates by studying HOIP interacting proteins,and further study their biological functions.Research purpose: To verify RabGEFl(Rabguanine nucleotide exchange factor 1)as a new substrate for linear ubiquitination modification.Research methods: The RabGEF1 protein,the interacting protein of the catalytic center HOIP of LUBAC,was selected from the BioGRID database.The RabGEF1 gene fragment was amplified by PCR technology,and digested with two restriction endonucleases,EcoRI and Bam HI,to obtain the RabGEF1 gene fragment and pEF6/Myc-His C vector with the same restriction site.In this system,the human RabGEF1 gene fragment was inserted into the pEF6/Myc-His C vector,and the pEF6/Myc-RabGEF1 plasmid was constructed to facilitate subsequent experiments.The interaction of RabGEFl and HOIP was verified by co-immunoprecipitation experiments(Co-IP).The interaction domain of RabGEF1 and HOIP was explored by GST-pulldown experiment.The interaction and subcellular localization of RabGEF1 and HOIP were verified by immunofluorescence experiments.The linear ubiquitination modification of RabGEF1 was detected by in vivo ubiquitination assay.The ubiquitinated protein samples of RabGEF1 were analyzed by mass spectrometry,and the RabGEF1 ubiquitination lysine site mutant plasmid was constructed according to the RabGEF1 ubiquitination site suggested by the mass spectrometry results.ubiquitination modification site.Findings: We found that RabGEF1 interacts with HOIP,and HOIP directly interacts with RabGEF1 through the ZF-NEF domain.RabGEF1 co-localizes with HOIP in the cytoplasm.LUBAC-mediated linear ubiquitination of RabGEF1 depends on LUBAC enzyme activity,and the ubiquitination modification site of RabGEF1 is K158.Conclusion: RabGEF1 is a new substrate for linear ubiquitination,and K158 is the site of linear ubiquitination.Figure 12 Table 16 Reference 42...