Functional And Mechanistic Study Of IRAK Inhibitors On Neurite Outgrowth

Background and Aims:Neurites are the structural basis of mutual information exchange between neurons.External trauma or the incomplete development of neurons can interrupt and damage neural network.Therefore,it is essential to discover the effective factors that regulate neurite outgrowth and to explore the underlying molecular mechanisms.According to the published work,kinases,E3 ubiquitin ligases,and microtubule activity associated proteins are important regulators that affect neurite formation.Cereblon(CRBN)is the substrate receptor of the Cullin-4 RING E3 ubiquitin ligase(CRL4)complex,which recruits substrate proteins for their ubiquitination and proteasome-mediated degradation.Either loss or mutation of CRBN can lead to different degree of autosomal recessive nonsyndromic intellectual disability(ARNS-ID).Two CRBN mutants have been found to be associated with intellectual disability.Based on the previous work,we speculate that CRBN may play an important role in neuronal development and post-traumatic nerve regeneration.Phosphorylation is one type of the most important post-translational modifications(PTMs)that are involved in the regulation of most cellular processes.Defect in phosphorylation signaling pathways is a hallmark of cancer,neurodegenerative diseases,and many other diseases.IRAK1 is a serine/threonine kinase,which mainly involves in inflammatory responses mediated by Toll-like receptors(TLRs)or interleukin-1 receptor(IL-1R)family.Phosphorylation of E3 ubiquitin ligase Pellino family by IRAK1 can enhance their E3 ubiquitin ligase activity.Moreover,it has been reported that phosphorylation of substrate receptors(such as Speckle-type POZ protein,SPOP)in E3 ubiquitin ligase complex can alter the ligase activity.Analysis of data from PhosphoSite and published work revealed that CRBN could be phosphorylated.Therefore,phosphorylation of CRBN may regulate its E3 substrate receptor function and downstream signaling pathways.In order to identify the protein kinases that interact with CRBN,previously we performed biotin-based proximity labeling and mass spectrometry analysis and found several CRBN interacting kinases,including IRAK1,PKN1,NEK9,and MAPK1.However,it is unknown whether these kinases affect the neurite extension,whether CRBN is involved in this regulation and what the underlying molecular mechanism is.Therefore,in this thesis,we try to find the kinases regulating neuronal differentiation,then examine the role of CRBN,and explore the downstream signaling molecules of IRAK1/4 inhibitor I through stable isotope labeling by amino acids in cell culture(SILAC)quantitative phosphoproteomics.Methods:Firstly,FLAG-IRAK1/PKN1/NEK9/MAPK1 were co-transfected with HA-CRBN in HEK293T cells.The interaction between these kinases and CRBN was verified by immunoprecipitation and immunoblotting.Secondly,FLAG-tagged IRAK1,PKN1,NEK9,and MAPK1 were expressed in HEK293T cells,respectively,to examine their effect on endogenous Nogo A protein level.Thirdly,N2a and SH-SY5Y cell lines and primary mouse cortical neurons were treated with different concentrations of IRAK inhibitors to explore the regulatory effect of IRAK inhibitors on axonal growth.Fourthly,siRNA was transfected into N2a and SH-SY5Y cell lines to knock down CRBN,and then the effect of IRAK inhibitors on axon length was studied under the differentiation conditions.Fifthly,N2a cells were cultured in SILAC medium for more than six passages.N2a cells cultured with light lysine and arginine(K0:12C614N2;R0:12C614N4)were treated with DMSO,while N2a cells cultured with heavy lysine and arginine(K8:13C615N2;R10:13C615N4)were treated with IRAK1/4 inhibitor I.After 24 h of treatment,the cells were harvested and lysed.Cell lysates were digested by trypsin,and the resulting tryptic peptides were fractionated by high performance liquid chromatography(HPLC).Phosphopeptides were purified with immobilized metal affinity chromatography(IMAC).The whole proteome and phosphoproteome were analyzed by liquid chromatography and tandem mass spectrometry(LC-MS/MS).Proteins and peptides were obtained by searching MS data with MaxQuant against the UniProt mouse database.In the future,biochemical experiments will be performed to verify the results of LC-MS/MS and explore the function of proteins and phosphorylation on the neuronal development.Results:In our previous work,we identified several kinases interacting with CRBN by biotin-based proximity labeling and mass spectrometry analysis.Then we confirmed the interaction between IRAK1/PKN1/NEK9/MAPK1 and CRBN by immunoprecipitation and immunoblotting,and further found that IRAK1 upregulated Nogo A protein level.Inhibiting GSK-3β and IRAK1 by kinase inhibitors in N2a and SH-SY5Y cell lines and primary mouse cortical neurons could promote neuronal differentiation under the differentiation conditions.However,IRAK1/4 inhibitor I could still slightly promotes neurite outgrowth after CRBN knockdown although the axon length in the CRBN knockdown group was significantly shorter than that in the control group.Finally,we identified the proteins and phosphorylated substrates regulated by IRAK 1/4 inhibitor I in N2a cells through SILAC quantitative phosphoproteomics.Conclusions:We confirmed that IRAK1 could interact with CRBN and upregulate Nogo A protein level.Inhibition of IRAK1 by IRAK1/4 inhibitor I promotes the neurite outgrowth,probably by regulating downstream proteins including H2A2C,ERF 1,SCFD1,and THUM1,and phosphorylated peptides in proteins such as HMCN1 and UHRF2,and CRBN may partially participates in this signaling pathway.