Proteomics Resolve The Complex Of Tlr2 Of The Innate Immune Response And Tnf-¦Á Stimulation Of 14-3-3 Proteins

The thesis was focused on quantitative proteomics platform of mass spectrometry based amino acid-coded tagging(AACT) technique.The main contributions were to dissect the agonist-specific TLR-mediated innate responses on macrophages at subcellular resolution and to study the tumor necrosis factor alpha (TNF-α) stimulated 14-3-3 protein complex.Meanwhile,different from typical digestion methods,trypsin immobilization with microfluidic chip was also investigated to realize the fast,efficient proteolysis of complex biological samples, and compared the results with traditional methods.In Chapter 1,recent research progresses on quantitative proteomics and protein-protein interaction were reviewed and summarized in the context of their respective strengths/weakness,especially emphasized the application of major amino acid-coded mass tagging based quantitative proteomics approaches.In the end of this part,the purpose and significance of our study were introduced.The research work of this dissertation was composed of three parts.The first parts involved in the differential proteins profile of immune response of immune cells; The second part involved in the interactome of protein complex;The third part was to utilize the microfluidic chip to realize the proteolysis of the practical samples.The details were described in the following:(1) Proteomic dissection of agonist-specific TLR-mediated innate responses on macrophages at subcellular resolutionUpon stimulation by distinct bacterial/viral products/agonists,APC cells including macrophages tend to express different TLR molecules to coordinate their innate immune responses through the signal transduction pathways governing the activity of downstream transcriptional factors that can regulate particular sets of gene expression.Importantly,these inflammatory signals ultimately target at chromatin to induce characteristic alterations in gene expression.To understand the regulatory mechanisms of agonist-specific responses,at subcellular resolution we analyzed Pam3CSK4-,the TLR2 agonist-induced proteome changes in living macrophages and identified the differentially expressed proteins in the cytosol and chromatin-associated parts respectively by using AACT/SILAC-based quantitative proteomic approach.In our investigation,we quantified 540 of the Pam3CSK4-induced proteins among more than 1000 proteins identified.In cytosol fraction,we found the proteins with notable Pam3CSK4-induced expression changes were primarily involved in post-translational events,energy metabolism,protein transporting,and apoptosis,respectively.For those regulated proteins identified in the nuclear fraction we further integrated bioinformatics tools to discover the interconnected relationship of certain chromatin-associated proteins which suggested their involvements in proteasome-ubiquitin pathway,DNA replication,and post-translational activity upon Pam3CSK4 stimulation.Furthermore,certain regulated proteins in our quantitative proteomic dataset showed the similar trend of up-regulation in both Pam3CSK4- and LPS-stimulated macrophages(Nature,447,(7147):972,2007),suggesting their belonging to the recently identified class of pro-inflammatory genes.The regulatory discrepancy between both datasets for other set of genes indicated their agonist-specific nature in innate immune responses.(2) TNF-αinduced dynamic changes of 14-3-3 epsilon protein complex14-3-3 proteins are a family of highly conserved cellular proteins that play key roles in the regulation of central physiological pathways,including cell growth, proliferation,apoptosis,signal transduction.While TNF-αis a biologically active factor that is found to have the strongest activity in directly killing tumor cells to date. It can activate different signal pathways to lead to cell survival or apoptosis and one of the most important discoveries was to activate MAP3K signal pathway,recruiting 14-3-3s,and initiating NF-kB and corresponding gene expressions.In this part,dual tagging approach integrated AACT and epitope tagging was adopted and stably expressed flag-14-3-3 epsilon 293T cells were used as the study object.Under the stimulation of TNF-α,14-3-3 epsilon protein complex changes were investigated through co-immunoprecipitation(Co-IP) experimental method and MS-based quantitative proteomics technology.The epitope tagging can increase the efficiency of IP and therefore enhance the identification possibilities of protein complex;At the same time,the quantitative method of MS-based AACT can effectively differentiate the specific protein partners from the non-specific ones.Recurring to the strategy,55 of specific 14-3-3εinteraction proteins were identified including known protein partners such as CDC-37,HSP90,TAK1,TBK1 during the stimulation of TNF-α except for the bait protein.Many unknown interaction proteins,such as DDX21, RuvBL2,PPM1B and some ribosomal proteins were also identified.Further confirmation experiments were done and biological functions were analyzed.First, HSP90,TAK1 and TBK1 as the known interaction proteins were chosen to do WB to verify the feasibility and accuracy of this strategy.WB results showed the consistency with the MS results.Then,for the unreported interaction proteins,DDX21,RuvBL2 and PPM1B were done WB to further confirm the results.On the other hand,the composition and function of 14-3-3εprotein complex were compared before and after the TNF-αstimulation.As a result,the identified interaction proteins without stimulation mainly involved in the signal transduction,RNA folding,protein folding, redox-regulation,stress response,actin cytoskeleton and cellular energy metabolism; Whereas,after the TNF-αstimulation,the identified protein partners mainly participated in the TNF-αsignal pathway and NF-κB transcriptional regulation.The changes displayed the specificity of TNF-αand would have a profound impact on the understanding the network of intra-cell signal transduction.Furthermore,14-3-3s are associated with many tumor diseases,our results would potentially break a new pathway for the therapy of certain 14-3-3 related diseases.(3) The application of Au nanoparticles-assembly microfluidic reactor for efficient proteolysis of complex,practical samplesDifferent from the traditional digestion methods,we collaborated with Prof.Liu research group and tried the microfluidic enzyme reactor for the proteolysis of practically biological samples.First,hydrophilic proteins extracted from mouse macrophages were digested by both Au nanoparticles-assembly microfluidic reactor and conventional solution method,respectively.The obtained peptides were separated and analyzed using 2D-LC-ESI-MS/MS.194 and 170 of proteins identified by chip reactor and solution method showed the comparable performance between the two methods.Interestingly,it only took several seconds for the chip reactor to finish the proteolysis and displayed the obvious advantage compared with solution method.In the following work,the microfluidic reactor was utilized to proteolyze the total proteins from mouse macrophages and got 497 of confident proteins at one experiments.Compared with in-gel digestion,although the identified proteins number was less than that,different extraction reagents and different mass spectrometry may account for part of the reasons.The proteolysis application in the complex samples of Au nanoparticles-assembly microfluidic reactor proved that chip reactor was absolutely competent for the on-line proteolysis of practical protein samples and it was very efficient,fast,low cost and low sample consumption.Further improving the technology will broaden the application in the proteomics fields.