Extablish SILAC Quantitative Technology To Find New Components Of CD40 Receptor Complex Recruited After Activation

Study of intracellular protein interaction is a hot area of life science.It helps us tounderstand protein function, clarify signaling pathway, then revealed the secret of life.With the advances in science and technology, there are a lot of technologies toresearch protein interaction, but they still have limitations.Therefore, how to study thedynamics interactions of protein is one of the core issues. Compared with the staticinteractions, dynamic protein interactions attract concerns of biologists.Many tumor necrosis factor receptor superfamily members play a vital role in theimmune response, among them,CD40 is an important promoter in MAPK,NFκB andnon- canonical NFκB pathway. CD40 mediated signaling pathway is essential notonly for the immune response, but also to autoimmune diseases, neurodegenerativediseases, cancer,et.al. Therefore, understanding of molecular mechanisms of CD40signaling pathways has become a research hotspot.The current study reveals the process of CD40 activation. First, CD40 recruitTNF receptor associated factors TRAFs directly. Second, TRAFs as adaptors,recruit molecules which are essential for downstream signal activation to form areceptor complex. Finally., some molecules of receptor complex activate downstreamsignaling pathway to exert CD40's biological effects. But CD40 on antigen-presentingcells activating signal mechanism is not entirely clear, Therefore, the study of CD40signaling complex composition and function will help to reveal molecular mechanismof transduction and modulation of CD40 signaling. The purpose of this paper is to use the latest technology of quantitativeproteomics to establish a set of technological system for studying dynamic proteininteraction,and identify the dynamic new composition of CD40 signal complexrecruited upon signal activation.Research methods of protein complex mainly include yeast two-hybrid, proteinchips, tandem affinity purification combined with mass spectrometry andbioinformatics-based analysis and other methods. In recent years, massspectrometry-based quantitative Proteomics technologies are developing rapidly,provides a new and reliable technologies for biologists to study protein interaction.The principle of these technologies is to enrich protein complex from control groupand experment group of protein, in which the background protein and non-specificprotein is equivalent exists, and the amount of specific complex components willchange(up or down) upon experimental treatment. So the background andnon-specific binding protein will be excluded, and the specific binding proteins willbe recognized.Quantitative proteomic technology based on Mass spectrometry include stableisotope labeling and lable free quantitative analysis method. The method of based onstable isotope labeling is divided into two class: one is chemical labeling technology,including Isobaric Tagging for Relative and Absolute protein Quantitation(iTRAQ)and Isotope-Coded Affinity Tagging( ICAT), another one is metabolic labelingtechnology. Such as Stable isotope labeling by amino acid in cell culture(SILAC).With characteristics such as high labeling efficiency, good reproducibility, smallcoefficient of variation and accurate quantitative results, SILAC is becoming a mosteffective way to analyse dynamic protein interactions. Its basic principle is using toincorporate stable isotope into cellular proteomes by adding isotope labeled aminoacids to growth medium-for quantitative analysis. For the analysis of proteininteractions, SILAC technology can overcome the limitations of traditional researchmethod such as yeast two hybrid, protein chip, etc., SILAC can further study theinteraction between endogenous proteins, discriminate effectively the background and real proteins interactions, identify dynamic binding protein, detected a weak orunstable protein interactions, and real-time analysis large-scale dynamic changes inprotein interactionsWe have Established SILAC quantitative technologies and used it identify newdynamic binding protein in CD40 signaling complex induced by CD40 activation. Weselected traditional SILAC experimental steps( PAM) and improved SILACexperimental steps(MAP), In three experiments, a total of 2,130 proteins wereidentified, among them 234 proteins were identified in all experiments, 310 proteinswewe quantified, 51 Up Regulated proteins (RATIO>1.3%)51proteins, 14 downRegulated proteins (RATIO<0.7%), In these proteins,some were reported to beinvolved in CD40 signal transcription pathway, such as MAPKK1, MAPKK3,TRAF2 and TRAF3, NFκBp100, NFκB Activator 1,Myb binding protein 1,Ubiquitin protease pathway related protein such as OTUB1, and NEDD4, also somehas other important function protein such as NEDD1, Sequestosome 1 and so on.Now proteins mentioned above are undergoing further validation as well as thefunction exploration. With Westen-blot, We verified the interaction between NEDD4and CD40, and provided an important clue for the CD40 signaling pathway.This study establishes a quantitative technology using SILAC for research ofCD40 signal complex, and identifies dynamic interaction of proteins in CD40signaling pathway activation, Not only provides key clues for the CD40 signalingpathway, more importantly, constructs preliminary a technology platform for studyingdynamic protein complex, and also offers a technological choice for other importantsignaling pathway.