| The thesis mainly focused on the quantitative proteomics study by using amino acid-coded tagging in cell culture (AACT, also known as SILAC) strategy with the help of the mass spectrum. Based on this platform, three systematical researches were studied as follows:1) Investigated the TNF-αstimulated 14-3-3εinteracting partners changes; 2) Studied the global profile of cells resistance to TNF-a prolonged stimulation, also the 14-3-3εcomplex changes under this situation, and through this study it would provide more profound meaning for exploring cancer escape mechanism; Meanwhile,3) Applied iTRAQ labeling strategy to study the dynamic changes of 14-3-3εinteracting partners at different time points under the stimulation of TNF-α.In Chapter 1, recent research results and developments of proteomics and protein-protein interaction in strategy, technology and application fields were reviewed, especially emphasized currently broadly applied amino acid-coded mass tagging (AACT/SILAC) based quantitative proteomics and interactomics approaches. Summarized the advantages/weaknesses of each technology, thus lead to the background of this thesis and illustrated the purpose and significance of our study.The research work of this dissertation was composed of three parts. The first part involved in TNF-αintroduced/introducible 14-3-3εinteracting partners, and in-depth study of particular complex in NF-κB activity regulation. The second part comprehensively studied cell tolerant response to TNF-αprolonged stimulation, and the 14-3-3εinteracting complex changes during this process. The third part studied the dynamic changes of 14-3-3εcomplex at different time points of TNF-αstimulation. The details were described in the fallowing: 1.14-3-3 epsilon dynamically interacts with key components of MAPK signal module for selective modulation of the TNF-α-induced time course-dependent NF-κB activityInflammation is tightly regulated by NF-κB if left unchecked excessive NF-κB activation for cytokine overproduction can lead to various pathogenic consequences including carcinogenesis. A proinflammatory cytokine, tumor necrosis factor-α(TNF-a), can be used to explore possible mechanisms whereby unknown functional pathways modulate the NF-κB activity for regulating TNF-α-induced inflammation. 14-3-3 proteins are highly conserved acidic soluble proteins, existing studies have shown that 14-3-3 proteins involve in the regulation of variety cellular biological process by mediating multiple intracellular signal transduction pathways, including inflammatory responses, mitogenic and cell survival signaling, cell cycle, apoptosis, growth, proliferation, DNA replication, DNA damage, signal transduction, etc. Given the multi-functional nature of 14-3-3 family proteins and recent finding of their presence in the TNF-α/NF-κB pathway network, we used a dual-tagging quantitative proteomic method, and stably expressed flag-tagged 14-3-3ε293T cells as the study object, using co-immunoprecipitation technology, to first profile the TNF-αinducible interacting partners of 14-3-3ε, the least characterized 14-3-3 isomer in the family. The epitope tag approach can increase the efficiency of co-immunoprecipitation and therefore enhance the possibilities of protein complex being identified; and the quantitative strategy of MS-based AACT/SILAC can effectively distinguish the specific protein partners from the non-specific backgrounds. Based on this dual-tagging strategy, we identified 55 TNF-αinducible 14-3-3εinteracting partners including the bait protein,17 proteins among them were the previously known interactors or the complex components with either 14-3-3 family members or 14-3-3ε, such as CDC37, Hsp90, RuvBL2, etc, new interactors such as TAK1, PPM1B, TBK1, etc were first reported by our study. We did immunoblot to validate the identified proteins and classified their biological process and functions. We chose the already known interactors Hsp90, RuvBL2, and also unknown ones such as DDX21, PPM1B, TAK1, TBK1 to do western blot, all results were consistent with the Mass data, fully proved the feasibility and reliability of mass-based dual tagging strategy in protein protein interaction study. And for the first time we found that TNF-αstimulation enhances the interactions between 14-3-3εand some key components in the MAPK signal module which is located at the immediate upstream of NF-κB, including transforming growth factor-beta activated kinase-1 (TAK1) and its interacting protein, protein phosphatase 2Cβ(PPM1B). By using confocal laser scanning we observed the TNF-α-induced co-localizations among 14-3-3ε, TAK1, and PPM1B and these interactions were also TNF-a-inducible in different cell types. Further, we found that during the full course of cellular response to TNF-αthe interactions between 14-3-3εand these two proteins were dynamic and were closely correlated with the time course-dependent changes in NF-κB activity, suggesting these 14-3-3εinteractions are the critical points of convergence for TNF-a signaling for modulating NF-κB activity. We then postulated a mechanistic view describing how 14-3-3εcoordinates its dynamic interactions with TAK1 and PPM1B for differentially modulating TNF-α-induced changes in NF-κB activity. By using bioinformatics tools we constructed the network involving most of 14-3-3εinteracting proteins identified in our proteomic study. We revealed that 14-3-3εcoordinate the cross talks between the MAPK signal module and other molecular pathways/biological processes primarily including protein metabolism and synthesis, DNA repair, and cell cycle regulation where pharmacological targets for therapeutic intervention could be systematically located.2. Quantitative study of cell tolerance to TNF-αprolonged stimulation and the 14-3-3εinteracting partners changes during this processInflammation is a complex response to infection and tissue injury. By far, TNF that was found to have the strongest activity in directly killing tumor cells, can lead cells to grow or apoptosis through different signal pathways. Because the inflammatory response causes marked changes in tissue physiology, dysregulated inflammation can lead to a variety of pathological conditions. Multiple mechanisms control the extent and duration time of inflammation response. Because of the regulations of these mechanisms, that contributes to the phenomenon ’tolerance’ :the transient unresponsiveness of cells or organisms to repeated or prolonged stimulation. With SILAC triple-labeling quantitative proteomics approach, we systematically studied the response of 293T cells to TNF-a prolonged stimulation. During the whole process of TNF-a prolonged stimulation, we finally identified 701 proteins changed. These proteins involved in metabolic process, cellular biosynthetic process, gene expression, transport, programmed cell death, regulation of catalytic activity, cell cycle, cellular response to stress, response to DNA damage stimulus, negative regulation of cellular function, negative regulation of cell death and other biological processes.225 proteins were resistant to TNF-a prolonged stimulation, mainly involved in metabolic process, cellular biosynthetic process, gene expression, transport, mitotic cell cycle, chromosome organization, negative regulation of cellular function, as well as the regulation of ubiquitin-protein ligase activity during mitotic cell cycle and so on. While there were 159 proteins continuing responsed to TNF-a prolonged stimulation, participating in transport, protein localization, cellular catabolic process, metabolic process, response to DNA damage stimulus, etc. Comparing the differences between tolerant proteins and non-tolerant proteins, we found that in order to survive from TNF-a prolonged stimulation, cell mobilized tolerant proteins to progress gene expression, biosynthesis, cell cycle and other similar functions, while the non-tolerant proteins involved in response to DNA damage stimulus and cellular catabolic process. All of these showed that these two groups of proteins might coordinate with each other to avoid apoptosis, which provides us an effective model and strategy to in-depth study the escape mechanism of cancer cells.Because of the multifunction of 14-3-3 proteins, they involve in many processes such as inflammation response, cell cycle, mitosis, apoptosis, etc. Therefore we applied mass spectrum-based in vivo dual-tagging strategy, using stably expressed flag-tagged 14-3-3ε293T cells to study 14-3-3εinteracting proteins’ changes during TNF-αprolonged stimulation. We identified 295 interacting proteins, they mainly involved in cellular metabolic process, gene expression, cellular biosynthetic process, complex assembly, chromosome organization, cellular response to stress, response to DNA damage stimulus, cell cycle, etc. Combined with the global profile data, we found that there were many overlaps between 14-3-3εinteracting proteins and whole cell proteins responsed to TNF-αprolonged stimulation in biological process. This suggests the multifunctional character of 14-3-3εand the crucial roles of 14-3-3εcomplex during TNF-αprolonged stimulation.3. The dynamic changes of 14-3-3εinteracting partners during different time points of TNF-αstimulationThe limitation of the past quantitative strategies is they only have two or three tags, which means you have to do more experiments to compare more than two samples. Whereas cellular biological process is dynamic, study protein interaction at a time point only shows the complex changes at that particular time, it’s just a small part of a cellular process, although it will reveal some phenomenon we still can’t get the whole picture. So we applied iTRAQ labeling strategy, using stably expressed flag-14-3-3ε293T cells, studied the dynamic changes of 14-3-3εinteracting partners during different time points of TNF-αstimulation. We found that proteins involved in regulation of cell death or mitotic cell cycle have a position in the changed proteins, indicating that 14-3-3εprobably plays key roles in cell surviving/dying through regulating its interactions with these two kinds of proteins during this process. |
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