Study On The Interaction Of 13-3-3ε Interaction Complex By Proteomics Combined With Biological Means

14-3-3s are a family of abundant,widely expressed28-33kDa acidic proteins. They are expressed in all eukaryotic cells and are highly conserved in protein sequence and function from yeast to mammals. There are seven isoforms have been identifed in human(β、γ、ε、η、σ、τ andζ) so far. Those14-3-3isoforms play redundant roles in widely biological processes including cell cycle control, intracellular signaling, stress response and apoptosis, transcriptional regulation, cellular metabolism and cytoskeletal integrity.14-3-3s involving in wide range of cellular processes is mainly via binding specific phospho-serine/threonine containing motifs on the target proteins.14-3-3s binding on target proteins can alter their catalytic activity, change their cellular localization, mediate them incorporating into protein complexes or induce their susceptibility to proteases and phosphatases.14-3-3s can bind and regulate many oncoproteins and tumor suppressor proteins, which indicates that14-3-3s might play important role in cancer. The conversion of a normal cell to a malignant, cancer forming cell is a multi-step process, including cell’s attainment of an uncontrolled or an autonomous proliferation capability, ability to evade immune system surveillance, ability to resist apoptosis and gaining of invasive and metastatic capabilities. Evidences showed that14-3-3s involve in every step of cancer cell forming and developing. For examples,14-3-3s interact with Raf, Bcr and Bcr-abl to regulate cell proliferation, interact with Bad, Bax and ASK-1to regulate cell survival and apoptosis, interact with Foxo to regulate gene transcription, interact with p53,TSC2and p27to suppress tumor growth, interact with CDC25,Weel and CHK1to regulate cell cycle progression and interact with integrins and Ron to regulate cell motility.As a general feature,14-3-3s usually positively regulate cell growth and survival and negatively regulate proteins involved in cell apoptosis and cell death, attributing them an oncogenic potential. However, various reports also showed that14-3-3s can negatively regulate signals from several growth factor receptors and attenuate cell cycle progression, especially following DNA damage, suggesting their tumor suppressing activity.Hepatoma is the most common malignancy in China. To date, the detailed pathogenies of Hepatoma are not very clear. On the other hand, more10percent of population in China is hepatitis B or hepatitis B virus carriers. Therefore, understanding the mechanism how hepatoma forming and developing is important and essential.Based on14-3-3s potential role in hepatoma forming and developing, we choose epsilon(ε) isoform, the most conserved member of14-3-3family as our bait protein to study its interacting network in hepatoma carcinoma cell(HCC)line-QGY-7703in native and bleomycin-induced DNA damage condition. To achieve the goal we set, we established the stable cell line expressing FLAG tagged14-3-3ε in HCC cells first. Then we used affinity purification combined with quantitative proteomics approach based on stable isotope labelling to dissect14-3-3ε interacting complexes in native and bleomycin-induced DNA damage conditions.The work summarized in this dissertation is divided into four parts. The detailed information of each part is described individually as below.(1) Studying cellular responses to bleomycin-induced DNA damage in different cell types.Bleomycin (BLM) is one kind of clinical chemotherapeutics used to treat testicular cancer and certain types of lymphoma. The mechanism of BLM inducing cancer cell death is attributing to it can generate DNA double-stand break(DSB) and DNA single-strand break(SSB)on chromosome. DSB is the major products induced by BLM.DSB is one of the most toxic and mutagenic type of DNA lesion.Therefore, cells evolved mechanisms including sensing DSB damage, inducing cell cycle arrest and activating DSB repair pathways to respond DSB lesions. Here, we used the optimized BLM dose to stimulate cells for different time points to observe the activated trends of potential DSB response markers. According to the experimental results, we observed the activation of H2AX and CHK1induced by BLM were time-dependent. H2AX activation is the commonly accepted biomarker for X-ray and Y-ray induced DSB lesion. BLM can induce DSB similar to X-ray and Y-ray, which indicates that H2AX activation can use as biomarker for BLM-induced DSB lesion. CHK1activation can be induced by SSB to arrest cell cycle progression. BLM can also induce SSB in cells, therefore, CHK1activation can also be considered as biomarker of BLM induced DNA damage.Our results also demonstrated that ERK1/2activation and IκB degradation induced by BLM is quite distinct among different cell types, which indicated that MAPKs activation induced by BLM were cell-type dependent.Our experiment is the first time to study BLM-induced DNA damage in a systematic manner. According to the results, we found the biomarkers responding to BLM-induced DNA damage which facilitates researchers involved in studying BLM to use those biomarkers more convincing. At the same time, those results provided us the optimized BLM stimulation time points for our following research.(2) Biological methods combined with quantitative proteomics approach to dissect14-3-3εinteracting complexes induced by bleomycin.DNA damage repair failure or repair pathways defect is the major factor contributing to induce cancer cell forming. Bleomycin(BLM)-induced cancer cell death is attributing to it can generate DSB and SSB lesion on cellular chromosomes. In cancer cells, DSB response mechanisms are also existing. BLM-induced DSB can activate DSB responses which include sensing DSB damage, arresting cell cycle and activating DSB repair pathways. During DSB responses, post-translational modifications on proteins are extremely wide. Phosphorylation is the highlighted one.14-3-3proteins prefer to bind on phosphorylated substrates. Therefore, It is reasonable to assume that14-3-3s play important role in response to BLM-induce DNA damage.Based on this assumption, we used affinity purification combined with Amino Acid-coded Mass tagging(AACT/SILAC)quantitative proteomcis appaoach to identify and quantitate the specifically BLM-induced14-3-3ε interacting complexes in HCC cell line.After data analysis, we identified32novel14-3-3ε interating partners. Then we used immunoprecipitation and the following immunoblotting to validate several potential14-3-3ε interacting partners including HDAC1,HDAC6, NONO, DDB1and TAB1. The results demonstrated that those proteins indeed are BLM-induced14-3-3ε interacting partners which were consistent with MS quantitation.Based on accuracy and reliability of our approach, we zoomed in another BLM-induced14-3-3ε interacting partner-TAK1to perform deep bilogical analysis. Our results demonstrated:1. TAK1kinase domain mediating its interacting with14-3-3ε;2. TAK1was activated by BLM and its activation resulted in increase of14-3-3ε binding;3.14-3-3ε binding on phosphorylated TAK1can maintain TAK1activation to promote cell survival upon BLM stress.(3) Studying MVP interacting with14-3-3ε induced by bleomycin.Vaults were characterised as the largest ribonucleoprotein particles (RNP) ever described. Vault comprises of at least one untranslated RNA and three major proteins-MVP,PARP4and TEP1, and assembles into a "capsule" shape with mass of about12.9MDa and a dimension of around41x41x71.5nm. Among those proteins, MVP represents more than70%of the vault mass and are responsible for assembling the whole vault outer shell. MVP lacks homologies to other proteins but itself is highly conserved among species. MVP/vault are widely expressed in many eukaryotic cells, due to MVP/vault’s distinct structure, it’s reasonable to consider it has conserved and important roles in cells. Unfortunately, MVP/vault function is less known so far.We were suprised to identify all components of vault when we used mass spectrometry to identify bleomycin-induced14-3-3ε interacting complexes, which attracted us to dig in depth to explore the biological consequences of14-3-3ε interacting with MVP/vault.Our data demonstrated:1.MVP indeed is bleomycin(BLM)induced14-3-3ε interacting parnter;2. According to protein sequence analysis and point mutation, we found two14-3-3ε binding motifs on MVP, T52and S864residues located on those motifs should be phosphorylated induced by BLM, and those two phosphorylated sites mediated14-3-3ε binding on MVP;3. MVP/vault functions as drug resistant proteins to inhibit BLM-induced DNA damage, while14-3-3ε can bind and inhibit MVP/vault to increase of BLM-induced DNA damage;4. The biological consequences of14-3-3ε interacting with MVP/vault are the common phenomenon in all tested MVP expressing cells.(4)Quantitative proteomics approach to dissect14-3-3ε native interacting complexes in hepatoma carcinoma cell(HCC).The conversion of normal cell to cancer forming cell is multiple-step process. Attaining uncontrolled proliferation ability, resisting apoptotic and gaining of invasive and metastatic capabilities are the major characteriscs of malignancy.14-3-3proteins are implicated in forming and developing of malignancy.Hepatoma carcinoma cell(HCC) is one kind of maligancy. To investigate the biological functions of14-3-3in HCC cells. We used the established stably expressing FLAG tagged14-3-3ε HCC cell line combined with Amino Acid-Coded Mass tagging(AACT/SILAC)quantiative proteomics approach to dissect14-3-3ε interacting complexes in native condition.After data analysis, We found36novel14-3-3ε interacting partners in HCC cells. Firstly, we used immuoprecipitation and immunoblotting to validate that14-3-3ε is14-3-3ε native interacting partner, which is consistent with MS quantitation. This results demonstrated that our experimental approach is accurate and reliable; Secondly, we classified those proteins according to their biological function, we found that more than10proteins interacting with14-3-3ε involved in cell motion and cytoskeletal organization,which indicated14-3-3ε incorporated into metastatic process of HCC cells; More than10proteins are involved in regulating cell death or cell apoptosis, which indicated that14-3-3ε contributes to help HCC cells resist apoptosis during malignization of HCC.