| Summary: | 博士 === 國立清華大學 === 生物資訊與結構生物研究所 === 103 === PART І. Proteomics study of oxidative stress, Src kinase inhibition (PP1) and Quercetin in H9C2 cardiomyocytes: a cell model of heart ischemia reperfusion injury and treatment.
Oxidative stress production of myocardial ischemia/reperfusion injury leads to protein phosphorylation in regulating gene expression, metabolism, cell adhesion and survival. In this thesis, we used hydrogen peroxide treatment of H9C2 rat cardiomyocytes as a model of oxidative stress in heart ischemia reperfusion injury. A proteomics approach using anti-phosphotyrosine affinity purification and LC-MS/MS was then used to identify the stress-induced protein phosphorylation. We showed that oxidative stress induces a robust tyrosine phosphorylation of multiple proteins in this cell type. Most of identified tyrosine phosphorylated proteins were relative to cell-cell junctions, the actin cytoskeleton and cell adhesion. This suggested that oxidative stress may have a profound effect on intercellular connections and the cytoskeleton to affect cell adhesion, morphology and survival. After stress-induced phosphotyrosine proteins were analyzed by STRING, Src kinase was shown to be a major upstream regulator of these events. Furthermore, immunofluorescence studies, fluorescent activated cell sorting and cell-based assays were used to demonstrate H2O2-induced modifications of cell adhesion structures and cytoskeleton, de-adhesion and apoptosis, which were reversed by treatment with the Src kinase inhibitor PP1 or quercetin. Moreover, quercetin likely blocked the H2O2-induced inflammatory response through STAT3 modulation, which also contributed in preventing ischemia/reperfusion injury in cardiomyocytes. These findings provide the critical role of Src kinase in oxidative stress-induced phosphorylation and cell damage in cardiomyocytes and suggested that targeting Src kinase or quercetin may be an effective strategy for preventing ischemia reperfusion injury in the heart.
PART П. The application of Proteomics for disease biomarker discovery
Cancer and diabetic are high incidence and mortality in worldwide; however, early detection, surgical resection and postoperative therapy can lead to survival improvement for cancer or diabetes. In recent study, body fluids of patient were used to screen markers, such as plasma, urine and cerebrospinal fluid. Here, plasma of critical limb ischemia (CLI), type 1 diabetic (T1DM), transition carcinoma cancer and uterine leiomyoma were collected and analyzed by 2D-DIGE and MALDI-TOF. Then, particular protein markers were found in specific diseases, such as dual adapter for phosphotyrosine and 3-phosphotyrosine and 3-phosphoinositide (DAPP1) in CLI, hemopexin in T1DM, selenocysteine-specific elongation factor in TCC and vitamin D-binding protein in uterine leiomyoma. Nevertheless, most identified plasma proteins are related to inflammatory responses and blood coagulation. Therefore, a cell-based platform was established to screen protein markers relating to gemcitabine (GEM)-induced drug resistance pancreatic cells and tumorigenic breast cells. In GEM-induced drug resistant pancreatic cells, ribonucleoside-diphosphate reductase large subunit significantly overexpressed and tumor suppressor protein p53 may interplay with GEM-induced pancreatic cell resistance. In addition, in breast cancer cells, the level of calcium-binding mitochondrial carrier protein SCaMC-1 in tumorigenetic breast cancer cells or breast cancer patients’ plasma was higher than that of normal cell or health donors’ plasma. These data demonstrate that plasma proteomics provides a lot of common proteins between various diseases, but a cell based strategy provides a good platform for specific protein markers discovery in particular disease and afterwards these protein markers are potential for disease screening.
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