京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12802号 / 論医博第2074号 / 新制||医||1001(附属図書館) / 80846 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 戸口田 淳也, 教授 松田 秀一 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>The reversible phosphorylation of proteins is central to regulating most aspects of cell function. Malfunction in this critical cellular process have been implicated to cause diseases such as diabetes, cancer, and Alzheimer’s. Recent advances in mass spectrometry have made it possible to study this important post translational modification on a proteome-wide scale. However, to be able to do so, enrichment of phosphorylated peptides is required. Pairwise comparison of individual steps in an enrichment procedure and simultaneous improvement of data analysis resulted in a protocol which allowed high confidence identification of 2,131 unique phosphorylated peptides from 1,026 proteins. Thereby not only establishing a working protocol for phosphopeptide enrichment in the Griffin Lab, but also generating the largest list of proteins phosphorylated under normal conditions in yeast to date.</p>

The reversible phosphorylation of proteins is central to regulating most aspects of cell function. Malfunction in this critical cellular process have been implicated to cause diseases such as diabetes, cancer, and Alzheimer’s. Recent advances in mass spectrometry have made it possible to study this important post translational modification on a proteome-wide scale. However, to be able to do so, enrichment of phosphorylated peptides is required. Pairwise comparison of individual steps in an enrichment procedure and simultaneous improvement of data analysis resulted in a protocol which allowed high confidence identification of 2,131 unique phosphorylated peptides from 1,026 proteins. Thereby not only establishing a working protocol for phosphopeptide enrichment in the Griffin Lab, but also generating the largest list of proteins phosphorylated under normal conditions in yeast to date.

Atherosclerosis is a chronic inflammatory disease leading to plaque buildup in the major arteries. The plaques consist of cholesterol, calcium, inflammatory cells, extracellular matrix and fibrous material. Under inflammatory conditions IFN-• stimulation of human monocytes and macrophages generates reduced pteridine, 7,8-dihydroneopterin (78NP) which has been shown to be an effective cytoprotective agent to some cell types against oxidative damage by reactive oxygen species (ROS). 7,8-dihydroneopterin is oxidized to fluorescent neopterin in the presence of hypochlorite (HOCl). Although a considerable amount of work has been published on the composition of neopterin in atherosclerotic plaques, very little is known about the variation of 78NP and other oxidative biomarkers across the length of the carotid and femoral and their contribution to plaque progression, which was researched in this work. Atherosclerotic plaques excised from patients with carotid and femoral plaques were sliced into 3-5 mm sections, and each section was analyzed for concentrations of neopterin, 7,8- dihydroneopterin, •-tocopherol, TBARS, DOPA, cholesterol, dityrosine, protein carbonyls •- aminoadipic semialdehyde (AAS) and •-glutamic semialdehyde (GGS), free and esterified 7- ketocholesterol (7-KC). Cultured live plaque as a source of 7,8-dihydroneopterin and neopterin was also investigated in this study. It was shown that carotid plaques significantly vary from femoral plaques, in the levels and range of most oxidative biomarkers. Carotid plaques showed a high variation in the biomarker concentrations between plaques but also between sections of an individual plaque. Femoral plaques on the other hand showed lower amounts of biomarkers with very little variation in biomarker concentrations. High variation with pterin concentrations and other biomarkers suggests dynamic and active changes in inflammation within the plaque. Collectively, it was observed that every plaque was unique with respect to its composition and correlations between the biomarkers. Though shown to be a well-known antioxidant and a radical scavenger, there is no published literature on 7,8-dihydroneopterin’s mode of entry into and out of the cell. To understand how it enters the cells could explain the difference in its protective ability of different cell types Abstract xxviii against oxidative stress-mediated cell death. Knowledge of transport of 7,8-dihydroneopterin will provide insights about its protection of monocyte/macrophage cell death which could potentially reduce atherosclerotic plaque growth and progression. As 7,8-dihydroneopterin is produced from guanosine, a nucleoside that is transported using specialized nucleoside transporters (equilibrative nucleoside transporters (ENT's) and concentrative nucleoside transporters (CNT's), their role was examined and characterized for 7,8-dihydroneopterin transport. It was found that 7,8-dihydroneopterin and neopterin are transported via nucleoside transporters in U937 cells, THP-1 cells and human monocytes. ENT 2 was the major transporter in U937 cells while ENT 1 transported bulk of 7,8-dihydroneopterin in THP-1 cells. Both ENT's and CNT's are involved in 7,8-dihydroneopterin uptake in human monocytes. In all the cell lines tested, 7,8-dihydroneopterin protection against AAPH mediated oxidative cell death was inhibited by nucleoside transport inhibitors, suggesting that nucleoside transporters are indispensible for 7,8-dihydroneopterin mediated intracellular protection against oxidative stress. Accurate measurement of neopterin, as a biomarker of inflammation in plaques and cells is critical aspect to assess disease progression. The current C18 HPLC method used in our laboratory for neopterin measurement lacks sensitivity due to interference of acetonitrile (ACN) over time. Acidic tri-iodide conversion of 7,8-dihydroneopterin to neopterin was also variable at times giving inconsistent measurement of neopterin so the manganese oxide (MnO2) method was looked at as an alternative. Electrochemical detector (ECD) was another option studied as it did not require any precolumn oxidation of 7,8-dihydroneopterin to neopterin. A new method using strong cation exchange (SCX) column was developed for a precise, sensitive neopterin assay which got rid of the ACN interference completely. The MnO2 method of 7,8-dihydroneopterin oxidation did not work with biological samples such as serum or plaque homogenates. Electrochemical detection was also found to be very unreliable and inconsistent.