探討端粒錨定作用和熱量限制調控的相關性路徑

細胞至產生後會經歷一連串複雜的原因而逐漸走向喪失生理功能的過程，此過程稱之為老化。為了維持基因的穩定性，酵母菌Saccharomyces cerevisiae藉由將端粒連接在細胞核膜上，以避免進行不正常的telomere-telomere recombination。過去的研究指出當酵母菌中的tlc1缺失時，端粒的縮短會促使細胞啟動telomere-telomere recombination來維持端粒的正常長度，並產生type I 和II survivors。所以我從兩個錨定(anchorage)作用途徑(telomearse-Mps3-Ku80和Sir4-Esc1)去探討影響 telomere 與細胞膜結合。其中我透過螢光共軛焦顯微鏡去觀察並評估過量表現Mps3的N端truncated蛋白在wild type及tlc1的酵母菌中，端粒連接在細胞核膜的程度與比例。此外再透過solid plate assay檢查esc1是否會影響telomere-telomere recombination。另一方面，之前的研究也發現藉由熱量限制調控老化相關的訊息傳遞路徑能有效地延長各物種的平均壽命和延緩老化。但是對於細胞為了適應外在環境改變來延長細胞壽命的確切調控機制目前仍有待探討。因此分別將酵母菌培養在含有不同葡萄糖濃度(2%，0.5%)的培養基中，並透過液相層析串聯式質譜儀和蛋白質體學來分析在熱量限制調控下蛋白的磷酸化位點及磷酸化狀態的變異。接著利用Gene ontology和SGD phenotype資料庫的分析，我們一共選擇了15個蛋白磷酸化位點做後續的研究和分析，並透過單一磷酸化位點或雙磷酸化位點點突變來模擬蛋白磷酸化狀態的改變，且觀察在不同的壓力下酵母菌的生長情形是否會被影響。上述研究的結果顯示皆無明顯差異和影響，然而細胞內可能還存在著其他調控老化相關的途徑，因此我們未來還需要做更進一步的探討和分析。

關鍵字

端粒；錨定；磷酸化；突變；熱量限制

並列摘要

Cell from born to death may face aging which was characterized by progressive loss of physiological functions. To avoid abnormal change of genome and telomere-telomere recombination, telomeres are usually anchored at the nuclear envelope in budding yeast. Previous studies indicated that once telomeres become critically short, telomere - telomere recombination is promoted to maintain the normal length of telomere and generate the type I or type II survivors in the telomerase-negative yeast. To investigate the influence of the anchorage between telomere and nuclear envelope, we use fluorescence and confocal microscope to examine that the influence of Mps3-N’ on telomere anchorage when yeast loses telomerase. Moreover, we also use the solid plate assay to examine whether Esc1 suppresses telomere-telomere recombination in telomerase-negative yeast. On the other hand, early studies indicated that the influence of calorie restriction (CR) on some transduction signaling pathways which are associated with mechanisms of aging could effectively extend the lifespan in many organisms but the precise mechanism is not well-known as yet. In this study, yeast cells which were treated with YPAD medium containing 2% or 0.5% glucose grown at 30℃. Then we employed the quantitative proteomics, LC–MS / MS, Gene ontology, and the SGD phenotype to analyze phosphorylation state of protein which may be modulated. In total, the phospho-mimic or phospho-abolishing mutants are generated from 15 candidates to test the growth of yeast under different sorts of stress. However, the results showed that no obvious difference. There may be some alternative pathways that regulate the mechanisms of aging which existing in yeast.

並列關鍵字

telomere ； anchorage ； mutation ； calorie restriction ； phosphorylation

參考文獻

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