探討雙羥奈酸及5,5'-亞甲基二水楊酸對細胞代謝之影響: 靶向人類粒線體蘋果酸酶

The functional effects of Embonic acid and 5,5'-Methylenedisalicylic acid on cellular metabolism: A focus on human mitochondrial NAD(P)+-dependent malic enzyme

葉婕安(Jie-An Ye)

指導教授 ： 劉光耀

中山醫學大學/醫學院/醫學研究所 (原生化微生物免疫研究所)/碩士(2022年)

https://doi.org/10.6834/csmu202200032

摘要

蘋果酸酶 (malic enzyme, ME) 為一種同源四聚體 (homo-tetramer) 蛋白，蘋果酸酶家族在自然界中廣泛分佈，其胺基酸序列具有高度保留性，可以在細菌、植物及哺乳動物中發現，根據其所在的胞器位置及特性可分為三種異構型 (isoform)，分別為c-NADP-ME (ME1)、m-NAD(P)-ME (ME2) 及m-NADP-ME (ME3)，其中m-NAD(P)-ME會表現在快速增殖的腫瘤中，例如: 肝癌、乳腺癌及黑色素瘤等。在實驗室之前的研究中，發現雙羥奈酸 (embonic acid, EA) 會抑制m-NAD(P)-ME的活性，而後續發現與雙羥奈酸化學結構相似的5,5'-亞甲基二水楊酸 (5,5'-methylenedisalicylic acid, MDSA) 同樣具有抑制m-NAD(P)-ME活性的功能，因此想進一步探討雙羥奈酸與5,5'-亞甲基二水楊酸對正常細胞代謝的影響，並藉由正常細胞中過量或敲低m-NAD(P)-ME與抑制劑的細胞代謝結果相互比較。為了瞭解m-NAD(P)-ME對細胞代謝的影響，使用檢測丙酮酸 (pyruvate)、還原態菸鹼醯胺腺嘌呤二核苷酸磷酸 (nicotinamide adenine dinucleotide phosphate, NADPH) 及菸鹼醯胺腺嘌呤二核苷酸 (nicotinamide adenine dinucleotide, NAD+/NADH) 之套組進行分析，進而瞭解雙羥奈酸與5,5'-亞甲基二水楊酸抑制m-NAD(P)-ME影響細胞代謝的能力。實驗結果顯示雙羥奈酸及5,5'-亞甲基二水楊酸抑制m-NAD(P)-ME後，的確改變了丙酮酸、NADPH、NAD+與NADH的狀態，表示未來此兩藥物有機會參與抗癌藥物的應用。

關鍵字

細胞代謝 ； 人類粒線體蘋果酸酶 ； 雙羥奈酸 ； 5,5'-亞甲基二水楊酸

並列摘要

Malic enzyme (ME) is a homo-tetramer protein, which family is abundant in nature and with highly conserved amino acid sequence, can be found in bacteria, plants, and mammals. It can be divided into three isoforms based on the location and characteristics of the organelle, namely c-NADP-ME (ME1), m-NAD(P)-ME (ME2), and m-NADP-ME (ME3), and m-NAD(P)-ME presents in rapidly proliferating tumors such as liver cancer, breast cancer, and melanoma. Previous laboratory studies revealed that embonic acid (EA) inhibits the enzyme activity of m-NAD(P)-ME. Subsequent research has revealed that 5,5'-methylenedisalicylic acid (MDSA), a similar chemical structure to embonic acid, also inhibits the activity of m-NAD(P)-ME. As a result, we evaluated the impacts of embonic acid and 5,5'-methylenedisalicylic acid on normal cell metabolism and compared the metabolism alterations in normal cells with excessive or pulled down m-NAD(P)-ME. To further understand how m-NAD(P)-ME affects cell metabolism, use the detection kits for pyruvate, nicotinamide adenine dinucleotide phosphate (NADPH), and nicotinamide adenine dinucleotide (NAD+/NADH) to assess the effect of m-NAD(P)-ME on cell metabolism, and then understand how embonic acid and 5,5'-methylenedisalicylic acid inhibit m-NAD(P)-ME and alter cell metabolism ability. According to the findings, embonic acid and 5,5'-methylenedisalicylic can change the states of pyruvate, NADPH, and NAD+/NADH by inhibiting the m-NAD(P)-ME enzyme activity, and had the potential to participating the application in cancer therapy.

並列關鍵字

cellular metabolism ； human mitochondrial NAD(P)+-dependent malic enzyme ； Embonic acid ； 5,5'-Methylenedisalicylic acid

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