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標題: 第一部分:探討人類第二型抗酶之關鍵泛素化位點 第二部分:探討人類粒線體NAD(P)+-依賴型蘋果酸酶在肺癌細胞A549重編程之研究
Part 1:Investigation of the Ubiquitination Sites on Human Antizyme 2 Part 2:Study of Human Mitochondrial NAD(P)+-Dependent Malic Enzyme Involving in the Metabolic Reprogramming of A549 Lung Cancer Cells
作者: 楊皓評
Hao-Ping Yang
關鍵字: 抗酶
Malic Enzyme
Metabolism Reprogramming
Lung Cancer
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摘要: 鳥胺酸脫羧酶抗酶 (antizyme, AZ) 為一多元胺 (polyamine) 生成抑制蛋白,利用與鳥胺酸脫羧酶 (ornithine decarboxylase, ODC) 的高親和性,結合形成異源雙聚體 (heterodimer),抑制ODC催化之多元胺生合成。多元胺為帶正電的有機分子,藉由與遺傳物質或蛋白質結合,調控細胞生長、分化、凋亡及癌化轉形…等過程。而抗酶蛋白表現的多寡,也是多元胺影響基因表現的一個例子。當多元胺濃度過高時,會發生+1鹼基位移 (frameshift) 因而轉譯出具有功能的全長抗酶蛋白,可調控鳥胺酸脫羧酶被26S蛋白酶體 (proteasome) 降解。目前發現人類AZ共具有三種異構型 (isoform),皆能抑制ODC活性,並阻止細胞攝入多元胺,但在調節降解ODC方面,各異構型間的能力則是大不相同。與大部分蛋白質相同,AZ也是泛素依賴型 (ubiquitin-dependent) 的蛋白,即藉由泛素的標定,成為蛋白酶體降解之目標。本研究主要藉由定點突變及體外降解分析 (in vitro degradation),確認第二型鳥胺酸脫羧酶抗酶 (AZ2) 之主要泛素化位點 (ubiquitination site)。經由與各異構型AZ胺基酸做序列比較,總共有6個離胺酸 (Lysine, K) 擁有較高程度的保留性,分別是第40、75、89、116、129、141號離胺酸。在體外降解分析的結果中,以AZ2-K116A及AZ2-K141A所減少的降解程度略有差異,但在AZ2-K40A/K75A/K89A/K116A/K129A/K141A中,並沒有造成降解程度上的差異。結果顯示,在本實驗架構中,此6個高保留性的離胺酸,並非是AZ2泛素依賴型降解過程中,重要的泛素化位點。 癌細胞的代謝重置被認為是一個在腫瘤發展過程中相當重要的指標,而有氧糖解 (Warburg effect) 以及麩醯胺酸 (glutaminolysis) 代謝則被認為是腫瘤能持續獲得能量供給的兩個重要關鍵。因此,以腫瘤代謝為目標的治療策略,也在近年來被認為是一相當有潛力的研究方向。粒線體NAD(P)+-依賴型蘋果酸酶 (m-NAD(P)+-ME, ME2) 為一種氧化還原酶,能在粒線體中催化蘋果酸 (malate) 形成丙酮酸 (pyruvate),並且伴隨NAD(P)+還原成NAD(P)H。由於催化過程後的產物丙酮酸以及NADH可以做為細胞的能量代謝來源,因此在最近的研究中被認為是在腫瘤發展中的一個重要角色。為了瞭解ME2在癌症代謝重置上的角色,以及其對於能量相關代謝路徑的影響,實驗利用肺癌細胞株A549,並以RNA干擾技術降低ME2表現量後,比較在不同能量供給下,對於能量相關代謝路徑的影響。結果顯示,在肺癌細胞A459細胞株中,當降低ME2表現量後,即使在不同能量供給狀態下,皆可增加葡萄醣轉運蛋白的表現;而在缺乏葡萄醣的狀態下,則是會增強五碳醣磷酸路徑的強度;最後在缺乏麩醯胺酸情況下,則是可以看到細胞傾向增加將丙酮酸還原成乳酸的能力。
Antizyme (AZ) is a protein that can inhibit polyamine production via interacting with ornithine decarboxylase (ODC), an enzyme catalyzing a reaction that coverts ornithine to putrescine. Due to its high affinity toward ODC, AZ interacts with ODC to form heterodimer and then directly inhibits the enzyme activity of ODC. Polyamines are a group of organic molecules with positive charges. By interacting with DNA or proteins, it can regulate various biological functions, including cell proliferation and differentiation. Uncontrolled ODC enzyme activities lead to abnormal cell growth then tumorigenesis. Until now, three kinds of AZ isoforms have been identified. Like most proteins, AZ degradation follows an ubiquitin-dependent 26S proteasomal pathway. In our study, we used in vitro degradation assay to identify the ubiquitination sites within antizyme isoform 2 (AZ2). According to the sequence alignments, six conserved Lys residues were found within AZ2 protein among three AZ isoforms, including K40, K75, K89, K116, K129, K141. Our data indicated that AZ2-K116A and AZ2-K141A were slightly decreased in its degradation level as compared with AZ2-WT. However, AZ2-6M didn't show any difference in protein degradation. As a result, we conclude that these six conserved Lys residues are not the ubiquitination site when AZ2 possessed an ubiquitin-dependent degradation. Metabolism reprogramming has been thought as a quite important hallmark of tumor progression. Aerobic glycolysis (Warburg effect) and glutaminolysis also have been considered as two critical keys for tumor to receive energy supply continuously. Therefore, targeting tumor metabolism as therapeutic strategy is considered to be a potential research direction. The mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME, ME2) is an oxidoreductase which can catalyze the reversible oxidative decarboxylation of L-malate to pyruvate with simultaneous reduction of NAD(P)+ to NAD(P)H. Recent publishes suggests that ME2 plays an crucial role in tumorigenesis by providing pyruvate and NADH as the energy source. In order to understand the role of ME2 in the cancer metabolism reprogramming and how it participates in the energy-associated metabolism pathway, we use RNA interference (RNAi) to knockdown the expression level of ME2 and compare the effects of energy-related metabolic pathways under different conditions of energetic supplies. The results showed that in the lung cancer cell A459 cell line, when the ME2 expression was decreased, the glucose transporter expression was increased even in the different energy supply states. In the absence of glucose, the pentose phosphate pathway was enhanced. Finally, in the absence of glutamine, it can be seen that cells tend to increase the ability to reduce pyruvate to lactic acid.
文章公開時間: 2021-08-28
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