砷促使細胞中MTF-1與PML 交互作用的機制研究

Study on the mechanism of Arsenic-induced interaction between MTF-1 and PML.

吳品慧(Wu, Pin Hui)

指導教授 ： 林立元

國立清華大學/生命科學院/分子與細胞生物研究所/碩士(2015年)

https://doi.org/10.6843/NTHU.2015.00028

摘要

金屬感應轉錄因子（MTF-1）調控細胞內與金屬恆定及抗氧化作用相關基因表現，為哺乳動物發育時期重要的調節因子。早幼粒細胞白血病蛋白（PML）的基因受到選擇性剪接，具不同等型（isoform），而PML能夠促進細胞凋亡、細胞老化以及DNA修復，為人所熟知之重要腫瘤抑制因子，近期研究指出PML能夠調節細胞內氧化壓力的反應，也與相關蛋白結合並且共同調控細胞內生理反應。實驗室過去研究發現MTF-1受砷刺激後，會降低類泛素蛋白的修飾作用；研究也指出PML能夠調控其相關蛋白的類泛素蛋白修飾作用，所以本實驗為研究細胞在受砷刺激後，MTF-1與PML彼此間是否存在交互影響之作用。本研究發現於細胞中加入砷（NaAsO2）刺激，隨著刺激時間以及濃度增加，更加促進MTF-1與PML進行交互作用。而不同等型之PML，PMLⅠ到PMLⅥ皆能夠與MTF-1結合，且此交互作用並非藉由MTF-1結構中的特定區域所達成。但這種現象不會發生在受到鋅或鎘刺激的細胞中，此外PML結構中與砷結合的胱胺酸無法影響MTF-1與PML的交互作用，且此交互作用也無法調節MTF-1下游基因MT2A的表現量以及砷刺激下所造成的細胞死亡。MTF-1與PML的交互作用主要位於細胞質中，所以MTF-1並非與PML核小體進行交互作用。我們探討此交互作用在細胞內生理反應上的重要性，發現抗氧化劑BHA能夠降低MTF-1與PML的交互作用，且在共同表現MTF-1與PML的情況下，BHA會將活性氧物質（ROS）的含量降低到未處理的細胞值以下，表示活性氧物質的增加促進MTF-1與PML的交互作用。最後分別或共同表現MTF-1與PML，在受砷刺激後偵測活性氧物質含量，結果發現MTF-1與PML能夠共同降低活性氧物質生成量。這個結果證實在砷刺激下會增加細胞內的活性氧物質生成，因而促進MTF-1與PML進行交互作用並降低砷所造成的活性氧物質生成量。

關鍵字

金屬感應轉錄因子 ； 早幼粒細胞白血病蛋白 ； 砷

並列摘要

Metal-responsive transcription factor (MTF-1) regulates the expression of genes that modulates metal homeostasis and oxidative stress. MTF-1 is an essential regulatory factor for embryonic development of mammals. Owing to alternative splicing of the gene, several promyelocytic leukemia protein (PML) isoform can be found in the cells. PML is a well-known tumor suppressor involving in the induction of apoptosis, senescence and DNA repair. Reportedly, PML participates in the regulation of cellular antioxidative pathway and interacts with partner protein to modulate cellular activity. Previous studies indicate that SUMO modification of MTF-1 and PML can be altered by arsenic (As) treatment, we investigated whether PML and MTF-1 interacts and regulates cellular activity. We found that As induced the interaction between MTF-1 and PML in a dose- and time-dependent manner. This interaction does not occur for specific PML isoform, but for the PMLⅠto PMLⅥ. On the other hand, PML neither interacts with a specific domain of MTF-1. Mutation at the As binding residues of PML has also no effect on the interaction with MTF-1. Noticeably, the interaction was not stimulating by zinc or cadmium. Interestingly, the MTF-1/PML interaction does not alter the expression of MT2A gene, the major downstream target gene of MTF-1, or cell viability. MTF-1 interacted with PML mainly in cytosol. It is thus speculated that MTF-1 does not interact with PML nuclear body. The As-induced MTF-1/PML interaction can be reduced by decreasing cellular oxidative stress via administration of BHA, an antioxidant. Finally, we found that MTF-1 and PML synergistically decreased the level of reactive oxygen species (ROS). We conclude that cellular ROS level increases with the addition of As and stimulates the interaction between MTF-1 and PML. Concurrently, the interaction of MTF-1 and PML reduced cellular oxidative stress generated by As.

並列關鍵字

MTF-1 ； PML ； Arsenic

參考文獻

陳俊豪.(2013)金屬感應轉錄因子與缺氧誘導因子之交互作用及對其轉錄活性的影響.Institute of Molecular and Cellular Biology, Department of Life Science. National Tsing Hua University

Andrews GK (2000) Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochemical pharmacology 59: 95-104

Andrews GK (2001) Cellular zinc sensors: MTF-1 regulation of gene expression. Biometals 14: 223-237

Bernardi R, Guernah I, Jin D, Grisendi S, Alimonti A, Teruya-Feldstein J, Cordon-Cardo C, Simon MC, Rafii S, Pandolfi PP (2006) PML inhibits HIF-1alpha translation and neoangiogenesis through repression of mTOR. Nature 442: 779-785

Brugnera E, Georgiev O, Radtke F, Heuchel R, Baker E, Sutherland GR, Schaffner W (1994) Cloning, chromosomal mapping and characterization of the human metal-regulatory transcription factor MTF-1. Nucleic Acids Res 22: 3167-3173