Pharmacogenomics Laboratory, Centre Hospitalier Universitaire de Québec Research Center, Faculty of Pharmacy, Laval University, Québec City, Québec, Canada.
Mol Pharmacol. 2014 Jan;85(1):29-36. doi: 10.1124/mol.113.089227. Epub 2013 Oct 18.
Transcripts of the UGT1A gene, encoding half of human UDP-glucuronosyltransferase (UGT) enzymes, undergo alternative splicing, resulting in active enzymes named isoforms 1 (i1s) and novel truncated isoforms 2 (i2s). Here, we investigated the effects of depleting endogenous i2 on drug response and attempted to unveil any additional biologic role(s) for the truncated novel UGT proteins. We used an integrated systems biology approach that combines RNA interference with unbiased global genomic and proteomic screens, and used HT115 colorectal cancer cells as a model. Consistent with previous evidence suggesting that i2s negatively regulate i1s through protein-protein interactions, i2-depleted cells were less sensitive to drug-induced cell death (IC50 of 0.45 ± 0.05 µM versus 0.22 ± 0.03 µM; P = 0.006), demonstrating that modulation of i2 levels meaningfully impacts drug bioavailability and cellular response. We also observed reduced production of reactive oxygen species by 30% (P < 0.05), and an enhanced expression (>1.2-fold; P < 0.05) of several proteins, such as hemoglobin α genes and superoxide dismutase 1, that have network functions associated with antioxidant properties. Interaction proteomics analysis of endogenous proteins from the cellular model, mainly in human intestine but also in kidney tissues, further uncovered interactions between i2s (but not i1s) and the antioxidant enzymes catalase and peroxiredoxin 1, which may influence antioxidant potential through sequestration of these novel partners. Our findings demonstrate for the first time dual roles for i2s in the cellular defense system as endogenous regulators of drug response as well as in oxidative stress.
UGT1A 基因的转录本，编码人类一半的 UDP-葡糖醛酸基转移酶 (UGT) 酶，经过选择性剪接，产生具有活性的酶，分别命名为同工型 1 (i1s) 和新型截断同工型 2 (i2s)。在这里，我们研究了耗尽内源性 i2 对药物反应的影响，并试图揭示截断的新型 UGT 蛋白的任何其他生物学作用。我们使用了一种综合系统生物学方法，该方法将 RNA 干扰与无偏全球基因组和蛋白质组筛选相结合，并使用 HT115 结肠癌细胞作为模型。与先前的证据一致，即 i2s 通过蛋白质-蛋白质相互作用负调节 i1s，i2 耗尽的细胞对药物诱导的细胞死亡的敏感性降低（IC50 为 0.45 ± 0.05 µM 对 0.22 ± 0.03 µM；P = 0.006），表明调节 i2 水平对药物生物利用度和细胞反应有重要影响。我们还观察到活性氧的产生减少了 30%（P < 0.05），并且几种蛋白质的表达增强了 >1.2 倍（P < 0.05），如血红蛋白 α 基因和超氧化物歧化酶 1，它们具有与抗氧化特性相关的网络功能。来自细胞模型的内源性蛋白质的相互作用蛋白质组学分析，主要在人肠中，但也在肾组织中，进一步揭示了 i2s（而不是 i1s）与抗氧化酶过氧化氢酶和过氧化物酶 1 之间的相互作用，这可能通过隔离这些新型伴侣来影响抗氧化潜力。我们的发现首次证明了 i2s 在细胞防御系统中的双重作用，既是药物反应的内源性调节剂，也是氧化应激的调节剂。
