Human intestinal acyl-CoA synthetase 5 is sensitive to the inhibitor triacsin C
Human intestinal acyl-CoA synthetase 5 is sensitive to the inhibitor triacsin C
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DOI：
10.3748/wjg.v17.i44.4883
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发表时间：
2011-11-28
影响因子：
4.3
通讯作者：
Gassler, Nikolaus
中科院分区：
文献类型：
--
作者：
Kaemmerer, Elke;Peuscher, Anne;Gassler, Nikolaus
目的：研究人酰基辅酶A合成酶5（ACSL 5）是否对ACSL抑制剂triacsin C敏感。方法：克隆大鼠ACSL亚型ACSL 1和ACSL 5以及人ACSL 5，并重组表达为6xHis标记的酶。在pH 7.5或pH 9.5存在或不存在三胞菌素C的情况下测定Ni（2+）亲和纯化的重组酶。此外，还监测了ACSL 5转染的CaC 〇 2细胞和人肠粘膜。ACSL 5在细胞系统中的表达使用蛋白质印迹和免疫荧光进行验证。ACSL试验混合物包括TrisHCl（pH 7.4）、ATP、CoA、EDTA、DTT、MgCl（2）、[9，10-（3）H]棕榈酸和Triton X-100。通过加入溶解的、纯化的重组蛋白或细胞裂解物启动200 μ L反应。结果：异丙基β-D-1-硫代半乳糖苷诱导表达可溶性重组ACSL蛋白，Ni（2+）亲和层析进一步纯化，纯化后的ACSL蛋白纯度接近均一。Triacsin C在pH 7.5和pH 9.5下选择性且强烈抑制重组人ACSL 5蛋白，以及重组大鼠ACSL 1（敏感对照），但不抑制重组大鼠ACSL 5（不敏感对照）。人ACSL 5的IC 50约为10 μ mol/L。对于不同的孵育时间（10、30和60分钟），抑制性三酸甘油酯C的作用是相似的，并且不被6xHis-标签的N-或C-末端位置改变。为了评价ACSL 5在细胞环境中对三酸甘油酯C的敏感性，分析了稳定的人ACSL 5 CaC 〇 2转染子和具有ACSL 5的高生理表达的机械解剖的正常人肠粘膜。在两种模型中，ACSL 5峰活性均在pH 7.5和pH 9.5下出现，与重组人ACSL 5蛋白的性质相对应。在三酸甘油酯C（25 μ mol/L）的存在下，总ACSL活性显着减少，在人类ACSL 5转染以及在ACSL 5丰富的人类肠mucosa.CONCLUSION：数据强烈表明，人类ACSL 5是敏感的三酸甘油酯C，并不补偿其他三酸甘油酯C敏感的ACSL亚型。(C)2011年百世登。All rights reserved.
AIM: To investigate whether human acyl-CoA synthetase 5 (ACSL5) is sensitive to the ACSL inhibitor triacsin C.METHODS: The ACSL isoforms ACSL1 and ACSL5 from rat as well as human ACSL5 were cloned and recombinantly expressed as 6xHis-tagged enzymes. Ni(2+)-affinity purified recombinant enzymes were assayed at pH 7.5 or pH 9.5 in the presence or absence of triacsin C. In addition, ACSL5 transfected CaCo2 cells and intestinal human mucosa were monitored. ACSL5 expression in cellular systems was verified using Western blot and immunofluorescence. The ACSL assay mix included TrisHCl (pH 7.4), ATP, CoA, EDTA, DTT, MgCl(2), [9,10-(3)H] palmitic acid, and triton X-100. The 200 mu L reaction was initiated with the addition of solubilized, purified recombinant proteins or cellular lysates. Reactions were terminated after 10, 30 or 60 min of incubation with Doles medium.RESULTS: Expression of soluble recombinant ACSL proteins was found after incubation with isopropyl beta-D-1-thiogalactopyranoside and after ultracentrifugation these were further purified to near homogeneity with Ni(2+)-affinity chromatography. Triacsin C selectively and strongly inhibited recombinant human ACSL5 protein at pH 7.5 and pH 9.5, as well as recombinant rat ACSL1 (sensitive control), but not recombinant rat ACSL5 (insensitive control). The IC50 for human ACSL5 was about 10 mu mol/L. The inhibitory triacsin C effect was similar for different incubation times (10, 30 and 60 min) and was not modified by the N- or C-terminal location of the 6xHis-tag. In order to evaluate ACSL5 sensitivity to triacsin C in a cellular environment, stable human ACSL5 CaCo2 transfectants and mechanically dissected normal human intestinal mucosa with high physiological expression of ACSL5 were analyzed. In both models, ACSL5 peak activity was found at pH 7.5 and pH 9.5, corresponding to the properties of recombinant human ACSL5 protein. In the presence of triacsin C (25 mu mol/L), total ACSL activity was dramatically diminished in human ACSL5 transfectants as well as in ACSL5-rich human intestinal mucosa.CONCLUSION: The data strongly indicate that human ACSL5 is sensitive to triacsin C and does not compensate for other triacsin C-sensitive ACSL isoforms. (C) 2011 Baishideng. All rights reserved.