Development of a selective fluorescence-based enzyme assay for glycerophosphodiesterase family members GDE4 and GDE7.
Development of a selective fluorescence-based enzyme assay for glycerophosphodiesterase family members GDE4 and GDE7.
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DOI：
10.1016/j.jlr.2021.100141
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发表时间：
2021
影响因子：
6.5
通讯作者：
Okamoto Y
中科院分区：
文献类型：
--
作者：
Kitakaze K;Tsuboi K;Tsuda M;Takenouchi Y;Ishimaru H;Okamoto Y
溶血磷脂酸（LPA）是一种调节多种过程的脂质介质，包括细胞迁移和癌症进展。Autotaxin （ATX）是一种溶血磷脂酶d型外酶，可产生细胞外LPA。相比之下，甘油磷酸二酯酶（GDE）家族成员GDE4和GDE7是形成LPA的细胞内溶血磷脂酶D，分别依赖于Mg2+和Ca2+。由于没有报道过这些GDE的荧光底物，在本研究中，我们研究了一种荧光ATX底物FS-3是否可以用于研究GDE的活性。我们发现，过表达GDE4-和gde7的人胚胎肾293T细胞的膜组分水解FS-3的方式几乎完全依赖于Mg2+和Ca2+。使用这些检测系统，我们发现几种ATX抑制剂，包括LPA的α-溴代膦酸类似物和3-碳环磷脂酸，也能有效抑制GDE4和GDE7的活性。相比之下，ATX抑制剂S32826几乎没有抑制这些活性。此外，FS-3被内源性表达GDE4的人前列腺癌LNCaP细胞的膜部分以Mg2+依赖的方式水解，而不被缺乏GDE4的LNCaP细胞的膜部分水解。在人乳腺癌MCF-7细胞中观察到类似的Ca2+依赖性GDE7活性，但在缺乏GDE7的MCF-7细胞中没有观察到。最后，我们的检测系统可以选择性地测量GDE4和GDE7过表达的人胚胎肾293T细胞膜组分混合物中GDE4和GDE7的活性，其中存在S32826。这些发现允许对GDE4和GDE7活性进行高通量测定，这可能导致选择性抑制剂和刺激剂的开发，以及更好地了解这些酶的生物学作用。
Lysophosphatidic acid (LPA) is a lipid mediator that regulates various processes, including cell migration and cancer progression. Autotaxin (ATX) is a lysophospholipase D-type exoenzyme that produces extracellular LPA. In contrast, glycerophosphodiesterase (GDE) family members GDE4 and GDE7 are intracellular lysophospholipases D that form LPA, depending on Mg2+ and Ca2+, respectively. Since no fluorescent substrate for these GDEs has been reported, in the present study, we examined whether a fluorescent ATX substrate, FS-3, could be applied to study GDE activity. We found that the membrane fractions of human GDE4- and GDE7-overexpressing human embryonic kidney 293T cells hydrolyzed FS-3 in a manner almost exclusively dependent on Mg2+ and Ca2+, respectively. Using these assay systems, we found that several ATX inhibitors, including α-bromomethylene phosphonate analog of LPA and 3-carbacyclic phosphatidic acid, also potently inhibited GDE4 and GDE7 activities. In contrast, the ATX inhibitor S32826 hardly inhibited these activities. Furthermore, FS-3 was hydrolyzed in a Mg2+-dependent manner by the membrane fraction of human prostate cancer LNCaP cells that express GDE4 endogenously but not by those of GDE4-deficient LNCaP cells. Similar Ca2+-dependent GDE7 activity was observed in human breast cancer MCF-7 cells but not in GDE7-deficient MCF-7 cells. Finally, our assay system could selectively measure GDE4 and GDE7 activities in a mixture of the membrane fractions of GDE4- and GDE7-overexpressing human embryonic kidney 293T cells in the presence of S32826. These findings allow high-throughput assays of GDE4 and GDE7 activities, which could lead to the development of selective inhibitors and stimulators as well as a better understanding of the biological roles of these enzymes.
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DOI：
10.1165/rcmb.2012-0004oc
发表时间：
2012-11-01
影响因子：
6.4
作者：
Oikonomou, Nikos;Mouratis, Marios-Angelos;Aidinis, Vassilis
通讯作者：
Aidinis, Vassilis
一种产生溶血磷脂酸的新型酰基甘油激酶可调节前列腺癌细胞中与 EGFR 的串扰。
DOI：
10.1083/jcb.200407123
发表时间：
2005-06-06
期刊：
The Journal of cell biology
影响因子：
--
作者：
Bektas M;Payne SG;Liu H;Goparaju S;Milstien S;Spiegel S
通讯作者：
Spiegel S
影响因子：
3.7
作者：
Pleli T;Martin D;Kronenberger B;Brunner F;Köberle V;Grammatikos G;Farnik H;Martinez Y;Finkelmeier F;Labocha S;Ferreirós N;Zeuzem S;Piiper A;Waidmann O
通讯作者：
Waidmann O
影响因子：
4.8
作者：
Dancs, Peter Tibor;Ruisanchez, Eva;Benyo, Zoltan
通讯作者：
Benyo, Zoltan
影响因子：
8.1
作者：
Kitakaze K;Oyadomari M;Zhang J;Hamada Y;Takenouchi Y;Tsuboi K;Inagaki M;Tachikawa M;Fujitani Y;Okamoto Y;Oyadomari S
通讯作者：
Oyadomari S