Validation of Trifluoromethylphenyl Diazirine Cholesterol Analogues As Cholesterol Mimetics and Photolabeling Reagents.
Validation of Trifluoromethylphenyl Diazirine Cholesterol Analogues As Cholesterol Mimetics and Photolabeling Reagents.
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DOI：
10.1021/acschembio.1c00364
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发表时间：
2021-08-20
影响因子：
4
通讯作者：
Covey DF
中科院分区：
文献类型：
--
作者：
Krishnan K;Qian M;Feltes M;Chen ZW;Gale S;Wang L;Sugasawa Y;Reichert DE;Schaffer JE;Ory DS;Evers AS;Covey DF
胆固醇的脂肪族二氮嘧啶类似物先前已被用于详细说明胆固醇蛋白质组和鉴定蛋白质上的胆固醇结合位点。含有三氟甲基苯基二嗪（TPD）基团的胆固醇类似物尚未报道。两类重氮嘧啶已被制备用于神经类固醇光标记研究，它们的联合使用提供了单独使用两类重氮嘧啶无法获得的信息。因此，我们制备了胆固醇TPD类似物，并将它们与先前报道的脂肪族重氮嘧啶类似物一起用作光亲和标记试剂，以获得关于五聚体离子通道GLIC的胆固醇结合位点的额外信息。我们首先验证了TPD类似物作为胆固醇替代品，并将其与先前报道的脂肪族嘧啶在细胞培养分析中的作用进行了比较。所有探针都结合到GLIC上相同的胆固醇结合位点，但在光标记效率和鉴定的残基方面存在差异。哺乳动物（HEK）细胞膜的光标记显示了两类探针标记的蛋白质模式的差异。总的来说，这些数据表明含有脂肪族重氮嘧啶或TPD基团的胆固醇光亲和标记试剂提供了补充信息，并将成为未来胆固醇生物学研究的有用工具。
Aliphatic diazirine analogues of cholesterol have been used previously to elaborate the cholesterol proteome and identify cholesterol binding sites on proteins. Cholesterol analogues containing the trifluromethylphenyl diazirine (TPD) group have not been reported. Both classes of diazirines have been prepared for neurosteroid photolabeling studies and their combined use provided information that was not obtainable with either diazirine class alone. Hence, we prepared cholesterol TPD analogues and used them along with previously reported aliphatic diazirine analogues as photoaffinity labeling reagents to obtain additional information on the cholesterol binding sites of the pentameric ion channel GLIC. We first validated the TPD analogues as cholesterol substitutes and compared their actions with those of previously reported aliphatic diazirines in cell culture assays. All the probes bound to the same cholesterol binding site on GLIC, but with differences in photolabeling efficiencies and residues identified. Photolabeling of mammalian (HEK) cell membranes demonstrated differences in the pattern of proteins labeled by the two classes of probes. Collectively, these date indicate that cholesterol photoaffinity labeling reagents containing an aliphatic diazirine or TPD group provide complementary information and will both be useful tools in future studies of cholesterol biology.
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影响因子：
6.8
作者：
Kowatsch, Christiane;Woolley, Rachel E.;Siebold, Christian
通讯作者：
Siebold, Christian
DOI：
10.1007/978-3-030-14265-0_8
发表时间：
2019-01-01
期刊：
DIRECT MECHANISMS IN CHOLESTEROL MODULATION OF PROTEIN FUNCTION
影响因子：
--
作者：
Cologna, Stephanie M.;Rosenhouse-Dantsker, Avia
通讯作者：
Rosenhouse-Dantsker, Avia
影响因子：
4.7
作者：
Jiang, Xiaoping;Shu, Hong-Jin;Mennerick, Steven
通讯作者：
Mennerick, Steven
影响因子：
6.5
作者：
Feltes, McKenna;Moores, Samantha;Schaffer, Jean E.
通讯作者：
Schaffer, Jean E.
影响因子：
4.3
作者：
Chen, Daniel M.;Ziolkowski, Luke;Mennerick, Steven
通讯作者：
Mennerick, Steven