Minitags for small molecules: detecting targets of reactive small molecules in living plant tissues using 'click chemistry'
Minitags for small molecules: detecting targets of reactive small molecules in living plant tissues using 'click chemistry'
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DOI：
10.1111/j.1365-313x.2008.03683.x
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发表时间：
2009-01-01
期刊：
影响因子：
7.2
通讯作者：
van der Hoorn, Renier A. L.
中科院分区：
文献类型：
--
作者：
Kaschani, Farnusch;Verhelst, Steven H. L.;van der Hoorn, Renier A. L.
小分子为植物研究提供了前所未有的机会，因为植物对各种各样的内源性和外源性小分子做出反应、代谢和反应。这些小分子中的许多与蛋白质共价连接。为了在植物中显示这些小分子靶标，我们介绍了一种两步标记小分子的方法。微型标签是小的化学部分（叠氮化物或炔），在生物条件下是惰性的，对小分子的膜渗透性和特异性几乎没有影响。标记后，在变性条件下提取蛋白质组，并通过“点击化学”反应将标记的蛋白质与报告标签偶联。我们通过研究小分子半胱氨酸蛋白酶抑制剂E-64的良好特征靶标，在植物中引入这两步标记程序。与生物素化的E-64相反，微标记的E-64在体内有效地标记液泡蛋白酶。我们展示，纯化和鉴定了一种针对活植物细胞中蛋白酶体和半胱氨酸蛋白酶的minitaged抑制剂的靶标。抑制剂的化学干扰试验表明，MG 132，一种常用的蛋白酶体抑制剂，优先抑制半胱氨酸蛋白酶在体内。两步标记程序可应用于离体叶片、细胞培养物、幼苗和其他活植物组织，并且当与光反应基团组合时，可用于鉴定除草剂、植物激素和选自化学遗传筛选的反应性小分子的靶标。
Small molecules offer unprecedented opportunities for plant research since plants respond to, metabolize, and react with a diverse range of endogenous and exogenous small molecules. Many of these small molecules become covalently attached to proteins. To display these small molecule targets in plants, we introduce a two-step labelling method for minitagged small molecules. Minitags are small chemical moieties (azide or alkyne) that are inert under biological conditions and have little influence on the membrane permeability and specificity of the small molecule. After labelling, proteomes are extracted under denaturing conditions and minitagged proteins are coupled to reporter tags through a 'click chemistry' reaction. We introduce this two-step labelling procedure in plants by studying the well-characterized targets of E-64, a small molecule cysteine protease inhibitor. In contrast to biotinylated E-64, minitagged E-64 efficiently labels vacuolar proteases in vivo. We displayed, purified and identified targets of a minitagged inhibitor that targets the proteasome and cysteine proteases in living plant cells. Chemical interference assays with inhibitors showed that MG132, a frequently used proteasome inhibitor, preferentially inhibits cysteine proteases in vivo. The two-step labelling procedure can be applied on detached leaves, cell cultures, seedlings and other living plant tissues and, when combined with photoreactive groups, can be used to identify targets of herbicides, phytohormones and reactive small molecules selected from chemical genetic screens.