Evaluation of substrate and inhibitor binding to yeast and human isoprenylcysteine carboxyl methyltransferases (Icmts) using biotinylated benzophenone-containing photoaffinity probes.
Evaluation of substrate and inhibitor binding to yeast and human isoprenylcysteine carboxyl methyltransferases (Icmts) using biotinylated benzophenone-containing photoaffinity probes.
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DOI：
10.1016/j.bbrc.2012.05.089
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发表时间：
2012-06-22
影响因子：
3.1
通讯作者：
Hrycyna CA
中科院分区：
文献类型：
--
作者：
Hahne K;Vervacke JS;Shrestha L;Donelson JL;Gibbs RA;Distefano MD;Hrycyna CA
异戊二烯半胱氨酸羧甲基转移酶（Isoprenylcysteine carboxymethyltransferases，Icmts）是一类位于真核生物内质网（endoplasmic reticulum，ER）膜上的膜蛋白甲基转移酶。人（hIcmt）和S. Cerevisae（Ste 14 p）催化CaaX蛋白（包括酵母a因子交配信息素以及人和酵母Ras蛋白）翻译后加工中的α-羧甲基酯化步骤。在此，我们评估了两个充分表征的Icmt底物，N-乙酰基-S-法呢基-L-半胱氨酸（AFC）和酵母α-因子肽交配信息素的合成类似物，其在分子的脂质或肽部分中含有光活性二苯甲酮部分。基于AFC的化合物是hIcmt和Ste 14 p的底物，而a-因子类似物仅是Ste 14 p的底物。然而，发现α-因子类似物是hIcmt的微摩尔抑制剂。总之，这些数据表明，Icmt底物结合位点是依赖于异戊二烯基部分和上游氨基酸组成的功能，hIcmt和Ste 14 p有重叠，但不同的底物特异性。用这些类似物进行的光交联和中性抗生物素蛋白-琼脂糖捕获实验表明，hIcmt和Ste 14 p都被所有测试化合物特异性地光标记到不同程度。这些数据表明，这些类似物将是有用的，为未来的ICMT底物结合位点的鉴定。
Isoprenylcysteine carboxyl methyltransferases (Icmts) are a class of integral membrane protein methyltransferases localized to the endoplasmic reticulum (ER) membrane in eukaryotes. The Icmts from human (hIcmt) and S. cerevisae (Ste14p) catalyze the α-carboxyl methyl esterification step in the post-translational processing of CaaX proteins, including the yeast a-factor mating pheromones and both human and yeast Ras proteins. Herein, we evaluated synthetic analogs of two well-characterized Icmt substrates, N-acetyl-S-farnesyl-L-cysteine (AFC) and the yeast a-factor peptide mating pheromone, that contain photoactive benzophenone moieties in either the lipid or peptide portion of the molecule. The AFC based-compounds were substrates for both hIcmt and Ste14p, whereas the a-factor analogs were only substrates for Ste14p. However, the a-factor analogs were found to be micromolar inhibitors of hIcmt. Together, these data suggest that the Icmt substrate binding site is dependent upon features in both the isoprenyl moiety and upstream amino acid composition and that hIcmt and Ste14p have overlapping, yet distinct, substrate specificities. Photocrosslinking and neutravidin-agarose capture experiments with these analogs revealed that both hIcmt and Ste14p were specifically photolabeled to varying degrees with all of the compounds tested. These data suggest that these analogs will be useful for the future identification of the Icmt substrate binding sites.
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影响因子：
3.6
作者：
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