GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation.
GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation.
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DOI：
10.1042/bj20090110
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发表时间：
2009-05-13
期刊：
影响因子：
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通讯作者：
van Aalten DM
中科院分区：
文献类型：
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作者：
Dorfmueller HC;Borodkin VS;Schimpl M;van Aalten DM
O-GlcNAc酰化是后生动物胞质蛋白质翻译后的一种必需的、动态的、可诱导的糖基化，并与蛋白质磷酸化相互作用。抑制OGA（O-GlcNAc酶）（从O-GlcNAc酰化蛋白质中去除O-GlcNAc的酶）是探测这种修饰在一系列细胞过程中的作用的有用策略。在本研究中，我们报告了GlcNAc抑制素的合理设计和评价，GlcNAc抑制素是一种有效的、竞争性的和选择性的人OGA抑制剂家族。重组人OGA的动力学实验表明，GlcNAc抑制素是迄今报道的最有效的人OGA抑制剂，在亚纳摩尔至纳摩尔范围内抑制酶。GlcNAc他汀N-乙酰基基团的修饰导致对采用类似底物辅助催化机制的人溶酶体氨基己糖苷酶的高达160倍的选择性。由GlcNAc抑制素复合物的结构指导的细菌OGA中的诱变研究提供了对人OGA活性位点中保守残基的作用的深入了解。GlcNAc抑制素是细胞渗透性的，并且在低纳摩尔浓度下，在一系列人细胞系中通过抑制OGA有效地调节细胞内O-GlcNAc水平。因此，这些化合物是研究O-GlcNAc的细胞生物学的有效选择性工具。
O-GlcNAcylation is an essential, dynamic and inducible post-translational glycosylation of cytosolic proteins in metazoa and can show interplay with protein phosphorylation. Inhibition of OGA (O-GlcNAcase), the enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, is a useful strategy to probe the role of this modification in a range of cellular processes. In the present study, we report the rational design and evaluation of GlcNAcstatins, a family of potent, competitive and selective inhibitors of human OGA. Kinetic experiments with recombinant human OGA reveal that the GlcNAcstatins are the most potent human OGA inhibitors reported to date, inhibiting the enzyme in the sub-nanomolar to nanomolar range. Modification of the GlcNAcstatin N-acetyl group leads to up to 160-fold selectivity against the human lysosomal hexosaminidases which employ a similar substrate-assisted catalytic mechanism. Mutagenesis studies in a bacterial OGA, guided by the structure of a GlcNAcstatin complex, provides insight into the role of conserved residues in the human OGA active site. GlcNAcstatins are cell-permeant and, at low nanomolar concentrations, effectively modulate intracellular O-GlcNAc levels through inhibition of OGA, in a range of human cell lines. Thus these compounds are potent selective tools to study the cell biology of O-GlcNAc.