Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, WI, USA.
Department of Chemistry - BMC, Uppsala University, Uppsala, Sweden.
Nat Chem Biol. 2023 Nov;19(11):1423-1431. doi: 10.1038/s41589-023-01422-2. Epub 2023 Aug 31.
The modification of intracellular proteins with O-linked β-N-acetylglucosamine (O-GlcNAc) moieties is a highly dynamic process that spatiotemporally regulates nearly every important cellular program. Despite its significance, little is known about the substrate recognition and regulation modes of O-GlcNAc transferase (OGT), the primary enzyme responsible for O-GlcNAc addition. In this study, we identified the intervening domain (Int-D), a poorly understood protein fold found only in metazoan OGTs, as a specific regulator of OGT protein-protein interactions and substrate modification. Using proteomic peptide phage display (ProP-PD) coupled with structural, biochemical and cellular characterizations, we discovered a strongly enriched peptide motif, employed by the Int-D to facilitate specific O-GlcNAcylation. We further show that disruption of Int-D binding dysregulates important cellular programs, including response to nutrient deprivation and glucose metabolism. These findings illustrate a mode of OGT substrate recognition and offer key insights into the biological roles of this unique domain.
细胞内蛋白质的 O 链接 β-N-乙酰氨基葡萄糖（O-GlcNAc）修饰是一个高度动态的过程，它在时空上调节着几乎每一个重要的细胞程序。尽管其意义重大，但人们对 O-连接 N-乙酰氨基葡萄糖转移酶（OGT）的底物识别和调节模式知之甚少，OGT 是负责 O-GlcNAc 添加的主要酶。在这项研究中，我们确定了居间结构域（Int-D），这是一种在后生动物 OGT 中才发现的尚未充分了解的蛋白质折叠，它是 OGT 蛋白-蛋白相互作用和底物修饰的特定调节剂。我们使用与结构、生化和细胞特性相结合的蛋白质组肽噬菌体展示（ProP-PD），发现了一个强烈富集的肽基序，该基序被 Int-D 用来促进特定的 O-GlcNAc 化。我们进一步表明，Int-D 结合的破坏会使包括对营养缺乏和葡萄糖代谢的反应在内的重要细胞程序失调。这些发现阐明了 OGT 底物识别的一种模式，并为这一独特结构域的生物学作用提供了关键的见解。
