综述题目：Regulating the Regulators: Mechanisms of Substrate Selection of the O-GlcNAc Cycling Enzymes OGT and OGA

作者：Hannah M. Stephen, Trevor M. Adams and Lance Wells1

Abstract:  Thousands of nuclear and cytosolic proteins are modified with a single β-N-acetylglucosamine on serine and threonine residues in mammals, a modification termed O-GlcNAc. This modification is essential for normal development and plays important roles in virtually all intracellular processes. Additionally, O-GlcNAc is involved in many disease states, including cancer, diabetes, and X-linked intellectual disability. Given the myriad of functions of the O-GlcNAc modification, it is therefore somewhat surprising that O-GlcNAc cycling is mediated by only two enzymes: the O-GlcNAc transferase (OGT), which adds O-GlcNAc, and the O-GlcNAcase (OGA), which removes it. A significant outstanding question in the O-GlcNAc field is how do only two enzymes mediate such an abundant and dynamic modification. In this review, we explore the current understanding of mechanisms for substrate selection for the O-GlcNAc cycling enzymes. These mechanisms include direct substrate interaction with specific domains of OGT or OGA, selection of interactors via partner proteins, posttranslational modification of OGT or OGA, nutrient sensing, and localization alteration. Altogether, current research paints a picture of an exquisitely regulated and complex system by which OGT and OGA select substrates. We also make recommendations for future work, toward the goal of identifying interaction mechanisms for specific substrates that may be able to be exploited for various research and medical treatment goals.

Keywords：O-GlcNAc, OGA, OGT, substrate selection  

摘要 : 哺乳动物中数千种核蛋白和细胞溶质蛋白的丝氨酸和苏氨酸残基上用单个β-N-乙酰氨基葡萄糖修饰，即O-GlcNAc糖基化修饰。这种修饰对正常发育至关重要，在几乎所有细胞内过程中都起着重要作用。此外，O-GlcNAc修饰与许多疾病状态有关，包括癌症、糖尿病和X相关智力残疾。O-GlcNAc修饰具有多种功能，但令人惊讶是O-GlcNA循环仅由两种酶介导：O-GlcNAc转移酶（OGT）（添加O-GlcNAc修饰）和O-GlcNAc糖苷酶（OGA）（去除O-GlcNAc修饰）。O-GlcNAc领域一个重要的突出问题是，仅有的两种酶是如何介导如此丰富的动态修饰。在这篇综述中，作者探讨了目前对O-GlcNAc循环酶底物选择机制的理解。这些机制包括与OGT或OGA特定结构域的直接底物相互作用、通过伴侣蛋白选择相互作用底物、OGT或OGA的翻译后修饰、营养感应和定位改变等。总之，目前的研究描绘了OGT和OGA通过一个精心调节的复杂系统来选择底物蛋白。作者还对未来的研究工作提出了建议：鉴定特定底物的相互作用机制，以用于开展各种研究和发展潜在的治疗靶标。

关键词: O-GlcNAc修饰；O-GlcNAc转移酶；O-GlcNAc糖苷酶；底物选择

OGT底物选择的可能机制：

Substrate selection: consensus sequence

Substrate selection: selection by OGT’s catalytic domain

Substrate selection: selection by OGT’s TPR domain

  Subhypothesis 1: the TPR domain directly modulates substrate interaction

  Subhypothesis 2: partner proteins assist in targeting the TPR domain to substrates

Substrate selection: OGT subcellular localization is altered to affect substrate access and selection

Substrate selection: post-translational modifications

Substrate selection: UDP-GlcNAc sensing

Summary of possible OGT substrate selection mechanisms

The nature of OGT as the sole enzyme modifying thousands of proteins with O-GlcNAc demands that the regulation of its substrate selection be elegant and complex. Many different possible mechanisms of substrate selection have been studied for a limited number of substrates. It is essential to note that the reality of OGT substrate selection is likely an intricate combination of those described above. The substrate selection mechanism is likely to be unique for each protein or protein class and exists as a combination of global interaction mechanisms, global cellular effects (from nutrient status), specific pathways designated for that protein (involving partner proteins and OGT posttranslational modifications), and interaction features unique to that interactor (specific amino acid interactions). In order to better understand OGT substrate selection, a combination of targeted research, like studies described above, and global assays must be performed.

To the end of understanding OGT interaction on a global scale, a limited number of attempts to define the OGT interactome have been performed. Most have focused on the full-length OGT interactome, which is helpful to define what proteins interact with OGT, but cannot determine domain effects on protein–protein interaction. These include Co-IP and microarray interactomes (Deng et al. 2014; Gao et al. 2018). One OGT TPR interactome, which identified 115 OGT-TPR–interacting proteins, has been recently defined by our group demonstrating that the TPR domain of OGT can select for substrates/partners (Stephen et al. 2020).

展望：未来的研究方向

Current understandings and recommendations for future study

Multiple studies have focused on the O-GlcNAc cycling enzymes since their discovery several decades ago. Much of this work has focused on the functional outcomes and disease roles that these enzymes and the modification play. However, one fundamental question still remains unanswered: How are only two enzymes capable of modulating such an abundant and finely-tuned protein modification with so many diverse roles? Most of the work in this area has focused on the transferase, OGT. Many probable and mutually inclusive hypotheses exist to explain how OGT can select substrates. Most evidence points strongly toward a significant role for the TPR domain of OGT in substrate selection, via a variety of mechanisms including direct substrate selection and/or partner protein interactions. However, other mechanisms have been identified and may play varying roles, including posttranslational modification of OGT, alterations in OGT localization and OGT nutrient sensing. OGA, to the contrary, has had comparatively much less research performed to identify how it selects substrates. Some evidence exists for selection via structural features of the OGA enzyme, and a few potential “partner proteins” have been identified. It is also possible that OGA regulation occurs uniquely on a more global scale; e.g. interacting proteins like those described above dampen its activity globally when necessary. However, many mysteries still exist regarding OGA substrate selection. One such large gap in knowledge is the function of the HAT-like domain.

Research into OGT’s substrate selection mechanism has reached a point where highly targeted studies are possible and will be beneficial. Several global interactor lists have been identified, and many specific interacting substrates have been studied as well. The next logical step is to focus on specific interactors and identify several characteristics.

重要的科学问题：

1) By what mechanism does the interactor bind OGT? What domain is responsible for the interaction? Do posttranslational modifications on OGT or the interactor play a role? Is the interactor O-GlcNAc modified?

2) How does the interaction affect OGT? Is localization altered? Does the interaction induce interaction with other substrates?

3) Under what conditions does the interaction occur? What is the functional outcome of the interaction?

One challenge that will come with validating and identifying individual interactions is that by nature many OGT interactions are transient, making them difficult to identify. A combination of biochemical techniques, including truncation mutations and in vitro binding assays to determine binding regions, and in cellulo methods like Förster resonance energy transfer (Brzostowski et al. 2009) and bimolecular fluorescence complementation (Kerppola 2008) will be useful in determining where and when interactions occur in cells. Methods used to determine functional outcomes will vary with the interactor, but since OGT interacts with many proteins involved in well-studied systems (e.g. transcription, chromatin regulation, heat shock), many assays are available for this purpose.

In order to understand how OGA selects its substrates, more preliminary work is needed. Truncation studies, like those performed for OGT, to determine what regions of OGA are responsible for its interaction with known substrates will be useful. With currently available proteomic and proximity labeling technology, it may also be extremely useful to use these approaches to identify interactors of the various domains of OGA. This approach can be used to distinguish between proteins that interact with the catalytic domain, the stalk domain, or the HAT-like domain. Structural studies focusing on the HAT-like domain may also be of use to determine if this domain plays a role in substrate selection and, if not, what function, if any, it serves. Once a more unified theory of OGA substrate interaction is identified, studies for individual interactors like described above for OGT can take place. It may also be useful to determine OGA’s interaction with OGT substrates concurrently, if the cycling of the O-GlcNAc modification on a given substrate is of particular interest.

The O-GlcNAc cycling enzymes present a fascinating story of an essential protein modification that plays a role in nearly every biological process within the cell but is regulated by only two enzymes. Uncovering the complicated and elegant mechanisms for how this regulation occurs is essential to understand how these enzymes and the resulting modification interplay with multiple biological processes related to diseases that affect millions of people worldwide.

参考资料：Stephen H M, Adams T M, Wells L. Regulating the regulators: mechanisms of substrate selection of the O-GlcNAc cycling enzymes OGT and OGA [J]. Glycobiology, 2021, 31(7): 724-733.

DOI: https://doi.org/10.1093/glycob/cwab005 

原文链接：https://academic.oup.com/glycob/article/31/7/724/6106259