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

Thousands of nuclear and cytosolic proteins are modified with a single ß-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.

Generation of an Interactome for the Tetratricopeptide Repeat Domain of O-GlcNAc Transferase Indicates a Role for the Enzyme in Intellectual Disability.

The O-GlcNAc transferase (OGT) modifies nuclear and cytoplasmic proteins with ß-N-acetyl-glucosamine (O-GlcNAc). With thousands of O-GlcNAc-modified proteins but only one OGT encoded in the mammalian genome, a prevailing question is how OGT selects its substrates. Prior work has indicated that the tetratricopeptide repeat (TPR) domain of OGT is involved in substrate selection. Furthermore, several variants of OGT causal for X-linked intellectual disability (XLID) occur in the TPR domain. Therefore, we adapted the BioID labeling method to identify interactors of a TPR-BirA* fusion protein in HeLa cells. We identified 115 interactors representing known and novel O-GlcNAc-modified proteins and OGT interactors (raw data deposited in MassIVE, Dataset ID MSV000085626). The interactors are enriched in known OGT processes (e.g., chromatin remodeling) as well as processes in which OGT has yet to be implicated (e.g., pre-mRNA processing). Importantly, the identified TPR interactors are linked to several disease states but most notably are enriched in pathologies featuring intellectual disability that may underlie the mechanism by which mutations in OGT lead to XLID. This interactome for the TPR domain of OGT serves as a jumping-off point for future research exploring the role of OGT, the TPR domain, and its protein interactors in multiple cellular processes and disease mechanisms, including intellectual disability.

Differences in Endothelin B Receptor Isoforms Expression and Function in Breast Cancer Cells.

The endothelins and their receptors are best known for their regulation of the vascular system. Their widespread expression in epithelial cells and their overexpression in some tumors has prompted investigation into their ability to regulate cancer progression. In this study, we assessed the mRNA expression of the major endothelin B receptor gene (EDNRB) isoforms and found differences in both mRNA and protein expression in normal breast cells and breast cancer cell lines. Knocking down the EDNRB gene in breast cancer cells altered invasiveness toward endothelin 3 (ET3), and we observed EDNRB isoform-specific regulation of breast cancer cell invasion and cell signaling, as well as isoform- and subtype-specific differences in breast cancer patient survival. The results reported in this study emphasize the importance of the endothelin B receptor in breast cancer. To our knowledge, this study is the first to clarify the differential expression and roles of specific EDNRB isoforms in breast cancer.

An intellectual disability syndrome with single-nucleotide variants in O-GlcNAc transferase.

Intellectual disability (ID) is a neurodevelopmental condition that affects ~1% of the world population. In total 5-10% of ID cases are due to variants in genes located on the X chromosome. Recently, variants in OGT have been shown to co-segregate with X-linked intellectual disability (XLID) in multiple families. OGT encodes O-GlcNAc transferase (OGT), an essential enzyme that catalyses O-linked glycosylation with ß-N-acetylglucosamine (O-GlcNAc) on serine/threonine residues of thousands of nuclear and cytosolic proteins. In this review, we compile the work from the last few years that clearly delineates a new syndromic form of ID, which we propose to classify as a novel Congenital Disorder of Glycosylation (OGT-CDG). We discuss potential hypotheses for the underpinning molecular mechanism(s) that provide impetus for future research studies geared towards informed interventions.