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The vast array of cell types of multicellular organisms must individually fine-tune their internal metabolism. One important metabolic and stress regulatory mechanism is the dynamic attachment/removal of glucose-derived sugar N-acetylglucosamine on proteins (O-GlcNAcylation). The number of proteins modified by O-GlcNAc is bewildering, with at least 7,000 sites in human cells. The outstanding challenge is determining how key O-GlcNAc sites regulate a target pathway amidst thousands of potential global sites. Innovative solutions are required to address this challenge in cell models and disease therapy. This Perspective shares critical suggestions for the O-GlcNAc field gleaned from the international O-GlcNAc community. Further, we summarize critical tools and tactics to enable newcomers to O-GlcNAc biology to drive innovation at the interface of metabolism and disease. The growing pace of O-GlcNAc research makes this a timely juncture to involve a wide array of scientists and new toolmakers to selectively approach the regulatory roles of O-GlcNAc in disease.The diversity of biological pathways that are regulated by protein post-translational modification with O-GlcNAc requires community engagement to develop and apply tools to probe the biological and disease-relevant functions of these modifications.

Post-translational modifications (PTMs) greatly expand the structures and functions of proteins in nature 1 , 2 . Although synthetic protein functionalization strategies allow mimicry of PTMs 3 , 4 , as well as formation of unnatural protein variants with diverse potential functions, including drug carrying 5 , tracking, imaging 6 and partner crosslinking 7 , the range of functional groups that can be introduced remains limited. Here we describe the visible-light-driven installation of side chains at dehydroalanine residues in proteins through the formation of carbon-centred radicals that allow C–C bond formation in water. Control of the reaction redox allows site-selective modification with good conversions and reduced protein damage. In situ generation of boronic acid catechol ester derivatives generates RH 2 C • radicals that form the native (β-CH 2 –γ-CH 2 ) linkage of natural residues and PTMs, whereas in situ potentiation of pyridylsulfonyl derivatives by Fe( ii ) generates RF 2 C • radicals that form equivalent β-CH 2 –γ-CF 2 linkages bearing difluoromethylene labels. These reactions are chemically tolerant and incorporate a wide range of functionalities (more than 50 unique residues/side chains) into diverse protein scaffolds and sites. Initiation can be applied chemoselectively in the presence of sensitive groups in the radical precursors, enabling installation of previously incompatible side chains. The resulting protein function and reactivity are used to install radical precursors for homolytic on-protein radical generation; to study enzyme function with natural, unnatural and CF 2 -labelled post-translationally modified protein substrates via simultaneous sensing of both chemo- and stereoselectivity; and to create generalized ‘alkylator proteins’ with a spectrum of heterolytic covalent-bond-forming activity (that is, reacting diversely with small molecules at one extreme or selectively with protein targets through good mimicry at the other). Post-translational access to such reactions and chemical groups on proteins could be useful in both revealing and creating protein function. A wide range of side chains are installed into proteins by addition of photogenerated alkyl or difluroalkyl radicals, providing access to new functionality and reactivity in proteins.

The elucidation and prediction of how changes in a protein result in altered activities and selectivities remain a major challenge in chemistry. Two hurdles have prevented accurate family-wide models: obtaining (i) diverse datasets and (ii) suitable parameter frameworks that encapsulate activities in large sets. Here, we show that a relatively small but broad activity dataset is sufficient to train algorithms for functional prediction over the entire glycosyltransferase superfamily 1 (GT1) of the plant Arabidopsis thaliana. Whereas sequence analysis alone failed for GT1 substrate utilization patterns, our chemical–bioinformatic model, GT-Predict, succeeded by coupling physicochemical features with isozyme-recognition patterns over the family. GT-Predict identified GT1 biocatalysts for novel substrates and enabled functional annotation of uncharacterized GT1s. Finally, analyses of GT-Predict decision pathways revealed structural modulators of substrate recognition, thus providing information on mechanisms. This multifaceted approach to enzyme prediction may guide the streamlined utilization (and design) of biocatalysts and the discovery of other family-wide protein functions.

Background Enhanced glucose metabolism is a feature of most tumors, but downstream functional effects of aberrant glucose flux are difficult to mechanistically determine. Metabolic diseases including obesity and diabetes have a hyperglycemia component and are correlated with elevated pre-menopausal cancer risk for triple-negative breast cancer (TNBC). However, determining pathways for hyperglycemic disease-coupled cancer risk remains a major unmet need. One aspect of cellular sugar utilization is the addition of the glucose-derived protein modification O-GlcNAc (O-linked N-acetylglucosamine) via the single human enzyme that catalyzes this process, O-GlcNAc transferase (OGT). The data in this report implicate roles of OGT and O-GlcNAc within a pathway leading to cancer stem-like cell (CSC) expansion. CSCs are the minor fraction of tumor cells recognized as a source of tumors as well as fueling metastatic recurrence. The objective of this study was to identify a novel pathway for glucose-driven expansion of CSC as a potential molecular link between hyperglycemic conditions and CSC tumor risk factors. Methods We used chemical biology tools to track how a metabolite of glucose, GlcNAc, became linked to the transcriptional regulatory protein tet-methylcytosine dioxygenase 1 (TET1) as an O-GlcNAc post-translational modification in three TNBC cell lines. Using biochemical approaches, genetic models, diet-induced obese animals, and chemical biology labeling, we evaluated the impact of hyperglycemia on CSC pathways driven by OGT in TNBC model systems. Results We showed that OGT levels were higher in TNBC cell lines compared to non-tumor breast cells, matching patient data. Our data identified that hyperglycemia drove O-GlcNAcylation of the protein TET1 via OGT-catalyzed activity. Suppression of pathway proteins by inhibition, RNA silencing, and overexpression confirmed a mechanism for glucose-driven CSC expansion via TET1-O-GlcNAc. Furthermore, activation of the pathway led to higher levels of OGT production via feed-forward regulation in hyperglycemic conditions. We showed that diet-induced obesity led to elevated tumor OGT expression and O-GlcNAc levels in mice compared to lean littermates, suggesting relevance of this pathway in an animal model of the hyperglycemic TNBC microenvironment. Conclusions Taken together, our data revealed a mechanism whereby hyperglycemic conditions activated a CSC pathway in TNBC models. This pathway can be potentially targeted to reduce hyperglycemia-driven breast cancer risk, for instance in metabolic diseases. Because pre-menopausal TNBC risk and mortality are correlated with metabolic diseases, our results could lead to new directions including OGT inhibition for mitigating hyperglycemia as a risk factor for TNBC tumorigenesis and progression.

Despite nature's prevalent use of metals as prosthetics to adapt or enhance the behaviour of proteins, our ability to programme such architectural organization remains underdeveloped. Multi-metal clusters buried in proteins underpin the most remarkable chemical transformations in nature, but we are not yet in a position to fully mimic or exploit such systems. With the advent of copious, relevant structural information, judicious mechanistic studies and the use of accessible computational methods in protein design coupled with new synthetic methods for building biomacromolecules, we can envisage a 'new dawn' that will allow us to build de novo metalloenzymes that move beyond mono-metal centres. In particular, we highlight the need for systems that approach the multi-centred clusters that have evolved to couple electron shuttling with catalysis. Such hybrids may be viewed as exciting mid-points between homogeneous and heterogeneous catalysts which also exploit the primary benefits of biocatalysis.

Despite nature’s prevalent use of metals as prosthetics to adapt or enhance the behaviour of proteins, our ability to programme such architectural organization remains underdeveloped. Multi-metal clusters buried in proteins underpin the most remarkable chemical transformations in nature, but we are not yet in a position to fully mimic or exploit such systems. With the advent of copious, relevant structural information, judicious mechanistic studies and the use of accessible computational methods in protein design coupled with new synthetic methods for building biomacromolecules, we can envisage a ‘new dawn’ that will allow us to build de novo metalloenzymes that move beyond mono-metal centres. In particular, we highlight the need for systems that approach the multi-centred clusters that have evolved to couple electron shuttling with catalysis. Such hybrids may be viewed as exciting mid-points between homogeneous and heterogeneous catalysts which also exploit the primary benefits of biocatalysis.

Selective Probes for Cytochrome P450 17A1 Suggest a Design Strategy Toward Improved Breast and Prostate Cancer Agents. Sex steroids stimulate the growth of hormone-responsive breast and prostate tumors, the two most commonly diagnosed cancers in America. Inhibiting the sex steroid biosynthetic pathway is a promising strategy to halt the progression of these cancers. Cytochrome P450 17A1 (CYP17A1) is a validated target in prostate cancer, but clinical agents fail to achieve selectivity over the highly similar CYP21A2, involved in mineralocorticoid production and blood pressure regulation. In this work, we devised and confirmed a strategy to allow the selective blockade of sex steroidogenesis with the continued production of corticosteroid hormones. To rationally achieve this aim, several co-crystal structures were solved and an assay to evaluate CYP17A1/CYP21A2 selectivity was developed to guide the synthesis of new compounds. A class of these probes achieved significant gains in selectivity over the currently marketed drug abiraterone and galaterone and orteronel, still in clinical trials. This design strategy represents a step toward selectively targeting sex steroidogenesis as a chemotherapeutic tactic for prostate and possibly breast cancers. Spatiotemporal Control of Reactivity via Visible Light-Mediated C-H Activation Creates Chemically Patterned Carbohydrate Surfaces. Reactivity in photochemistry can be restricted to a desired area of a reaction medium by controlling the exposure of reagents to light. This can create patterned surfaces of chemical functionality, which can be used in numerous applications in chemical biology. To demonstrate this utility, we formulated a light-mediated photo-Meerwein arylation reaction to occur on a surface encoded with the reaction substrate. In this proof-of-concept study, reactants were chosen that would undergo a visible color change upon successful reaction. Potential surfaces to display these reactants were then tested. Paper sheets proved optimal for visualization, in which the cellulose C6-position was covalently modified by functionalized coumarin reactants. Use of a photomask allowed these surfaces to be selectively modified by exposure to visible light in the presence of various aryl diazonium reagents and a photocatalyst. These reactions resulted in a strikingly visible color change over the target area. This represents a rapid, cost-effective strategy to selectively encode desired chemical functionality on cellulose medium. This technology could be applied toward surfaces bearing protein capture resins, biosensors, or other agents for chemical biology. Studies Toward Overcoming Product Inhibition in Catalysis of the Intramolecular Schmidt Reaction. Transformations that efficiently generate molecular complexity are useful in drug design, polymer chemistry, and natural product synthesis. The intramolecular Schmidt reaction allows access to amides and lactams from ketone starting materials, and has seen extensive use in the above applications. This reaction is promoted by strongly acidic conditions. Since the reaction creates amide products with increased Lewis basicity over the ketone reactants, most acids are readily sequestered upon successful reaction. In this chapter, we describe the optimization of conditions that promote the intramolecular Schmidt reaction with substoichiometric Sc(OTf)3, which turns over to allow a catalytic cycle in response to heat. The scope, temperature dependence, and kinetics of this transformation were characterized. Additionally, several strategies to expand this strategy were screened. This work, with additional results from a co-worker, ultimately led to the development of vastly improved conditions and scope for catalytic Schmidt reactions.

Sialylation, the addition of negatively charged sialic acid sugars to terminal ends of glycans, is upregulated in most cancers. Hypersialylation supports multiple pro-tumor mechanisms such as enhanced migration and invasion, resistance to apoptosis and immune evasion. A current gap in knowledge is the lack of understanding on how the tumor microenvironment regulates cancer cell sialylation. The adipose niche is a main component of most peritoneal cancers' microenvironment. This includes ovarian cancer (OC), which causes most deaths from all gynecologic cancers. In this report, we demonstrate that the adipose microenvironment is a critical regulator of OC cell sialylation. adipose conditioning led to an increase in both ⍺2,3- and ⍺2,6-linked cell surface sialic acids in both human and mouse models of OC. Adipose-induced sialylation reprogramming was also observed from intra-peritoneal OC tumors seeded in the adipose-rich omentum. Mechanistically, we observed upregulation of at least three sialyltransferases, ST3GAL1, ST6GAL1 and ST3GALNAC3. Hypersialylated OC cells consistently formed intra-peritoneal tumors in both immune-competent mice and immune-compromised athymic nude mice. In contrast, hyposiaylated OC cells persistently formed tumors only in athymic nude mice demonstrating that sialylation impacts OC tumor formation in an immune dependent manner. To our knowledge, this is the first demonstration of the effect of adipose microenvironment on OC tumor sialylation. Our results set the stage for translational applications targeting sialic acid pathways in OC and other peritoneal cancers.

A fundamental mechanism that all eukaryotic cells use to adapt to their environment is dynamic protein modification with monosaccharide sugars. In humans, O-linked N-acetylglucosamine (O-GlcNAc) is rapidly added to and removed from diverse protein sites as a response to fluctuating nutrient levels, stressors, and signaling cues. Two aspects remain challenging for tracking functional O-GlcNAc events with chemical strategies: spatial control over subcellular locations and time control during labeling. The objective of this study was to create intracellular proximity labeling tools to identify functional changes in O-GlcNAc patterns with spatiotemporal control. We developed a labeling strategy based on the TurboID proximity labeling system for rapid protein biotin conjugation that we directed to O-GlcNAc protein modifications inside cells, a set of tools we called 'GlycoID.' Localized variants to the nucleus and cytosol, nuc-GlycoID and cyt-GlycoID, labeled O-GlcNAc proteins and their interactomes in subcellular space. Labeling during insulin as well as serum stimulation revealed functional changes in O-GlcNAc proteins as soon as 30 minutes of signaling. We demonstrated using proteomic analysis that the GlycoID strategy captured O-GlcNAcylated 'activity hubs' consisting of O-GlcNAc proteins and their associated protein-protein interactions. The ability to follow changes in O-GlcNAc hubs during physiological events like insulin stimulation poises these tools to be used for determining mechanisms of glycobiological cell regulation. Our functional O-GlcNAc datasets in human cells will be a useful resource for O-GlcNAc-driven mechanisms. Competing Interest Statement The authors have declared no competing interest.

Enhanced glucose metabolism is a feature of almost all cancers, but downstream functional effects of aberrant glucose flux are difficult to mechanistically determine. The objective of this study is to characterize a mechanism by which elevated glucose level drives a tumorigenic pathway in triple negative breast cancer (TNBC). We used chemical biology methods to track how a metabolite of glucose, N-acetylglucosamine (GlcNAc), is linked to the transcriptional regulatory protein tet-methylcytosine dioxygenase 1 (TET1) as an O-linked GlcNAc post translational modification (O-GlcNAc). In this work, we revealed that intracellular protein glycosylation by O-GlcNAc is driven by high glucose levels in TNBC models, including on TET1. A single enzyme, O-GlcNAc transferase (OGT), is responsible for catalyzing protein modification of O-GlcNAc. We showed that OGT activity is higher in TNBC cell lines compared to non-tumor breast cell lines and is associated with hyperglycemia. Furthermore, enhanced OGT activity activated a pathway for cancer stem-like cell (CSC) reprogramming in TNBC cells. In our model, O-GlcNAcylated TET1 upregulated expression of splicing factor TAR-DNA binding protein (TARDBP), which drives CSC induction as well as higher OGT levels. We show that this OGT-TET1-TARDBP axis 'feeds-forward' in hyperglycemic conditions both in cell lines and diet-induced obese mice, which displayed higher blood glucose levels and tumor O-GlcNAc levels than lean littermates. This data converges on a novel pathway whereby hyperglycemia drives aberrant OGT activity, activating a pathway for CSC induction in TNBC. Our findings partially explain a key aspect of how obesity is associated with TNBC risk and negative outcomes. Competing Interest Statement The authors have declared no competing interest.