Mutations in glucocerebrosidase cause the lysosomal storage disorder Gaucher's disease and are the most common risk factor for Parkinson's disease. Therapies to restore the enzyme's function in the ...brain hold great promise for treating the neurological implications. Thus, we developed blood-brain barrier penetrant therapeutic molecules by fusing transferrin receptor-binding moieties to β-glucocerebrosidase (referred to as GCase-BS). We demonstrate that these fusion proteins show significantly increased uptake and lysosomal efficiency compared to the enzyme alone. In a cellular disease model, GCase-BS rapidly rescues the lysosomal proteome and lipid accumulations beyond known substrates. In a mouse disease model, intravenous injection of GCase-BS leads to a sustained reduction of glucosylsphingosine and can lower neurofilament-light chain plasma levels. Collectively, these findings demonstrate the potential of GCase-BS for treating GBA1-associated lysosomal dysfunction, provide insight into candidate biomarkers, and may ultimately open a promising treatment paradigm for lysosomal storage diseases extending beyond the central nervous system.
•Study of substoichiometric O-GlcNAc modifications requires enrichment from biological samples.•Metabolic labeling by O-GlcNAc analogues bearing bioorthogonal chemical handles.•Chemoenzymatic ...labeling and chemical probes enable protein ID and site mapping.•Photochemical cross-linking agent enables identification of O-GlcNAc-modified protein binding partners in cells.•Mass spectrometry-based quantitative proteomics can be applied to O-GlcNAc.
Intracellular glycosylation of nuclear and cytoplasmic proteins involves the addition of N-acetylglucosamine (O-GlcNAc) to serine and threonine residues. This dynamic modification occurs on hundreds of proteins and is involved in various essential biological processes. Because O-GlcNAc is substoichiometric and labile, identifying proteins and sites of modification has been challenging and generally requires proteome enrichment. Here we review recent advances on the implementation of chemical tools to perturb, to detect, and to map O-GlcNAc in living systems. Metabolic and chemoenzymatic labels along with bioorthogonal reactions and quantitative proteomics are enabling investigation of the role of O-GlcNAc in various processes including transcriptional regulation, neurodegeneration, and cell signaling.
The cream of the crop: With the world facing a projected shortfall of crops by 2050, new approaches are needed to boost crop yields. Metabolic feeding of plants with photocaged trehalose‐6‐phosphate ...(Tre6P) can increase levels of the signaling metabolite Tre6P in the plant. Reprogramming of cellular metabolism by Tre6P stimulates a program of plant growth and enhanced crop yields, while boosting starch content.
The
O-GlcNAc modification is found on many nucleocytoplasmic proteins. The dynamic nature of
O-GlcNAc, which in some ways is reminiscent of phosphorylation, has enabled investigators to modulate the ...stoichiometry of
O-GlcNAc on proteins in order to study its function. Although several genetic and pharmacological methods for manipulating
O-GlcNAc levels have been described, one of the most direct approaches of increasing global
O-GlcNAc levels is by using small-molecule inhibitors of
O-GlcNAcase (OGA). As the interest in increasing
O-GlcNAc levels has grown, so too has the number of OGA inhibitors. This review provides an overview of the available methods of increasing
O-GlcNAc levels, with a special emphasis on inhibition of OGA by small molecules. Known inhibitors of OGA are discussed with particular attention on those most suitable for cell-based biological studies. Several examples in which OGA inhibitors have been used to study the functional role of the
O-GlcNAc modification in biological systems are discussed, highlighting the pros and cons of different inhibitors.
Glycosyltransferases carry out important cellular functions in species ranging from bacteria to humans. Despite their essential roles in biology, simple and robust activity assays that can be easily ...applied to high‐throughput screening for inhibitors of these enzymes have been challenging to develop. Herein, we report a bead‐based strategy to measure the group‐transfer activity of glycosyltransferases sensitively using simple fluorescence measurements, without the need for coupled enzymes or secondary reactions. We validate the performance and accuracy of the assay using O‐GlcNAc transferase (OGT) as a model system through detailed Michaelis–Menten kinetic analysis of various substrates and inhibitors. Optimization of this assay and application to high‐throughput screening enabled screening for inhibitors of OGT, leading to a novel inhibitory scaffold. We believe this assay will prove valuable not only for the study of OGT, but also more widely as a general approach for the screening of glycosyltransferases and other group‐transfer enzymes.
A robust fluorescence‐based assay accurately reports on the sugar‐transfer activity of glycosyltransferases. This assay is amenable to high‐throughput screening and should be applicable to a diverse set of group‐transfer enzymes. This assay is validated by screening of a library of known bioactive molecules to identify a new O‐GlcNAc transferase antagonist.
Owing to its roles in human health and disease, the modification of nuclear, cytoplasmic, and mitochondrial proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) has emerged as a topic of ...great interest. Despite the presence of O-GlcNAc on hundreds of proteins within cells, only two enzymes regulate this modification. One of these enzymes is O-GlcNAcase (OGA), a dimeric glycoside hydrolase that has a deep active site cleft in which diverse substrates are accommodated. Chemical tools to control OGA are emerging as essential resources for helping to decode the biochemical and cellular functions of the O-GlcNAc pathway. Here we describe rationally designed bicyclic thiazolidine inhibitors that exhibit superb selectivity and picomolar inhibition of human OGA. Structures of these inhibitors in complex with human OGA reveal the basis for their exceptional potency and show that they extend out of the enzyme active site cleft. Leveraging this structure, we create a high affinity chemoproteomic probe that enables simple one-step purification of endogenous OGA from brain and targeted proteomic mapping of its post-translational modifications. These data uncover a range of new modifications, including some that are less-known, such as O-ubiquitination and N-formylation. We expect that these inhibitors and chemoproteomics probes will prove useful as fundamental tools to decipher the mechanisms by which OGA is regulated and directed to its diverse cellular substrates. Moreover, the inhibitors and structures described here lay out a blueprint that will enable the creation of chemical probes and tools to interrogate OGA and other carbohydrate active enzymes.
Glycosyltransferases are a superfamily of enzymes that are notoriously difficult to inhibit. Here we apply an mRNA display technology integrated with genetic code reprogramming, referred to as the ...RaPID (random non‐standard peptides integrated discovery) system, to identify macrocyclic peptides with high binding affinities for O‐GlcNAc transferase (OGT). These macrocycles inhibit OGT activity through an allosteric mechanism that is driven by their binding to the tetratricopeptide repeats of OGT. Saturation mutagenesis in a maturation screen using 39 amino acids, including 22 non‐canonical residues, led to an improved unnatural macrocycle that is ≈40 times more potent than the parent compound (Kiapp=1.5 nM). Subsequent derivatization delivered a biotinylated derivative that enabled one‐step affinity purification of OGT from complex samples. The high potency and novel mechanism of action of these OGT ligands should enable new approaches to elucidate the specificity and regulation of OGT.
Using the random non‐standard peptides integrated discovery (RaPID) system led to macrocyclic peptide ligands of O‐GlcNAc transferase (OGT). The most potent of these ligands bind to the tetratricopeptide repeat region of OGT and inhibit this enzyme in an allosteric manner, making them promising tool compounds for studying OGT.
Visualization of the reaction coordinate undertaken by glycosyltransferases has remained elusive but is critical for understanding this important class of enzyme. Using substrates and substrate ...mimics, we describe structural snapshots of all species along the kinetic pathway for human O-linked β-N-acetylglucosamine transferase (O-GlcNAc transferase), an intracellular enzyme that catalyzes installation of a dynamic post-translational modification. The structures reveal key features of the mechanism and show that substrate participation is important during catalysis.
The aggregation of the microtubule-associated protein tau into paired helical filaments to form neurofibrillary tangles constitutes one of the pathological hallmarks of Alzheimer's disease. Tau is ...post-translationally modified by the addition of N-acetyl-d-glucosamine O-linked to several serine and threonine residues (O-GlcNAc). Previously, increased O-GlcNAcylation of tau has been shown to block the accumulation of tau aggregates within a tauopathy mouse model. Here we show that O-GlcNAc modification of full-length human tau impairs the rate and extent of its heparin-induced aggregation without perturbing its activity toward microtubule polymerization. O-GlcNAcylation, however, does not impact the “global-fold” of tau as measured by a Förster resonance energy transfer assay. Similarly, nuclear magnetic resonance studies demonstrated that O-GlcNAcylation only minimally perturbs the local structural and dynamic features of a tau fragment (residues 353–408) spanning the last microtubule binding repeat to the major GlcNAc-acceptor Ser400. These data indicate that the inhibitory effects of O-GlcNAc on tau aggregation may result from enhanced monomer solubility or the destabilization of fibrils or soluble aggregates, rather than by altering the conformational properties of the monomeric protein. This work further underscores the potential of targeting the O-GlcNAc pathway for potential Alzheimer's disease therapeutics.
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•O-GlcNAcylation of full-length human tau impairs its aggregation.•O-GlcNAc does not impact the global-fold of tau.•O-GlcNAc on tau induces only minor local structural and dynamic perturbations.•O-GlcNAc may enhance monomer solubility or destabilize tau aggregates.
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