Biology subjects

van Aalten, D. M. F.

Publications and source records attributed to van Aalten, D. M. F..

8 recordsLinked to original sources

bioRxiv2025

Multi-domain O-GlcNAcase structures reveal allosteric regulatory mechanisms

Nucleocytoplasmic protein O-GlcNAcylation is an essential modification catalysed by O-GlcNAc transferase (OGT) and reversed by O-GlcNAc hydrolase (OGA), a multi-domain enzyme that also contains a C-terminal pseudo-histone acetyltransferase (pHAT) domain. OGA and OGT are tightly regulated using O-GlcNAc-dependent feedback mechanisms that are largely unknown. Although the structure of the OGA homodimeric catalytic domain has been reported, the structure and function of the pHAT domain remains poorly understood. We describe a crystal structure of the Trichoplax adhaerens pHAT domain and cryo-EM data of the multi-domain T. adhaerens and human OGAs, together with surface plasmon resonance and small-angle X-ray scattering studies. Our findings show that the eukaryotic OGA pHAT domains affect O-GlcNAc homeostasis, and form catalytically incompetent, symmetric homodimers, projecting a partially conserved putative peptide binding site available for interactions with binding partners. While there is evidence for symmetric OGA multi-domain dimers in solution, interactions between the linkers to the pHAT domains allow these to adopt a limited range of positions. In hOGA, the positions of the pHAT domains determine the wider active site environment through a key conformational change involving a tryptophan in a flexible arm region. Taken together, these multi-domain OGA structures reveal allosteric mechanisms of regulation.

molecular biology↗

bioRxiv2024

O-GlcNAcylation of the intellectual disability protein DDX3X exerts proteostatic cell cycle control

O-GlcNAcylation is an evolutionary conserved post-translational modification implicated in neurodevelopment. Missense variants of O-GlcNAc transferase (OGT) are causal for the intellectual disability syndrome OGT Congenital Disorder of Glycosylation (OGT-CDG). The observation of microcephaly in OGT-CDG patients suggests that dysregulation of the cell cycle and aberrant neurogenesis may contribute to disease aetiology. Here, we identify Ser584 O-GlcNAcylation of DDX3X, a known intellectual disability and microcephaly associated protein, as a key regulator of G1/S-phase transition, inhibiting proteasome-dependent degradation of DDX3X. DDX3X levels are reduced in a mouse model of OGT-CDG, alongside the DDX3X-target gene and synaptogenic regulator cyclin E1. These data reveal how a single DDX3X O-GlcNAc site exerts control of the cell cycle and highlights dysregulation of DDX3X-dependent translation, and concomitant impairments in cortical neurogenesis, as a possible pathway disrupted in OGT-CDG.

cell biology↗

bioRxiv2023

Neurodevelopmental defects in a mouse model of O-GlcNAc transferase intellectual disability

O-GlcNAcylation is a protein modification that is critical for vertebrate development, catalysed by O-GlcNAc transferase (OGT) and reversed by O-GlcNAcase (OGA). Missense mutations in OGT have recently been shown to segregate with a syndromic form of intellectual disability, OGT-linked Congenital Disorder of Glycosylation (OGT-CDG). Although OGT-CDG suggests a critical role of O-GlcNAcylation in neurodevelopment and/or cognitive function, the underlying pathophysiologic mechanisms remain unknown. Here we report three mouse lines that carry three different catalytically impaired OGT-CDG variants. These mice show altered O-GlcNAc homeostasis with decreased global O-GlcNAcylation and OGT/OGA levels in the brain. Phenotypic characterization of the mice revealed microcephaly and cognitive deficits including hyperactivity, anxiety and altered spatial working memory. These mouse models will serve as an important tool to study genotype-phenotype correlation in OGT-CDG in vivo and for the development of possible treatment avenues for this disorder. Significant statementMutations in O-GlcNAc transferase (OGT), the sole enzyme that installs O-GlcNAc sugar on proteins, lead to intellectual disability through unknown mechanisms. We have generated mouse models carrying OGT mutations that show reduction in brain size, hyperactivity and defects in memory. These mouse models will serve as a valuable tool to further investigate disease mechanism and propose future treatment avenues.

neuroscience↗

bioRxiv2023

Exploiting O-GlcNAc Transferase promiscuity to dissect site-specific O-GlcNAcylation

Protein O-GlcNAcylation is an evolutionary conserved post-translational modification catalysed by the nucleocytoplasmic O-GlcNAc transferase (OGT) and reversed by O-GlcNAcase (OGA). How site-specific O-GlcNAcylation modulates a diverse range of cellular processes is largely unknown. A limiting factor in studying this is the lack of accessible techniques capable of producing homogeneously O-GlcNAcylated proteins, in high yield, for in vitro studies. Here, we exploit the tolerance of OGT for cysteine instead of serine, combined with a co-expressed OGA to achieve site-specific, highly homogeneous mono-glycosylation. Applying this to DDX3X, TAB1, and CK2, we demonstrate that near-homogeneous mono-S-GlcNAcylation of these proteins promotes DDX3X and CK2 solubility and enables production of mono-S-GlcNAcylated TAB1 crystals, albeit with limited diffraction. Taken together, this work provides a new approach for functional dissection of protein O-GlcNAcylation.

biochemistry↗

bioRxiv2023

An O-GlcNAc transferase pathogenic variant that affects pluripotent stem cell self-renewal

O-linked {beta}-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential enzyme that modifies proteins with O-GlcNAc. Inborn OGT genetic variants were recently shown to mediate a novel type of Congenital Disorder of Glycosylation (OGT-CDG) which is characterized by X-linked intellectual disability (XLID) and developmental delay. Here, we report an OGTC921Y variant which co-segregates with XLID and epileptic seizures, and results in loss of catalytic activity. Colonies formed by mouse embryonic stem cells carrying OGTC921Y show decreased levels of protein O-GlcNAcylation accompanied by decreased levels of Oct4, Sox2 and extracellular alkaline phosphatase (ALP), implying reduced self-renewal capacity. These data establish a link between OGT-CDG and embryonic stem cell self-renewal, providing a foundation for examining the developmental aetiology of this syndrome. Summary statementWe show that the C921Y O-GlcNAc transferase variant found in patients with intellectual disability leads to a defect in pluripotent stem cell self-renewal and decreased levels of stem cell markers.

molecular biology↗

bioRxiv2022

A mutant bacterial O-GlcNAcase visualizes a progressive decline of protein O-GlcNAcylation in early Drosophila embryos critical for neurodevelopment

Protein O-GlcNAcylation, a monosaccharide posttranslational modification maintained by two evolutionarily conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), is a major nutrient sensor integrating key metabolic pathways. While mutations in OGT have recently been associated with neurodevelopmental disorders, the dynamics and function of protein O-GlcNAcylation during early embryogenesis remain elusive. Here we develop a new fluorescent probe to visualize O-GlcNAcylation levels in live Drosophila early embryos. Our study shows that protein O-GlcNAcylation declines as the embryos develop to the mid-blastula transition when the facultative heterochromatin makes its first appearance. Lowering O-GlcNAcylation levels by exogenous OGA activity promotes the polycomb group O-GlcNAc protein Polyhomeotic (Ph) to form nuclear foci and K27 trimethylation of histone H3. This enhanced facultative heterochromatin formation fine-tunes the expression of several neurodevelopmental genes including short of gastrulation (sog). We provide evidence that perturbation of O-GlcNAcylation during early embryogenesis affects learning ability in adulthood, highlighting the importance of O-GlcNAcylation homeostasis for the development of the nervous system.

developmental biology↗

bioRxiv2022

Intellectual disability-associated disruption of O-GlcNAcylation impairs neuronal development and cognitive function in Drosophila

O-GlcNAcylation is a reversible co-/post-translational modification involved in a multitude of cellular processes. The addition and removal of O-GlcNAc modification is controlled by two conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Mutations in OGT have recently been discovered to cause a novel Congenital Disorder of Glycosylation (OGT-CDG) that is characterized by intellectual disability. The mechanisms by which OGT-CDG mutations affect cognition remain unclear. We manipulated O-GlcNAc transferase and O-GlcNAc hydrolase activity in Drosophila and demonstrate an important role of O-GlcNAcylation in habituation learning and synaptic development at the larval neuromuscular junction. Introduction of patient-specific missense mutations into Drosophila O-GlcNAc transferase using CRISPR/Cas9 gene editing, leads to deficits in locomotor function and habituation learning. The habituation deficit can be corrected by blocking O-GlcNAc hydrolysis, indicating that OGT-CDG mutations affect cognitive function via reduced protein O-GlcNAcylation. This study establishes a critical role for O-GlcNAc cycling and disrupted O-GlcNAc transferase activity in cognitive dysfunction. These findings suggest that blocking O-GlcNAc hydrolysis is a potential treatment strategy for OGT-CDG. Author summaryAttachment of single N-acetylglucosamine (GlcNAc) sugars to intracellular proteins has recently been linked to neurodevelopment and cognition. This link has been strengthened by discovery of O-GlcNAc transferase (OGT) missense mutations in intellectual disability. Most of these mutations lie outside the catalytic O-GlcNAc transferase domain and it is unclear how they affect cognitive function. Using the fruit fly Drosophila melanogaster as a model organism, we found that a balance in O-GlcNAc cycling is required for learning and neuronal development. Habituation, a fundamental form of learning, is affected in flies that carry patient-specific OGT mutations, and increasing O-GlcNAcylation genetically corrects the habituation deficit. Our work establishes a critical role for O-GlcNAc cycling in a cognition-relevant process, identifies defective O-GlcNAc transferase activity as a cause of intellectual disability, and proposes underlying mechanisms that can be further explored as treatment targets.

genetics↗

bioRxiv2020

Targeting a critical step in fungal hexosamine biosynthesis

Aspergillus fumigatus is a human opportunistic fungal pathogen with a cell wall that protects it from the extracellular environment. Chitin, an essential cell wall component, is synthesised from UDP-GlcNAc that is produced by the hexosamine biosynthetic pathway. Here, we provide genetic and chemical evidence that glucosamine 6-phosphate N-acetyltransferase (Gna1), a key enzyme in this pathway, is an exploitable antifungal drug target. Deletion of GNA1 results in loss of viability and disruption of the cell wall, phenotypes that can be rescued by the product of the enzyme. In a murine model of aspergillosis, the{Delta} gna1 mutant strain attenuates virulence. Using a fragment-based approach, we discovered a small heterocyclic scaffold that binds proximal to the active site and can be optimised to a selective sub-micromolar binder. Taken together, we have provided genetic, structural and chemical evidence for Gna1 as an antifungal target in Aspergillus fumigatus.

microbiology↗