Biology subjects

O'Neill, A. G.

Publications and source records attributed to O'Neill, A. G..

4 recordsLinked to original sources

bioRxiv2025

An IMPDH2 variant associated with neurodevelopmental disorder disrupts purine biosynthesis and somitogenesis

IMP dehydrogenase (IMPDH) controls a key regulatory node in purine biosynthesis. Gain-of-function mutations in human IMPDH2 are associated with neurodevelopmental disorders and neuromuscular symptoms including dystonia, but the developmental mechanisms underlying these defects are unknown. We previously showed that these mutants are insensitive to GTP inhibition and hypothesized that their hyperactivity would affect nucleotide metabolism in vivo. Here, we characterize the metabolic and developmental consequences of the neurodevelopmental disorder-associated IMPDH2 mutant, S160del, in Xenopus tropicalis. We show that expressing S160del but not WT human IMPDH2 disrupts purine pools and somitogenesis in the developing tadpole. We also show that S160del disrupts in vivo IMPDH filament assembly, a well-described IMPDH regulatory mechanism. Cryo-EM structures show that S160del disrupts filament assembly by destabilizing the dimerization of regulatory Bateman domains. Dimerization of Bateman domains and subsequent filament formation can be restored with a high affinity ligand, but this does not restore sensitivity to GTP inhibition, suggesting S160del also disrupts allostery of IMPDH2 filaments. This work demonstrates that the structural effects of patient IMPDH2 variants can cause disruptions both to nucleotide levels and to the normal development of sensorimotor structures, helping us better understand the physiological basis of disease in these patients. SIGNIFICANCE STATEMENTIMPDH2 is a critical enzyme for de novo purine biosynthesis, regulating the balance between adenine and guanine nucleotides. Under purine stress, it forms filaments that resist feedback inhibition by GTP. Patients with gain-of-function variants of this enzyme suffer from early-onset neuromotor symptoms including dystonia. Here, we express one gain-of-function variant of IMPDH2, S160del, in Xenopus tropicalis. S160del is particularly powerful for structural and developmental studies, as it impedes filament formation and also is insensitive to feedback inhibition by GTP. Here, we show S160del can perturb vertebrate development, metabolism, and filament formation in a dominant fashion. Insights from this work will open the door to a new suite of studies defining the function of purine metabolism in development and disease.

biochemistry↗

bioRxiv2024

Appendage regeneration requires IMPDH2 and creates a sensitized environment for enzyme filament formation

Regeneration of lost tissue requires biosynthesis of metabolites needed for cell proliferation and growth. Among these are the critical purine nucleotides ATP and GTP. The abundance and balance of these purines is regulated by inosine monophosphate dehydrogenase 2 (IMPDH2), which catalyzes the committing step of GTP synthesis. IMPDH2 assembles into filaments that resist allosteric inhibition under conditions of high GTP demand. Here we asked whether IMPDH2 is required in the highly proliferative context of regeneration, and whether its assembly into filaments takes place in regenerating tissue. We find that inhibition of IMPDH2 leads to impaired tail regeneration and reduced cell proliferation in the tadpole Xenopus tropicalis. We find that both endogenous and fluorescent fusions of IMPDH2 robustly assemble into filaments throughout the tadpole tail, and that the regenerating tail creates a sensitized condition for filament formation. These findings clarify the role of purine biosynthesis in regeneration and reveal that IMPDH2 enzyme filament formation is a biologically relevant mechanism of regulation in vertebrate regeneration. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/605679v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1006d64org.highwire.dtl.DTLVardef@10ddf85org.highwire.dtl.DTLVardef@1cdc99eorg.highwire.dtl.DTLVardef@16fc4cb_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2023

Light-sensitive phosphorylation regulates enzyme activity and filament assembly of human IMPDH1 retinal splice variants

Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo guanosine triphosphate (GTP) synthesis and is controlled by feedback inhibition and allosteric regulation. IMPDH assembles into micron-scale filaments in cells, which desensitizes the enzyme to feedback inhibition by GTP and boosts nucleotide production. The vertebrate retina expresses two tissue-specific splice variants IMPDH1(546) and IMPDH1(595). IMPDH1(546) filaments adopt high and low activity conformations, while IMPDH1(595) filaments maintain high activity. In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of phosphorylation in IMPDH1 variants. The S477D mutation re-sensitized both variants to GTP inhibition, but only blocked assembly of IMPDH1(595) filaments and not IMPDH1(546) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of the high activity assembly interface, still allowing assembly of low activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, phosphorylation at S477 acts as a mechanism for downregulating retinal GTP synthesis in the dark, when nucleotide turnover is decreased. Like IMPDH1, many other metabolic enzymes dynamically assemble filamentous polymers that allosterically regulate activity. Our work suggests that posttranslational modifications may be yet another layer of regulatory control to finely tune activity by modulating filament assembly in response to changing metabolic demands. SIGNIFICANCE STATEMENTOver 20 different metabolic enzymes form micron-scale filaments in cells, suggesting that filament assembly is a conserved mechanism for regulating diverse metabolic pathways. Filament assembly regulates catalytic activity of many of these enzymes, including inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in de novo GTP biosynthesis. The vertebrate retina expresses two IMPDH1 splice variants that are critical for maintaining nucleotide levels required for phototransduction. Here, we show that filament assembly by these variants is itself controlled by phosphorylation at a single residue, adding further complexity to the tight regulation of nucleotide metabolism in the retina. Phosphorylation and other posttranslational modifications are likely to be a general regulatory mechanism controlling filament assembly by enzymes in many different metabolic pathways.

biochemistry↗

bioRxiv2023

Point mutations in IMPDH2 which cause early-onset neurodevelopmental disorders disrupt enzyme regulation and filament structure

Inosine 5 monophosphate dehydrogenase (IMPDH) is a critical regulatory enzyme in purine nucleotide biosynthesis that is inhibited by the downstream product GTP. Multiple point mutations in the human isoform IMPDH2 have recently been associated with dystonia and other neurodevelopmental disorders, but the effect of the mutations on enzyme function has not been described. Here, we report identification of two additional affected individuals with missense variants in IMPDH2 and show that all of the disease-associated mutations disrupt GTP regulation. Cryo-EM structures of one IMPDH2 mutant suggest this regulatory defect arises from a shift in the conformational equilibrium toward a more active state. This structural and functional analysis provides insight into IMPDH2-associated disease mechanisms that point to potential therapeutic approaches and raises new questions about fundamental aspects of IMPDH regulation. Significance StatementPoint mutations in the human enzyme IMPDH2, a critical regulator of nucleotide biosynthesis, are linked to neurodevelopmental disorders, such as dystonia. Here, we report two additional IMPDH2 point mutants associated with similar disorders. We investigate the effects of each mutation on IMPDH2 structure and function in vitro and find that all mutations are gain of function, preventing allosteric regulation of IMPDH2 activity. We report high resolution structures of one variant and present a structure-based hypothesis for its dysregulation. This work provides a biochemical basis for understanding diseases caused by IMPDH2 mutation and lays a foundation for future therapeutic development.

biochemistry↗