IMPDH2 forms spots at branching sites and distal ends of astrocyte stem processes.

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in the de novo GTP biosynthesis pathway. Recent studies suggest that IMPDH2, an isoform of IMPDH, can localize to specific subcellular compartments under certain conditions and regulate site-specific GTP availability and small GTPase activity in invasive cancer cells. However, it is unclear whether IMPDH2 plays a site-specific regulatory role in subcellular functions in healthy cells. In this study, we focused on brain cells and examined the localization pattern of IMPDH2. We discovered that IMPDH2 forms localized spots in the astrocytes of the adult mouse hippocampus. Further analysis of spot distribution in primary astrocyte cultures revealed that IMPDH2 spots are predominantly localized on branching sites and distal ends of astrocyte stem processes. Our findings suggest a potential unidentified role for IMPDH2 and GTP synthesis specifically at specialized nodes of astrocyte branches.

IMPDH1 retinal variants control filament architecture to tune allosteric regulation.

Inosine-5'-monophosphate dehydrogenase (IMPDH), a key regulatory enzyme in purine nucleotide biosynthesis, dynamically assembles filaments in response to changes in metabolic demand. Humans have two isoforms: IMPDH2 filaments reduce sensitivity to feedback inhibition, while IMPDH1 assembly remains uncharacterized. IMPDH1 plays a unique role in retinal metabolism, and point mutants cause blindness. Here, in a series of cryogenic-electron microscopy structures we show that human IMPDH1 assembles polymorphic filaments with different assembly interfaces in extended and compressed states. Retina-specific splice variants introduce structural elements that reduce sensitivity to GTP inhibition, including stabilization of the extended filament form. Finally, we show that IMPDH1 disease mutations fall into two classes: one disrupts GTP regulation and the other has no effect on GTP regulation or filament assembly. These findings provide a foundation for understanding the role of IMPDH1 in retinal function and disease and demonstrate the diverse mechanisms by which metabolic enzyme filaments are allosterically regulated.

The Bateman domain of IMP dehydrogenase is a binding target for dinucleoside polyphosphates.

IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the rate-limiting step in the de novo guanine nucleotide biosynthetic pathway. Because of its involvement in the control of cell division and proliferation, IMPDH represents a therapeutic for managing several diseases, including microbial infections and cancer. IMPDH must be tightly regulated, but the molecular mechanisms responsible for its physiological regulation remain unknown. To this end, we recently reported an important role of adenine and guanine mononucleotides that bind to the regulatory Bateman domain to allosterically modulate the catalytic activity of eukaryotic IMPDHs. Here, we have used enzyme kinetics, X-ray crystallography, and small-angle X-ray scattering (SAXS) methodologies to demonstrate that adenine/guanine dinucleoside polyphosphates bind to the Bateman domain of IMPDH from the fungus Ashbya gossypii with submicromolar affinities. We found that these dinucleoside polyphosphates modulate the catalytic activity of IMPDHs in vitro by efficiently competing with the adenine/guanine mononucleotides for the allosteric sites. These results suggest that dinucleoside polyphosphates play important physiological roles in the allosteric regulation of IMPDHs by adding an additional mechanism for fine-tuning the activities of these enzymes. We propose that these findings may have important implications for the design of therapeutic strategies to inhibit IMPDHs.

Interfilament interaction between IMPDH and CTPS cytoophidia.

Inosine monophosphate dehydrogenase (IMPDH) and cytidine triphosphate synthase (CTPS) are two metabolic enzymes that perform rate-limiting steps in the de novo synthesis of purine and pyrimidine nucleotides, respectively. It has been shown that IMPDH and CTPS can comprise a filamentous macrostructure termed the cytoophidium, which may play a role in regulation of their catalytic activity. Although these two proteins may colocalise in the same cytoophidium, how they associate with one another is still elusive. As reported herein, we established a model HeLa cell line coexpressing OFP-tagged IMPDH2 and GFP-tagged CTPS1 and recorded the assembly, disassembly and movement of the cytoophidium in live cells. Moreover, by using super-resolution confocal imaging, we demonstrate how IMPDH- and CTPS-based filaments are aligned or intertwined in the mixed cytoophidium. Collectively, our findings provide a panorama of cytoophidium dynamics and suggest that IMPDH and CTPS cytoophidia may coordinate by interfilament interaction.

Ultrastructure of cytoplasmic and nuclear inosine-5'-monophosphate dehydrogenase 2 "rods and rings" inclusions.

Inosine-5'-monophosphate dehydrogenase catalyzes the critical step in the de novo synthesis of guanosine nucleotides: the oxidation of inosine monophosphate to xanthosine monophosphate. This reaction can be inhibited by specific inhibitors, such as ribavirin or mycophenolic acid, which are widely used in clinical treatment when required to inhibit the proliferation of viruses or cells. However, it was recently found that such an inhibition affects the cells, leading to a redistribution of IMPDH2 and the appearance of IMPDH2 inclusions in the cytoplasm. According to their shape, these inclusions have been termed "Rods and Rings" (R&R). In this work, we focused on the subcellular localization of IMPDH2 protein and the ultrastructure of R&R inclusions. Using microscopy and western blot analysis, we show the presence of nuclear IMPDH2 in human cells. We also show that the nuclear pool has an ability to form Rod structures after inhibition by ribavirin. Concerning the ultrastructure, we observed that R&R inclusions in cellulo correspond to the accumulation of fibrous material that is not surrounded by a biological membrane. The individual fibers are composed of regularly repeating subunits with a length of approximately 11 nm. Together, our findings describe the localization of IMPDH2 inside the nucleus of human cells as well as the ultrastructure of R&R inclusions.

Automated particle correspondence and accurate tilt-axis detection in tilted-image pairs.

Tilted electron microscope images are routinely collected for an ab initio structure reconstruction as a part of the Random Conical Tilt (RCT) or Orthogonal Tilt Reconstruction (OTR) methods, as well as for various applications using the "free-hand" procedure. These procedures all require identification of particle pairs in two corresponding images as well as accurate estimation of the tilt-axis used to rotate the electron microscope (EM) grid. Here we present a computational approach, PCT (particle correspondence from tilted pairs), based on tilt-invariant context and projection matching that addresses both problems. The method benefits from treating the two problems as a single optimization task. It automatically finds corresponding particle pairs and accurately computes tilt-axis direction even in the cases when EM grid is not perfectly planar.