A highly conserved zebrafish IMPDH retinal isoform produces the majority of guanine and forms dynamic protein filaments in photoreceptor cells

Cleghorn, Whitney M.; Burrell, Anika; Giarmarco, Michelle M.; Brock, Daniel C; Wang, Yekai; Chambers, Zachary S.; Du, Jianhai; Kollman, Justin M.; Brockerhoff, Susan E.

doi:10.1016/j.jbc.2021.101441

Cited by 9 publications

(7 citation statements)

References 62 publications

(107 reference statements)

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“…In retinal cells, the presence of cytoophidium is tightly regulated by light conditions, as demonstrated in previous studies ( Plana-Bonamaiso et al, 2020 ; Cleghorn et al, 2022 ). This regulation is of particular significance in photoreceptor cells where IMPDH1 is highly overexpressed.…”

Section: Discussionsupporting

confidence: 74%

Effect on cell survival and cytoophidium assembly of the adRP-10-related IMPDH1 missense mutation Asp226Asn

Keppeke,

Chang,

Zhang

et al. 2023

Front. Cell Dev. Biol.

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Introduction: Inosine monophosphate dehydrogenase 1 (IMPDH1) is a critical enzyme in the retina, essential for the correct functioning of photoreceptor cells. Mutations in IMPDH1 have been linked to autosomal dominant retinitis pigmentosa subtype 10 (adRP-10), a genetic eye disorder. Some of these mutations such as the Asp226Asn (D226N) lead to the assembly of large filamentous structures termed cytoophidia. D226N also gives IMPDH1 resistance to feedback inhibition by GDP/GTP. This study aims to emulate the adRP-10 condition with a long-term expression of IMPDH1-D226N in vitro and explore cytoophidium assembly and cell survival. We also assessed whether the introduction of an additional mutation (Y12C) to disrupt the cytoophidium has an attenuating effect on the toxicity caused by the D226N mutation.Results: Expression of IMPDH1-D226N in HEp-2 cells resulted in cytoophidium assembly in ∼70% of the cells, but the presence of the Y12C mutation disrupted the filaments. Long-term cell survival was significantly affected by the presence of the D226N mutation, with a decrease of ∼40% in the cells expressing IMPDH1-D226N when compared to IMPDH1-WT; however, survival was significantly recovered in IMPDH1-Y12C/D226N, with only a ∼10% decrease when compared to IMPDH1-WT. On the other hand, the IMPDH1 expression level in the D226N-positive cells was <30% of that of the IMPDH1-WT-positive cells and only slightly higher in the Y12C/D226N, suggesting that although cell survival in Y12C/D226N was recovered, higher expression levels of the mutated IMPDH1 were not tolerated by the cells in the long term.Conclusion: The IMPDH1-D226N effect on photoreceptor cell survival may be the result of a sum of problems: nucleotide unbalance plus a toxic long-life cytoophidium, supported by the observation that by introducing Y12C in IMPDH1 the cytoophidium was disrupted and cell survival significantly recovered, but not the sensibility to GDP/GTP regulation since higher expression levels of IMPDH1-D226N were not tolerated.

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Section: Discussionsupporting

confidence: 74%

Effect on cell survival and cytoophidium assembly of the adRP-10-related IMPDH1 missense mutation Asp226Asn

Keppeke,

Chang,

Zhang

et al. 2023

Front. Cell Dev. Biol.

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“…We recently characterized IMPDH1 filaments in the zebrafish retina, finding a circadian pattern to filament assembly, with elongated filaments forming in photoreceptors during the day and smaller punctate structures at night (Cleghorn et al, 2022). We also showed that the zebrafish IMPDH1 retinal variants are very similar to human IMPDH1 in terms of structure, filament assembly, and regulation (Cleghorn et al, 2022). This is consistent with our finding here that the S477D mutation, which mimics dark-dependent phosphorylation, disrupts filament assembly and reduces enzyme activity in the presence of GTP.…”

Section: Discussionmentioning

confidence: 99%

“…Recombinant human IMPDH1 retinal variant proteins were expressed and purified as described previously (Anthony et al, 2017; Burrell et al, 2022; Cleghorn et al, 2022). Briefly, BL21 (DE3) E. coli cells were transformed with wildtype or S477D mutant IMPDH1(546) or IMPDH1(595) expression vectors.…”

Section: Methodsmentioning

confidence: 99%

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

Calise,

O’Neill,

Burrell

et al. 2023

Preprint

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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.

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“…Cyclic GMP is the key signaling molecule in the phototransduction cascade [62][63][64] while photoreceptors have an exceptionally high demand for ATP [65]. On top of this, there is a lack of redundancy in purine nucleotide production as there is very little expression of both IMPDH2 [53,66] and the major purine salvage enzyme HPRT [67,68]. Imbalanced purine pools in photoreceptors can lead to cell death [69,70].…”

Section: Impdh1-associated Retinitis Pigmentosamentioning

confidence: 99%

“…Treatment of IMPDH1-associated RP poses a unique challenge not only because it is autosomal dominant but also because there are only low levels of expression of the other isoform [ 53 , 66 ] in the retina to maintain purine biosynthesis. Therapeutic delivery to the retina is most commonly achieved by intravitreal injection.…”

Section: Introductionmentioning

confidence: 99%

IMPDH dysregulation in disease: a mini review

Burrell

2022

Biochemical Society Transactions

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Inosine-5′-monophosphate dehydrogenase (IMPDH) is a highly conserved enzyme in purine metabolism that is tightly regulated on multiple levels. IMPDH has a critical role in purine biosynthesis, where it regulates flux at the branch point between adenine and guanine nucleotide synthesis, but it also has a role in transcription regulation and other moonlighting functions have been described. Vertebrates have two isoforms, IMPDH1 and IMPDH2, and point mutations in each are linked to human disease. Mutations in IMPDH2 in humans are associated with neurodevelopmental disease, but the effects of mutations at the enzyme level have not yet been characterized. Mutations in IMPDH1 lead to retinal degeneration in humans, and recent studies have characterized how they cause functional defects in regulation. IMPDH1 is expressed as two unique splice variants in the retina, a tissue with very high and specific demands for purine nucleotides. Recent studies have revealed functional differences among splice variants, demonstrating that retinal variants up-regulate guanine nucleotide synthesis by reducing sensitivity to feedback inhibition by downstream products. A better understanding of the role of IMPDH1 in the retina and the characterization of an animal disease model will be critical for determining the molecular mechanism of IMPDH1-associated blindness.

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A highly conserved zebrafish IMPDH retinal isoform produces the majority of guanine and forms dynamic protein filaments in photoreceptor cells

Cited by 9 publications

References 62 publications

Effect on cell survival and cytoophidium assembly of the adRP-10-related IMPDH1 missense mutation Asp226Asn

Effect on cell survival and cytoophidium assembly of the adRP-10-related IMPDH1 missense mutation Asp226Asn

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

IMPDH dysregulation in disease: a mini review

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