Recombinant Danio rerio Inosine-5'-monophosphate dehydrogenase 1a (impdh1a), partial

What is the primary function of IMPDH1a in zebrafish?

IMPDH1a catalyzes the rate-limiting step in the de novo synthesis of guanine nucleotides, significantly impacting cellular pools of GMP, GDP, and GTP. In zebrafish, this enzyme is particularly important in photoreceptor cells, where guanine nucleotide homeostasis is central to visual function. The predominant variant (transcript variant X1) is expressed exclusively in rod and cone photoreceptors due to gene subfunctionalization resulting from ancestral duplication . This enzyme plays a crucial role in maintaining purine nucleotide balance, with loss of Impdh1a resulting in substantial reduction of guanine levels in photoreceptor cells .

How does zebrafish IMPDH1a compare structurally to human IMPDH1?

Zebrafish IMPDH1a is structurally and functionally similar to the human IMPDH1 retinal variant. Both enzymes share the ability to form protein filaments both in vitro and in vivo. When examining the proteins using negative stain electron microscopy, both human and zebrafish versions form filaments when ATP or GTP is added, with these filaments consisting of stacked octamers . Similar to human IMPDH1, zebrafish IMPDH1a octamers adopt an extended conformation in the active state and a compressed, inactive conformation when bound to GTP. The zebrafish retinal variant (Impdh1a_tvX1) and human retinal variant both demonstrate reduced sensitivity to GTP-mediated inhibition compared to their canonical counterparts .

What splice variants of IMPDH1a are present in zebrafish retina?

In zebrafish retina, transcript variant X1 is the predominant isoform, expressed approximately 150 times more abundantly than variants X2-X4. Using N-terminal and C-terminal primers to distinguish the predicted variants, researchers confirmed that X1 is significantly more expressed than other variants. Antibody validation further confirmed that only the X1 variant (624 amino acids) is expressed at significant levels in the zebrafish retina . Additionally, a new transcript variant, designated as X4, was identified through this research, expanding our understanding of the IMPDH1a gene expression profile in zebrafish .

What are the most effective methods for expressing and purifying recombinant zebrafish IMPDH1a?

For recombinant expression and purification of zebrafish IMPDH1a, researchers typically use bacterial expression systems with His-tagged constructs. The protocol involves:

• Cloning the IMPDH1a gene (preferably the predominant X1 transcript variant) into a suitable expression vector

• Transforming E. coli with the expression construct

• Inducing protein expression using IPTG

• Lysing cells and purifying the recombinant protein using nickel affinity chromatography

• Further purification via size exclusion chromatography

To maintain enzyme activity, buffers should include stabilizing agents such as glycerol and DTT. For specific experimental purposes, researchers may introduce point mutations (such as the Y12A non-assembly mutation) to prevent filament formation and study the effects on enzyme activity . This non-assembly mutation has been shown to affect the enzyme's sensitivity to GTP inhibition, providing valuable insights into structure-function relationships.

How can I visualize IMPDH1a filaments in zebrafish photoreceptors?

IMPDH1a forms prominent protein filaments in zebrafish photoreceptor cell bodies, synapses, and to a lesser degree, in outer segments. These filaments can be visualized using:

• Immunohistochemistry (IHC) with specific antibodies against the C-terminus of IMPDH1a_tvX1

• Confocal microscopy with image deconvolution to resolve filaments clearly

• Distinguishing between rod and cone photoreceptors using 4C12 antibody (for rods) and GFP-expressing transgenic strains (for cones)

What techniques are available for measuring IMPDH1a enzymatic activity in zebrafish tissues?

To measure IMPDH1a enzymatic activity in zebrafish tissues, researchers can employ several complementary approaches:

• Spectrophotometric assays that monitor the production of NADH, which absorbs at 340 nm, as IMPDH converts IMP to XMP

• HPLC-based methods to quantify nucleotide levels (IMP, XMP, GMP, GDP, and GTP)

• Metabolomic approaches to assess broader changes in purine metabolism

For accurate assessment of enzyme kinetics, it's important to consider the regulatory mechanisms affecting IMPDH1a activity. The enzyme demonstrates different sensitivity to GTP inhibition depending on the variant, with Impdh1a having an IC50 for GTP of 460 μM, while Impdh1a_tvX1 is more than tenfold less sensitive (IC50 of 4900 μM) . The introduction of the Y12A non-assembly mutation to Impdh1a_tvX1 significantly increases sensitivity to GTP inhibition (IC50 decreases to approximately 780 μM), highlighting the importance of filament formation in regulating enzymatic activity.

How does IMPDH1a contribute to photoreceptor function in zebrafish?

IMPDH1a plays a crucial role in photoreceptor function by maintaining guanine nucleotide pools necessary for phototransduction. In zebrafish, the predominant retinal variant (Impdh1a_tvX1) is exclusively expressed in rod and cone photoreceptors . This variant forms dynamic protein filaments that change in length and distribution throughout the day, suggesting regulation tied to circadian rhythms and visual activity cycles.

The importance of IMPDH1a in photoreceptor function is evidenced by studies showing that loss of this enzyme results in substantial reduction of guanine levels in the retina. Interestingly, despite this reduction, cellular morphology and cGMP levels remain normal, suggesting compensatory mechanisms that maintain critical aspects of photoreceptor function in the short term . The enzyme's ability to form filaments that resist allosteric inhibition by GTP suggests a mechanism to maintain guanine nucleotide synthesis in regions of high GTP demand, such as photoreceptor outer segments where cGMP serves as the signal transducing molecule in the light response .

What is known about the diurnal regulation of IMPDH1a in zebrafish retina?

IMPDH1a exhibits significant diurnal regulation in the zebrafish retina at multiple levels:

• Filament dynamics: IMPDH1a filaments change length and cellular distribution throughout the day in both rod and cone photoreceptors

• Expression levels: Both mRNA and protein levels of IMPDH1a show diurnal changes

• Enzymatic activity: The rate of GTP synthesis increases in response to light exposure

This dynamic regulation appears linked to changing metabolic demands in photoreceptors during light and dark cycles. When exposed to bright light, there is an increase in GTP and ATP synthesis in the retina, concomitant with IMPDH1a aggregate formation at the outer segment layer . This suggests that IMPDH1a filament formation and activity are regulated in response to the changing energetic and signaling needs of photoreceptors during visual processing.

How does IMPDH1a filament formation relate to its enzymatic function in vivo?

The relationship between IMPDH1a filament formation and enzymatic function appears to be multifaceted:

• Regulation of allosteric inhibition: Filament formation makes the enzyme less sensitive to GTP-mediated inhibition, allowing continued guanine nucleotide synthesis even in environments with high GTP concentration

• Subcellular localization: Filaments may help localize the enzyme to regions of high GTP demand

• Temporal regulation: Changes in filament length and distribution throughout the day suggest a mechanism to match guanine nucleotide synthesis with cyclical metabolic demands

Experimental evidence shows that the Y12A mutation, which prevents filament assembly, increases sensitivity to GTP inhibition . In the zebrafish retina, IMPDH1a filaments are dynamic structures that respond to changing metabolic conditions. The retinal variant's resistance to GTP inhibition (which depends on filament assembly) suggests that filament formation is a physiological adaptation that maintains guanine nucleotide synthesis in photoreceptors where these nucleotides are in high demand .

What are the key similarities and differences between zebrafish IMPDH1a and human IMPDH1?

Zebrafish IMPDH1a and human IMPDH1 share numerous key similarities and differences:

Similarities:

• Both enzymes catalyze the same biochemical reaction in guanine nucleotide synthesis

• Both form protein filaments in vitro and in vivo

• Both have retinal-specific isoforms with reduced sensitivity to GTP inhibition

• The filament structure is preserved, with both forming stacked octamers when bound to ATP or GTP

• Both exhibit active/extended conformations (with ATP) and inhibited/compressed conformations (with GTP)

Differences:

• Gene duplication in zebrafish resulted in subfunctionalization, with IMPDH1a being the predominant retinal variant

• The specific regulatory mechanisms may differ between species

• The patterns of expression across tissues may vary between zebrafish and humans

From a structural perspective, negative stain electron microscopy reveals remarkably similar filament arrangements between zebrafish and human enzymes. When bound to ATP, both form active/extended filaments with a rise of approximately 111 Å for zebrafish Impdh1a and 107 Å for Impdh1a_tvX1. When bound to GTP, both form inhibited/compressed filaments with a rise of 86 Å and 100 Å, respectively .

Can zebrafish IMPDH1a serve as a model for studying human IMPDH1-related diseases?

Zebrafish IMPDH1a serves as an excellent model for studying human IMPDH1-related diseases for several reasons:

• Functional conservation: The fundamental enzymatic function and regulatory mechanisms appear conserved between species

• Structural similarity: Filament formation and conformational changes in response to nucleotides are comparable

• Expression pattern: Like human IMPDH1, zebrafish IMPDH1a is prominently expressed in photoreceptors

• Genetic tractability: Zebrafish are amenable to genetic manipulation to model disease mutations

Zebrafish are widely used to study human mutations causing retinal degeneration . The structural and functional similarity between zebrafish and human retinal variants of IMPDH1 makes this model particularly valuable for studying the retinitis pigmentosa type 10 (RP10) form of autosomal dominant retinitis pigmentosa, which is caused by mutations in IMPDH1 . At least nine mutations in human IMPDH1 have been associated with RP10, including R224P, D226N, R231P, T116M, V268I, G324D, H372P, K238E, and K238R .

What post-translational modifications regulate IMPDH1 activity in vivo?

Post-translational modifications play a crucial role in regulating IMPDH1 activity in vivo. Most notably, light-dependent phosphorylation has been observed:

• Light-dependent phosphorylation: IMPDH1 undergoes phosphorylation at Thr159/Ser160 in the Bateman domain in response to light exposure

• Functional consequence: This phosphorylation desensitizes the enzyme to allosteric inhibition by GDP/GTP

• Physiological impact: When exposed to bright light, there is an increase in GTP and ATP synthesis in the retina

This post-translational modification mechanism appears to be a critical regulatory process that allows photoreceptors to maintain appropriate levels of guanine nucleotides during changing light conditions . The phosphorylation occurs in the Bateman domain, which is involved in nucleotide binding and allosteric regulation of the enzyme. This mechanism likely ensures adequate GTP supply for photoreceptor function during periods of high metabolic demand triggered by light exposure.

How can IMPDH1a mutations be modeled in zebrafish to study retinal degeneration?

Modeling IMPDH1a mutations in zebrafish to study retinal degeneration can be accomplished through several approaches:

• CRISPR/Cas9 gene editing to introduce specific point mutations that correspond to human disease mutations

• Transgenic expression of mutant forms of IMPDH1a under the control of photoreceptor-specific promoters

• Morpholino knockdown followed by rescue with wild-type or mutant forms of human IMPDH1

• Characterization of existing zebrafish mutant strains, such as the impdh1a^sa23234 strain that contains a splice site mutation introducing a premature stop codon

For comprehensive analysis of retinal degeneration phenotypes, researchers should examine:

• Photoreceptor morphology and survival using histology and immunohistochemistry

• Visual function using electroretinography (ERG) and behavioral assays

• Biochemical consequences, including changes in guanine nucleotide levels

• IMPDH1a filament formation and localization

Zebrafish are particularly suitable for these studies as they are widely used to study human mutations causing retinal degeneration, and the structural and functional similarity between zebrafish and human IMPDH1 facilitates translational research .

What is the relationship between IMPDH1 and cancer progression?

While the focus of the provided search results is primarily on the retinal function of IMPDH1, there is significant evidence linking IMPDH1 to cancer progression:

Specifically, high IMPDH1 expression is associated with poor prognosis in hepatocellular carcinoma (LIHC), bladder cancer (BLCA), cervical squamous cell carcinoma (CESC), glioblastoma multiforme (GBM), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), brain lower grade glioma (LGG), mesothelioma (MESO), uveal melanoma (UVM), and adrenocortical carcinoma (ACC) .

How might IMPDH1 be targeted for cancer immunotherapy?

IMPDH1 shows potential as a target for cancer immunotherapy based on several findings:

• Immune checkpoint correlation: IMPDH1 expression correlates with immune checkpoint molecules PD-1, TIGIT, and CTLA4

• Immunotherapy response: High expression of IMPDH1 is associated with poor response to immunotherapy and worse prognosis in patients receiving immune checkpoint inhibitors

• Immune cell infiltration: IMPDH1 expression correlates with infiltration of monocytes, macrophages, CD8+ T cells, and gamma delta T cells in tumors

In hepatocellular carcinoma, tumors with high IMPDH1 expression show significantly increased infiltrating CD8+ T lymphocytes and immune checkpoint expression . This suggests that IMPDH1 might influence the tumor immune microenvironment in ways that affect immunotherapy sensitivity. Targeting IMPDH1 in combination with existing immunotherapies could potentially enhance treatment efficacy, particularly in cancers where high IMPDH1 expression correlates with poor immunotherapy outcomes, such as non-small cell lung cancer, urothelial carcinoma, and renal clear cell carcinoma .

How do IMPDH1a filaments interact with other cellular structures in photoreceptors?

This area represents a frontier of IMPDH1a research. Current evidence shows that IMPDH1a forms prominent protein filaments in rod and cone photoreceptor cell bodies, synapses, and to a lesser extent, in outer segments . These filaments likely interact with various cellular structures, but the specific interactions and their functional significance remain to be fully elucidated.

Key research approaches to address this question include:

• Super-resolution microscopy to visualize spatial relationships between IMPDH1a filaments and other cellular structures

• Proximity labeling techniques (BioID, APEX) to identify proteins that interact with IMPDH1a filaments

• Co-immunoprecipitation studies to isolate IMPDH1a complexes

• Electron microscopy to examine the ultrastructural relationships between filaments and cellular organelles

A particularly intriguing aspect is the relationship between IMPDH1a filaments and the cytoskeleton, as well as potential interactions with mitochondria, which are abundant in photoreceptor inner segments where IMPDH1a filaments are most prominent.

What is the role of IMPDH1a in zebrafish development beyond the retina?

While the predominant focus of IMPDH1a research has been on retinal function, the enzyme likely plays important roles in other tissues during development. To investigate these broader roles, researchers could:

• Perform comprehensive expression analysis of IMPDH1a across tissues and developmental stages

• Generate tissue-specific knockdown or knockout models

• Use metabolomic approaches to assess changes in guanine nucleotide pools in various tissues

• Investigate potential compensatory mechanisms involving other IMPDH family members

Understanding the wider developmental roles of IMPDH1a could provide insights into the tissue-specific manifestations of IMPDH1 mutations in humans. While IMPDH1 mutations primarily cause retinal degeneration, the enzyme is expressed in multiple tissues, suggesting either functional redundancy or compensatory mechanisms that protect other tissues from the consequences of IMPDH1 dysfunction.

How does IMPDH1a activity coordinate with other enzymes in nucleotide metabolism pathways?

IMPDH1a functions within a complex network of enzymes involved in purine nucleotide metabolism. Advanced research questions in this area include:

• How is the activity of IMPDH1a coordinated with other enzymes in the de novo purine synthesis pathway?

• What is the relationship between the de novo pathway (involving IMPDH1a) and the salvage pathway for guanine nucleotide synthesis?

• How do changes in IMPDH1a activity affect the broader purine nucleotide balance in cells?

• What feedback mechanisms exist between IMPDH1a and downstream enzymes in guanine nucleotide synthesis?

Metabolic flux analysis using stable isotope-labeled precursors could help elucidate these complex interactions. Researchers might also investigate the spatial organization of purine metabolism enzymes, as the formation of enzyme clusters or "metabolons" could facilitate efficient substrate channeling through sequential enzymatic reactions.

What are the optimal conditions for measuring IMPDH1a enzymatic activity in vitro?

For optimal measurement of IMPDH1a enzymatic activity in vitro, researchers should consider the following conditions:

Buffer Composition:

• 50 mM Tris-HCl, pH 8.0

• 100 mM KCl

• 1 mM DTT

• 3 mM EDTA

• 1% glycerol

Substrate Concentrations:

• IMP: 0.5-1 mM

• NAD+: 0.5-1 mM

Regulatory Factors:

• ATP (1-2 mM) promotes filament formation and may affect activity

• GTP (0.1-5 mM) for inhibition studies

Temperature and Time:

• Reactions typically performed at 25-37°C

• Linear phase of reaction usually occurs within first 30 minutes

Detection Method:

• Spectrophotometric monitoring of NADH production at 340 nm

• Coupling to secondary reactions for increased sensitivity

It's important to consider that the different variants of IMPDH1a show different sensitivities to GTP inhibition, with Impdh1a having an IC50 for GTP of 460 μM and Impdh1a_tvX1 having an IC50 of 4900 μM . Therefore, regulatory studies should account for these variant-specific differences in inhibition profiles.

What techniques are most effective for analyzing IMPDH1a phosphorylation states?

To effectively analyze IMPDH1a phosphorylation states, researchers should employ multiple complementary techniques:

• Phospho-specific antibodies:

  • Develop antibodies specific to phosphorylated Thr159/Ser160 in the Bateman domain

  • Use for Western blotting and immunohistochemistry to detect phosphorylation in vivo

• Mass spectrometry:

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for identification and quantification of phosphorylation sites

  • Phosphopeptide enrichment using titanium dioxide (TiO2) or immobilized metal affinity chromatography (IMAC)

• Phosphomimetic mutations:

  • Generate T159D/S160D mutants to mimic constitutive phosphorylation

  • Generate T159A/S160A mutants to prevent phosphorylation

  • Analyze effects on enzymatic activity and filament formation

• In vivo labeling:

  • Use 32P-orthophosphate labeling in cultured cells or tissues

  • Perform immunoprecipitation followed by autoradiography

Since phosphorylation of IMPDH1 at Thr159/Ser160 in the Bateman domain is light-dependent and desensitizes the enzyme to allosteric inhibition by GDP/GTP , it's crucial to carefully control light conditions during sample preparation and analysis.

How should experimental data on IMPDH1a be integrated with computational models of purine metabolism?

Integrating experimental data on IMPDH1a with computational models of purine metabolism involves several key steps:

• Data collection and standardization:

  • Ensure kinetic parameters (Km, Vmax, Ki) are determined under comparable conditions

  • Standardize concentration units and experimental conditions

  • Include data on regulatory mechanisms (phosphorylation, filament formation)

• Model selection and development:

  • Choose between kinetic models (e.g., ordinary differential equations) or constraint-based models (e.g., flux balance analysis)

  • Include compartmentalization of purine metabolism

  • Incorporate regulatory mechanisms specific to IMPDH1a

• Parameter estimation and validation:

  • Use experimental data to estimate unknown parameters

  • Validate model predictions against independent experimental datasets

  • Perform sensitivity analysis to identify key parameters

• Simulation and prediction:

  • Simulate the effects of IMPDH1a mutations or inhibition

  • Predict metabolic responses to changes in light conditions

  • Model the impact of fluctuations in nucleotide levels

• Integration with broader cellular models:

  • Connect purine metabolism models with models of photoreceptor function

  • Link to models of energy metabolism and redox homeostasis

What emerging technologies could advance our understanding of IMPDH1a regulation in vivo?

Several emerging technologies hold promise for advancing our understanding of IMPDH1a regulation in vivo:

• Cryo-electron tomography:

  • Visualize IMPDH1a filaments in their native cellular context

  • Determine how filaments interact with other cellular structures

• Optogenetic tools:

  • Develop light-activated IMPDH1a variants to control enzyme activity with temporal precision

  • Study the effects of rapid changes in GTP synthesis on photoreceptor function

• Live-cell imaging of IMPDH1a dynamics:

  • Generate fluorescently tagged IMPDH1a to monitor filament formation in real-time

  • Track changes in filament structure in response to metabolic perturbations

• Single-cell metabolomics:

  • Measure nucleotide levels in individual photoreceptors

  • Correlate IMPDH1a activity with metabolite concentrations at the single-cell level

• CRISPR-based screening:

  • Identify genes that modify IMPDH1a function or filament formation

  • Discover novel regulatory pathways affecting IMPDH1a activity

These technologies would provide unprecedented insights into the dynamic regulation of IMPDH1a in photoreceptors and potentially reveal new therapeutic targets for IMPDH1-associated retinal degenerations.

How might insights from zebrafish IMPDH1a research translate to human therapy development?

Insights from zebrafish IMPDH1a research can translate to human therapy development through several pathways:

• Drug discovery:

  • Screen for compounds that specifically modulate IMPDH1 activity without affecting IMPDH2

  • Identify molecules that stabilize or disrupt IMPDH1 filaments

• Gene therapy approaches:

  • Develop strategies to replace mutant IMPDH1 in photoreceptors

  • Test expression of engineered IMPDH1 variants resistant to disease-causing mutations

• Metabolic interventions:

  • Design supplements or dietary modifications to compensate for altered guanine nucleotide metabolism

  • Test the effects of guanosine supplementation on retinal degeneration progression

• Precision medicine approaches:

  • Correlate specific IMPDH1 mutations with phenotypic outcomes

  • Develop mutation-specific therapeutic strategies

The zebrafish model offers significant advantages for translational research, including rapid development, transparent embryos allowing direct visualization of the retina, and genetic tractability. These features facilitate high-throughput screening of potential therapeutic compounds and genetic interventions that could be translated to human clinical applications.

What are the implications of IMPDH1a research for understanding broader metabolic regulation in photoreceptors?

IMPDH1a research has significant implications for understanding broader metabolic regulation in photoreceptors:

• Integration of nucleotide metabolism with energy homeostasis:

  • IMPDH1a activity requires NAD+ and produces NADH, linking guanine synthesis to redox balance

  • Bright light exposure increases both GTP and ATP synthesis, suggesting coordinated regulation

• Compartmentalization of metabolism:

  • IMPDH1a filaments show specific subcellular localization patterns

  • This suggests spatial organization of metabolic pathways within photoreceptors

• Temporal regulation of metabolism:

  • IMPDH1a filaments change in length and distribution throughout the day

  • This indicates dynamic adaptation of metabolic capacity to changing demands

• Stress responses:

  • Understanding how IMPDH1a responds to metabolic stress could reveal broader stress response mechanisms in photoreceptors

  • This may be relevant to multiple retinal degenerative diseases

• Signaling network integration:

  • GTP is both a metabolite and signaling molecule

  • IMPDH1a regulation may represent a node connecting metabolism with cellular signaling networks