Recombinant Xenopus tropicalis Phosphatidylinositol 5-phosphate 4-kinase type-2 gamma (pip4k2c)

What is Xenopus tropicalis PIP4K2C and what are its basic functions?

PIP4K2C (Phosphatidylinositol 5-phosphate 4-kinase type-2 gamma) belongs to the PI5P4K family of lipid kinases that phosphorylate phosphatidylinositol 5-phosphate (PI5P) to generate phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). In mammalian systems, PIP4K2C functions as a regulator of mTORC1 signaling pathways, which control cellular growth and metabolism . PIP4K2C is considered part of the "dark kinome" – the approximately 25% of kinases with unknown or poorly understood functions .

In Xenopus tropicalis, PIP4K2C shares functional homology with its mammalian counterparts, making this amphibian model valuable for studying conserved PIP4K2C functions. Research methodologies to study its basic functions typically include:

• Gene expression analysis using RT-PCR and RNA-seq

• Protein localization studies using immunofluorescence

• Loss-of-function studies using morpholinos or CRISPR-Cas9

• Gain-of-function studies using mRNA microinjection

The advantages of using X. tropicalis include its diploid genome with remarkable synteny to mammalian genomes, often in stretches of a hundred genes or more, which is far greater than that seen between fish and mammals .

How is PIP4K2C gene expression regulated during Xenopus tropicalis development?

PIP4K2C expression in X. tropicalis follows temporal and spatial patterns during embryonic development. While specific X. tropicalis PIP4K2C expression data is limited in the provided search results, methodologies to study its expression include:

• Temporal expression analysis: Using RT-qPCR or RNA-seq at different developmental stages to track expression changes over time. This approach is similar to the temporal expression analysis conducted for UV defense genes in the Tibetan frog using STEM clustering and WGCNA analysis .

• Spatial expression analysis: Whole-mount in situ hybridization (WISH) can visualize tissue-specific expression patterns.

• Regulatory element identification: Promoter analysis and enhancer mapping through reporter assays and chromatin immunoprecipitation (ChIP).

• Epigenetic regulation: Investigating methylation patterns and histone modifications using bisulfite sequencing and ChIP-seq.

Researchers should consider including multiple developmental stages in their analysis, from early cleavage to tadpole stages, to fully characterize expression dynamics.

What experimental systems are available for studying PIP4K2C in Xenopus tropicalis?

Several experimental systems and methodologies are available for studying PIP4K2C in X. tropicalis:

X. tropicalis has significant advantages as a model organism for genetics and genomics. The species has a diploid genome (unlike the allotetraploid X. laevis), which simplifies genetic analysis . Additionally, researchers have developed various inbred lines of X. tropicalis with the intent of creating strains more suitable for stable genetic screens and gene mapping . The availability of BAC libraries and genome sequencing data further enhances the utility of this model system .

How does Xenopus tropicalis PIP4K2C compare structurally and functionally to human PIP4K2C?

When comparing X. tropicalis PIP4K2C to human PIP4K2C, researchers should consider both structural and functional aspects:

Structural comparison:

• Sequence homology assessment using bioinformatic tools

• Domain architecture analysis

• 3D structure prediction and comparison

• Active site conservation evaluation

Functional comparison:

• Substrate specificity using in vitro kinase assays

• Protein-protein interaction networks

• Subcellular localization patterns

• Response to inhibitors and regulatory factors

While specific comparison data is not provided in the search results, the methodology for such analysis would typically involve recombinant protein expression and biochemical characterization. The enzymatic activities of both proteins can be compared using ADP-Glo reporter assays similar to those used for human PI5P4Kγ mutants . Such assays have been used to measure the conversion of PI5P to PI(4,5)P₂ by monitoring ADP production.

Researchers should note that the wild-type PI5P4Kγ has particularly low enzymatic activity, which can be challenging to measure directly. Comparable mutations to those made in human PI5P4Kγ (insertion of QAR at position 139 plus additional mutations) might be needed to enhance the activity of X. tropicalis PIP4K2C for robust assay development .

What methods are optimal for recombinant expression and purification of Xenopus tropicalis PIP4K2C?

Researchers seeking to produce recombinant X. tropicalis PIP4K2C should consider the following methodological approach:

Expression systems:

• Bacterial expression (E. coli): Using BL21(DE3) or similar strains with pET, pGEX, or pMAL vectors for fusion proteins

• Insect cell expression: Baculovirus expression in Sf9 or Hi5 cells for improved folding and post-translational modifications

• Mammalian cell expression: HEK293 or CHO cells for native-like modifications

Optimization strategies:

• Codon optimization: Adapting the coding sequence to the expression host

• Fusion tags: Testing His6, GST, MBP, or SUMO tags to improve solubility

• Expression conditions: Optimizing temperature, inducer concentration, and duration

• Co-expression: Including chaperones or binding partners to enhance folding

Purification methodology:

• Affinity chromatography: Using tag-specific resins (Ni-NTA, glutathione, etc.)

• Ion exchange chromatography: Based on protein's predicted isoelectric point

• Size exclusion chromatography: Final polishing step for homogeneity

• Tag removal: Using specific proteases (TEV, PreScission, etc.) if necessary

For functional studies, researchers should assess protein quality using:

• Thermal shift assays to evaluate stability

• Dynamic light scattering to confirm monodispersity

• Activity assays to verify functionality

For PI5P4K family members, researchers have developed cell-free thermal shift assays to determine compound binding and protein stability . Similar methodologies would be applicable to X. tropicalis PIP4K2C.

How can researchers effectively assess PIP4K2C kinase activity in Xenopus tropicalis models?

Assessing PIP4K2C kinase activity requires specialized methodologies tailored to its specific substrate preferences and enzymatic properties:

In vitro activity assays:

• ADP-Glo reporter assay: Measures ADP production as a result of kinase activity, similar to assays developed for human PI5P4Kγ . This luminescence-based assay provides a sensitive readout of enzyme activity.

• Radioactive assays: Using ³²P-ATP incorporation into PI5P substrate, followed by thin-layer chromatography or filter binding.

• ELISA-based assays: Utilizing specific antibodies against phosphorylated products.

• Mass spectrometry: For direct quantification of phospholipid products.

Cellular activity assessment:

• Phospholipidomics: Quantifying changes in cellular PI5P and PI(4,5)P₂ levels.

• Downstream signaling: Monitoring mTORC1 pathway activation through phosphorylation of substrates like S6K or 4E-BP1 .

• Cellular phenotypes: Assessing hypertrophy or fibrosis in cardiac models .

Important considerations:

It's critical to note that wild-type PI5P4Kγ has particularly low enzymatic activity. Researchers studying human PI5P4Kγ have developed a mutant form (PI5P4Kγ+) by converting the catalytic site to correspond to PI5P4Kα G loop sequence, which increases kinase functional activity . A similar approach might be necessary for X. tropicalis PIP4K2C.

To validate assay specificity, researchers should include:

• Kinase-dead mutants as negative controls

• Isoform-specific inhibitors when available

• siRNA/CRISPR knockdown confirmation in cellular assays

What are the most effective approaches for studying PIP4K2C-mediated signaling pathways in Xenopus tropicalis?

Studying PIP4K2C-mediated signaling pathways in X. tropicalis requires integrated approaches spanning from molecular to organismal levels:

Molecular approaches:

• Interactome analysis: Using BioID, APEX proximity labeling, or immunoprecipitation followed by mass spectrometry to identify binding partners.

• Phosphoproteomics: Global analysis of phosphorylation changes upon PIP4K2C manipulation.

• Lipid binding assays: Identifying proteins that interact with PI5P and PI(4,5)P₂ using lipid beads or liposome flotation assays.

Cellular approaches:

• Live imaging: Using fluorescent sensors for PI5P or PI(4,5)P₂ to visualize dynamic changes.

• Pathway reporter assays: Employing luciferase reporters for mTORC1 or other downstream pathways .

• Single-cell transcriptomics: Identifying cell-specific responses to PIP4K2C modulation.

In vivo approaches:

• Tissue-specific manipulation: Using targeted CRISPR or tissue-specific promoters.

• Phenotypic analysis: Assessing developmental, morphological, or physiological outcomes.

• Rescue experiments: Testing pathway specificity through genetic epistasis.

A significant advantage of the X. tropicalis system is the ability to create tissue chimeras, combining mutant and wildtype tissues to determine whether phenotypes are due to tissue-autonomous or non-autonomous mechanisms . This approach has proven valuable in other X. tropicalis research and could be applied to PIP4K2C studies.

For temporal analysis of signaling dynamics, researchers could employ methods similar to those used in time-course experiments for UV defense genes in the Tibetan frog, utilizing STEM clustering and WGCNA analysis to identify co-expressed genes activated at specific time points .

How does PIP4K2C function in disease models using Xenopus tropicalis?

PIP4K2C has been implicated in several disease processes, particularly cardiac hypertrophy, fibrosis, and cancer. Xenopus tropicalis can serve as a valuable model for investigating these disease connections:

Cardiac disease models:
PIP4K2C is significantly downregulated in the hearts of cardiac hypertrophy and heart failure patients compared to non-injured hearts . To study this in X. tropicalis:

• Pharmacological induction: Using isoproterenol or angiotensin II to induce cardiac hypertrophy

• Genetic models: CRISPR-mediated knockout or knockdown of PIP4K2C

• mRNA therapy approach: Testing therapeutic delivery of PIP4K2C-modified mRNA to attenuate cardiac pathology, similar to approaches in other models

• Phenotypic analysis: Assessing heart size, cardiomyocyte area, fibrosis markers, and cardiac function

Cancer models:
PIP4K2C acts in both cancer cells and immune cells to affect tumor antigen presentation . X. tropicalis approaches may include:

• Xenograft models: Implanting cancer cells into tadpoles with manipulated PIP4K2C levels

• Transgenic cancer models: Creating X. tropicalis lines with cancer-predisposing mutations

• PIP4K2C degraders: Testing compounds like LRK-A, which has shown anti-tumor activity in colorectal cancer models

• Immune response analysis: Assessing T-cell activation and tumor infiltration

Table: PIP4K2C Disease Associations and X. tropicalis Model Applications

It's worth noting that Larkspur Biosciences is developing a first-in-class PIP4K2C protein degrader (LRK-A) that drives monotherapy anti-tumor activity, including complete tumor regressions in colorectal cancer patient samples . Similar approaches could be adapted for X. tropicalis disease models.

What are the latest inhibitors and degraders targeting PIP4K2C, and how might they be tested in Xenopus tropicalis?

Recent advances in developing PIP4K2C-targeting compounds provide exciting opportunities for research applications in X. tropicalis models:

Current PIP4K2C-targeting compounds:

• Small molecule inhibitors: Several compounds have been identified with activity against PI5P4K family members :

  • Compound 2: IC₅₀ = 16 μM in a ³²P-ATP/PI5P incorporation assay, selective for PI5P4Kγ among 442 kinases tested

  • Compound 3: Showed 91% inhibition of PI5P4Kγ with a Kᴅ of 4.8 nM

  • Compound 4: IC₅₀ of 1.3 μM against PI5P4Kα and 9.9 μM against PI5P4Kβ, but only 22% inhibition of PI5P4Kγ at 1 μM

  • Compound 5: Similar activity profile to compounds 3 and 4

• Protein degraders:

  • LRK-A: A first-in-class PIP4K2C protein degrader developed by Larkspur Biosciences that drives monotherapy anti-tumor activity, including complete tumor regressions in colorectal cancer patient samples

Testing methodology in X. tropicalis:

• In vitro validation:

  • Biochemical assays with recombinant X. tropicalis PIP4K2C

  • Thermal shift assays to confirm direct binding

  • Cross-species comparison of inhibition profiles

• Cell-based validation:

  • X. tropicalis primary cell cultures

  • Cell-free thermal shift assays to determine compound binding to overexpressed PIP4K2C, similar to the InCell Pulse assay used for human PI5P4Kγ

  • Western blotting to confirm target degradation (for degraders)

• In vivo applications:

  • Microinjection of compounds into embryos

  • Compound treatment of tadpoles via water exposure

  • Direct injection into tissues of interest

  • Assessment of phenotypic rescue in PIP4K2C-overexpression models

• Disease model applications:

  • Testing in cardiac hypertrophy models to evaluate rescue of pathology

  • Cancer xenograft studies to assess anti-tumor activity

  • Evaluation of immune response modulation

For patient selection strategies, researchers could consider adapting approaches like those developed by Larkspur Biosciences, which used their proprietary bioinformatics platform (LarkX CRC) to develop patient biomarker strategies to select individuals most likely to respond to PIP4K2C degradation .

How can findings from Xenopus tropicalis PIP4K2C research be translated to human disease applications?

Translating findings from X. tropicalis PIP4K2C research to human applications requires systematic approaches to bridge model organism insights with clinical relevance:

Cross-species validation strategies:

• Comparative genomics and proteomics:

  • Sequence homology and conservation analysis

  • Structure-function relationship mapping

  • Identification of conserved binding partners and signaling pathways

• Parallel validation in human systems:

  • Studies in human cell lines and primary cells

  • Patient-derived organoids

  • Examination of patient tissue samples for PIP4K2C expression and pathway activation

• Disease-specific translation pipelines:

  For cardiac applications:

  • PIP4K2C is significantly downregulated in hearts of cardiac hypertrophy and heart failure patients

  • Modified mRNA delivery of PIP4K2C has shown promise in attenuating cardiac hypertrophy and fibrosis

  • X. tropicalis findings could inform optimal delivery mechanisms and dosing

  For cancer applications:

  • PIP4K2C functions in both cancer cells and immune cells to affect how tumor antigens are presented

  • Degraders like LRK-A show monotherapy anti-tumor activity

  • X. tropicalis models can help elucidate mechanisms and identify biomarkers

Methodological considerations:

The key advantage of X. tropicalis for translational research is its remarkable degree of synteny with mammalian genomes, often in stretches of a hundred genes or more, which far exceeds that seen between fish and mammals . This genomic conservation suggests that regulatory mechanisms and pathway interactions may be similarly conserved.

Researchers should employ a "bedside-to-bench-to-bedside" approach:

• Identify clinical observations (e.g., PIP4K2C downregulation in heart failure)

• Develop X. tropicalis models to study mechanisms

• Test interventions in X. tropicalis

• Validate findings in higher mammalian models

• Design human clinical applications

What genomic and bioinformatic approaches are most valuable for PIP4K2C research in Xenopus tropicalis?

Advanced genomic and bioinformatic approaches can significantly enhance PIP4K2C research in X. tropicalis:

Genomic resources and approaches:

• High-quality genome utilization:

  • The X. tropicalis genome has been sequenced with high continuity (contig N50 of 2.3 Mb and scaffold N50 of 269 Mb)

  • Researchers can leverage this high-integrity genome (87.9% of conserved vertebrate BUSCO genes identified)

  • Integration with long-range PacBio RS II, short-read Illumina sequencing, chromatin conformation capture (Hi-C), and genetic mapping data

• Transcriptomic analysis:

  • RNA-seq from different tissues and developmental stages

  • Single-cell transcriptomics to identify cell-specific expression patterns

  • Time-course expression analysis using STEM clustering and WGCNA methods

• Functional genomics:

  • CRISPR-Cas9 screening for PIP4K2C-interacting genes

  • Chromatin immunoprecipitation sequencing (ChIP-seq) to identify regulatory elements

  • ATAC-seq for chromatin accessibility analysis

Bioinformatic strategies:

• Pathway analysis:

  • Gene Ontology (GO) functional enrichment analysis of PIP4K2C-associated genes

  • Identification of co-expressed gene networks using WGCNA

  • Integrated multi-omics analysis (transcriptomics, proteomics, lipidomics)

• Biomarker development:

  • Machine learning approaches for predicting PIP4K2C activity

  • Patient stratification methodologies similar to Larkspur's approach with their LarkX CRC platform

  • Development of computational models for drug response prediction

• Evolutionary analysis:

  • Comparative genomics across species to identify conserved functional domains

  • Phylogenetic analysis of PIP4K2C across vertebrates

  • Identification of species-specific adaptations

The high degree of synteny between X. tropicalis and mammalian genomes makes this model particularly valuable for translational genomics approaches . Researchers should take advantage of the continually improving genomic resources for this species, including BAC libraries that are important for both genome assembly and as resources for projects examining gene organization .