Recombinant Xenopus tropicalis Protein phosphatase 1 regulatory subunit 3C (ppp1r3c)

What is the function of ppp1r3c in Xenopus tropicalis?

Protein phosphatase 1 regulatory subunit 3C (ppp1r3c) functions as a glycogen-targeting subunit for Protein Phosphatase 1 (PP1) and regulates its activity in Xenopus tropicalis. It plays a crucial role in glycogen metabolism by activating glycogen synthase, reducing glycogen phosphorylase activity, and limiting glycogen breakdown. As a member of the glycogen targeting subunits (GTSs), it belongs to the larger group of regulatory subunits of PP1, which is a major eukaryotic serine/threonine protein phosphatase that regulates diverse cellular processes .

How is ppp1r3c expression regulated during Xenopus development?

Ppp1r3c expression in Xenopus shows specific temporal and spatial patterns during development. In Xenopus laevis, in situ hybridization data reveals expression at Nieuwkoop and Faber (NF) stage 29/30, as documented in the Xenbase database . Like many genes involved in metamorphosis, its expression may be regulated by thyroid hormone (TH), which binds to TH receptors (TRs) to regulate gene expression programs underlying morphogenesis during amphibian development . Researchers investigating developmental regulation should consider analyzing expression at multiple developmental stages (premetamorphosis, prometamorphosis, metamorphic climax, and completion of metamorphosis) to understand its role in developmental processes.

How can I effectively manipulate ppp1r3c expression in Xenopus tropicalis embryos?

To manipulate ppp1r3c expression in Xenopus tropicalis embryos, several approaches can be employed:

• Morpholino knockdown: Use antisense morpholino oligonucleotides (MOs) targeting either the translation start site or splice junctions of ppp1r3c. For translation-blocking MOs, design them to target the 5' UTR and/or start codon region. For splice-blocking MOs, target exon-intron boundaries to disrupt proper splicing.

• CRISPR/Cas9 genome editing: This is highly efficient in X. tropicalis and allows generation of F0 mosaic mutants or stable transgenic lines.

  • Design sgRNAs targeting exonic regions of ppp1r3c

  • Inject Cas9 protein along with sgRNAs into one-cell stage embryos

  • Verify mutations by sequencing PCR products of the targeted region

• mRNA overexpression: Synthesize capped mRNA from a ppp1r3c expression construct (typically using pCS2+ vector) and inject into embryos.

  • Initial dosage should be 1-5pg to avoid toxicity

  • Always include proper controls (uninjected, control morpholino, or control mRNA)

For all approaches, targeted injections can be performed up to the 32-cell stage to achieve tissue-specific manipulation, taking advantage of the known fate map of Xenopus blastomeres .

What is the recommended protocol for reconstituting recombinant Xenopus tropicalis ppp1r3c?

For optimal reconstitution of recombinant Xenopus tropicalis ppp1r3c:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended) for long-term storage

• Aliquot to avoid repeated freeze-thaw cycles

• Store working aliquots at 4°C for up to one week

• For long-term storage, keep at -20°C/-80°C (liquid form has a shelf life of approximately 6 months, while lyophilized form can be stored for 12 months)

Remember that repeated freezing and thawing is not recommended as it may compromise protein integrity and function .

How can I validate ppp1r3c knockdown efficiency in Xenopus experiments?

To validate ppp1r3c knockdown efficiency in Xenopus experiments, employ multiple complementary approaches:

• Western blotting: Use antibodies against ppp1r3c to quantify protein levels in control versus knockdown samples. This provides direct evidence of reduced protein expression.

• RT-qPCR: Measure mRNA levels using primers specific to ppp1r3c. For splice-blocking morpholinos, design primers flanking the targeted exon to detect altered splicing products.

• Functional assays: Measure glycogen content in tissues, as ppp1r3c regulates glycogen metabolism. Similar to observations in PPP1R3F knockout cells, ppp1r3c knockdown may affect glycogen sensitivity to changes in extracellular glucose levels .

• Rescue experiments: Co-inject morpholino-resistant ppp1r3c mRNA (containing silent mutations in the MO target site) along with the morpholino. Rescue of the phenotype confirms specificity of the knockdown .

• Histological analysis: Examine tissues where ppp1r3c is normally expressed for morphological or functional changes consistent with altered glycogen metabolism.

Statistical analysis should be performed using appropriate tests, such as one-way ANOVA followed by Fisher's least significant difference (LSD) post hoc test (α = 0.05) .

How does ppp1r3c function differ between Xenopus tropicalis and Xenopus laevis?

The functional comparison between ppp1r3c in Xenopus tropicalis and Xenopus laevis reveals important evolutionary and mechanistic insights:

Xenopus tropicalis has a diploid genome, making genetic studies more straightforward compared to the allotetraploid genome of Xenopus laevis . This genomic difference may impact the regulation and function of ppp1r3c between the two species. The amino acid sequence differences (as seen in the recombinant protein datasheets ) suggest potential functional divergence that may have occurred following the evolutionary divergence of these species.

When designing experiments to study ppp1r3c function, researchers should consider these differences, especially when translating findings between the two Xenopus species or extrapolating to human biology .

What is the relationship between ppp1r3c and thyroid hormone-regulated metamorphosis in Xenopus?

The relationship between ppp1r3c and thyroid hormone (TH)-regulated metamorphosis in Xenopus presents an interesting research direction. While ppp1r3c is not directly mentioned among the well-characterized TH-responsive genes in the search results, its potential role can be investigated based on the following observations:

• Amphibian metamorphosis is controlled by thyroid hormone (TH), which binds TH receptors (TRs) to regulate gene expression programs underlying morphological changes .

• Key TH-responsive genes identified in Xenopus include thrb, thibz, klf9, gadd45g, tet2, and tet3 .

• RNA-seq analyses of the preoptic area/thalamus/hypothalamus region during different metamorphic stages (NF50, NF56, NF62, NF66) and in response to T3 treatment have been performed .

To investigate ppp1r3c's relationship with TH-regulated metamorphosis:

• Analyze existing RNA-seq datasets to determine if ppp1r3c expression changes during metamorphosis or in response to T3 treatment

• Perform ChIP-seq analysis to determine if TR binds to regulatory regions of ppp1r3c

• Conduct functional studies to assess whether ppp1r3c knockdown or overexpression affects TH-dependent metamorphic processes

• Examine whether ppp1r3c regulates glycogen metabolism during the energy-demanding process of metamorphosis

This research could provide new insights into the metabolic regulation during amphibian metamorphosis and potentially identify ppp1r3c as a novel player in TH-regulated developmental processes.

How can I use the Xenopus tropicalis ppp1r3c model to understand human glycogen metabolism disorders?

Xenopus tropicalis offers significant advantages for modeling human glycogen metabolism disorders related to ppp1r3c dysfunction:

• Genomic conservation: X. tropicalis has a diploid genome with high conservation of gene synteny with the human genome, making it suitable for studying orthologous gene function . The genome shows approximately 79% sharing of identified human disease genes .

• Disease modeling approach:

  • Generate ppp1r3c knockouts using CRISPR/Cas9

  • Introduce patient-specific variants using homology-directed repair

  • Analyze glycogen metabolism in relevant tissues (liver, muscle, brain)

  • Compare phenotypes to those observed in human patients with glycogen metabolism disorders

• Translational relevance: Based on findings with the related protein PPP1R3F, which when mutated leads to neurological disorders including developmental delay, intellectual disability, and seizures, researchers should examine:

  • Brain-specific expression and function of ppp1r3c

  • Effects of ppp1r3c mutations on glycogen content in astrocytes

  • Response to fluctuating glucose levels, which may be particularly relevant to neurological phenotypes

• Experimental advantages: X. tropicalis offers rapid external development, transparency of tadpoles, high fecundity, and ease of genomic manipulation . Additionally, the efficiency of CRISPR/Cas9 allows phenotype analysis in F0 generations, accelerating the research timeline .

By studying how mutations in ppp1r3c affect glycogen metabolism in X. tropicalis, researchers can gain insights into human disorders of glycogen metabolism and potentially identify new therapeutic targets.

What expression system is used to produce recombinant Xenopus tropicalis ppp1r3c?

Recombinant Xenopus tropicalis ppp1r3c is produced using an E. coli expression system as indicated in the product information . This bacterial expression system is chosen for several reasons:

• High yield: E. coli can produce substantial amounts of recombinant protein

• Cost-effectiveness: Bacterial culture is less expensive than mammalian or insect cell culture systems

• Simplicity: Well-established protocols for transformation, induction, and protein purification

• Lack of post-translational modifications: For functional studies where glycosylation is not critical

The expression construct typically includes the complete coding sequence (amino acids 1-223 for X. tropicalis ppp1r3c) with potential fusion tags to facilitate purification . The specific tag type may vary and is determined during the manufacturing process. After expression, the protein is purified to >85% purity as determined by SDS-PAGE .

What are the optimal storage conditions for maximizing recombinant ppp1r3c stability?

To maximize recombinant ppp1r3c stability, follow these storage recommendations based on the product information:

• Short-term storage (up to one week):

  • Store working aliquots at 4°C

  • Avoid repeated freeze-thaw cycles

• Long-term storage:

  • For liquid form: Store at -20°C/-80°C (shelf life approximately 6 months)

  • For lyophilized form: Store at -20°C/-80°C (shelf life approximately 12 months)

  • Add glycerol to a final concentration of 50% for liquid storage

• Reconstitution considerations:

  • Use deionized sterile water for reconstitution

  • Prepare at concentration of 0.1-1.0 mg/mL

  • Centrifuge vial briefly before opening to bring contents to bottom

  • Create small single-use aliquots to prevent repeated freeze-thaw cycles

The shelf life is influenced by multiple factors including storage state, buffer ingredients, storage temperature, and the inherent stability of the protein itself . Researchers should monitor protein activity periodically if stored for extended periods.

How can I use recombinant ppp1r3c to study protein-protein interactions in Xenopus glycogen metabolism?

To study protein-protein interactions involving ppp1r3c in Xenopus glycogen metabolism, consider these methodological approaches:

• Pull-down assays: Use the recombinant ppp1r3c protein as bait to identify interacting partners from Xenopus tissue lysates.

  • Immobilize the recombinant protein on an appropriate matrix

  • Incubate with tissue lysates (particularly from tissues with high glycogen metabolism)

  • Wash to remove non-specific interactions

  • Elute and identify binding proteins by mass spectrometry

• Co-immunoprecipitation: Use antibodies against ppp1r3c to pull down protein complexes from Xenopus tissues.

  • This can reveal physiologically relevant interactions in vivo

• Yeast two-hybrid screening: Use ppp1r3c as bait to screen for interacting proteins from a Xenopus cDNA library.

  • Can identify direct protein-protein interactions

• Bimolecular Fluorescence Complementation (BiFC): Express fragments of fluorescent proteins fused to ppp1r3c and potential interacting partners in Xenopus embryos.

  • Fluorescence is reconstituted when the proteins interact

• Surface Plasmon Resonance (SPR): Measure binding kinetics between recombinant ppp1r3c and known or suspected binding partners, particularly PP1 catalytic subunits.

From the related research on PPP1R3F, important interactions to investigate include:

• Binding to PP1 catalytic subunits

• Interactions with glycogen synthase

• Binding to glycogen phosphorylase

• Potential protein interactions affected by pathogenic variants

These approaches can provide insights into how ppp1r3c functions within the glycogen metabolism regulatory network in Xenopus and potentially identify novel therapeutic targets for glycogen metabolism disorders.

What techniques can I use to investigate the spatial and temporal expression of ppp1r3c during Xenopus development?

To comprehensively investigate the spatial and temporal expression patterns of ppp1r3c during Xenopus development, researchers can employ the following techniques:

• Whole-mount in situ hybridization (WISH):

  • Design antisense RNA probes targeting ppp1r3c mRNA

  • Analyze expression at different developmental stages (NF stages)

  • Document spatial expression patterns in intact embryos

  • Previous data indicates expression at NF stage 29/30

• Quantitative RT-PCR (RT-qPCR):

  • Collect samples from different developmental stages

  • Use stage-specific or tissue-specific RNA extraction

  • Design primers specific to ppp1r3c

  • Normalize to appropriate housekeeping genes

  • Perform statistical analysis using one-way ANOVA followed by Fisher's LSD post hoc test

• RNA-seq analysis:

  • Perform transcriptome analysis at key developmental stages

  • Include stages covering premetamorphosis (NF50), prometamorphosis (NF56), metamorphic climax (NF62), and completion of metamorphosis (NF66)

  • Analyze data for ppp1r3c expression patterns and correlation with other genes

• Transgenic reporter lines:

  • Generate transgenic Xenopus lines expressing fluorescent reporters under the ppp1r3c promoter

  • Use methods established for X. tropicalis transgenesis

  • Monitor expression in living embryos throughout development

• Immunohistochemistry:

  • Use antibodies against ppp1r3c protein

  • Perform on tissue sections to precisely localize protein expression

  • Co-stain with markers for specific cell types or tissues

Combining these approaches provides a comprehensive understanding of when and where ppp1r3c is expressed during development, offering insights into its potential developmental roles and regulation.

How do research findings on ppp1r3c in Xenopus tropicalis translate to understanding human metabolic disorders?

Research findings on ppp1r3c in Xenopus tropicalis can significantly advance our understanding of human metabolic disorders through several translational pathways:

• Conservation of glycogen metabolism pathways: The core machinery of glycogen metabolism is highly conserved from amphibians to humans. Studies with the related protein PPP1R3F demonstrate that mutations in these regulatory subunits can cause neurological disorders associated with abnormal glycogen metabolism in humans .

• Functional conservation validation:

  • Human PPP1R3C variants can be expressed in ppp1r3c-deficient Xenopus to test functional conservation

  • Patient-specific variants can be introduced into Xenopus to create disease models

  • Approximately 79% of identified human disease genes are shared with Xenopus

• Translational insights:

  Xenopus Finding	Human Disease Relevance
  Tissue-specific expression patterns	Predicts which human tissues might be affected by PPP1R3C mutations
  Developmental regulation	Suggests critical periods when PPP1R3C dysfunction might impact development
  Response to metabolic stress	Informs how PPP1R3C mutations might affect response to fasting or exercise
  Interaction with PP1 and other proteins	Identifies potential therapeutic targets

• Disease modeling advantages: Xenopus tropicalis offers unique advantages for modeling human metabolic disorders:

  • Transparent tadpoles allow visualization of internal organs

  • External development facilitates manipulation and observation

  • Diploid genome simplifies genetic analysis compared to X. laevis

  • CRISPR/Cas9 editing allows efficient introduction of patient-specific variants

• Clinical applications: Understanding ppp1r3c function in Xenopus can guide:

  • Identification of novel genes involved in human glycogen storage diseases

  • Development of diagnostic tests for mutations in PPP1R3C and related genes

  • Design of targeted therapies for glycogen metabolism disorders

  • Understanding how glycogen metabolism affects neurological development and function

The research with PPP1R3F demonstrates how variants in PP1 regulatory subunits can lead to human disease, suggesting that similar studies with ppp1r3c could yield valuable insights into other glycogen metabolism disorders .

What controls should I include when performing functional studies of ppp1r3c in Xenopus embryos?

When conducting functional studies of ppp1r3c in Xenopus embryos, including appropriate controls is essential for result validity and interpretation:

• For morpholino knockdown experiments:

  • Uninjected control embryos (wild-type development baseline)

  • Control morpholino-injected embryos (controls for injection procedure and non-specific effects)

  • Dose-response series (to determine optimal morpholino concentration)

  • Rescue controls: co-injection of morpholino with morpholino-resistant ppp1r3c mRNA (confirms specificity)

• For CRISPR/Cas9 gene editing:

  • Uninjected control embryos

  • Cas9-only injected embryos (without sgRNA)

  • Non-targeting sgRNA controls

  • Multiple guide RNAs targeting different regions of ppp1r3c (controls for off-target effects)

  • Sequencing verification of mutations

• For overexpression studies:

  • Uninjected control embryos

  • Control mRNA injection (e.g., GFP mRNA)

  • Dose-response series of ppp1r3c mRNA (1-5pg initially, to avoid toxicity)

  • Functional mutant versions of ppp1r3c (to identify critical domains)

• For tissue-specific manipulations:

  • Lineage tracers (e.g., fluorescent dextran) to confirm targeting

  • Tissue-specific markers to assess effects on particular tissues

• For biochemical assays:

  • Measurement of glycogen content in multiple tissues

  • Glycogen phosphorylase activity assays

  • Glycogen synthase activity assays

  • Western blots to confirm protein expression levels

Statistical analysis should employ appropriate tests such as one-way ANOVA followed by Fisher's LSD post hoc test (α = 0.05), with log transformation of data if variances are heterogeneous .

How can I design experiments to investigate the role of ppp1r3c in glycogen metabolism during metamorphosis?

To investigate the role of ppp1r3c in glycogen metabolism during Xenopus metamorphosis, researchers can design a comprehensive experimental approach:

• Expression analysis across metamorphic stages:

  • Collect tissue samples at key developmental stages: premetamorphosis (NF50), prometamorphosis (NF56), metamorphic climax (NF62), and completion of metamorphosis (NF66)

  • Focus on tissues with high glycogen content (liver, muscle, brain)

  • Perform RT-qPCR and Western blotting to quantify ppp1r3c expression

  • Conduct in situ hybridization to determine spatial expression patterns

• Thyroid hormone responsiveness:

  • Treat premetamorphic tadpoles (NF54) with T3 (5 nM in aquarium water) for 16 hours

  • Analyze ppp1r3c expression changes in response to T3

  • Perform ChIP-seq or ChIP-qPCR to determine if TR binds to ppp1r3c regulatory regions

  • Compare with known T3-responsive genes (thrb, thibz, klf9, gadd45g, tet2, tet3)

• Functional manipulation during metamorphosis:

  • Generate stage-specific ppp1r3c knockdown using inducible CRISPR/Cas9 systems

  • Create tissue-specific knockdowns using targeted injections

  • Analyze resulting phenotypes during metamorphosis

  • Measure changes in glycogen content and metabolism

• Metabolic measurements:

  • Quantify glycogen content in tissues at different metamorphic stages

  • Measure glycogen synthase and phosphorylase activities

  • Assess glucose tolerance and utilization

  • Compare wild-type to ppp1r3c-deficient animals

• Integration with energy demands of metamorphosis:

  • Monitor energy consumption during metamorphic climax

  • Track changes in feeding behavior and metabolism

  • Analyze how ppp1r3c regulation affects energy availability during this high-demand process

This experimental design allows for comprehensive characterization of ppp1r3c's role in regulating glycogen metabolism during the energetically demanding process of metamorphosis, potentially revealing novel insights into metabolic regulation during dramatic developmental transitions.

How can I compare ppp1r3c function across vertebrate model systems to identify evolutionarily conserved mechanisms?

To identify evolutionarily conserved mechanisms of ppp1r3c function across vertebrate model systems, researchers should employ a comprehensive comparative approach:

• Sequence analysis:

  • Perform multiple sequence alignments of ppp1r3c proteins from various vertebrates (fish, amphibians, reptiles, birds, mammals)

  • Identify conserved domains and motifs

  • Calculate evolutionary rates to detect regions under selective pressure

  • Map known human mutations onto conserved regions

• Expression pattern comparison:

  • Compare tissue-specific and developmental expression patterns across species

  • Analyze expression in homologous tissues and developmental stages

  • Use consistent methodologies (RNA-seq, in situ hybridization) for valid comparisons

• Functional conservation testing:

  • Perform cross-species rescue experiments:

    • Express human PPP1R3C in Xenopus ppp1r3c knockout

    • Test if Xenopus ppp1r3c can rescue mammalian cell models

  • Compare protein interaction networks across species

  • Assess conservation of regulatory mechanisms

• CRISPR/Cas9 comparative phenotyping:

  • Generate equivalent mutations in multiple model systems

  • Compare phenotypes focusing on:

    • Glycogen metabolism parameters

    • Developmental outcomes

    • Tissue-specific effects

    • Response to metabolic challenges

• Regulatory genomics:

  • Compare promoter and enhancer elements across species

  • Identify conserved transcription factor binding sites

  • Test regulatory element function across species

The Xenopus model offers unique advantages for this comparative approach:

• As a tetrapod, it has closer evolutionary relationships to mammals than zebrafish

• Its genome shows high synteny with humans

• The availability of both X. tropicalis (diploid) and X. laevis (allotetraploid) provides insight into gene function after genome duplication events

• The efficiency of genome editing techniques allows rapid generation of comparable models

This multi-faceted approach enables identification of truly conserved aspects of ppp1r3c function versus species-specific adaptations, providing valuable insights for translational research.

What bioinformatic approaches can help identify potential regulatory networks involving ppp1r3c in Xenopus tropicalis?

To identify potential regulatory networks involving ppp1r3c in Xenopus tropicalis, researchers can employ these bioinformatic approaches:

• Co-expression network analysis:

  • Utilize existing RNA-seq datasets from Xenopus development, particularly those covering metamorphosis stages

  • Identify genes with expression patterns correlated with ppp1r3c

  • Construct co-expression networks to visualize relationships

  • Tools: WGCNA (Weighted Gene Co-expression Network Analysis), CEMiTool

• Transcription factor binding site prediction:

  • Analyze the promoter and enhancer regions of ppp1r3c

  • Identify potential transcription factor binding sites

  • Focus on factors known to regulate glycogen metabolism and developmental processes

  • Cross-reference with ChIP-seq data if available

  • Tools: JASPAR, TRANSFAC, MEME

• Pathway enrichment analysis:

  • Perform Gene Ontology (GO) and pathway enrichment on genes co-expressed with ppp1r3c

  • Identify biological processes, molecular functions, and cellular components associated with ppp1r3c network

  • Tools: DAVID, g:Profiler, EnrichR

• Protein-protein interaction prediction:

  • Use sequence-based and structure-based approaches to predict protein interactions

  • Compare with known interactions from related proteins (e.g., PPP1R3F )

  • Tools: STRING, IntAct, BioGRID

• Comparative genomics:

  • Compare ppp1r3c regulatory networks across species

  • Identify evolutionarily conserved interactions

  • Tools: Ensembl Compara, UCSC Genome Browser

• Integration with epigenomic data:

  • Analyze chromatin accessibility (ATAC-seq) and histone modification data

  • Identify potential epigenetic regulators of ppp1r3c

  • Examine how these change during development or in response to thyroid hormone

• Motif analysis for RNA-binding proteins:

  • Identify potential regulatory elements in ppp1r3c mRNA

  • Predict RNA-binding protein interactions that may regulate translation or stability