Recombinant Xenopus laevis Acetyl-CoA acetyltransferase B, mitochondrial (acat1-b)

What is Acetyl-CoA acetyltransferase B, mitochondrial (acat1-b) in Xenopus laevis and what are its key functions?

Acetyl-CoA acetyltransferase B, mitochondrial (acat1-b) is an enzyme involved in ketone body metabolism and fatty acid oxidation in Xenopus laevis. The protein consists of approximately 420 amino acids, with recombinant forms typically including amino acids 34-420 as shown in commercially available preparations . The enzyme catalyzes the reversible formation of acetoacetyl-CoA from two molecules of acetyl-CoA, representing a critical step in ketogenesis and cholesterol metabolism. In Xenopus laevis, as a tetraploid species, acat1-b is one of the homeologous genes that resulted from genome duplication, potentially providing metabolic redundancy and specialized functions during development .

The mitochondrial localization of ACAT1-B distinguishes it from cytosolic thiolases and indicates its role in mitochondrial metabolic pathways rather than cytosolic cholesterol synthesis. During embryonic development, its expression follows patterns typical of mitochondrial genes, with significant regulation during key developmental transitions .

How is ACAT1-B expression regulated during Xenopus laevis development?

Mitochondrial gene expression in Xenopus laevis follows a specific pattern during embryonic development that likely includes ACAT1-B regulation. According to studies of mitochondrial gene expression during development, mitochondrial mRNAs show a dramatic decrease after fertilization (by a factor of 5-10), maintain very low levels through the late neurula stages, and then increase significantly during organogenesis . This pattern suggests complete inactivation of the mitochondrial genome at the beginning of embryonic development.

The mitochondrial DNA content remains constant during this period, indicating that regulation occurs at the transcriptional level rather than through changes in gene copy number . For nuclear-encoded mitochondrial proteins like ACAT1-B, this pattern suggests a coordinated regulation between nuclear and mitochondrial genomes during development. The resumption of mitochondrial RNA accumulation coincides with the general increase in transcription in the embryo, occurring before DNA replication resumes in the organelle .

What are the optimal expression systems for producing active recombinant Xenopus laevis ACAT1-B?

Based on available data, several expression systems have been successfully used to produce recombinant Xenopus laevis ACAT1-B, each with distinct advantages:

• Yeast expression systems: Commercial preparations of recombinant Xenopus laevis ACAT1-B are successfully produced in yeast, which provides proper eukaryotic protein folding machinery and post-translational modifications . This system represents a balance between yield and proper protein processing.

• Bacterial expression (E. coli): While not the primary system used for the commercial ACAT1 preparation described in the search results, bacterial expression can be considered for higher yield, though potentially with compromised folding or activity .

• Alternative systems: For specialized applications, researchers might consider mammalian cell expression or baculovirus-infected insect cell systems, particularly when native-like glycosylation or other post-translational modifications are critical.

The optimal choice depends on the experimental requirements, with the following comparison table to guide selection:

How can CRISPR/Cas9 be utilized to study ACAT1-B function in Xenopus laevis?

CRISPR/Cas9 technology offers a powerful approach for investigating ACAT1-B function through targeted gene disruption in Xenopus laevis. Research has demonstrated that CRISPR/Cas9 can achieve high sequence disruption efficiency in X. laevis, with clear phenotypes observable in G0 embryos without requiring the generation of stable lines .

A methodological workflow for ACAT1-B functional studies using CRISPR/Cas9 would include:

• Guide RNA design:

  • Target conserved functional domains within the ACAT1-B coding sequence

  • Account for potential homeologs in the tetraploid X. laevis genome

  • Use algorithms to minimize off-target effects

  • Consider targeting both homeologs simultaneously if complete knockout is desired

• Microinjection protocol:

  • Synthesize sgRNAs targeting ACAT1-B

  • Prepare Cas9 protein or mRNA

  • Inject into one-cell stage X. laevis embryos (typically 2-4 ng of sgRNA and 8 ng of Cas9 protein)

  • Include appropriate controls (uninjected and control sgRNA injected embryos)

• Validation and analysis:

  • Confirm targeting efficiency by DNA extraction and sequencing

  • Assess mRNA and protein levels to confirm knockdown

  • Perform enzymatic activity assays to evaluate functional consequences

  • Monitor developmental phenotypes, focusing on metabolic parameters

This approach allows for rapid assessment of ACAT1-B function directly in G0 embryos, similar to successful targeting of other genes such as ptf1a/p48 and tyrosinase in X. laevis .

What methods are most effective for measuring ACAT1-B enzymatic activity in Xenopus samples?

Measuring ACAT1-B enzymatic activity in Xenopus samples requires specialized biochemical approaches to accurately quantify its thiolase activity. The following methods have proven effective:

• Spectrophotometric assays:

  • Direct measurement of acetoacetyl-CoA formation at 303 nm

  • Coupled enzyme assays with 3-hydroxyacyl-CoA dehydrogenase to monitor NADH oxidation

  • Optimal buffer conditions: 100 mM Tris-HCl (pH 8.0), 25 mM MgCl₂, 50 mM KCl

• Sample preparation considerations:

  • Tissue homogenization in ice-cold buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT)

  • Mitochondrial enrichment through differential centrifugation

  • Addition of protease inhibitors to prevent degradation

  • Gentle solubilization using mild detergents

• Activity calculation:

  • Determine specific activity (μmol/min/mg protein)

  • Calculate kinetic parameters (Km, Vmax) through Lineweaver-Burk plots

  • Normalize to mitochondrial markers for cross-tissue comparisons

• Developmental considerations:

  • Account for the developmental regulation of mitochondrial gene expression

  • Consider the dramatic decrease in mitochondrial mRNAs after fertilization and subsequent increase during organogenesis when interpreting activity data

  • Use stage-matched controls when comparing experimental conditions

How does recombinant Xenopus laevis ACAT1-B compare to human ACAT1 for research on metabolic diseases?

Recombinant Xenopus laevis ACAT1-B serves as a valuable comparative model for human ACAT1 research, particularly for metabolic disease studies. Key similarities and differences include:

The use of recombinant Xenopus ACAT1-B can complement human ACAT1 studies in several ways:

• Comparative enzymology: Understanding evolutionary conservation of catalytic mechanisms can highlight essential functional residues.

• Disease modeling: While human ACAT has been investigated for roles in immune responses, viral infections, and cancer , Xenopus ACAT1-B can provide a model system for investigating fundamental mechanisms.

• Developmental context: The clear pattern of mitochondrial gene regulation during Xenopus development provides a unique framework for understanding how metabolic enzymes are deployed during key developmental transitions.

What insights can Xenopus laevis ACAT1-B research provide about mitochondrial metabolism during embryonic development?

Research on Xenopus laevis ACAT1-B offers unique insights into mitochondrial metabolism during embryonic development:

• Temporal regulation: Mitochondrial gene expression in Xenopus embryos shows a distinctive pattern with mRNAs decreasing dramatically after fertilization (by 5-10 fold), remaining low until late neurula stages, then increasing during organogenesis . This pattern likely applies to nuclear-encoded mitochondrial proteins like ACAT1-B, suggesting coordinated regulation between nuclear and mitochondrial genomes.

• Metabolic remodeling: The decrease in mitochondrial gene expression post-fertilization suggests a period of reduced mitochondrial biogenesis and activity. For ACAT1-B, this indicates potential metabolic remodeling during early development, with a shift away from active ketone metabolism until organogenesis begins.

• Mitochondrial biogenesis: The resumption of mitochondrial RNA accumulation coincides with the general increase in transcription in the embryo and occurs before DNA replication resumes in the organelle . This suggests that transcriptional activation precedes mitochondrial proliferation, with implications for the timing of ACAT1-B expression.

• Evolutionary perspective: The tetraploid nature of the Xenopus laevis genome provides an opportunity to study the functional divergence of ACAT1 homeologs, potentially revealing subspecialization of mitochondrial metabolic pathways following genome duplication.

These insights are particularly valuable because Xenopus embryonic development represents "a quite clear example of regulation of the mitochondrial expression at the level of transcription" , providing a model system for understanding mitochondrial biogenesis and metabolic adaptation during development.

How can protein-protein interactions of ACAT1-B be effectively investigated to understand its role in broader metabolic networks?

Investigating protein-protein interactions of ACAT1-B requires specialized approaches to understand its integration into mitochondrial metabolic networks:

• Affinity purification methods:

  • Co-immunoprecipitation using recombinant His-tagged ACAT1-B as bait

  • Tandem affinity purification to reduce non-specific interactions

  • Proximity-based labeling approaches (BioID or APEX) to capture transient interactions

  • Cross-linking mass spectrometry to map interaction interfaces

• Imaging-based approaches:

  • Fluorescence resonance energy transfer (FRET) with fluorescently tagged proteins

  • Split-GFP complementation assays to visualize interactions in living cells

  • Super-resolution microscopy to detect co-localization at sub-mitochondrial resolution

• Functional interaction studies:

  • Enzyme activity assays in the presence of potential interactors

  • Metabolic flux analysis to identify functional metabolic partners

  • Genetic interaction screens using CRISPR/Cas9 technology in Xenopus

• Computational analysis:

  • Network modeling to predict interactions based on metabolic pathways

  • Structural modeling to identify potential protein-protein interfaces

  • Evolutionary analysis to identify co-evolving proteins

The His-tagged recombinant ACAT1-B protein described in the search results would be particularly useful for affinity purification approaches, allowing researchers to identify interaction partners through pull-down experiments followed by mass spectrometry analysis.

What are common challenges in expressing and purifying recombinant Xenopus laevis ACAT1-B, and how can they be addressed?

Researchers working with recombinant Xenopus laevis ACAT1-B may encounter several challenges during expression and purification, each requiring specific troubleshooting approaches:

• Expression yield limitations:

  • Challenge: Low protein expression levels, particularly in eukaryotic systems

  • Solutions: Optimize codon usage for expression host, test different promoters, adjust induction conditions

  • Validation approach: Quantify expression by Western blot before proceeding to large-scale purification

• Protein solubility issues:

  • Challenge: Tendency to form inclusion bodies, especially in bacterial systems

  • Solutions: Lower expression temperature (16-20°C), add solubility tags, co-express with chaperones

  • Validation approach: Compare protein in soluble fraction versus inclusion bodies by SDS-PAGE

• Purification complications:

  • Challenge: Co-purification of contaminating proteins despite His-tag affinity purification

  • Solutions: Implement multiple chromatography steps (ion exchange, size exclusion), optimize imidazole concentration

  • Validation approach: Assess purity by SDS-PAGE, verify identity by mass spectrometry

• Activity preservation:

  • Challenge: Loss of enzymatic activity during purification or storage

  • Solutions: Include glycerol (10-20%) and reducing agents in buffers, avoid freeze-thaw cycles

  • Validation approach: Monitor specific activity throughout purification process

• Tetraploid genome complications:

  • Challenge: Presence of homeologous genes in Xenopus laevis potentially producing similar proteins

  • Solutions: Design purification strategies that can distinguish between highly similar proteins

  • Validation approach: Peptide mass fingerprinting to confirm identity of purified protein

A systematic approach to optimization can help achieve the >90% purity reported for commercial recombinant Xenopus laevis ACAT1 protein .

How can researchers distinguish between the effects of ACAT1-B disruption and off-target effects when using CRISPR/Cas9?

Distinguishing between specific ACAT1-B disruption effects and off-target effects when using CRISPR/Cas9 requires rigorous controls and validation:

• Guide RNA design and validation:

  • Use multiple prediction algorithms to select guides with minimal off-target potential

  • Test multiple independent guide RNAs targeting different regions of ACAT1-B

  • Sequence validate the on-target modifications at the ACAT1-B locus

• Phenotypic validation strategies:

  • Compare phenotypes between embryos targeted with different guide RNAs against ACAT1-B

  • Perform rescue experiments by co-injecting wild-type ACAT1-B mRNA with CRISPR components

  • Use graduated doses of CRISPR components to establish dose-dependence of phenotypes

• Genomic validation:

  • Perform targeted sequencing of predicted off-target sites

  • Consider whole-genome sequencing for comprehensive off-target analysis

  • Examine expression of related genes to rule out compensatory effects

• Biochemical validation:

  • Directly measure ACAT1-B enzyme activity in targeted versus control embryos

  • Perform metabolic profiling to confirm pathway disruption

  • Use Western blotting to verify protein knockdown

Research on CRISPR/Cas9 in Xenopus laevis has demonstrated high sequence disruption efficiency with clear phenotypes observable in G0 embryos , suggesting that careful application of this technique can produce reliable results for ACAT1-B functional studies.

What considerations are important when interpreting data about ACAT1-B expression during Xenopus development?

Interpreting data on ACAT1-B expression during Xenopus development requires careful consideration of several factors:

• Developmental timing and staging:

  • Precisely stage embryos according to established Xenopus developmental tables

  • Consider that mitochondrial mRNAs decrease dramatically after fertilization and remain low until late neurula stages

  • Account for the fact that mitochondrial gene expression resumes during organogenesis

• Tissue-specific expression patterns:

  • Distinguish between whole-embryo measurements and tissue-specific analyses

  • Consider differential timing of organogenesis across tissues

  • Account for potential metabolic specialization in developing organs

• Technical considerations:

  • When using RT-PCR or RNA-seq, ensure proper normalization given the global changes in transcription during development

  • For protein analysis, consider the time lag between mRNA expression and protein accumulation

  • For enzymatic assays, account for potential post-translational regulation

• Evolutionary context:

  • Consider the tetraploid nature of Xenopus laevis genome

  • Distinguish between homeologous ACAT1 genes when possible

  • Compare expression patterns to those in diploid relatives like Xenopus tropicalis

• Mitochondrial-nuclear coordination:

  • Consider that the mitochondrial genome appears completely inactivated at the beginning of embryonic development

  • Recognize that nuclear-encoded mitochondrial proteins like ACAT1-B may follow different regulatory patterns

  • Evaluate the coordination between mitochondrial and nuclear genome expression

These considerations will help researchers accurately interpret ACAT1-B expression data within the complex context of Xenopus development, where mitochondrial gene expression follows distinctive regulatory patterns .

How might research on Xenopus laevis ACAT1-B contribute to understanding human metabolic disorders?

Research on Xenopus laevis ACAT1-B has significant potential to advance our understanding of human metabolic disorders through several approaches:

• Evolutionary conservation analysis:

  • Identifying functionally conserved residues between Xenopus and human ACAT1 can highlight critical sites for enzyme function

  • Mutations found in human metabolic disorders can be introduced into Xenopus ACAT1-B to test functional consequences

  • The clear developmental regulation of mitochondrial genes in Xenopus provides context for understanding when and how metabolic pathways become disrupted

• Disease modeling:

  • CRISPR/Cas9 technology in Xenopus laevis enables creation of models with ACAT1 disruption

  • The rapid development and transparent nature of Xenopus embryos facilitates real-time visualization of metabolic consequences

  • Human ACAT inhibition has been explored in the context of immune responses and viral infections , suggesting parallel applications for Xenopus studies

• Therapeutic development:

  • Recombinant Xenopus ACAT1-B can serve as a platform for screening potential therapeutic compounds

  • The effects of ACAT inhibitors, shown to impact human immune cells and viral infections , could be tested in Xenopus models

  • Rescue experiments with modified ACAT1 variants can test potential gene therapy approaches

• Developmental metabolism insights:

  • Understanding how ACAT1-B expression and activity change during normal development provides context for metabolic disorders that manifest at specific developmental stages

  • The coordinated regulation of mitochondrial biogenesis during Xenopus development offers insights into metabolic adaptation mechanisms

What innovative experimental approaches could enhance our understanding of ACAT1-B function and regulation?

Several innovative experimental approaches could significantly advance our understanding of ACAT1-B function and regulation:

• Single-cell omics technologies:

  • Single-cell RNA-seq to map ACAT1-B expression in specific cell types during development

  • Single-cell metabolomics to correlate ACAT1-B activity with metabolite profiles

  • Spatial transcriptomics to visualize ACAT1-B expression patterns in the context of tissue architecture

• Advanced genome editing approaches:

  • Prime editing or base editing for precise modification of ACAT1-B regulatory elements

  • Conditional CRISPR systems for temporal control of ACAT1-B disruption

  • Simultaneous targeting of multiple metabolic enzymes to map pathway interactions

• Real-time imaging techniques:

  • Development of activity-based probes for live imaging of ACAT1-B activity

  • FRET-based biosensors to monitor substrate/product levels in real-time

  • Label-free imaging techniques to track metabolic changes in living embryos

• Integrative multi-omics:

  • Combined analysis of transcriptomics, proteomics, and metabolomics data

  • Network modeling to place ACAT1-B in the broader context of developmental metabolism

  • Machine learning approaches to identify regulatory patterns across multiple datasets

• Xenopus organoid models:

  • Development of tissue-specific organoids to study ACAT1-B in defined cellular contexts

  • Co-culture systems to investigate intercellular metabolic cooperation

  • Microfluidic systems for controlled manipulation of metabolic environments

These approaches build upon the established strengths of Xenopus as a model system, including the efficient application of CRISPR/Cas9 technology and the well-characterized patterns of mitochondrial gene regulation during development .

How might the tetraploid nature of the Xenopus laevis genome influence research approaches to studying ACAT1-B?

The tetraploid nature of the Xenopus laevis genome presents both challenges and opportunities for ACAT1-B research:

• Gene redundancy considerations:

  • Xenopus laevis contains homeologous copies of genes due to genome duplication

  • Researchers must design experiments that account for potential functional redundancy between ACAT1 homeologs

  • CRISPR/Cas9 targeting strategies may need to disrupt multiple homeologs simultaneously for complete loss-of-function

• Subfunctionalization analysis:

  • The presence of homeologs enables investigation of potential subfunctionalization

  • Targeted disruption of individual homeologs can reveal specialized functions

  • Expression pattern analysis may show differential regulation of ACAT1 homeologs during development

• Methodological adaptations:

  • PCR primers and probes must be designed to distinguish between highly similar homeologs

  • Antibodies may need validation for specificity against different homeologous proteins

  • Mass spectrometry approaches must account for slight sequence variations between homeologs

• Evolutionary insights:

  • Comparison between tetraploid X. laevis and diploid X. tropicalis provides a natural experiment in gene dosage

  • Analysis of selection pressure on different homeologs can reveal evolutionary trajectories

  • Examination of regulatory element divergence may explain differential expression patterns

• Application advantages:

  • The presence of multiple homeologs can provide internal controls for experimental manipulations

  • The ability to manipulate gene dosage by targeting specific homeologs offers nuanced experimental design

  • Successful CRISPR/Cas9 targeting in Xenopus laevis demonstrates that technical challenges can be overcome

This genomic feature makes Xenopus laevis a particularly interesting model for studying the evolution and regulation of metabolic pathways, potentially revealing how genome duplication events influence metabolic network robustness and adaptation.