Recombinant Xenopus tropicalis Acyl-CoA-binding domain-containing protein 5 (acbd5)

What is the fundamental role of ACBD5 in cellular metabolism?

Acyl-CoA-binding domain-containing protein 5 (ACBD5) is a member of the acyl-CoA binding protein family that plays a crucial role in the transport and distribution of long chain acyl-Coenzyme A molecules within cells . The protein contains a conserved acyl-CoA binding domain that allows it to sequester acyl-CoA esters in the cytoplasm, thus regulating their availability for various metabolic processes. ACBD5 has essential functions in lipid metabolism across species, as demonstrated by studies in various organisms including insects and amphibians . In its functional state, ACBD5 demonstrates binding affinity to acyl-CoA molecules of different chain lengths, with studies on related proteins showing nanomolar affinity to specific acyl-CoA molecules such as lauroyl-CoA . This high-affinity binding capacity enables ACBD5 to effectively regulate intracellular lipid distribution and metabolism.

How does Xenopus tropicalis ACBD5 compare structurally to ACBD5 in other species?

Xenopus tropicalis ACBD5 shares significant homology with ACBD5 proteins from other vertebrates, particularly in the acyl-CoA binding domain. While the search results don't provide the exact percentage of identity, comparative studies with other ACBD family members suggest conservation of functional domains across species. For example, similar to human ACBD5, the Xenopus tropicalis protein is likely to contain both an acyl-CoA binding domain at its N-terminus and transmembrane domains at its C-terminus that facilitate its localization to cellular organelles . The functional conservation of ACBD proteins is evident from studies in diverse species, including insects like Rhodnius prolixus, where ACBP family proteins show conserved binding capabilities . Xenopus tropicalis serves as an excellent model organism for ACBD5 studies due to its diploid genome, which simplifies genetic manipulations compared to other amphibian models .

What are the known interaction partners of ACBD5 in Xenopus development?

Based on studies of ACBD5 in mammalian systems, the protein is known to interact with vesicle-associated membrane protein-associated protein B (VAPB) to form tethers between peroxisomes and the endoplasmic reticulum . This interaction is regulated by phosphorylation of specific residues in ACBD5, particularly in the acidic tract and FFAT motif . While specific Xenopus tropicalis ACBD5 interaction partners are not explicitly detailed in the search results, the conserved nature of these proteins suggests similar interactions occur in amphibian systems. The importance of these interactions in Xenopus development is underscored by studies of related proteins like ACBD6, where knockout experiments have demonstrated severe developmental consequences including gastrulation failure and brain defects . The interaction network likely involves proteins associated with peroxisomal function, lipid metabolism, and potentially developmental signaling pathways.

What are the optimal expression systems for producing recombinant Xenopus tropicalis ACBD5?

For producing functional recombinant Xenopus tropicalis ACBD5, wheat germ cell-free expression systems have proven effective for related proteins . This eukaryotic expression system offers advantages for amphibian proteins by providing appropriate post-translational modifications and protein folding mechanisms. Alternative expression systems include E. coli for basic structural studies, insect cell systems (Sf9, Sf21) for more complex functional analyses, and mammalian cell lines for studies requiring native-like post-translational modifications. When expressing Xenopus tropicalis ACBD5, researchers should consider adding affinity tags (such as GST or His tags) at the N-terminus rather than the C-terminus to avoid interfering with potential transmembrane domains . Purification protocols typically involve affinity chromatography followed by size exclusion chromatography to isolate monomeric protein. Protein yield and solubility can be optimized through adjustments in induction temperature, expression time, and the addition of specific detergents when solubilizing membrane-associated forms of the protein.

How can CRISPR/Cas9 technology be effectively applied to generate Xenopus tropicalis ACBD5 knockouts?

CRISPR/Cas9 technology has been successfully used to generate Xenopus tropicalis gene knockouts, including for ACBD family members. The process begins with the design of guide RNAs (gRNAs) targeting exonic regions of the acbd5 gene, preferably early exons to ensure complete disruption of protein function . For Xenopus tropicalis, injection of the CRISPR/Cas9 components is typically performed at the one-cell or two-cell stage to ensure germline transmission. The protocol involves:

• Design of specific gRNAs (typically 2-3 different guides) targeting conserved regions of acbd5

• In vitro transcription of gRNAs

• Preparation of Cas9 protein or mRNA

• Microinjection into fertilized Xenopus tropicalis eggs

• Validation of editing efficiency using T7 endonuclease assays or direct sequencing

• Phenotypic characterization of F0 "crispant" animals

• Establishment of stable knockout lines through selective breeding of founders

Validation of knockout efficiency should include both genomic analysis and protein level verification using Western blotting with anti-ACBD5 antibodies . This approach has been successfully used for related genes in Xenopus tropicalis, generating phenotypes that recapitulate aspects of human disease conditions associated with these proteins .

What immunodetection methods are most reliable for studying ACBD5 expression patterns in Xenopus tissues?

For studying ACBD5 expression patterns in Xenopus tissues, both Western blotting and immunofluorescence techniques have proven effective for related proteins. When selecting antibodies, those raised against conserved epitopes of the acyl-CoA binding domain show better cross-reactivity across species . The immunodetection protocol should include:

• Tissue fixation: 4% paraformaldehyde for immunofluorescence or flash freezing for Western blotting

• Permeabilization: 0.1-0.5% Triton X-100 for immunofluorescence studies

• Blocking: BSA or serum-based blockers to reduce non-specific binding

• Primary antibody incubation: Using validated anti-ACBD5 antibodies at optimized dilutions

• Detection: Fluorescently-labeled secondary antibodies for imaging or HRP-conjugated antibodies for Western blotting

For developmental studies, whole-mount immunostaining of Xenopus embryos can reveal spatiotemporal expression patterns . When analyzing expression in regenerating tissues, such as tadpole limbs or tails, it's important to include markers of cellular proliferation (like PCNA) and apoptosis (like active Caspase3) to correlate ACBD5 expression with specific cellular processes . Quantitative analysis of expression should include normalization to appropriate housekeeping proteins and statistical analysis of biological replicates to ensure reproducibility.

How is ACBD5 expression regulated during Xenopus development and regeneration?

Based on studies of related proteins in Xenopus, ACBD5 expression is likely dynamically regulated during development and regeneration processes. For instance, developmental expression patterns have been observed for other genes involved in regeneration, with specific upregulation during key developmental transitions . In regeneration contexts, expression may follow a pattern similar to that observed for genes like evi5, which peaks at specific time points after amputation (for example, 5 days post-amputation) and then returns to baseline levels as regeneration progresses .

The regulation of ACBD5 likely involves both transcriptional and post-translational mechanisms. At the transcriptional level, developmental stage-specific expression has been observed for related genes in Xenopus, with tissue-specific patterns emerging during organogenesis . Post-translationally, ACBD5 activity and localization are regulated by phosphorylation of specific residues, as demonstrated in mammalian systems . This phosphorylation affects the protein's ability to interact with binding partners such as VAPB, thus modulating its function in establishing organelle contacts . The phosphorylation status of ACBD5 appears to be particularly important for its function, with studies showing that phosphatase treatment significantly reduces the interaction between ACBD5 and VAPB .

What is the role of ACBD5 phosphorylation in regulating its function?

ACBD5 function is intricately regulated by phosphorylation at multiple sites, affecting its protein-protein interactions and subcellular activities. Studies in mammalian systems have demonstrated that the ACBD5-VAPB interaction, which mediates peroxisome-ER contacts, is highly sensitive to phosphorylation status . Specific serine residues in the acidic tract surrounding the FFAT motif of ACBD5 serve as phosphorylation sites that enhance binding to VAPB when phosphorylated . These include threonine-258 (T258), serine-259 (S259), S261, and S263 in the human protein, which when mutated to non-phosphorylatable alanine residues result in reduced binding to VAPB .

Additionally, phosphorylation of serine-269 (S269) within the FFAT core appears critical, as mutation to alanine completely abolishes VAPB interaction . The phosphorylation of these residues likely contributes to the acidic environment necessary for the initial interaction with VAPB as part of a two-step binding model . This phospho-regulation represents a sophisticated mechanism for controlling organelle contacts in response to cellular signals. While these specific phosphorylation sites have been identified in human ACBD5, conservation analysis suggests similar regulatory mechanisms likely exist in Xenopus tropicalis ACBD5, though the exact residue positions may differ based on sequence alignment.

How does ACBD5 contribute to peroxisome-ER tethering in Xenopus cells?

ACBD5 plays a crucial role in establishing and maintaining contacts between peroxisomes and the endoplasmic reticulum (ER) through its interaction with VAPB, an ER-resident protein . While the specific details of this interaction in Xenopus cells aren't explicitly detailed in the search results, studies in mammalian systems provide a model that likely applies to amphibian cells as well.

The tethering mechanism involves the FFAT-like motif in ACBD5, which interacts with the MSP domain of VAPB at the ER surface . This interaction follows a two-step binding model: first, the acidic tract surrounding the FFAT motif establishes an initial electrostatic interaction, followed by specific binding of the FFAT core . The strength and regulation of this tethering are modulated by phosphorylation of serine/threonine residues in both the acidic tract and the FFAT core of ACBD5 .

In Xenopus cells, this tethering mechanism would facilitate metabolic cooperation between peroxisomes and the ER, particularly for lipid metabolism pathways that require coordination between these organelles. Disruption of this tethering through mutation or altered phosphorylation would likely impact peroxisomal functions and lipid homeostasis, potentially contributing to developmental abnormalities observed in ACBD family protein deficiencies .

What developmental abnormalities result from ACBD5 deficiency in Xenopus models?

While the search results don't provide specific information about developmental abnormalities resulting from ACBD5 deficiency in Xenopus tropicalis, studies of related ACBD family proteins provide insights into potential phenotypes. For instance, ACBD6 deficiency in Xenopus tropicalis results in severe developmental abnormalities including gastrulation failure, brain defects, and reduced locomotion . These phenotypes reflect the essential roles of ACBD proteins in early development and neurological function.

By extrapolation, ACBD5 deficiency in Xenopus would likely manifest as defects in processes requiring proper lipid metabolism and peroxisome function. These might include:

• Neurological development abnormalities due to impaired myelination and membrane synthesis

• Disrupted organogenesis, particularly in lipid-rich tissues like brain and liver

• Metabolic disturbances affecting energy homeostasis

• Potential defects in regenerative capacity, as observed with knockdown of other genes in tadpole limb and tail regeneration

The phenotypes would likely vary in severity depending on whether the deficiency represents a complete knockout or partial knockdown, with compensatory mechanisms potentially mitigating effects in the latter case. As observed with other ACBD family members, functional overlap between family proteins might mask some phenotypes in single gene knockouts .

How does ACBD5 function compare between regenerative and non-regenerative contexts in Xenopus?

While specific information about ACBD5 in regenerative contexts is not directly provided in the search results, insights can be drawn from studies of other proteins in Xenopus regeneration. In regenerative contexts such as tadpole limb and tail regeneration, genes like evi5 show dynamic expression patterns with specific upregulation during blastema formation and proliferation phases . ACBD5 likely follows a similar pattern, with potential roles in lipid metabolism supporting membrane synthesis during the high-growth phases of regeneration.

The function of ACBD5 may differ between regenerative and non-regenerative contexts primarily in terms of its regulation and interaction partners. In regenerative tissues, ACBD5 might interact with regeneration-specific factors to facilitate the rapid lipid metabolism and membrane formation required for blastema cell proliferation. In non-regenerative contexts, ACBD5 would maintain its housekeeping functions in lipid metabolism and peroxisome-ER tethering.

Studies of regeneration in Xenopus tadpole limbs have shown that knockdown of certain genes results in reduced proliferation of blastema cells and increased apoptosis, effectively blocking regeneration . If ACBD5 plays a similar role in supporting the proliferative capacity of blastema cells through its functions in lipid metabolism, its knockdown would potentially show comparable regeneration defects.

What human disease models can be recapitulated using Xenopus tropicalis ACBD5 knockouts?

Xenopus tropicalis ACBD5 knockouts can serve as valuable models for human diseases associated with ACBD5 dysfunction. Studies of ACBD6 variants have already demonstrated that Xenopus knockouts can recapitulate aspects of human neurodevelopmental syndromes . For ACBD5 specifically, knockout models would be relevant for several human conditions:

• Peroxisomal disorders: Human mutations in ACBD5 have been associated with a spectrum of peroxisomal disorders characterized by abnormal very long-chain fatty acid metabolism.

• Neurodevelopmental conditions: Given the importance of proper lipid metabolism for neurological development, ACBD5 knockouts could model aspects of neurodevelopmental disorders.

• Movement disorders: Related ACBD protein deficiencies result in complex and progressive movement disorder phenotypes in humans, which might be modeled in Xenopus through appropriate behavioral assays .

• Thrombocytopenia: Human ACBD5 has been implicated in megakaryocyte differentiation and platelet formation, with mutations associated with autosomal dominant thrombocytopenia .

The advantages of using Xenopus tropicalis for these disease models include its diploid genome (simplifying genetic manipulations), external fertilization and development (facilitating embryonic interventions), and the ability to generate large numbers of embryos for statistical analyses . Additionally, the evolutionary conservation of ACBD5 function makes findings in Xenopus potentially translatable to human disease contexts.

How can mass spectrometry be optimized for identifying ACBD5 interactome changes during Xenopus development?

To optimize mass spectrometry for identifying the ACBD5 interactome during Xenopus development, researchers should implement a comprehensive approach combining co-immunoprecipitation (co-IP) with quantitative proteomics. The protocol should include:

• Sample preparation stages:

  • Collection of Xenopus tissues from key developmental stages

  • Expression of tagged ACBD5 (FLAG-tag or BioID) or use of validated anti-ACBD5 antibodies

  • Cross-linking to stabilize transient interactions (using DSP or formaldehyde)

  • Stringent controls including IgG pulldowns and reverse IP validations

• MS optimization parameters:

  • Use of high-resolution mass spectrometry (Orbitrap or QTOF instruments)

  • Implementation of SILAC or TMT labeling for quantitative comparison across developmental stages

  • Data-independent acquisition (DIA) methods to improve coverage of low-abundance interactors

  • Multiple fragmentation methods (HCD, ETD) to improve peptide identification

• Data analysis considerations:

  • Stringent statistical filtering using both fold-change and p-value cutoffs

  • Network analysis to identify interaction clusters changing across development

  • Validation of top candidates using reciprocal co-IPs and proximity ligation assays

  • Integration with phosphoproteomic data to correlate with ACBD5 phosphorylation states

This approach would reveal how the ACBD5 interactome changes during developmental processes, providing insights into stage-specific functions and regulatory mechanisms. Particular attention should be paid to interactions that might be regulated by phosphorylation, given the known importance of phosphorylation in modulating ACBD5-VAPB interactions .

What strategies can resolve conflicting data between ACBD5 knockout phenotypes and biochemical activity assays?

Resolving conflicts between ACBD5 knockout phenotypes and biochemical activity assays requires a multi-faceted approach addressing both technical and biological factors. Researchers should consider:

• Compensatory mechanism investigation:

  • Perform combinatorial knockdowns of multiple ACBD family members to address functional redundancy, as seen with RpACBP-1 and RpACBP-5 in Rhodnius prolixus

  • Analyze expression changes in other ACBD family members following ACBD5 knockout

  • Conduct temporal knockout studies using inducible systems to bypass developmental compensation

• Technical validation approaches:

  • Verify knockout efficiency at both mRNA and protein levels using qPCR and Western blotting

  • Confirm specificity of antibodies used in biochemical assays

  • Perform rescue experiments with wild-type and mutant ACBD5 to establish causality

  • Use multiple independent knockout/knockdown methods (CRISPR, morpholinos, RNAi) to validate phenotypes

• Contextual analyses:

  • Examine tissue-specific effects rather than only whole-organism phenotypes

  • Investigate phenotypes under metabolic stress conditions that might reveal cryptic defects

  • Perform detailed lipidomic analyses to detect subtle metabolic alterations

  • Analyze specific developmental timepoints and regenerative contexts separately

By systematically addressing these aspects, researchers can resolve apparent discrepancies between organismal phenotypes and biochemical findings. The resolution may reveal that the discrepancies stem from biological compensatory mechanisms rather than technical issues, as demonstrated by the lack of detectable phenotypes in some ACBP family protein knockdowns despite proven biochemical activity .

How can phosphoproteomics be integrated with functional studies of ACBD5 in Xenopus systems?

Integration of phosphoproteomics with functional studies of ACBD5 in Xenopus systems requires a comprehensive experimental design that connects phosphorylation events with biological outcomes. A strategic approach would include:

• Phosphosite mapping and dynamics:

  • Identify all phosphorylation sites in Xenopus tropicalis ACBD5 using mass spectrometry

  • Track phosphosite occupancy across developmental stages and regeneration timepoints

  • Create phosphosite mutants (phosphomimetic and non-phosphorylatable) corresponding to key regulatory sites identified in mammalian ACBD5 (especially in the FFAT motif region)

• Kinase and phosphatase identification:

  • Perform kinase prediction analysis for identified phosphosites

  • Conduct kinase inhibitor screens to identify enzymes regulating ACBD5 phosphorylation

  • Test specific candidates like GSK3β, which has been implicated in regulating peroxisome-ER contacts via the ACBD5-VAPB tether

• Functional correlation studies:

  • Express phosphosite mutants in ACBD5-knockout backgrounds to assess rescue efficiency

  • Analyze peroxisome morphology, distribution, and contacts with ER in cells expressing different phosphovariants

  • Measure lipid metabolism parameters in tissues expressing wild-type versus phosphosite mutant ACBD5

  • Correlate phosphorylation changes with developmental transitions or regeneration phases

• Integration with interactome data:

  • Create dynamic interactome maps showing how phosphorylation status affects protein interactions

  • Identify interaction partners whose binding is phospho-dependent

  • Develop computational models predicting how phosphorylation cascades regulate ACBD5 function throughout development

This integrated approach would establish mechanistic links between specific phosphorylation events and the biological functions of ACBD5 in Xenopus systems, providing insights applicable to both developmental biology and human disease mechanisms .

What are the key unanswered questions regarding ACBD5 function in Xenopus development?

Despite significant advances in understanding ACBD5 and related proteins, several critical questions remain unanswered regarding its specific functions in Xenopus development. These knowledge gaps represent important areas for future investigation:

• The precise spatial and temporal expression pattern of ACBD5 throughout Xenopus tropicalis development has not been fully characterized, particularly in relation to tissue-specific functions.

• The direct contribution of ACBD5 to organogenesis and tissue differentiation in Xenopus remains unclear, especially regarding its potential roles in neurogenesis and neural crest development.

• The extent of functional redundancy between ACBD5 and other ACBD family members in Xenopus development is not fully established, though studies in other organisms suggest considerable overlap may exist .

• The mechanisms by which ACBD5 phosphorylation regulates developmental processes in Xenopus are largely unexplored, despite evidence for the importance of phosphorylation in regulating ACBD5 function in mammalian systems .

• The potential roles of ACBD5 in regenerative processes in Xenopus tadpoles and whether its function differs between regeneration-competent and incompetent stages remain to be elucidated.

Addressing these questions will require interdisciplinary approaches combining developmental biology, biochemistry, and systems biology to fully understand the complex roles of ACBD5 in amphibian development and evolution.

How might emerging technologies enhance our understanding of ACBD5 function in Xenopus models?

Emerging technologies offer exciting opportunities to address current knowledge gaps regarding ACBD5 function in Xenopus models. Several cutting-edge approaches show particular promise:

• Single-cell multi-omics technologies will enable characterization of ACBD5 expression and function with unprecedented cellular resolution, revealing cell type-specific roles during development. Integrating single-cell transcriptomics, proteomics, and metabolomics can identify how ACBD5 functions vary across different cell populations during organogenesis.

• Live-cell super-resolution microscopy techniques like PALM, STORM, and lattice light-sheet microscopy will permit visualization of ACBD5-mediated peroxisome-ER contacts with nanometer resolution in living Xenopus cells. This will reveal the dynamics of these interactions during development and in response to metabolic challenges.

• Optogenetic and chemogenetic tools can be adapted for rapid, reversible modulation of ACBD5 function in specific tissues during Xenopus development. These approaches would overcome limitations of conventional knockout strategies where compensatory mechanisms may mask phenotypes .

• CRISPR base editing and prime editing technologies will enable precise modification of individual phosphorylation sites without disrupting the entire protein, allowing more nuanced investigation of phosphoregulation than conventional knockouts .

• Spatial metabolomics and lipidomics techniques will map the distribution of lipid species in Xenopus tissues with high spatial resolution, correlating ACBD5 function with specific metabolic outputs during development and regeneration.

These technologies, applied systematically to Xenopus models, will provide multidimensional insights into ACBD5 function that extend beyond what conventional approaches have revealed.

What comparative studies between Xenopus and mammalian ACBD5 would yield the most translational insights?

Comparative studies between Xenopus and mammalian ACBD5 offer valuable opportunities for translational insights into both fundamental biology and disease mechanisms. The most productive comparative approaches would include:

• Structure-function conservation analysis:

  • Compare phosphorylation patterns and their functional consequences between species

  • Evaluate conservation of interaction interfaces, particularly in the FFAT motif and acyl-CoA binding domain

  • Determine if regulatory mechanisms, such as the phosphorylation-dependent interaction with VAPB, are evolutionarily conserved

• Cross-species rescue experiments:

  • Test whether human ACBD5 can rescue phenotypes in Xenopus ACBD5 knockouts

  • Identify domains or residues essential for cross-species functionality

  • Evaluate disease-associated human variants through expression in Xenopus models

• Developmental timing comparison:

  • Map expression patterns of ACBD5 across developmental stages in both species

  • Identify conserved vs. divergent developmental processes requiring ACBD5 function

  • Compare tissue-specific functions, particularly in the nervous system and regenerative contexts

• Disease model validation:

  • Generate Xenopus models of human disease-associated ACBD5 variants

  • Compare phenotypes with patient data to validate model fidelity

  • Test therapeutic approaches in Xenopus before advancing to mammalian models

• Metabolomic profiling:

  • Compare lipid profiles affected by ACBD5 deficiency across species

  • Identify conserved metabolic pathways dependent on ACBD5 function

  • Evaluate species-specific metabolic adaptations that might explain differences in phenotypic severity