Recombinant Rat Acyl-CoA-binding domain-containing protein 5 (Acbd5)

What is the subcellular localization of ACBD5 and how has it been confirmed experimentally?

ACBD5 is primarily localized to peroxisomes, as confirmed through multiple experimental approaches. Proteomic studies of isolated mammalian peroxisomes first identified ACBD5 as a peroxisomal protein . This localization has been further verified through immunofluorescence microscopy in HeLa cells, which confirmed the peroxisomal localization of endogenous ACBD5 .

For researchers seeking to validate ACBD5 localization, differential permeabilization techniques can be applied. This method involves:

• Transiently expressing tagged ACBD5 (e.g., N-terminally FLAG-tagged and C-terminally HA-tagged ACBD5)

• Performing immunostaining after either full permeabilization of cellular membranes with Triton X-100 or selective permeabilization of the plasma membrane with digitonin

• Using antibodies against intraperoxisomal markers like catalase as controls

This approach allows determination of not only localization but also membrane topology, revealing that ACBD5 is a tail-anchored protein with its N-terminal domain facing the cytosol .

What is the structural organization of ACBD5 protein and which domains are critical for its function?

ACBD5 is structurally characterized as a tail-anchored membrane protein with distinct functional domains:

Experimental evidence demonstrates that mutations in the ACB domain significantly impair VLCFA metabolism, while interestingly, mutations in the FFAT motif (which disrupt ER tethering) do not affect VLCFA metabolism to the same extent . This suggests that the lipid-binding capacity of ACBD5 is more critical for VLCFA metabolism than its ER-tethering function .

To explore domain functionality, researchers can employ targeted mutagenesis approaches and complementation assays in ACBD5-deficient cell lines to evaluate the specific contribution of each domain to various ACBD5-dependent cellular processes.

How does ACBD5 contribute to peroxisome-ER membrane contact sites, and what experimental approaches can be used to study these interactions?

ACBD5 serves as a key tethering protein that mediates physical contacts between peroxisomes and the endoplasmic reticulum. These contact sites play critical roles in lipid metabolism and peroxisome biogenesis.

Experimental approaches to study ACBD5-mediated peroxisome-ER contacts include:

• Electron microscopy to visualize the ultrastructural contacts between organelles

• Live-cell imaging with fluorescently tagged organelle markers to monitor contact dynamics

• Proximity ligation assays to detect protein-protein interactions at contact sites

• CRISPR/Cas9-mediated knockout of ACBD5 followed by rescue experiments with wild-type or mutant forms

Research has shown that loss of ACBD5 results in:

• Reduction of physical tethering between the ER and peroxisomes

• Increased peroxisomal movement

• Reduced expansion of the peroxisomal membrane

• Altered metabolism of very long-chain fatty acids (VLCFAs)

Interestingly, while ACBD4 can partially compensate for ACBD5 loss in terms of restoring peroxisome-ER contacts when overexpressed, ACBD4 knockout cells do not show significant differences in peroxisome-ER contacts compared to controls . This suggests a primary role for ACBD5 in establishing these contacts in HEK293 cells.

What is the role of ACBD5 in peroxisomal fatty acid metabolism, particularly regarding very long-chain fatty acids (VLCFAs)?

ACBD5 plays a crucial role in peroxisomal β-oxidation of very long-chain fatty acids (VLCFAs). Evidence for this includes:

• ACBD5-deficient patient fibroblasts show accumulation of VLCFAs, particularly C26:0

• CRISPR/Cas9-generated ACBD5 knockout HeLa cells demonstrate similar VLCFA accumulation

• Metabolic profiling reveals increased C26:0/C22:0 and C24:0/C22:0 ratios in patients with ACBD5 deficiency

For researchers investigating the role of ACBD5 in fatty acid metabolism, the following methodological approaches are recommended:

• VLCFA analysis: Measure C26:0, C24:0, and C22:0 levels using liquid chromatography-tandem mass spectrometry (LC-MS/MS)

• β-oxidation assays: Utilize D3-C22:0 loading tests to track metabolic processing of VLCFAs

• Complementation studies: Express wild-type or mutant ACBD5 in knockout cells to identify domains critical for function

Data from complementation studies indicate that the acyl-CoA binding (ACB) domain is essential for proper VLCFA metabolism, as expression of ACBD5 with a mutated ACB domain failed to restore normal VLCFA levels in ACBD5 knockout cells . Surprisingly, the FFAT motif (which mediates ER tethering) appears dispensable for this specific function, as FFAT-mutant ACBD5 successfully complemented the VLCFA metabolism defect .

What are the clinical manifestations of ACBD5 deficiency and how do they relate to the protein's molecular function?

ACBD5 deficiency is associated with a distinct clinical syndrome known as ACBD5-related retinal dystrophy with leukodystrophy (RDLKD). The cardinal features include:

The pathophysiology appears to be linked to ACBD5's role in VLCFA metabolism. Deficiency leads to:

• Accumulation of VLCFAs, particularly C26:0 lysophosphatidylcholine (C26:0 lysoPC)

• Impaired peroxisomal β-oxidation

• Potential alterations in membrane lipid composition affecting neuronal and retinal function

Long-term follow-up of patients reveals a progressive neurodegenerative course, with the oldest reported patient (36 years) developing significant cognitive decline, upper extremity weakness, difficulty with fine motor movements, sphincter incompetence, neurogenic bladder, and severe dysphagia requiring gastrostomy .

What neuroimaging findings are characteristic of ACBD5 deficiency, and how can they be used in research models?

Neuroimaging studies of patients with ACBD5 deficiency reveal a consistent pattern of abnormalities that can serve as biomarkers in research:

• Diffuse hyperintense T2 and FLAIR signal abnormality in cerebral white matter

• Relative sparing of subcortical U fibers

• Extension of signal abnormalities along cerebrospinal tracks involving bilateral cerebral peduncles up to the brain stem

• Progressive atrophic changes in bilateral cerebellar hemispheres, brainstem, and thoracic spinal cord in older patients

Magnetic resonance spectroscopy (MRS) shows characteristic metabolite changes:

• Decreased N-acetylaspartate (NAA)

• Increased choline

These neuroimaging findings can be applied in research through:

• Correlation of imaging markers with biochemical parameters (VLCFA levels) in patient samples

• Development of comparable neuroimaging protocols for animal models of ACBD5 deficiency

• Longitudinal studies to track disease progression and potential therapeutic responses

• Translation of imaging biomarkers to cellular or organoid models through related biochemical assays

Researchers should note that these changes appear to correlate with the progressive neurodegenerative nature of the disease and may serve as objective markers for evaluating experimental therapeutics.

What are the validated approaches for detecting and quantifying ACBD5 expression in different experimental systems?

Several validated approaches can be employed to detect and quantify ACBD5 in experimental systems:

• Western Blot (WB):

  • Commercially available antibodies such as 21080-1-AP have been validated for ACBD5 detection

  • Optimal dilution ranges: 1:5000-1:50000

  • Successfully tested in multiple cell lines including HeLa, L02, and MCF-7 cells, as well as mouse skeletal muscle tissue

• Immunohistochemistry (IHC):

  • Recommended dilution: 1:500-1:2000

  • Validated in human ovary cancer tissue and human stomach tissue

  • Antigen retrieval with TE buffer pH 9.0 or citrate buffer pH 6.0

• CRISPR/Cas9 gene editing:

  • Successfully employed to generate ACBD5 knockout cell lines using specific guide RNAs (gRNAs)

  • Validation of knockout typically performed via immunoblotting

• Quantitative PCR:

  • For mRNA expression analysis

  • Requires careful primer design to distinguish ACBD5 from other ACBD family members

• Mass spectrometry:

  • For detection of endogenous ACBD5 in proteomic studies

  • Particularly useful for organelle proteomics studies of peroxisomes

When working with recombinant rat ACBD5, researchers should consider species-specific detection methods and validate antibodies for cross-reactivity, as most published research has focused on human and mouse ACBD5.

How can researchers generate and validate ACBD5-deficient cellular models for functional studies?

Generation of ACBD5-deficient cellular models can be achieved through several approaches, each with specific validation requirements:

CRISPR/Cas9 Gene Editing:

• Design specific gRNAs targeting exonic regions of ACBD5 (published studies have successfully used multiple independent gRNAs)

• Transfect cells with CRISPR/Cas9 components and select potential knockout clones

• Validate knockouts through:

  • Genomic DNA sequencing to confirm mutations

  • Immunoblotting to verify absence of ACBD5 protein

  • Functional assays such as peroxisome morphology assessment and VLCFA analysis

Complementation Systems:
For controlled expression studies, the FlpIn system has been successfully used to generate stable cell lines expressing:

• Wild-type ACBD5

• ACBD5 with mutations in the ACB domain

• ACBD5 with mutations in the FFAT motif

Validation Criteria:
A comprehensive validation protocol should include:

It's important to note that ACBD5 deficiency does not affect general peroxisome biogenesis or the import of peroxisomal matrix proteins, making these parameters useful negative controls for model validation .

How do ACBD5 and ACBD4 differ functionally despite their structural similarities, and what experimental approaches can distinguish their roles?

Despite structural similarities between ACBD5 and ACBD4, research has revealed important functional differences that can be distinguished through specific experimental approaches:

Key Differences:

Experimental Approaches to Distinguish Their Roles:

• Differential Expression Analysis:

  • Tissue-specific expression patterns may provide insights into specialized functions

  • Single-cell RNA sequencing to identify cell types preferentially expressing each protein

• Domain Swap Experiments:

  • Generate chimeric proteins with swapped domains between ACBD4 and ACBD5

  • Assess the ability of each chimera to complement ACBD5 deficiency

• Interaction Proteomics:

  • Proximity labeling coupled with mass spectrometry to identify unique interaction partners

  • Co-immunoprecipitation with domain-specific antibodies

• Substrate Specificity Assays:

  • In vitro binding assays with different acyl-CoA species to determine differential substrate preferences

  • Lipidomic analysis of cells expressing either ACBD4 or ACBD5

• Double Knockout Studies:

  • Generate ACBD4/ACBD5 double knockout cells to assess potential compensatory mechanisms

  • Compare phenotypes with single knockouts to identify synergistic effects

These approaches would help clarify whether ACBD4 and ACBD5 have truly distinct functions or merely different expression patterns or substrate preferences that result in the observed phenotypic differences.

What are the challenges and considerations when developing therapeutic approaches targeting ACBD5-related disorders?

Developing therapeutic approaches for ACBD5-related disorders presents several challenges and considerations that researchers should address:

1. Understanding Disease Mechanisms:

• The precise mechanisms linking ACBD5 deficiency to retinal and neurological manifestations remain incompletely understood

• Research suggests multiple potential pathogenic pathways:

  • Direct effects of VLCFA accumulation on membrane properties

  • Impaired peroxisome-ER communication affecting lipid transfer

  • Secondary effects on other peroxisomal functions

2. Therapeutic Target Selection:

• Potential approaches include:

  • Gene therapy to restore ACBD5 expression

  • Replacement of functional domains (e.g., the critical ACB domain)

  • Pharmacological modulation of VLCFA metabolism

  • Enhancement of compensatory pathways (e.g., ACBD4 upregulation)

3. Delivery Challenges:

• Blood-brain barrier penetration for CNS manifestations

• Retinal delivery systems for vision-related symptoms

• Targeting both neuronal and glial cells in the CNS

4. Model Systems Considerations:

• Patient-derived fibroblasts provide a cellular model but don't recapitulate tissue-specific pathology

• Animal models need to replicate both biochemical and clinical features

• iPSC-derived neurons or organoids might provide better disease models

5. Biomarker Development:

• VLCFA levels (particularly C26:0 and C26:0 lysoPC) can serve as biochemical biomarkers

• Neuroimaging markers including white matter signal changes and spectroscopic alterations

• Visual evoked potentials and electroretinography for retinal function

6. Clinical Trial Design:

• Progressive nature of the disease requires long-term outcome measures

• Rare disease status necessitates innovative trial designs

• Consideration of age-dependent intervention windows

Researchers should consider a multidisciplinary approach, combining expertise in peroxisome biology, lipid metabolism, neurology, and ophthalmology to address these challenges effectively.

What emerging technologies could advance our understanding of ACBD5 function in complex cellular environments?

Several cutting-edge technologies hold promise for deepening our understanding of ACBD5 function:

• Advanced Imaging Technologies:

  • Super-resolution microscopy to visualize peroxisome-ER contacts at nanometer resolution

  • Live-cell imaging with optogenetic tools to manipulate ACBD5 interactions in real-time

  • Correlative light and electron microscopy (CLEM) to link functional observations with ultrastructural details

• Single-Cell Multi-omics:

  • Combining transcriptomics, proteomics, and lipidomics at single-cell resolution

  • Spatial transcriptomics to map ACBD5 expression patterns in complex tissues

  • Cell-type specific interactome analysis in brain and retinal tissue

• CRISPR-Based Technologies:

  • CRISPRi/CRISPRa for tunable modulation of ACBD5 expression

  • Base editing for precise introduction of patient-specific mutations

  • CRISPR screens to identify genetic modifiers of ACBD5 function

• Organoid and Advanced In Vitro Models:

  • Brain organoids to model neurodevelopmental aspects

  • Retinal organoids to study vision-related pathology

  • Microfluidic systems for studying cell-type specific responses

• Computational Approaches:

  • Molecular dynamics simulations of ACBD5-lipid interactions

  • Systems biology modeling of peroxisome-ER communication networks

  • AI-driven prediction of small molecule modulators of ACBD5 function

These technologies could help address key questions about tissue-specific roles of ACBD5, dynamic regulation of its function, and its integration into broader cellular networks controlling lipid homeostasis.

How might differential expression or regulation of ACBD5 contribute to tissue-specific manifestations in disease states?

The tissue-specific manifestations of ACBD5 deficiency, particularly in retina and white matter, raise important questions about differential expression and regulation:

Current Understanding:

• ACBD5 deficiency primarily affects retina and central nervous system white matter

• Different tissues may have varying requirements for VLCFA metabolism

• Compensatory mechanisms (e.g., ACBD4) may vary across tissues

Research Approaches to Address This Question:

• Tissue-Specific Expression Profiling:

  • Comparative transcriptomics and proteomics across tissues

  • Single-cell analysis in retina and brain to identify cell types with highest ACBD5 expression

  • Analysis of alternative splicing patterns across tissues

• Regulatory Mechanisms:

  • Identification of tissue-specific transcription factors controlling ACBD5 expression

  • Epigenetic profiling to identify regulatory elements

  • miRNA regulation patterns across different cell types

• Metabolic Requirements Analysis:

  • Tissue-specific lipidomics to characterize VLCFA distribution

  • Metabolic flux analysis using isotope-labeled precursors

  • Comparison of peroxisome abundance and activity across tissues

• Conditional Knockout Models:

  • Generation of tissue-specific ACBD5 knockout animals

  • Temporal control of ACBD5 deletion to identify critical developmental windows

  • Comparison of phenotypes between tissue-specific knockouts

• Interaction Network Mapping:

  • Tissue-specific interactome analysis

  • Identification of tissue-restricted binding partners

  • Characterization of compensatory mechanisms in different tissues

Understanding tissue-specific functions could inform more targeted therapeutic approaches and explain the selective vulnerability of certain tissues in ACBD5-related disorders.