ABHD5 Antibody, HRP conjugated

What is the biological role of ABHD5 in cellular metabolism?

ABHD5 functions as a coenzyme A-dependent lysophosphatidic acid acyltransferase that catalyzes the transfer of acyl groups to lysophosphatidic acid molecules . This enzyme plays a critical role in phosphatidic acid biosynthesis pathways and demonstrates substrate preferences with regard to both lipid acceptors and acyl group donors . Functionally, ABHD5 preferentially catalyzes reactions with 1-oleoyl-lysophosphatidic acid as the primary lipid acceptor, followed by 1-palmitoyl-lysophosphatidic acid, 1-stearoyl-lysophosphatidic acid, and 1-arachidonoyl-lysophosphatidic acid in decreasing order of preference . For acyl donors, ABHD5 shows highest activity with arachidonoyl-CoA, followed by oleoyl-CoA .

Beyond its enzymatic function, ABHD5 also regulates cellular triacylglycerol storage through activation of the phospholipase PNPLA2 . Additionally, research has established ABHD5's involvement in keratinocyte differentiation and lipid droplet fusion mechanisms . Recent studies have also identified an important role for ABHD5 in autophagy regulation and potential tumor suppression activity, suggesting previously unrecognized functions independent of its interaction with PNPLA2 .

How does the structure of ABHD5 relate to its function?

The structure of ABHD5 follows the canonical alpha/beta hydrolase fold composed of six α helices, eight β sheets, and two insertion regions—an α helix (α1) near the N-terminal before β1 and helical insertions (α2–α6) between β6 and αD . Computational modeling has identified several potential structures for ABHD5, with the most promising models created using deep learning-based methods .

Despite maintaining structural elements similar to other hydrolases, ABHD5 lacks the conserved catalytic serine typically found in related α-β hydrolases, which explains its lack of hydrolase activity . The protein contains three catalytic triad residues—N155, H329, and D303—positioned in close proximity to each other, yet the absence of hydrolase activity suggests these residues may serve alternative functions .

Structure-function studies have identified critical regions for ABHD5's cellular activities. For example, the interaction between residues E41 and R116 plays a key role in stabilizing the protein structure and facilitates membrane binding through an extended amphipathic helix . Mutation studies (R116N or E41A) have demonstrated defective membrane binding, validating the importance of these structural elements .

What are the known aliases and alternative names for ABHD5?

When searching literature and databases for ABHD5, researchers should be aware of several alternative names and identifiers for this protein :

• CGI-58 (Comparative Gene Identification-58)

• NCIE2 (Neutral Lipid Storage Disease with Ichthyosis, Type 2)

• 1-acylglycerol-3-phosphate O-acyltransferase ABHD5

• Abhydrolase domain-containing protein 5

• Lipid droplet-binding protein CGI-58

Recognizing these alternative designations is essential for comprehensive literature searches and database queries to ensure complete coverage of relevant research findings.

What are the validated applications for ABHD5 antibodies with HRP conjugation?

HRP-conjugated ABHD5 antibodies are validated for multiple research applications, each with specific methodological considerations :

The HRP conjugation offers direct enzymatic detection without the need for secondary antibody incubation, thereby streamlining experimental workflows and potentially reducing background signal . This direct detection approach is particularly advantageous in multi-labeling experiments where antibody cross-reactivity might otherwise be problematic.

How can researchers verify ABHD5 antibody specificity?

Verification of antibody specificity is crucial for experimental validity. For ABHD5 antibodies, several approaches are recommended:

• Affinity probe competition assay: NBD-HE-HP affinity probe has been shown to covalently modify ABHD5 in transfected cells . Researchers can conduct competition assays where the antibody and NBD-HE-HP compete for binding, confirming target specificity .

• Knockout/knockdown controls: Compare antibody signal between wild-type samples and those with ABHD5 genetically depleted to confirm signal specificity.

• Peptide blocking: Pre-incubate the antibody with the immunizing peptide (such as the PrEST antigen sequence for polyclonal antibodies) to block specific binding sites before sample application .

• Cross-species reactivity testing: While many ABHD5 antibodies are verified for human reactivity , testing across species should be empirically validated when extending research to animal models.

• Point mutation analysis: Using known functional mutations (e.g., R116N or E41A) can provide both specificity confirmation and functional insights, as these mutations show altered cellular localization patterns .

How does ABHD5 interact with binding partners and what methods can detect these interactions?

ABHD5 forms important protein-protein interactions that regulate its function in lipid metabolism and other cellular processes. Several methodologies have been developed to study these interactions:

• Luciferase complementation assay: This technique has been successfully employed to study the interaction between ABHD5 and perilipin proteins (PLIN1 and PLIN5) . The assay involves fusing ABHD5 and its binding partners to complementary fragments of luciferase, which generate measurable luminescence when the proteins interact .

• Ligand-induced disruption of protein interactions: Researchers can quantify ABHD5-perilipin interactions through ligand-induced inhibition of luciferase complementation. This approach has been used to calculate IC50 values for various ABHD5 ligands .

• Affinity labeling with competition: The NBD-HE-HP affinity probe covalently modifies ABHD5 at Y330, which can be blocked by co-incubation with specific ligands like TTU and SPZ compounds . This technique provides insights into binding domains and ligand specificity.

• Molecular modeling and mutation analysis: Computational techniques combined with experimental validation through point mutations (like R116N and E41A) have revealed crucial structural determinants of ABHD5 interactions .

These methodologies have revealed that ABHD5 interacts with multiple proteins including PLIN1, PLIN5, BECN1 (in autophagy regulation), and PNPLA2, with each interaction playing distinct roles in cellular physiology .

What are the challenges in studying ABHD5 localization and how can HRP-conjugated antibodies help?

Studying ABHD5 localization presents several challenges due to its dynamic subcellular distribution and association with different cellular compartments. The protein localizes to lipid droplets under basal conditions through interactions with perilipins, but can relocalize upon various stimuli .

HRP-conjugated antibodies offer several advantages for studying ABHD5 localization:

• Enhanced sensitivity: The enzymatic amplification provided by HRP increases detection sensitivity, which is particularly valuable for detecting low-abundance protein pools or studying redistribution dynamics.

• Subcellular resolution: When used in immunoelectron microscopy, HRP-conjugated antibodies enable ultra-structural localization studies with nanometer-scale resolution.

• Quantitative analysis: HRP enzymatic activity can be measured quantitatively, allowing for precise comparisons of ABHD5 abundance across different subcellular compartments.

Research has shown that ABHD5 localization is regulated by specific structural elements, including the interaction between E41 and R116 that facilitates membrane binding . When designing localization studies, researchers should consider these regulatory mechanisms and incorporate appropriate controls for antibody specificity.

How does ABHD5 regulate autophagy and what methodologies are optimal for studying this function?

Recent research has uncovered a PNPLA2-independent function of ABHD5 in regulating autophagy and tumorigenesis . This finding expands our understanding of ABHD5 beyond its canonical role in lipid metabolism. To study ABHD5's function in autophagy, researchers can employ several methodologies:

• Interaction studies with autophagy proteins: Investigating the interaction between ABHD5 and BECN1 (Beclin 1), a key regulator of autophagy initiation, can provide insights into how ABHD5 influences the autophagy machinery .

• Autophagosome formation assays: Monitoring autophagosome formation through fluorescent markers (e.g., LC3-GFP) in the presence and absence of ABHD5 can help quantify its impact on autophagy dynamics.

• Autophagic flux measurements: Combining HRP-conjugated ABHD5 antibodies with autophagy markers in dual immunofluorescence studies can reveal co-localization patterns during different stages of autophagy.

• Tumor suppressor activity evaluation: Since ABHD5 appears to function as a tumor suppressor through its regulation of autophagy, researchers can design experiments to assess tumor growth and development in models with altered ABHD5 expression or activity .

When designing these experiments, it's important to distinguish between ABHD5's direct effects on autophagy and those mediated through its lipid metabolism functions, which may require careful experimental controls and multiple methodological approaches.

What are common issues with ABHD5 antibody experiments and how can they be resolved?

Researchers working with ABHD5 antibodies may encounter several technical challenges. Here are common issues and their solutions:

• Weak or absent signal:

  • Verify protein expression in your sample type

  • Optimize antibody concentration (working range 0.04-0.4 μg/ml for WB, 1:50-1:200 for IHC)

  • Ensure appropriate antigen retrieval (HIER pH6 for IHC)

  • Check HRP activity with appropriate substrate

• High background signal:

  • Increase blocking time/concentration

  • Reduce antibody concentration

  • Include additional washing steps

  • Use more selective detection substrates for HRP

• Unexpected molecular weight bands:

  • Consider post-translational modifications of ABHD5

  • Evaluate potential splice variants

  • Verify specificity using knockout/knockdown controls

  • Test alternative lysis conditions to preserve protein integrity

• Inconsistent results across experiments:

  • Standardize sample preparation protocols

  • Use consistent antibody lots when possible

  • Include positive controls in each experiment

  • Maintain consistent incubation times and temperatures

How can researchers distinguish between ABHD5's multiple cellular functions in experimental data?

ABHD5 participates in multiple cellular processes including lipid metabolism, membrane binding, lipid droplet regulation, and autophagy . Distinguishing between these functions requires careful experimental design:

• Function-specific mutants: Utilize specific mutations that selectively disrupt one function while preserving others. For example, the R116N mutation specifically affects membrane binding without directly impacting catalytic activity .

• Pathway-specific inhibitors: Combine ABHD5 studies with inhibitors targeting specific downstream pathways to isolate function-specific effects.

• Co-localization studies: Employ dual-labeling approaches to simultaneously visualize ABHD5 and markers for different cellular compartments or processes (lipid droplets, autophagosomes, etc.).

• Interaction-specific readouts: Design assays that specifically measure one interaction, such as the luciferase complementation assay for ABHD5-perilipin interactions .

• Temporal analysis: Some ABHD5 functions may operate on different time scales, so temporal analysis of cellular responses can help disentangle overlapping functions.

By carefully designing experiments that can isolate specific aspects of ABHD5 function, researchers can better interpret complex data and avoid attributing observations to the wrong cellular mechanism.

What are emerging areas of ABHD5 research where HRP-conjugated antibodies might be valuable?

Several promising research directions for ABHD5 could benefit from the application of HRP-conjugated antibodies:

• Structural biology integration: Combining antibody-based detection with emerging structural data from computational modeling could validate predicted binding domains and interaction surfaces in cellular contexts.

• Cancer research applications: Given ABHD5's emerging role as a tumor suppressor through regulation of autophagy , HRP-conjugated antibodies could facilitate studies examining altered ABHD5 localization or expression in tumor samples.

• High-resolution imaging: The sensitivity of HRP-conjugated antibodies makes them ideal for super-resolution microscopy techniques to visualize ABHD5's dynamic association with subcellular structures.

• Therapeutic target validation: As synthetic ligands that disrupt ABHD5-perilipin interactions are developed , HRP-conjugated antibodies could help validate target engagement and downstream effects in complex biological systems.

• Metabolic disease models: ABHD5 plays a crucial role in lipid metabolism , making HRP-conjugated antibodies valuable tools for studying altered ABHD5 function in metabolic disorders through sensitive detection in tissue samples.

How can researchers design experiments to investigate the dual role of ABHD5 in lipid metabolism and autophagy?

To investigate ABHD5's dual functionality in lipid metabolism and autophagy, researchers should consider these experimental approaches:

• Domain-specific mutations: Create mutations that selectively disrupt ABHD5's interaction with either lipid metabolism partners (PLIN1, PLIN5, PNPLA2) or autophagy regulators (BECN1) to dissect pathway-specific functions .

• Conditional expression systems: Employ inducible expression systems to control ABHD5 levels with temporal precision, allowing observation of immediate versus delayed effects that may correspond to different cellular functions.

• Metabolic stress conditions: Compare ABHD5 localization and function under different metabolic conditions (starvation, lipid loading) using HRP-conjugated antibodies to visualize potential redistribution between lipid droplets and autophagy-related compartments.

• Dual reporter systems: Develop systems that simultaneously monitor lipid droplet dynamics and autophagy flux to correlate ABHD5 activity with both processes in real-time.

• Proximity labeling techniques: Combine HRP-conjugated ABHD5 antibodies with proximity labeling approaches to identify novel interaction partners that may link lipid metabolism and autophagy regulation.

These experimental strategies will help untangle the complex roles of ABHD5 in cellular homeostasis and potentially identify new therapeutic opportunities targeting this multifunctional protein.