Recombinant Mouse Abhydrolase domain-containing protein 3 (Abhd3)

What is the basic structure and function of mouse Abhd3?

Mouse Abhd3 is a 46.7 kDa protein (full length 411 amino acids) that functions as a phospholipase with predominant phospholipase 1 activity, cleaving preferentially acyl groups in the sn1 position. It may also possess minor phospholipase 2 activity acting on acyl groups in the sn2 position. Structurally, it's predicted to be a single-pass type II membrane protein, although this has not been experimentally confirmed . Unlike some other ABHD family members, Abhd3 does not possess an HX4D acyltransferase motif, which impacts its functional properties .

What are the primary substrates for mouse Abhd3?

Mouse Abhd3 selectively cleaves medium-chain phosphatidylcholines, particularly those containing myristate (C14). Research has demonstrated that Abhd3 exhibits high specificity for C14-containing phospholipids as well as oxidatively-truncated phospholipids . This substrate specificity distinguishes Abhd3 from other phospholipases and suggests a specialized role in phospholipid remodeling.

What is the tissue distribution pattern of Abhd3 in mice?

Abhd3 is ubiquitously expressed in mice, with highest expression observed in brain and small intestine . Additional studies using activity-based protein profiling (ABPP) and gene expression profiling have also established high expression in liver and kidney tissues . This distribution pattern suggests potential physiological roles across multiple organ systems.

How can researchers assess Abhd3 enzymatic activity in experimental systems?

Several methodological approaches can be employed to assess Abhd3 activity:

• Overexpression systems: Comparing wild-type Abhd3 with catalytically dead mutants (ABHD3-S220A) in cell lines allows assessment of enzymatic activity through metabolite profiling .

• Activity-based protein profiling (ABPP): Using probes like fluorophosphonate (FP)-rhodamine that covalently label the conserved serine nucleophile in the active sites of serine hydrolases .

• Metabolite analysis by LC-MS: Untargeted liquid chromatography-mass spectrometry can identify substrate and product metabolites that change in response to Abhd3 activity or inhibition .

• Targeted multiple reaction monitoring (MRM): Allows precise quantification of specific lipid species affected by Abhd3 activity or inhibition .

What expression systems are most effective for producing recombinant mouse Abhd3?

Based on the available literature, HEK293T cells have been successfully used for expressing recombinant mouse Abhd3 with various tags . When expressing recombinant mouse Abhd3:

• C-terminal tags like MYC/DDK can be employed without compromising protein function

• The expressed protein typically yields >80% purity as determined by SDS-PAGE and Coomassie blue staining

• For optimal storage, a buffer containing 25 mM Tris.HCl (pH 7.3), 100 mM glycine, and 10% glycerol is recommended

• The recombinant protein should be stored at -80°C to maintain stability, with repeated freeze-thaw cycles avoided

How can researchers effectively detect and quantify Abhd3 in tissue samples?

Multiple complementary approaches can be used for detection and quantification:

• Untargeted MS-based proteomics: Allows identification of Abhd3 protein in complex tissue samples.

• Activity-based protein profiling (ABPP): Using the probe fluorophosphonate (FP)-rhodamine enables detection of active Abhd3 enzyme.

• Western blotting: Using antibodies against the native protein or epitope tags if using recombinant versions.

• Immunohistochemistry: For localization studies within tissues.

For quantitative assessment, MS-based approaches with labeled internal standards provide the most accurate quantification .

What are the key phenotypic characteristics of Abhd3 knockout mice?

Abhd3−/− mice have been characterized as viable, fertile, and normal in their general cage behavior. The most notable biochemical phenotype is the elevation of myristoyl (C14)-phospholipids in multiple tissues. Specifically:

• Elevated C14-lysophosphatidylcholine (C14-LPC) across brain, liver, and kidney tissues

• Increased levels of phosphatidylcholines with C14/18:2, C14/20:4, and C14/22:6 acyl chains, particularly in kidney

• These lipid changes are not accompanied by alterations in other LPC species, myristic acid, or C14-CoA levels

These findings confirm the physiological role of Abhd3 in regulating medium-chain phospholipid metabolism in vivo.

What metabolomic approaches are most effective for analyzing changes in phospholipid profiles in Abhd3 studies?

Based on the research literature, several complementary metabolomic approaches have proven effective:

• Untargeted LC-MS: Provides broad coverage of the lipidome to identify novel metabolites affected by Abhd3 manipulation.

• Tandem MS experiments: Essential for structural characterization of lipids, particularly through identification of characteristic fragments (e.g., phosphocholine with m/z of 184).

• Multiple Reaction Monitoring (MRM): Enables precise quantification of specific lipid species across different tissues and experimental conditions.

• Comparative analysis across tissues: Analysis of brain, liver, and kidney reveals tissue-specific effects of Abhd3 deficiency, with the greatest fold-increases typically observed in kidney .

A comprehensive table showing metabolite changes in Abhd3−/− mice tissues would include:

What are the most effective known inhibitors of Abhd3 and their mechanisms of action?

Several inhibitors with varying selectivity for Abhd3 have been identified:

• β-aminocyano(MIDA)boronate compound 2: The most selective Abhd3 inhibitor with an IC50 value of 0.14 μM in vitro. Structure-activity relationship analysis revealed that the phenylamide portion, the cyano group, and the fluorine atom are crucial for inhibition potency. The boron atom is essential for covalent inhibition, while the MIDA boronate portion increases cell permeability or stability .

• N-hydroxyhydantoin carbamates (compounds 5/ABC47 and 6/ABC34): These demonstrate good activity against Abhd3 (IC50 = 0.1 and 7.6 μM, respectively) but also inhibit ABHD4 more potently (IC50 = 0.03 and 0.1 μM, respectively). Additional off-targets include ABHD6, hormone-sensitive lipase (HSL), phospholipase A2 Group VII (PLA2G7), and carboxylesterase 2 (CES2) .

• Organophosphorus agents: These have been identified as useful tools to inhibit Abhd3 serine hydrolase activity through activity-based protein profiling (ABPP) .

What experimental approaches should be used to assess inhibitor selectivity against Abhd3?

Multiple complementary approaches should be employed to thoroughly assess inhibitor selectivity:

• In vitro enzyme assays: Using purified recombinant Abhd3 to determine direct inhibition potency.

• SDS-PAGE analysis of tissue proteomes: Provides initial assessment of inhibitor activity against multiple serine hydrolases.

• MS-based ABPP using SILAC: Stable isotope labeling with amino acids in cell culture combined with activity-based protein profiling provides comprehensive assessment of inhibitor selectivity across the serine hydrolase family. This approach confirmed β-aminocyano(MIDA)boronate compound 2 selectivity with >95% blockade of Abhd3 at 0.5 μM without affecting 60 additional serine hydrolases in human colon cancer cell line SW620 .

• Metabolomic profiling: Confirms functional inhibition by demonstrating increased levels of medium-chain phosphatidylcholines similar to the phenotype observed in Abhd3−/− mice .

• ABPP-SILAC experiments in relevant cell lines: Identifies potential off-target effects against other serine hydrolases in a cellular context .

How can researchers effectively use cell-based screens to identify novel Abhd3 substrates?

A systematic approach to identify novel Abhd3 substrates through cell-based screening involves:

• Untargeted metabolomics: Transfect cells with Abhd3 expression constructs alongside control vectors (including catalytically inactive Abhd3-S220A mutant).

• Broad metabolite profiling: Analyze cellular lipid extracts using positive and negative polarity modes across a wide m/z range (e.g., 200-1200 Da).

• Comparative analysis: Identify metabolite peaks selectively altered in Abhd3-expressing cells compared to controls.

• Validation across cell lines: Reproduce findings in multiple cell lines to confirm consistency.

• Structural characterization: Perform tandem MS experiments to identify the chemical structures of altered metabolites.

• In vitro validation: Test identified candidate substrates using purified recombinant Abhd3 protein .

This approach successfully identified Abhd3's activity toward medium-chain and oxidatively truncated phospholipids in previous studies .

What is the relationship between Abhd3 function and potential roles in disease pathology?

While the precise role of Abhd3 in disease pathology remains under investigation, several associations have been identified:

• Cancer biology: Abhd3 is upregulated in the early response to chemotherapy treatment in human ovarian cancer cell lines and has been identified in screens for pro-apoptotic genes in breast cancer tumors .

• Tumor suppression: Abhd3 is upregulated in osteosarcoma cell lines overexpressing HIC1 (Hypermethylated in Cancer 1), a tumor suppressor that is silenced in many human tumors .

• Inflammatory conditions: Abhd3 is downregulated in peripheral blood mononuclear cells from patients with Crohn's disease. The promoter contains binding sites for transcription factors T-bet and Early Growth Response (EGRF) .

• Neurological conditions: Abhd3 expression in the optic nerve is downregulated by early optic nerve injury in a rat model of glaucoma .

These associations suggest potential roles in disease processes, though mechanistic studies are needed to establish causal relationships.

How does the experimental design for studying Abhd3 differ between in vitro and in vivo systems?

Effective experimental design varies significantly between in vitro and in vivo Abhd3 research:

In vitro systems:

• Overexpression systems: Use of recombinant Abhd3 in cell lines allows controlled manipulation of enzyme levels and activity.

• Catalytic comparisons: Side-by-side comparison with catalytically inactive mutants (Abhd3-S220A) enables clear attribution of effects to enzymatic activity.

• Inhibitor screening: Direct assessment of inhibitor potency and specificity through biochemical assays.

• Substrate profiling: Identification of candidate substrates through metabolite profiling of cellular extracts.

• Control considerations: Must include proper controls for transfection efficiency, protein expression levels, and potential non-specific effects .

In vivo systems:

• Genetic models: Abhd3−/− mice provide the gold standard for studying physiological roles.

• Tissue-specific analysis: Examination of multiple tissues reveals differential effects across organ systems.

• Metabolic characterization: Comprehensive metabolomic profiling to identify changes in phospholipid species.

• Phenotypic assessment: Evaluation of physiological parameters and behavioral phenotypes to identify systemic effects.

• Control considerations: Must include appropriate littermate controls and account for potential compensatory mechanisms in knockout models .

The integration of findings from both approaches provides the most comprehensive understanding of Abhd3 biology.

What are the optimal conditions for storage and handling of recombinant mouse Abhd3?

Based on the literature, the following conditions are recommended:

• Storage buffer: 25 mM Tris.HCl (pH 7.3), 100 mM glycine, 10% glycerol provides optimal stability.

• Storage temperature: Store at -80°C for long-term storage.

• Aliquoting: To avoid repeated freeze-thaw cycles, prepare working aliquots and store at 4°C for up to one week.

• Stability: Under proper storage conditions, the protein remains stable for approximately 12 months .

What quality control measures should be implemented when working with recombinant mouse Abhd3?

Several quality control measures should be routinely implemented:

• Purity assessment: SDS-PAGE with Coomassie blue staining should confirm >80% purity.

• Concentration determination: Protein concentration should be >50 μg/mL as determined by microplate BCA method.

• Activity verification: Functional assays comparing wild-type Abhd3 with catalytically inactive mutants (Abhd3-S220A) should confirm enzymatic activity.

• Mass spectrometry validation: Confirm protein identity and integrity through MS-based proteomics.

• Stability monitoring: Regular testing of protein activity after various storage durations to ensure preservation of function .

What are the key considerations for designing experiments using recombinant mouse Abhd3 in phospholipid metabolism studies?

When designing experiments with recombinant mouse Abhd3 for phospholipid metabolism studies, researchers should consider:

• Substrate selection: Focus on medium-chain phosphatidylcholines (particularly C14-containing) and oxidatively truncated phospholipids as primary substrates based on established specificity.

• Activity controls: Include catalytically inactive Abhd3-S220A mutant as a negative control to distinguish enzymatic from non-enzymatic effects.

• Detection methods: Employ mass spectrometry-based approaches for comprehensive detection of substrate conversion and product formation.

• Physiological relevance: Compare results with observations from Abhd3−/− mouse tissues to establish relevance to in vivo conditions.

• Reaction conditions: Optimize reaction conditions including pH, temperature, and cofactor requirements for maximal activity.

• Experimental design considerations: Follow structured experimental design principles with appropriate controls, replication, and statistical analysis to ensure reliable and reproducible results .