Recombinant Mouse Abhydrolase domain-containing protein 13 (Abhd13)

What is the molecular function of mouse ABHD13 in relation to other ABHD family proteins?

Mouse ABHD13 is a member of the α/β-hydrolase domain (ABHD) family of serine hydrolases that shares structural similarities with other ABHD proteins. While specific ABHD13 functions are still being elucidated, other ABHD family members (such as ABHD3, ABHD6, and ABHD12) serve as lipid hydrolases with distinct substrate specificities . Based on structural homology, ABHD13 likely possesses hydrolase activity toward specific lipid substrates, potentially including phospholipids or endocannabinoids. Unlike the better-characterized ABHD12, which acts as a major lyso-PS lipase in mouse brain, or ABHD16A with prominent PS lipase activity, the precise lipid substrates for ABHD13 require further investigation using activity-based protein profiling (ABPP) methodologies similar to those used for other ABHD proteins .

What expression systems are most effective for producing recombinant mouse ABHD13?

For recombinant mouse ABHD13 expression, prokaryotic systems using E. coli BL21(DE3) strain with TB (Terrific Broth) culture medium have shown optimal results for similar hydrolase proteins . Based on optimization studies with other mouse proteins, induction with 0.25 mM IPTG at lower temperatures (15°C) for extended periods (24 hours) typically yields higher amounts of properly folded recombinant protein . For ABHD proteins specifically, mammalian expression systems using HEK293T cells have proven effective for maintaining native catalytic activity, as demonstrated with ABHD16A expression . The choice between prokaryotic and mammalian expression systems should be guided by downstream applications, with bacterial systems providing higher yields but mammalian systems potentially preserving native folding and post-translational modifications critical for enzymatic activity.

What purification strategy produces the highest yield and purity for recombinant mouse ABHD13?

An optimized purification protocol for mouse ABHD13 would involve:

• Cell lysis using buffers containing 2% sarkosyl, which has been shown to significantly improve both yield and purity of similar recombinant proteins

• Initial purification using immobilized metal affinity chromatography (IMAC) with His-tagged constructs

• Secondary purification via size exclusion chromatography to remove aggregates and impurities

For ABHD13, buffer optimization is critical, as demonstrated in similar hydrolase purification protocols. The following buffer composition table provides recommended starting conditions:

What analytical methods are most effective for confirming the identity and activity of purified recombinant mouse ABHD13?

For comprehensive characterization of recombinant mouse ABHD13:

• Identity confirmation: Western blotting using specific anti-ABHD13 antibodies or anti-tag antibodies when working with tagged recombinant proteins

• Purity assessment: SDS-PAGE analysis with Coomassie staining and ELISA-based methods

• Enzymatic activity: Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes that react with the active-site serine residue common to hydrolases . This approach has been successfully employed for other ABHD family members

• Mass spectrometry validation: LC-MS/MS analysis to confirm protein identity and assess post-translational modifications, following protocols similar to ABPP-MudPIT (Multidimensional Protein Identification Technology) used for analyzing ABHD16A

Integrating these complementary approaches provides comprehensive validation of recombinant ABHD13 identity, purity, and functional activity.

How should researchers design specificity studies to distinguish ABHD13 activity from other ABHD family members?

When designing specificity studies for ABHD13:

• Competitive ABPP screening: Employ competitive activity-based protein profiling similar to methods used for ABHD3 inhibitor characterization . This approach allows evaluation of enzyme activity in complex proteomes.

• Substrate panel testing: Develop a comprehensive substrate panel including various mono- and diacyl lipid substrates to determine ABHD13's substrate preference profile. This methodology has successfully differentiated ABHD16A's preferential activity toward PS substrates .

• Selective inhibition validation: Use the following experimental design template to establish ABHD13 specificity:

• Cross-reactivity assessment: Evaluate potential inhibitors against at least 60 additional serine hydrolases to confirm selectivity, following the approach used for ABHD3 inhibitor characterization with boronate compound 2 .

What are the most effective methods for determining the physiological substrates of mouse ABHD13?

To identify physiological substrates of mouse ABHD13:

• Metabolomic profiling: Compare lipid profiles between wild-type and ABHD13-knockout or ABHD13-inhibited mouse tissues using liquid chromatography-mass spectrometry (LC-MS). Substrates will accumulate in tissues lacking ABHD13 activity, as demonstrated for ABHD3 inhibition leading to increased medium-chain phosphatidylcholines (PCs) .

• In vitro substrate screening: Test purified recombinant ABHD13 against a library of potential lipid substrates, measuring hydrolysis products via LC-MS/MS. Begin with phospholipids, lysophospholipids, and neutral lipids based on known substrates of other ABHD enzymes.

• Cell-based validation: Overexpress or knock down ABHD13 in relevant cell lines and measure changes in candidate substrate levels. This approach can validate findings from metabolomic profiling in a controlled cellular environment.

• Structure-based predictions: Use computational docking and molecular dynamics simulations with homology models based on crystal structures of related ABHD proteins to predict likely substrate binding modes and preferences.

What approaches should be used to develop selective inhibitors for mouse ABHD13?

Development of selective ABHD13 inhibitors should follow these strategies:

• Chemical scaffold selection: Based on successful ABHD inhibitor development, consider these chemical classes:

  • β-aminocyano(MIDA)boronates, which have shown high selectivity for ABHD3

  • 1,3-dicarbonyl derivatives, identified in screening efforts for ABHD inhibitors

  • Piperidyl-1,2,3-triazole urea compounds, which have demonstrated efficacy against other ABHD proteins

• Structure-activity relationship studies: Systematic modification of chemical scaffolds to optimize potency and selectivity, focusing on key structural elements such as:

  • The importance of phenylamide portions for binding

  • The role of cyano groups and halogen atoms in enhancing activity

  • The potential benefit of MIDA boronate portions for improving cell permeability or stability

• Inhibitor validation pipeline:

  • Initial screening using competitive ABPP in mouse brain proteome

  • Secondary validation in cell lysates overexpressing ABHD13

  • Selectivity profiling using SDS-PAGE analysis and MS-based ABPP with SILAC

  • In-cell activity confirmation through metabolomic studies to confirm target engagement

• Covalent versus reversible inhibitor design: Consider both covalent inhibitors targeting the active site serine and reversible inhibitors for varied research applications. The boron atom in certain ABHD inhibitors has proven fundamental for covalent inhibition .

How can researchers investigate the potential role of mouse ABHD13 in neurological disorders based on findings from other ABHD proteins?

Investigating ABHD13's potential role in neurological disorders should include:

• Comparative genetic studies: Analysis of ABHD13 mutations/polymorphisms in neurological disease cohorts, informed by the connection between ABHD12 mutations and PHARC (Polyneuropathy, Hearing loss, Ataxia, Retinitis pigmentosa, and Cataract) . Focus on disorders with similar symptomatology but unknown genetic basis.

• Mouse model development and characterization: Generate ABHD13 knockout or conditional knockout mice to investigate:

  • Behavioral phenotypes (motor, sensory, cognitive)

  • Histopathological changes, particularly in the nervous system

  • Alterations in lipid profiles in brain and peripheral tissues

  • Inflammatory markers, given the role of other ABHD enzymes in immunomodulatory lipid regulation

• Tissue-specific expression analysis: Quantify ABHD13 expression in different brain regions and at different developmental stages using:

  • Quantitative PCR for mRNA expression

  • Western blotting and immunohistochemistry for protein localization

  • Single-cell RNA sequencing to identify cell type-specific expression patterns

• Functional studies in neural cell models: Investigate ABHD13's role in:

  • Myelination processes, based on the demyelination observed in PHARC patients

  • Neuroinflammatory responses

  • Neuronal differentiation pathways, which have been linked to other ABHD enzyme activities

What are the most promising approaches for studying ABHD13 protein-protein interactions in mouse tissues?

To investigate ABHD13 protein-protein interactions:

• Affinity purification-mass spectrometry (AP-MS): Express tagged ABHD13 in mouse cell lines or tissues and identify interacting proteins through pull-down experiments followed by mass spectrometry analysis.

• Proximity labeling approaches: Use BioID or APEX2 fusion proteins to identify proximal proteins in living cells, providing insight into the spatial organization of ABHD13 in cellular compartments.

• Chemical crosslinking: Employ chemical crosslinkers to stabilize transient interactions before immunoprecipitation and mass spectrometry analysis.

• Co-immunoprecipitation validation: Confirm key interactions identified through high-throughput methods with targeted co-immunoprecipitation experiments using specific antibodies.

• Functional validation: Assess the functional significance of identified interactions through mutagenesis of interaction interfaces or pharmacological disruption of protein complexes.

How can researchers effectively employ activity-based protein profiling (ABPP) to characterize mouse ABHD13 in complex proteomes?

For effective ABPP characterization of ABHD13:

• Probe selection and optimization: Utilize fluorophosphonate (FP) probes that react with the conserved active-site serine nucleophile in ABHD proteins . Consider both fluorescent and biotin-tagged probes for different analytical readouts.

• Competitive ABPP: Pre-treat samples with candidate inhibitors or substrates before adding activity-based probes to identify compounds that compete for the active site.

• MS-based ABPP with SILAC: Implement stable isotope labeling with amino acids in cell culture combined with mass spectrometry to quantitatively measure ABHD13 activity and inhibition across diverse experimental conditions .

• Click chemistry applications: Design probes with terminal alkyne or azide groups for subsequent click chemistry reactions to introduce reporter tags, facilitating visualization of targets as demonstrated for other ABHD inhibitors .

• In situ versus in vitro profiling: Compare ABHD13 activity profiles in living cells versus cell lysates to understand the influence of cellular context on enzyme activity.