Recombinant Mouse Abhydrolase domain-containing protein 1 (Abhd1)

What is the expression pattern of ABHD1 in mouse tissues?

ABHD1 is ubiquitously expressed in mice, with the highest expression observed in heart and small intestine . Real-time PCR analysis has shown variable expression across tissues, with notable expression in skeletal muscle . Interestingly, in the testis, ABHD1 expression exceeds that of Sec12 (its antisense gene partner), while in most other tissues, ABHD1 expression averages about 7% of Sec12 expression levels .

What is the gene structure of ABHD1 and are there any unique genomic features?

The human and mouse ABHD1 genes have overlapping antisense genes. The mouse ABHD1 gene overlaps with Sec12/PREB in a tail-to-tail manner, sharing part of their 3'UTRs . This overlapping gene structure is significant because it can potentially lead to the formation of double-stranded RNA that might trigger destruction of homologous mRNAs. The gene pair represents an example of overlapping polyadenylation signal sequences, which is a rare genomic feature .

How does ABHD1 contribute to cellular lipid metabolism?

ABHD1 appears to play a role in lipid droplet (LD) formation and biogenesis. In Chlamydomonas, overexpression of ABHD1 induces LD formation and increases triacylglycerol (TAG) content . The protein has a dual function in this process:

• Enzymatic function: Hydrolyzing lyso-DGTS on the LD surface

• Structural function: Promoting LD emergence through distinct biophysical properties that facilitate lipid droplet budding

This suggests ABHD1 may be an important regulator of cellular lipid storage and energy homeostasis, though its specific role in mammalian cells requires further investigation.

What is the relationship between ABHD1 and oxidative stress?

Multiple studies have suggested that ABHD1 may function as a regulator of oxidative stress. Overexpression of ABHD1 in renal cell lines reduces the generation of reactive oxygen species by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase . Additionally, kidney ABHD1 expression is significantly upregulated in mouse models of oxidative stress-induced hypertension, suggesting it may serve as a protective response . ABHD1 is also upregulated in cellular models of Huntington's disease, concurrent with activation of the anti-oxidant Nrf2-ARE pathway, further supporting its potential role in oxidative stress management .

What are the optimal storage and handling conditions for recombinant mouse ABHD1?

Recombinant full-length mouse ABHD1 protein is typically supplied as a lyophilized powder. For optimal stability and activity:

• Store the lyophilized protein at -20°C/-80°C upon receipt

• For reconstitution, centrifuge the vial briefly before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended) for long-term storage

• Aliquot to avoid repeated freeze-thaw cycles, which can compromise protein activity

• Store working aliquots at 4°C for up to one week

The protein is provided in a Tris/PBS-based buffer with 6% Trehalose at pH 8.0, which helps maintain stability during lyophilization and reconstitution .

What assays can be used to measure ABHD1 enzymatic activity?

Based on the confirmed lipase activity of ABHD1 in Chlamydomonas, researchers can develop assays to measure its hydrolytic activity using:

• Lipid hydrolysis assays: Using lyso-DGTS as a substrate and measuring the release of free fatty acids or glyceryltrimethylhomoserine moieties

• Activity-based protein profiling (ABPP): This technique has been successfully used to characterize other ABHD family enzymes and could be adapted for ABHD1

• Fluorogenic substrate assays: Using substrates that release fluorescent products upon hydrolysis to monitor enzymatic activity in real-time

• ELISA-based quantification: Commercial ELISA kits are available for measuring mouse ABHD1 protein levels in tissue homogenates, cell lysates, and other biological fluids, with a detection range of 0.156-10 ng/ml

When designing activity assays, consider that the optimal pH and temperature conditions for mouse ABHD1 may differ from those of other species or family members.

What expression systems are recommended for producing recombinant mouse ABHD1?

Recombinant full-length mouse ABHD1 protein has been successfully expressed in E. coli with an N-terminal His tag . This system allows for efficient purification using affinity chromatography. Key considerations for expression include:

• Protein construct design: The full-length protein (1-412 amino acids) with an N-terminal His tag has proven successful

• Expression conditions: Optimize temperature, IPTG concentration, and induction time to maximize soluble protein expression

• Purification strategy: Affinity chromatography using Ni-NTA or similar matrices, followed by size exclusion chromatography if higher purity is required

• Protein characterization: Verify purity by SDS-PAGE (>90% purity is achievable) and confirm identity by Western blotting or mass spectrometry

Mammalian expression systems might be considered if post-translational modifications are critical for the research application.

What role does ABHD1 play in diabetic retinopathy?

Recent research has identified ABHD1 as a potential regulator in diabetic retinopathy (DR) pathogenesis. Key findings include:

• ABHD1 expression is increased in both retinal tissues of DR patients and in high-glucose-treated human retina endothelial cells

• Inhibition of ABHD1 reduces endothelial cell proliferation and migration, key processes in pathological neovascularization

• Gene set enrichment analysis (GSEA) revealed that ABHD1 knockdown reduces endothelial cell chemotaxis, potentially through regulation of intermediate filament proteins keratin 1 (KRT1) and keratin 2 (KRT2)

These findings suggest ABHD1 may contribute to endothelial dysfunction and pathological neovascularization in DR, positioning it as a potential therapeutic target for DR treatment .

How is ABHD1 expression regulated in response to physiological and pathological stimuli?

ABHD1 expression is dynamically regulated under various physiological and pathological conditions:

• Oxidative stress: Upregulated in mouse models of oxidative stress-induced hypertension

• Neurodegenerative conditions: Upregulated in cellular models of Huntington's disease, concurrent with Nrf2-ARE pathway activation

• Infectious challenges: Liver ABHD1 is upregulated in mice challenged with parasitic infection

• Developmental signaling: Downregulated in mouse liver and small intestine by transgenic activation of Notch signaling

• Aging and exercise: Hippocampal ABHD1 expression is downregulated by age and upregulated by exercise in mice

• Injury response: Downregulated in regenerative neurons following spinal cord injury in rats

• Hyperglycemic conditions: Upregulated in high-glucose-treated human retina endothelial cells

This transcriptional plasticity suggests ABHD1 may serve as an adaptive response element across multiple physiological and stress conditions.

How does ABHD1 compare functionally with other members of the ABHD protein family?

The ABHD protein family consists of 19 members in mammals with diverse functions in lipid metabolism. Key functional comparisons between ABHD1 and other family members include:

Unlike ABHD6 and ABHD12, which hydrolyze 2-arachidonoylglycerol in the endocannabinoid system, ABHD1 appears to have distinct substrate specificity and cellular functions . Also notably, ABHD1 lacks the HX₄D motif found in many ABHD proteins, suggesting it has different catalytic mechanisms .

What knockout or knockdown approaches have been used to study ABHD1 function?

Studies in Chlamydomonas have used knockout mutants to investigate ABHD1 function, revealing that these mutants contained similar amounts of triacylglycerols (TAG) but their lipid droplets showed increased content of lyso-derivatives of DGTS . This approach helped establish ABHD1's role in lipid droplet formation and lipid metabolism.

For mouse ABHD1 studies, researchers can consider:

• CRISPR/Cas9 gene editing: To generate complete knockout cell lines or animal models

• RNAi approaches: Using siRNA or shRNA to achieve transient or stable knockdown

• Antisense oligonucleotides: Particularly useful given ABHD1's overlapping antisense gene structure with Sec12/PREB

When designing knockdown experiments, consider the potential effects on the overlapping antisense gene (Sec12/PREB) to ensure phenotypic changes can be specifically attributed to ABHD1 depletion rather than disruption of the antisense gene.

What are the current challenges in developing selective inhibitors for ABHD1?

Development of selective ABHD1 inhibitors faces several challenges:

• Limited structural information: Unlike some other ABHD family members, detailed structural data for ABHD1 is lacking, hampering structure-based drug design

• Substrate specificity: The natural substrates of mammalian ABHD1 are not fully characterized, making it difficult to design substrate-competitive inhibitors

• Selectivity across ABHD family: The ABHD family shares structural similarities, making selective targeting challenging; for example, many inhibitors targeting ABHD6 also inhibit ABHD12

• Physicochemical properties: ABHD1 is predicted to be a membrane-associated protein, requiring inhibitors with appropriate physicochemical properties to access the active site

• Overlapping antisense gene: The unique genomic arrangement with Sec12/PREB must be considered when developing genetic tools or inhibitors to avoid off-target effects

Current research on ABHD family inhibitors, particularly those targeting ABHD6 and ABHD12, might provide valuable scaffolds and strategies for developing selective ABHD1 inhibitors .

How might ABHD1's role in lipid droplet formation be exploited for metabolic disorder treatments?

Given ABHD1's role in lipid droplet formation, as demonstrated in Chlamydomonas , it presents several potential therapeutic strategies for metabolic disorders:

• Obesity management: Targeting ABHD1 might modulate lipid storage capacity, potentially reducing excessive lipid accumulation in adipose and non-adipose tissues

• Non-alcoholic fatty liver disease (NAFLD): Modulating hepatic ABHD1 activity could potentially reduce lipid accumulation in the liver

• Lipid metabolism disorders: ABHD1 modulation might help correct dysregulated lipid storage and utilization in various metabolic conditions

• Drug delivery systems: Understanding ABHD1's role in lipid droplet formation could inform the development of lipid-based drug delivery systems targeting metabolic tissues

Research should focus on validating these potential applications in mammalian models, as current evidence for ABHD1's role in lipid droplet formation comes primarily from Chlamydomonas studies .

What is the significance of ABHD1's potential dual function as both an enzyme and a structural protein?

The discovery that ABHD1 possesses both enzymatic activity (hydrolyzing lyso-DGTS) and structural properties that promote lipid droplet budding represents an intriguing dual functionality that could have several implications:

• Therapeutic targeting: This dual functionality suggests multiple potential avenues for therapeutic intervention—targeting either the enzymatic activity or the structural properties

• Evolutionary significance: This dual role might represent an efficient evolutionary solution for coordinating lipid metabolism and storage

• Regulatory mechanisms: The balance between these two functions might be regulated differently under various physiological conditions

• Protein-protein interactions: ABHD1 might interact with different protein partners depending on whether it's functioning enzymatically or structurally

Understanding how these two functions are coordinated and regulated could provide fundamental insights into lipid droplet biogenesis and cellular lipid homeostasis mechanisms.

How do the findings in Chlamydomonas models of ABHD1 function translate to mammalian systems?

While recent research has provided valuable insights into ABHD1 function in Chlamydomonas , several key questions remain regarding translation to mammalian systems:

• Substrate specificity: Chlamydomonas ABHD1 hydrolyzes lyso-DGTS, but DGTS is not a major lipid in mammals. The mammalian substrates of ABHD1 need to be identified, potentially including phosphatidylcholine or other phospholipids

• Lipid droplet association: Confirmation of ABHD1's localization to lipid droplet surfaces in mammalian cells is needed to validate its proposed role in lipid droplet biogenesis

• Interaction with mammalian lipid metabolism pathways: How ABHD1 interacts with established mammalian lipid metabolism regulators requires investigation

• Role in oxidative stress: The connection between ABHD1's lipid metabolism functions and its apparent role in oxidative stress response needs clarification in mammalian systems

• Tissue-specific functions: Given its differential expression across tissues , ABHD1 may have tissue-specific functions in mammals that differ from its role in Chlamydomonas

Comparative studies between algal and mammalian ABHD1 would help elucidate conserved and divergent aspects of its function across evolution.

What are the recommended controls when performing ABHD1 enzymatic assays?

When designing enzymatic assays for ABHD1, incorporate these essential controls:

• Negative controls:

  • Heat-inactivated ABHD1 (95°C for 10 minutes)

  • Reaction buffer without enzyme

  • Samples treated with serine hydrolase inhibitors like PMSF

• Positive controls:

  • If available, a known functioning batch of recombinant ABHD1

  • If studying lipase activity, include a well-characterized lipase with similar substrate preferences

• Specificity controls:

  • Other ABHD family members to assess substrate specificity

  • Site-directed mutants of the putative catalytic residues to confirm enzymatic mechanism

• Assay validation:

  • Linearity assessment over time and enzyme concentration ranges

  • Substrate concentration curve to determine Km and Vmax

  • pH and temperature optimization curves

These controls will help ensure results are specific to ABHD1 activity rather than contaminating enzymes or non-enzymatic effects.

What considerations should be made when designing antibodies against mouse ABHD1?

When designing or selecting antibodies against mouse ABHD1, consider:

• Epitope selection:

  • Avoid the predicted transmembrane region (amino acids involved in membrane association)

  • Select regions with low sequence homology to other ABHD family members

  • Consider accessibility of epitopes in the native protein conformation

• Antibody format:

  • Monoclonal antibodies for consistent results and specificity

  • Polyclonal antibodies for higher sensitivity but potential lower specificity

  • Consider application needs (Western blot, immunoprecipitation, immunohistochemistry)

• Validation requirements:

  • Test specificity against recombinant ABHD1

  • Validate in ABHD1-knockout or knockdown samples

  • Check for cross-reactivity with other ABHD family members

• Species cross-reactivity:

  • If cross-species detection is desired, select epitopes conserved across species

  • Check for potential cross-reactivity with human ABHD1 if translational research is planned

The mouse ABHD1 protein sequence provided in the search results can guide epitope selection for optimal antibody design.

What are the key considerations when interpreting ABHD1 expression data across different tissue types?

When analyzing ABHD1 expression across tissues, consider these important factors:

• Reference gene selection:

  • β-actin has been used as a reference gene , but expression levels vary across tissues

  • Consider using multiple reference genes for more accurate normalization

  • Skeletal muscle typically shows lower reference gene expression per μg cDNA

• Antisense gene interaction:

  • ABHD1 expression correlates positively with its antisense gene partner Sec12/PREB (r=0.73)

  • In most tissues, ABHD1 expression is approximately 7% of Sec12 expression

  • Testis is an exception where ABHD1 expression exceeds Sec12

• Detection sensitivity:

  • Some tissues (e.g., spleen) may show ABHD1 expression detectable by SYBR green fluorescence but below the level of detection by ethidium bromide agarose gel electrophoresis

• Protein vs. mRNA correlation:

  • Verify if protein levels correlate with mRNA expression

  • Consider post-transcriptional regulation mechanisms

• Cellular heterogeneity:

  • Whole tissue expression may mask cell-type specific expression patterns

  • Consider single-cell approaches for tissues with complex cellular composition

These considerations will help ensure accurate interpretation of ABHD1 expression patterns and their biological significance.