Recombinant Putative 1-acyl-sn-glycerol-3-phosphate acyltransferase acl-1 (acl-1)

What is 1-acyl-sn-glycerol-3-phosphate acyltransferase (ACL-1) and what is its primary function?

1-acyl-sn-glycerol-3-phosphate acyltransferase (ACL-1) is an enzyme that catalyzes the acylation of lysophosphatidic acid, responsible for the de novo production of phosphatidic acid, which serves as a precursor for the synthesis of various membrane glycerophospholipids . The enzyme plays a critical role in lipid metabolism, particularly in glycerolipid synthesis and regulation . In different organisms, this enzyme family exists in multiple isoforms that catalyze the same biochemical reaction but display distinct tissue expression patterns and regulatory mechanisms .

How does ACL-1 differ from other members of the AGPAT family?

The AGPAT family exists in at least five isoforms in humans (AGPAT1, AGPAT2, AGPAT3, AGPAT4, and AGPAT5), all catalyzing the same biochemical reaction but with different tissue expression profiles and regulations . Comparative studies in murine models show that AGPAT1 and AGPAT3 are ubiquitously expressed in most tissues, whereas AGPAT2, AGPAT4, and AGPAT5 demonstrate tissue-specific expression patterns . When comparing amino acid sequences of the five mAGPATs, researchers have identified three highly conserved motifs, including a novel motif/pattern KX₂LX₆GX₁₂R . The differences in expression patterns suggest specialized physiological roles for each isoform despite their similar catalytic functions.

What structural motifs are conserved in ACL-1 across species?

Amino acid sequence comparisons of ACL-1 and related enzymes across species have revealed three highly conserved motifs, with particular significance for the novel motif/pattern KX₂LX₆GX₁₂R identified in murine AGPATs . These conserved regions likely play crucial roles in substrate binding or catalytic activity. For instance, in Caenorhabditis elegans, the full-length ACL-1 protein (262 amino acids) contains these conserved regions within its sequence . These motifs are essential for function and represent potential targets for structure-function studies or drug development.

What are the most effective methods for expressing and purifying active recombinant ACL-1?

Purification of active recombinant ACL-1 remains challenging because it is an integral membrane protein that is difficult to solubilize without causing inactivation . Recent advances have demonstrated success using 6-cyclohexyl-1-hexyl-β-d-maltoside as a detergent for solubilization and purification of active recombinant enzyme . The methodological approach includes:

• Recombinant expression in an appropriate host system (E. coli is commonly used)

• Careful solubilization with specialized detergents that maintain enzyme activity

• Affinity purification using tags such as His-tag for ease of isolation

• Buffer optimization to maintain stability during storage (e.g., Tris/PBS-based buffer with 6% Trehalose, pH 8.0)

• Addition of glycerol (recommended 5-50% final concentration) for long-term storage at -20°C/-80°C

This approach has been successfully applied to purify active ACL-1 from various organisms, including bacterial homologs like PlsC from Shewanella livingstonensis Ac10 .

How can researchers effectively measure ACL-1 enzyme activity in different experimental systems?

Measuring ACL-1 activity requires specialized assays that monitor the acylation of lysophosphatidic acid. The following methodological approach has proven effective:

• In vitro translated enzyme activity assay: Using a coupled transcription and translation system (e.g., TnT coupled Reticulocyte Lysate System) to generate enzyme protein for activity measurements .

• Assay components for acyltransferase activities:

  • 100 mM Tris/HCl (pH 7.4)

  • 3 mM MgCl₂

  • Radiolabeled substrate: [¹⁴C]oleoyl-CoA

  • Appropriate substrate: 1-oleoyl-sn-glycerol 3-phosphate for AGPAT activity

  • Enzyme preparation (10 μl of reticulocyte lysate containing translated enzyme)

• Techniques for cell-based assays: When evaluating ACL-1 activity in cells, transfection of expression constructs into suitable cell lines (e.g., COS-1 cells) followed by extraction and enzyme assays has proven effective .

What approaches can be used to investigate substrate specificity of ACL-1?

Investigating ACL-1 substrate specificity requires systematic testing with different acyl donors and acceptors. Research has shown that some ACL-1 homologs, like the one from Shewanella livingstonensis Ac10, have a substrate preference for acyl donors with polyunsaturated fatty acyl groups, such as eicosapentaenoyl group . Methodological approaches include:

• Comparative substrate utilization assays:

  • Testing various lysophospholipid acceptors

  • Evaluating different acyl-CoA donors with varying chain lengths and saturation levels

  • Measuring reaction rates under standardized conditions

• Structural analysis and modeling:

  • Using purified enzyme for crystallography or cryo-EM studies

  • Computational modeling of substrate binding sites

  • Directed mutagenesis of putative substrate-binding residues to confirm their roles

• In vivo labeling studies:

  • Incorporating labeled fatty acids in cell culture

  • Analyzing resulting phospholipid profiles to determine preferred substrates in cellular context

How is ACL-1 expression regulated in different tissues and under various physiological conditions?

ACL-1 expression shows tissue-specific patterns and responds to metabolic regulators. Studies in murine models demonstrate that mAGPAT1 and mAGPAT3 are ubiquitously expressed in most tissues, whereas mAGPAT2, mAGPAT4, and mAGPAT5 are expressed in tissue-specific patterns . Regulatory mechanisms include:

• Transcriptional regulation by PPARα: Cardiac mAGPAT activities were 25% lower in PPARα null mice compared with wild-type, and 50% lower in PPARα null mice fed clofibrate compared with clofibrate-fed wild-type animals . This modulation of AGPAT activity was accompanied by significant enhancement/reduction of the mRNA levels of mAGPAT3/mAGPAT2 respectively .

• Tissue-specific expression profiles: The differential expression across tissues suggests specialized roles in lipid metabolism depending on tissue requirements .

• Response to metabolic state: Expression levels may change in response to nutritional status, metabolic disease states, or pharmacological interventions that alter lipid metabolism.

What experimental approaches can be used to study the interaction of ACL-1 with other proteins in lipid metabolism pathways?

Studying protein-protein interactions involving ACL-1 requires multiple complementary approaches:

• Proximity labeling techniques:

  • TurboID system has been effectively used to identify protein interactions in related systems

  • BioID or APEX2 proximity labeling to identify proteins in close proximity to ACL-1 in living cells

• Biochemical assays for interaction confirmation:

  • Co-immunoprecipitation with tagged ACL-1

  • Pull-down assays with recombinant proteins

  • Surface plasmon resonance for quantitative binding measurements

• Structural studies of protein complexes:

  • Cross-linking mass spectrometry to identify interaction interfaces

  • Cryo-EM of protein complexes to visualize interaction architecture

• Functional assays:

  • Competition assays to study homo-dimer and hetero-dimer formation

  • Enzyme activity measurements in the presence of potential interacting partners

How does ACL-1 function differ between species, and what can comparative studies tell us about its evolutionary conservation?

Comparative studies of ACL-1 across species reveal both conserved functions and species-specific adaptations:

• Conserved catalytic function: The basic enzymatic activity of ACL-1 in phospholipid synthesis is conserved across species from bacteria to mammals, highlighting its fundamental role in lipid metabolism .

• Specialized adaptations: Some organisms have evolved specialized versions of the enzyme with unique substrate preferences. For example, the PlsC from Shewanella livingstonensis Ac10 shows preference for polyunsaturated fatty acyl donors, which may be related to the organism's ability to produce eicosapentaenoic acid .

• Evolutionary conserved motifs: Three highly conserved motifs have been identified across species, including the novel motif KX₂LX₆GX₁₂R, suggesting these regions are critical for function .

• Isoform diversification: Higher organisms typically have multiple isoforms with specialized functions and expression patterns, indicating evolutionary diversification to meet complex metabolic needs .

What is the role of ACL-1 in disease models, and how can this inform therapeutic approaches?

Research on the role of ACL-1 in disease models is emerging, with several implications for therapeutic development:

• Lipid metabolism disorders: Linkage studies in humans have revealed that AGPAT2 contributes to glycerolipid synthesis and plays an important role in regulating lipid metabolism . Mutations in AGPAT2 cause congenital generalized lipodystrophy, suggesting critical roles in adipose tissue development.

• Metabolic regulation: Cardiac mAGPAT activities were significantly modulated by PPARα status and clofibrate feeding , indicating a potential regulatory role in cardiac metabolism that could be relevant to cardiac disease.

• Therapeutic considerations:

  • Targeting specific AGPAT isoforms might allow for tissue-specific interventions

  • The conserved motifs identified in AGPATs could serve as specific targets for drug development

  • Understanding substrate specificity could inform the design of inhibitors or activators with therapeutic potential

How does the confusion between different proteins abbreviated as "ACL-1" impact research, and how can researchers address this?

The research literature contains multiple proteins abbreviated as "ACL-1," which can lead to confusion. Researchers should be aware of these distinctions:

• 1-acyl-sn-glycerol-3-phosphate acyltransferase (ACL-1): The enzyme involved in lipid metabolism and phospholipid synthesis discussed in this FAQ .

• Abaxially curled leaf1 (ACL1): A gene in rice that negatively regulates brown planthopper resistance and drought tolerance by modulating cuticular wax content and bulliform cell development . This ACL1 interacts with rice outermost cell-specific (ROC) proteins and forms ACL1-ROC4/5 complexes.

• ATP-citrate lyase (ACL): An enzyme that generates acetyl-CoA from citrate, with ACL subunit A2 (ACLA2) involved in nuclear acetyl-CoA accumulation and histone acetylation .

To address this confusion, researchers should:

• Always use the full name of the protein/gene in addition to the abbreviation

• Clearly specify the organism and protein function in publications

• Include relevant database identifiers (e.g., UniProt ID: Q93841 for C. elegans ACL-1)

• Consider using more specific nomenclature in new research to avoid perpetuating confusion

What are the key considerations for designing experiments to study ACL-1 function in vivo?

When designing experiments to study ACL-1 function in vivo, researchers should consider:

How can researchers address the challenges of studying membrane-bound enzymes like ACL-1?

Studying membrane-bound enzymes like ACL-1 presents unique challenges that can be addressed through specialized approaches:

• Solubilization and purification strategies:

  • Use of specialized detergents like 6-cyclohexyl-1-hexyl-β-d-maltoside that maintain enzyme activity

  • Nanodiscs or liposome reconstitution systems to maintain a lipid environment

  • Fusion with solubility-enhancing tags or domains

• Alternative approaches to avoid solubilization:

  • Studying enzyme activity in crude membrane preparations

  • In situ activity assays in intact cells or organelles

  • Using intact cell systems with complementation approaches

• Structural studies considerations:

  • Cryo-EM approaches for membrane proteins

  • Computational modeling based on homologous structures

  • Limited proteolysis combined with mass spectrometry for domain identification

• Expression systems optimization:

  • Testing multiple expression systems (bacterial, insect cell, mammalian cell)

  • Co-expression with chaperones or stabilizing proteins

  • Optimization of growth conditions to enhance proper folding and membrane insertion

What controls and validations are essential when working with recombinant ACL-1 proteins?

When working with recombinant ACL-1 proteins, the following controls and validations are essential:

• Expression verification:

  • Western blotting to confirm expression at expected molecular weight

  • Mass spectrometry to verify protein identity

  • For labeled proteins, monitoring incorporation of [³⁵S]methionine to confirm successful translation

• Activity validation:

  • Enzyme activity assays with appropriate substrates

  • Comparison to known enzyme standards or tissue preparations

  • Substrate specificity profiles to confirm expected biochemical properties

• Experimental controls:

  • Empty vector or inactive mutant controls

  • Positive controls with well-characterized related enzymes

  • Verification of activity across different preparation batches

• Storage and stability considerations:

  • Regular testing of enzyme activity after storage

  • Optimization of buffer conditions to maintain stability

  • Addition of stabilizers such as glycerol (5-50%) for long-term storage

Table 1: Comparison of AGPAT Isoform Properties

Table 2: Experimental Approaches for ACL-1 Research